caryologia. international journal of cytology, cytosystematics and cytogenetics 74(4): 51-58, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1129 caryologia international journal of cytology, cytosystematics and cytogenetics citation: melika tabasi, masoud sheidai, fahimeh koohdar, darab hassani (2021) a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.). caryologia 74(4): 51-58. doi: 10.36253/ caryologia-1129 received: november 06, 2020 accepted: november 25, 2021 published: march 08, 2022 copyright: © 2021 melika tabasi, masoud sheidai, fahimeh koohdar, darab hassani. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) melika tabasi1, masoud sheidai1,*, fahimeh koohdar1, darab hassani2 1 department of plant sciences and biotechnology, faculty of life sciences and biotechnology, shahid beheshti university, tehran, iran 2 temperate fruits research center, horticultural science research institute, agricultural research, education and extension organizations, karaj, iran *corresponding author. e-mail: msheidai@sbu.ac.ir abstract. persian walnut (juglans regia l.), is very auspicious plant species of iran from both economical and food points of views. both wildly grown as well as cultivated forms of this plant species are scattered in different geographical regions of the country and are a valuable source for edible nut as well as job employment. scattered published data on genetic diversity of this important plant species are mainly based on different molecular data analyses; therefore a meta-analysis of the same cultivars based on several different molecular markers including dna-sequences and multi-locus markers was conducted to provide a detailed insight on genetic structure of walnuts. the results indicated a moderated genetic variability of about 40 percent in the studied cultivars; however these cultivars are genetically differentiated as revealed by fst and amova. hgt analyses revealed that the cultivars phylogeny differs to some degree by different markers and therefore a heat map was constructed to reveal the cultivars relationships based on combined molecular data. a higher pst value was obtained compared to that of fst genetic differentiation, therefore, it seems that local adaptation and selection have played role in the walnut cultivars’ morphological divergence. lfmm analysis identified some adaptive multi-locus alleles in the studied walnut cultivars. keyword: adaptive alleles, hgt, lfmm, persian walnut, pst. introduction persian walnut (juglans regia l.), is one of the main economically cultivated crop plants in iran. we have a rich walnut germplasm with extensive cultivar genetic variability (tabasi et al. 2020). both wildly grown as well as cultivated forms of this valuable plant species are scattered in different geographical regions of the country. now and then we encounter new and limited reports on genetic features some of these populations by using different molecular markers (see for example, maghsoodi et al. 2018; tabasi et al. 2020). we therefore carried out the present meta-analysis study based on a set of persian walnut cultivars which were used in previous investigation to pro52 melika tabasi et al. vide data for: 1how much genetic diversity is present in these cultivars based on combined data, 2-determine the phylogenetic relationship of these cultivars based on combined molecular data, 3-provide data on phenotypic plasticity of these cultivars. a meta-analysis is a statistical analysis that combines the results of multiple scientific studies addressing the same question. each individual study reporting measurements that are expected to have some degree of error, and therefore, the aim is to derive a pooled estimate closest to the unknown common truth based on how this error is perceived (nakaoka et al. 2009). meta-analysis is mainly a method for systematically combining pertinent qualitative and quantitative data from several selected studies to develop a single conclusion that has greater statistical power. this conclusion is statistically stronger than the analysis of any single study, due to increased numbers of subjects, greater diversit y a mong subjects, or accumu lated effects and results (greenland et al. 2008; walker et al. 2008). meta-analysis would be used for the following purposes: to establish statistical significance with studies that have conflicting results, to develop a more correct estimate of effect magnitude, to provide a more complex analysis of harms, safety data, and benefits (nakaoka et al. 2009). material and methods plant materials morphological, issr, irap, remap, scot, trnhpsba and its data of 6 populations used (table 1) are collected according to following studies: maghsoodi et al. 2018; aghaei et al. 2019 in press; tabasi et al. 2020. data analyses genetic analyses detrented correspondance analysis (dca) was performed to study the suitability of molecular markers for population genetic study as performed in past version 2.17 (podani 2000). genetic distance and fst values were obtained from published data and also determined by us for dna sequences. we used dna sequence comparison non-parametric kruskal–wallis test as performed in past, and also by dnasp program (rozas et al. 2019). horizontal gene transfer (hgt) was used to reveal the phylogenetic trees discordance as performed in trex online (boc et al. 2012). similarly, the mantel test was used to indicate association between genetic distance and geographical distance in the studied cultivars. past program was used for this purpose. a heat map was constructed based on combined genetic data by r-package. 4.2. we used lfmm program (frichot. et al. 2013) to identify multi-locus alleles with potential adaptive value. this program includes an integrated framework based on population genetics, ecological modeling and statistical techniques using latent factor mixed model. quantitative morphological characters for pst (phenotypic plasticity) analysis, we used four quantitative morphological characters in persian walnut cultivars. these characters are: 1-nut length (mm), 2-nut diameter (mm), 3-nut length/diameter ratio, and, 4-thickness of shell (mm). pst values were determined by r-package 4.2. table 1. juglans regia populations. no type name of population province locality longitude latitude altitude 1 wild nahavand hamadan nahavand 23°48’ 23°48’ 1627 2 cultivated soozani markazi tafresh 59°49’ 42°34’ 1838 3 cultivated basloghi markazi tafresh 59°49’ 42°34’ 1837 4 wild astara gilan astara 52°48’ 20°38’ -26 5 cultivated kaghazi markazi tafresh 59°49’ 42°34’ 1838 6 wild khoy west azarbaijan khoy 57°44’ 33°38’ 1135 53a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) results genetic divergence dca (dentrented correspondance analysis) plot (fig. 1) of combined molecular markers (issr, irap, remap, scot, cp-dna, and its data), produced a well scattered diagram, indicating that these molecular markers in combination can differentiate walnut populations well-enough. in maghsoodi et al. (2018) study on walnut populations, the mean nei’ genetic distance for issr data was 0.3, while the mean genetic distance based on chloroplast-dna as well as nuclear its (maghsoodi et al. 2018) sequence analysis were 0.1. the fst analysis for all genetic markers produced a significant difference among walnut cultivars (p<0.05). for example, the kruskal-wallis test for equal medians in nuclear its dna sequences as well as chloroplast dna, produced a significant difference. (p = 0.03, and p<0.001, respectively). similarly, dnasp population’ genetic differentiation test produced significant fst value for most of the pair-wise population compared (see for example table 2). the mantel test produced significant correlation (r = 0.14, p = 0.04) between genetic distance and geographical distance of the studied populations. these results indicate that an extensive genetic changes have occurred during walnut cultivar divergence. cultivar phylogeny cultivar grouping obtained by different molecular markers differed in some degree from each other. the hgt (horizontal gene transfer) analysis between chloroplast dna and combined multi locus data (issr, remap, and scot), revealed in total two hgt events due to phylogenetic dis-agreement (fig. 2). similarly, hgt analysis revealed that three hgt events occurred between cp-dna and nuclear its-dna sequences (fig. 3). the mantel test performed among chloroplast and nuclear its-dna sequences as well as combined multilocus molecular markers, after 10000 permutation, did not produce significant association between phylogenetic trees obtained from these (partial correlation r: 0.50, p = 0.14). moreover, robinson and foulds distance between phylogenetic trees produced rf =6. these results are in agreement with hgt results, and indicate that phylogenetic relationship of walnut cultivars based on different molecular markers differ to some extent, and care should be taken for drawing concrete conclusions in these types of investigations. figue 1. dca plot of walnut cultivars based on combined sequence and multi-locus data. 54 melika tabasi et al. a phylogenetic heat map constructed based on combined molecular data (fig. 4), revealed that the cultivar 1 is genetically stands far from the others due to genetic difference. genetic diversity and local adaptation the lfmm did not produce a significant association between irap and remap loci with geographical distribution of the studied walnut cultivars. however, a significant association was obtained between three issr loci as well as two scot loci with geographical features (longitude, latitude and altitude) in walnut populations (table 3, fig. 5). phenotypic plasticity versus genetic divergence we obtained a significant correlation (p<0.01) between morphological characters studied (table 4). moreover, anova revealed that the studied cultivars differ significantly in these morphological features (p = 0.01). the pst values obtained for the studied quantitative morphological characters were much higher than genetic fst value (mean = 0.25). for example, representative pst values for the nut height and width are provided in tables 4 and 5. the higher pst values compared to that of fst indicate that the studied morphological characters diverged among walnut cultivars under influence of either local environmental conditions, or due to cultivating and selection practice available in the region (table 6). table 2. pair-wise fst value for its dna sequences among walnut cultivars (the number of cultivars are according to table1). 1 2 3 4 5 6 1 — 2 0.47 — 3 0.05 0.29 — 4 0.05 0.29 1 — 5 0.24 0.30 0.43 0.43 — 6 0.04 0.08 0.09 0.09-0 0.04 — figure 2. hgt between cp dna and combined multi locus markers in walnut cultivars. figure 3. hgt tree between chloroplast and its-dna sequences in walnut cultivars. figure 4. heat map of walnut cultivars based on combined molecular data. 55a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) discussion genetic diversity and cultivars phylogeny we obtained almost close value for genetic diversity of persian walnut cultivars by both dna-sequences analysis as well as multi-locus molecular markers data. a significant genetic difference observed in both kinds of data indicates genetic differentiation of the studied cultivars which can be utilized in future breeding and hybridization projects. detailed data on genetic structure and phylogenetic relationship of economically crop plants like the persian walnut is of immediate importance for genetic conservation and breeding programs. moreover, knowledge about the effect of environmental/geographical features on genetic diversity as well as agronomic features of crop plants can improve above mentioned tasks. meta-analysis is an approach which either increase the number of observation in a particular study, or combine scattered data from several sources on a similar subject. both cases can improve insight about a particular problem or task (heidari et al. 2018). the present study revealed that a combined data analysis based on different molecular markers may improve our insight on both genetic structure as well as phylogenetic relationship of important crop plants like persian walnut. we also noticed that walnut cultivar agronomic features may be affected by both artificial as well as natural selection. a proper qtl (quantitative trait locus) study can also reveal association of important agronomic characters. the present study identified some of multi-locus molecular markers are either adaptive or are the genomic sites in the vicinity of adaptive genes. this is an important finding for qtl investigation of persian walnut. meta-analysis concerned with plant genetic studies have been performed with regard to genetic correlations between plant resistances to multiple natural enemies (leimu et al. 2006). these studies are considered important to determine the mode of selection that natural enemies impose on a host plant, the structure of herbivore and pathogen communities, all of which determine the success of plant breeding for resistance to multiple diseases and pests (leimu et al. 2006). similarly, a metaanalysis was performed to understand the genetic control of flavor in tomato cultivars (zhao et al. 2019). these authors used data of genome-wide association studies (gwas) using 775 tomato accessions and 2,316,117 snps from three gwas panels and reported several significant associations for the contents of sugars, acids, amino acids, and flavor-related volatiles. they also concluded that fruit citrate and malate contents were affected by selection during domestication and improvement. table 3. lfmm results for issr data in walnut populations. issr loci zscore -log10(p-value) p-value snp_1 0.215472 0.0812363 0.829399 snp_2 1.07686 0.550458 0.281541 snp_3 0.257655 0.0987196 0.796673 snp_4 0.640301 0.282349 0.521977 snp_5 0.090856 0.032636 0.927607 snp_6 1.69642 1.04669 0.0898066 snp_7 0.309536 0.120954 0.756914 snp_8 3.24327 2.92751 0.001181 snp_9 0.369296 0.147577 0.711907 snp_10 0.014174 0.00493936 0.988691 snp_11 0.00968666 0.00336958 0.992271 snp_12 0.651046 0.288179 0.515017 snp_13 0.162106 0.0598711 0.871222 snp_14 0.502927 0.211114 0.615015 snp_15 0.538672 0.229065 0.590113 snp_16 3.17389 2.82271 0.0015041 snp_17 0.551459 0.235586 0.581319 snp_18 2.34492 1.72054 0.019031 snp_19 0.63401 0.278953 0.526074 snp_20 0.477576 0.198629 0.632952 snp_21 2.72343 2.18971 0.00646084 snp_22 0.498368 0.208854 0.618224 snp_23 0.383171 0.153915 0.701593 snp_24 0.590575 0.255859 0.554805 snp_25 0.798828 0.372235 0.42439 snp_26 0.449796 0.185181 0.652858 snp_27 0.083641 0.0299592 0.933342 snp_28 1.82734 1.16974 0.0676484 snp_29 0.838597 0.396103 0.401695 snp_30 0.0258046 0.00903406 0.979413 snp_31 1.89246 1.23337 0.0584291 snp_32 1.44793 0.830805 0.147637 snp_33 1.45811 0.839203 0.144809 snp_34 1.38228 0.777582 0.166885 snp_35 0.0207987 0.00726703 0.983406 snp_36 0.451128 0.185821 0.651898 snp_37 1.03731 0.523472 0.299591 snp_38 1.55968 0.925052 0.118836 snp_39 0.576949 0.248741 0.563974 snp_40 1.16006 0.609022 0.246024 snp_41 0.0331206 0.0116291 0.973578 snp_42 1.06123 0.539726 0.288585 snp_43 1.23962 0.667327 0.215116 snp_44 0.928131 0.451808 0.35334 snp_45 0.906725 0.43824 0.364552 snp_46 0.908105 0.43911 0.363823 snp_47 0.643651 0.284162 0.519802 56 melika tabasi et al. in a meta-analysis with respect to plant crop cultivars genetic diversity (van de wouw et al. 2010), it was concluded that in the long run no substantial reduction in the regional diversity of crop varieties which were released by plant breeders has taken place, and a gradual narrowing of the genetic base of the varieties was not observed. genetic versus phenotypic differentiation we obtained a higher value for pst versus fst, in quantitative morphological characters of fruit among representative walnut cultivars studied. the pst is taken as index for morphological local adaptation under the influence of natural selection imposed by environment (brommer 2011). when pst = fst, it is believed that morphological divergence is due to genetic drift; but while pst > fst, it indicates the role of directional selection among the studied populations. however, a lower figure 5. lfmm manhattan plots showing three issr loci. (a) and two scot loci (b), with -log10(p) >2, which indicate association with geographical features in walnut populations. table 4. pearson’ coefficient of correlation among morphological characters studied. (characters a-d are: the nut height, nut width, ration of nut height/nut width, and the nut diameter. all values are in mm). a b c d a — 2.5062e-05 0.026767 0.31372 b 0.77027 — 1.0055e-10 0.021356 c -0.53937 -0.92392 — 0.17431 d -0.38474 -0.55061 0.42827 — bellow diagonal = r value, above diagonal = p value. table 5. pst values for the nut height among studied walnut cultivars. (the mean fst value = 0.25). 1 2 3 4 5 6 7 8 9 1 -2 0.05 — 3 0.88 0.70 — 4 0.73 0.40 0.70 — 5 0.88 0.70 0.04 0.71 — 6 0.84 0.65 0.07 0.56 0.16 — table 6. pst values for the nut width (mm) among studied walnut cultivars. (the mean fst value = 0.25). 1 2 3 4 5 6 7 8 9 1 -2 0.18 — 3 0.77 0.77 — 4 0.76 0.74 0.20 — 5 0.89 0.87 0.46 0.73 — 6 0.90 0.86 0.17 0.73 0.38 — 57a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) value of pst in contrast to fst, indicates that the same phenotypes are favored in different populations due to stabilizing selection. we may therefore, conclude that, due to some local environmental conditions/geographical coordinates, or local practices of cultivation or selection, some adaptive changes have occurred in walnut cultivars. similar studies have shown that morphological and agronomical traits divergence have been occurred in many taxa (see for example, leinonen et al. 2013; stojanova et al. 2018; caré et al. 2018). stojanova et al. (2018), found an adaptive differentiation in phenotypic traits across the climatic gradient in different populations of festuca rubber. similarly, caré et al. (2018), reported morphological differentiation in crown architecture in geographical populations of german norway spruce. they reported a high pst value (0.952–0.989) between the neighboring autochthonous and allochthonous stands of similar age in contrast to a very low neutral genetic differentiation (fst = 0.002– 0.007; g”st = 0.002–0.030) probably due to the effect of directional selection on adaptive gene loci involved in phenotypic differentiation. in conclusion, the meta-analysis based on published data on different molecular markers concerned with the same persian walnut cultivars, revealed that a combined analysis of molecular data matrix (including chloroplast and nuclear dna, as well as multi-locus markers, like issr, irap, remap, and scot markers), produce more accurate and significantly improved result for genetic diversity analysis as well as finding the cultivars’ phylogenetic relationship. data archiving statement the data that support the findings of this study are available on request from the corresponding author. the data are not publicly available due to privacy or ethical restrictions. authors’ contributions m.t: data collection and lab work, m.sh and f.k: conceptualization of the project and analyses of data, d.h: providing samples. the authors accept responsibility for releasing this material references bitton f, bauchet g, liu d, huang s, tieman d.m, klee hj, causse m (2019). meta-analysis of genome-wide association studies provides insights into genetic control of tomato flavor. nat commun 10: 1534. boc a, diallo a b, makarenkov v (2012) t-rex: a web server for inferring, validating and visualizing phylogenetic trees and networks, nucleic acids research, 40(w1), w573-w579. brommer je (2011) whither pst? the approximation of qst by pst in evolutionary and conservation biology. j. evol. biol. 24(6): 1160-1168. caré o, müller m, vornam b, höltken am, kahlert k, krutovsky kv, gailing o, leinemann l (2018) high morphological differentiation in crown architecture contrasts with low population genetic structure of german norway spruce stands. forests 9(12): 752. frichot e, schoville, sd, bouchard g, françois o (2013) testing for associations between loci and environmental gradients use latent factor mixed models. mol biol evol 30(7): 1687–1699. greenland s, o’ rourke k (2008) meta-analysis. in: rothman kj, greenland s, lash t(eds) modern epidemiology, lippincott williams and wilkins pp.652. heidari horestani m, atri roozbahani g, sheidai m (2018) the potential role of tnf-α (rs361525 and rs1800629) in hepatocellular carcinoma: multivariate analysis (meta-analysis). j. gastrointest. cancer 50:744–749 leimu r, koricheva j. a (2006) meta-analysis of genetic correlations between plant resistances to multiple enemies. am nat 168(1): e15-e37. leinonen t, mccairns rjs, o’hara rb, meril€a j (2013) qst – fst comparisons: evolutionary and ecological insights from genomic heterogeneity. nat rev genet. 14: 179–190. maghsoodi m, sheidai m, koohdar f (2018) population genetic study in juglans regia l. 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(caprifoliaceae), using scot molecular markers fengzhen chen1, dongmei li2,* , mohsen farshadfar3 the new chromosomal data and karyotypic variations in genus salvia l. (lamiaceae): dysploidy, polyploidy and symmetrical karyotypes halil erhan eroğlu1,*, esra martin2, ahmet kahraman3, elif gezer aslan4 cytogenetic survey of eight ant species from the amazon rainforest luísa antônia campos barros1, gisele amaro teixeira2, paulo castro ferreira1, rodrigo batista lod1, linda inês silveira3, frédéric petitclerc4, jérôme orivel4, hilton jeferson alves cardoso de aguiar1,5,* molecular phylogeny and morphometric analyses in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f. neda atazadeh1,*, masoud sheidai1, farideh attar2, fahimeh koohdar1 a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) melika tabasi1, masoud sheidai1,*, fahimeh koohdar1, darab hassani2 genetic diversity and relationships among glaucium (papaveraceae) species by issr markers: a high value medicinal plant lu feng1,*, fariba noedoost2 morphometric analysis and genetic diversity in rindera (boraginaceae-cynoglosseae) using sequence related amplified polymorphism xixi yao1, haodong liu2,*, maede shahiri tabarestani3 biosystematics, fingerprinting and dna barcoding study of the genus lallemantia based on scot and remap markers fahimeh koohdar*, neda aram, masoud sheidai karyotype analysis in 21 plant families from the qinghai–tibetan plateau and its evolutionary implications ning zhou1,2, ai-gen fu3, guang-yan wang1,2,*, yong-ping yang1,2,* some molecular cytogenetic markers and classical chromosomal features of spilopelia chinensis (scopoli, 1786) and tachybaptus ruficollis (pallas, 1764) in thailand isara patawang1,*, sarawut kaewsri2, sitthisak jantarat3, praween supanuam4, sarun jumrusthanasan2, alongklod tanomtong5 centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates) simona ceraulo, vanessa milioto, francesca dumas* repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil marina souza cunha1,2,*,#, silvana melo1,3,#, filipe schitini salgado1,2, cidimar estevam assis1, jorge abdala dergam1,* karyomorphology of some crocus l. taxa from uşak province in turkey aykut yilmaz*, yudum yeltekin variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l. egizia falistocco1,*,+, gianpiero marconi1,+, lorenzo raggi1, daniele rosellini1, marilena ceccarelli2, emidio albertini1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(4): 77-83, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1163 caryologia international journal of cytology, cytosystematics and cytogenetics citation: fahimeh koohdar, neda aram, masoud sheidai (2021) biosystematics, fingerprinting and dna barcoding study of the genus lallemantia based on scot and remap markers. caryologia 74(4): 77-83. doi: 10.36253/caryologia-1163 received: december 14, 2021 accepted: december 17, 2021 published: march 08, 2022 copyright: © 2021 fahimeh koohdar, neda aram, masoud sheidai. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. biosystematics, fingerprinting and dna barcoding study of the genus lallemantia based on scot and remap markers fahimeh koohdar*, neda aram, masoud sheidai department of plant sciences and biotechnology, faculty of life sciences and biotechnology, shahid beheshti university, tehran, iran *corresponding author. e-mail: f_koohdar@yahoo.com abstract. lallemantia is a medicinally important plant in the world. due to interspecific hybridization and horizontal gene transfer, spices relationship and delimitation on the genus lallemantia is difficult based on different molecular markers. therefore, selecting the appropriate marker can be important. fingerprinting techniques continue to be used for genomic profiling for the characterization of germplasm and the establishment of the identity of varieties/hybrids/parental sources of aromatic and medicinal plants. for this, we need to produce detailed information on genetic diversity available in lallemantia as well as investigate spices relationship and delimitation. therefore, the present study was performed on lallemantia species in iran. we used the start codon targeted and retrotransposon-microsatellite amplified polymorphism molecular marker for our genetic investigation with the following aims: 1to reveal the species delimitation and species relationship in lallemantia, and 2to investigate discriminating power of the start codon targeted and retrotransposon-microsatellite amplified polymorphism markers by gst and nm analysis. the results obtained revealed that the start codon targeted marker is the best to show the relationships between species while the retrotransposon-microsatellite amplified polymorphism marker is the best for species delimitation. we found the loci with the high value of gst (1.00) in start codon targeted and retrotransposon-microsatellite amplified polymorphism markers that can be used in barcoding and fingerprinting of l. royleana. keyword: lallemantia, fingerprinting, scot, rema, iran. introduction the genus lallemantia (lamiaceae) is composed of 5 species (lallemantia royleana (benth.) benth., l. canescens (l.) fisch. & c.a.mey.,l. baldschuanica gontsch., l. iberica (m.bieb.) fisch. &c.a. mey. and l. peltata(l.) fisch. & c. a. mey.) that are widely distributed in afghanistan, china, india, kazakhstan, kyrgyzstan, iran, russia, tajikistan, turkmenistan, uzbekistan and europe (sheidai et al. 2018). there are all five species in iran (rechinger 1982). lallemantia species are herbaceous with simple leaves, interrupted inflorescence, aristate-toothed bracteoles, and oblong, trigonous, smooth, and mucilaginous nutlets (harley et al. 2004). these species are well known 78 fahimeh koohdar, neda aram, masoud sheidai as a source of food and medicine plant. for example, l.ibericais used as an oil seed plant in iran and ussr (rivera-nunez and obonde-gastro 1992, dinç et al. 2009), l. royleana seeds have considerable anti bacterial properties and is a suitable remedy for skin diseases and gastrointestinal diseases and also l. peltata that grows in limited area in iran is known as medicinal plant that contains volatile and essential oil (mahmood et al. 2013). the molecular systematic study of plants is performed with different purposes like: species delimitation, population divergence, species relationships, date of divergence determination, etc (broadhurst et al., 2004; millar et al., 2011). various molecular markers have been used to perform the above tasks such as, amplified fragments length polymorphism (aflp), simple sequence repeats (ssrs), inter-simple sequence repeats (issrs), start codon targeted (scots), retrotransposon-microsatellite amplified polymorphism (remaps) etc. (e.g., sheidai et al. 2012, 2013, 2014, minaeifar et al. 2015, saboori et al. 2019). dna barcoding is a sequence of dna that can help in rapid and accurate recognition of species. it has been used in the identification of medicinal plants and has been able to detect actual and original products from its fake type (heubl et al. 2010, sheidai et al. 2018). nuclear and chloroplast dna have been examined for their suitability as barcodes through dna fingerprinting and dna sequencing-based approaches. in principle, both approaches can be used to differentiate between individuals, species, and populations and to detect the presence of adulterants. notwithstanding the increasing use of dna sequence-based approaches, fingerprinting techniques continue to be used for genomic profiling for characterization of germplasm and establishment of the identity of varieties/hybrids/parental sources of aromatic and medicinal plants (sheidai et al., 2019). among dna markers, there are ubiquitous retro elements in the plant genome like irap and remap (retrotransposon-microsatellite amplif ied polymorphism). the remap is produced by amplifying the fragments between a retrotransposon insertion site and a microsatellite site and employed in fingerprinting, linkage analysis, mapping, analysis of genome evaluation and genetic diversity. remap describe the profile of a population, discriminate between species or genotypes and analyze population diversity (kumar et al. 2010). start codon targeted (scot) polymorphisms (collard and mackill 2009) are dominant and reproducible markers based on the short conserved region flanking the atg start codon in plant genes and use a single 18-mer primer in the polymerase chain reaction (pcr) assays and high annealing temperature (50 °c). these markers could have potential in genotyping and to reveal polymorphisms that might be directly related to gene function. scot markers have been used to assess genetic diversity and structure, in bulked segregant analysis, and for quantitative trait loci (qtl) mapping and dna fingerprinting (collard and mackill 2009, luo et al. 2010). due to the morphological similarity of lallemantia species and sell them in the market as seed, the present study was performed with the following aims: 1to reveal the species delimitation and species relationship in lallemantia by scot and remap markers, and 2to investigate discriminating power of the scot and remap markers by gst and nm analysis for barcoding and fingerprinting of medicinal spices in lallemantia. material and methods plant materials extensive field investigations and collections were under taken during 2013–2015. forty-two specimens of five species, lallemantia royleana, l. canescens, l. baldschuanica gontsch., l. iberica and l. peltata were randomly collected from different geographic populations for molecular study. scot and remap assay fresh leaves were put to dry in silica gel powder. cetyltrimethyl-ammonium bromide -activated charcoal protocol (ctab) was applied to extract the genomic dna. the extraction was done by activating charcoal and poly vinyl pyrrolidone (pvp) for binding of polyphenolics during extraction; for mild extraction and precipitation conditions, the high-molecular weight dna isolation was boosted without the interference of impurities. the extracted dna was examined in terms of quality by running on 0.8% agarose (sheidai et al. 2013). three remap primer combinations, derived from one single irap primer (nikita) with 3 issr primers ((ca)7gt, (ga)9t, (ga)9c) were tested on plants samples. using a 25 µl volume containing 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany); 50 mmkcl; 10 mmtris-hcl buffer at ph;8 1.5mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 µm of each primer, polymerase chain reaction (pcr) was implemented. the following program was used for amplification of nuclear region in a pcr reaction: 5 min initial denatura79biosystematics and fingerprinting study of the genus lallemantia based on scot and remap markers tion step 94°c, followed by 40 cycles of 1 min at 94°c; 1 min at 53.5°c and 2 min at 72°c. the reaction was completed by a final extension step of 7 min at 72°c. the amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated by using a 100 bp molecular size ladder (fermentas, germany). four primers (scot1, scot2, scot36, and scot41) based on collard and mackill (2009) for monocotyledons plants were selected (collard and mackill 2009). these primer sequences are: scot1: caacaatggctaccacca, scot2: caacaatggctaccaccc , scot36: gca aca atggctaccacc and scot41: caatggctaccactgaca. pcr reaction mixture with total volume of 25 μl contained 10 mmtris-hcl buffer (ph = 8), 50 mmkcl, 1.5 mm mgcl2, 0.2 mm of dntp (bioron, germany), 0.2 μm of primer, 20 ng genomic dna and 1u of taq dna polymerase (bioron, germany). the amplification reactions were performed in techne thermocycler (germany) with the following program: 5 min at 94 ºc, 40 cycles of 1 min at 94 ºc, 1 min at 49–58 ºc (scot1 50 ºc, scot2 49 ºc, scot36 50 ºc, scot41 58 ºc) and 1 min at 72 ºc and a final cycle of 7 min at 72 ºc. the amplification products were visualized by running on 2% agarose gel, stained with syber green (powerload, kosar co. iran). the fragment size was estimated by using a 100 bp molecular size ladder (fermentas, germany). data analyses the scot and remap bands obtained were treated as binary characters and coded accordingly (presence = 1, absence = 0). the number of private bands versus common bands and genetic diversity parameters like: the percentage of allelic polymorphism, allele diversity (weising, 2005), nei’ gene diversity (he), and shannon information index (i) (weising 2005), were determined. we used genalex 6.4 for these analyses (peakall and smouse 2006). discriminating power of remap and scot markers investigated by gst and nm analysis as implemented in popgene32. grouping of the species was done by different clustering and ordination methods such as unweighted paired group using average (upgma), multidimensional scaling (mds), and principal components analysis (pca) (podani 2000). past version 2.17 (hammer et al., 2012) was used for multivariate analysis. results scot results almost all the scot primers produced bands were used and finally a data matrix of 70 × 42 was formed for further analysis. based on band pattern in lallemantia genus, the highest number of privet bands were observed in l. iberica and l. baldschuanica had the lowest value (fig. 1). genetic variation parameters were investigated in 5 species of lallemantia genus. highest level of shannon index (0.336), expected heterozygosity (0.218), and percentage of polymorphism (70.15) in l. iberica and lowest level of shannon index (0.105), expected heterozygosity (0.072) and percentage of poly morphism (17.91) were observed in l. baldschuanic species (table 1). the amova test showed significant genetic differences among lallemantia species (p = 0.001). the results show that the species of this genus have been genetically distinguished from each other using scot marker. different ordination and clustering methods like pca, mds and upgma produced similar results; therefore, only upgma plot is presented here. upgma figure 1. band patterns of scot marker in lallemantia spices. 1: l. royleana, 2: l. iberica, 3: l. canescens, 4: l. peltata, 5: l. baldschuanica. 80 fahimeh koohdar, neda aram, masoud sheidai plot of scot markers(fig. 2) grouped the specimens of l. platata, l. canescens and l. baldshuanica together in a single cluster, separated from each other but in l. iberica and l. royleana the specimens were divided in two separate clades. in this plot, l. royleana and l. baldshuanica as well as l. peltata were placed close to each other while l. iberica and l. canescens was placed far from them. remap results almost all the remap primers produced bands were used and finally a data matrix of 55 (number of bands) × 42 (number of samples) was formed for further analysis. based on band pattern in lallemantia genus, the highest number of privet bands were observed in l. iberica and l. baldschuanica had the lowest value (fig. 3). genetic variation parameters and band patterns were investigated in 5 species of lallemantia genus. highest level of shannon index (0.111), expected heterozygosity (0.073), and percentage of polymorphism (21.88%) in l. baldschuanic and lowest level of shannon index (0.017), expected heterozygosity (0.011) and percentage of poly morphism (0.012) were observed in l. royleana species (table 2). amova showed significant genetic differences among lallemantia species (p = 0.001). the amova test showed 45% inter-species diversity and 55% intra-species diversity. the results show that the species of this genus table 1. genetic diversity parameters in lallemantia species based on scot marker. species n na ne i he uhe p l. royleana 10.000 0.925 1.204 0.201 0.128 0.135 44.78% l. iberica 10.000 1.522 1.354 0.336 0.218 0.230 70.15% l. peltata 5.000 0.761 1.191 0.163 0.110 0.122 29.85% l. canescens 7.000 0.821 1.207 0.183 0.121 0.130 35.82% l. bahdchuanic 5.000 0.493 1.128 0.105 0.072 0.080 17.91% abbreviations: n = no of plants studied; na = no. of alleles; ne = effective no. of alleles; he = gene diversity; uhe = unbiased gene diversity; p = polymorphism percentage. figure 2. upgma plot of scot marker in lallemantia. r: l. royleana, ib: l.iberica, c: l. canescens, p: l. peltata, b: l. baldschuanica. 81biosystematics and fingerprinting study of the genus lallemantia based on scot and remap markers have been genetically distinguished from each other using scot marker. different ordination and clustering methods like pca, mds and upgma produced similar results; therefore, only upgma plot is presented here. upgma plot of molecular markers (fig. 4) grouped the specimens of all species together in a single cluster, separated from the other species. this means that remap molecular markers are of taxonomic value and can delimit the lallemantia species. in this plot, l. royleana and l. baldschuanica as well as l. peltata and l. canescens were placed close to each other while l. iberica was placed far from them. barcoding and fingerprinting discriminating power analysis of molecular markers is important for fingerprinting and barcoding. for this purpose, gst and nm parameters was measured. the value of gst in seven loci in scot and 13loci in rema marker in lallemantia was 1 .00 while the mean nm value was 0.00 which indicated that these markers can be used in l. royleana differentiation of other species. comparison of scot and remap markers in this study, scot marker was able to separate only three species of five species, while remap marker was able to demonstrate the boundary between any five species (figs 1 and 2). according to amova result, scot marker was more successful in showing intraspecific diversity. discussion the present study revealed that species delimitation based on remap markers was more successful than scot marker. however, this marker was more successful in showing intraspecific diversity. these results are consisting with previous findings (al-qurainy et al. 2015, saboori et al. 2019). l. baldschuanica and l. royleana as well as l. canescens and l. iberica are similar to each other based on morphological and micro morphological (nutlet and pollen structure) studies (talebi and rezakhanlou 2010; kamrani et al. 2018). lamiaceae family has many phylogenetically unresolved genera and therefore many species are of not determined relationship due to the conf lict between molecular data and potential inter-specific hybridization as well as horizontal gene transfer. sheidai et al. 2018, revealed that the relationships between lallemantia species based on cpdna, its and issr molecular markers differed from morphological and micromorphological as well as to each other due to inter-specific hybridization and horizontal gene transfer. table 2. genetic diversity parameters in lallemantia species based on remap marker. species n na ne i he uhe p l. royleana 5.000 0.594 1.017 0.017 0.011 0.012 3.13% l. iberica 5.000 0.656 1.075 0.075 0.048 0.054 15.63% l. canescens 5.000 0.656 1.036 0.053 0.030 0.033 15.63% l. peltata 5.000 0.594 1.109 0.090 0.061 0.068 15.63% l. baldschuanic 5.000 0.719 1.123 0.111 0.073 0.081 21.88% abbreviations: n = no of plants studied; na = no. of alleles; ne = effective no. of alleles; he = gene diversity; uhe = unbiased gene diversity; p = polymorphism percentage. figure 3. band patterns of remap marker in lallemantia spices. 1: l. royleana, 2: l.iberica, 3: l. canescens, 4: l. peltata, 5: l. baldschuanica. 82 fahimeh koohdar, neda aram, masoud sheidai our result suggested conf lict between scot and remap marker in relationship of lallemantia species but in both markers, l. baldschuanica and l. royleana were placed to each other. the scot result was 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(lamiaceae): dysploidy, polyploidy and symmetrical karyotypes halil erhan eroğlu1,*, esra martin2, ahmet kahraman3, elif gezer aslan4 cytogenetic survey of eight ant species from the amazon rainforest luísa antônia campos barros1, gisele amaro teixeira2, paulo castro ferreira1, rodrigo batista lod1, linda inês silveira3, frédéric petitclerc4, jérôme orivel4, hilton jeferson alves cardoso de aguiar1,5,* molecular phylogeny and morphometric analyses in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f. neda atazadeh1,*, masoud sheidai1, farideh attar2, fahimeh koohdar1 a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) melika tabasi1, masoud sheidai1,*, fahimeh koohdar1, darab hassani2 genetic diversity and relationships among glaucium (papaveraceae) species by issr markers: a high value medicinal plant lu feng1,*, fariba noedoost2 morphometric analysis and genetic diversity in rindera (boraginaceae-cynoglosseae) using sequence related amplified polymorphism xixi yao1, haodong liu2,*, maede shahiri tabarestani3 biosystematics, fingerprinting and dna barcoding study of the genus lallemantia based on scot and remap markers fahimeh koohdar*, neda aram, masoud sheidai karyotype analysis in 21 plant families from the qinghai–tibetan plateau and its evolutionary implications ning zhou1,2, ai-gen fu3, guang-yan wang1,2,*, yong-ping yang1,2,* some molecular cytogenetic markers and classical chromosomal features of spilopelia chinensis (scopoli, 1786) and tachybaptus ruficollis (pallas, 1764) in thailand isara patawang1,*, sarawut kaewsri2, sitthisak jantarat3, praween supanuam4, sarun jumrusthanasan2, alongklod tanomtong5 centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates) simona ceraulo, vanessa milioto, francesca dumas* repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil marina souza cunha1,2,*,#, silvana melo1,3,#, filipe schitini salgado1,2, cidimar estevam assis1, jorge abdala dergam1,* karyomorphology of some crocus l. taxa from uşak province in turkey aykut yilmaz*, yudum yeltekin variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l. egizia falistocco1,*,+, gianpiero marconi1,+, lorenzo raggi1, daniele rosellini1, marilena ceccarelli2, emidio albertini1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(3): 91-97, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1082 caryologia international journal of cytology, cytosystematics and cytogenetics citation: ciler kartal, nuran ekici, almina kargacıoğlu, hazal nurcan ağırman (2021) development of female gametophyte in gladiolus italicus miller (iridaceae). caryologia 74(3): 91-97. doi: 10.36253/caryologia-1082 received: september 24, 2020 accepted: july 20, 2021 published: december 21, 2021 copyright: © 2021 ciler kartal, nuran ekici, almina kargacıoğlu, hazal nurcan ağırman. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid ne: 0000-0003-2005-7293 çk: 0000-0002-8621-7889 development of female gametophyte in gladiolus italicus miller (iridaceae) ciler kartal1, nuran ekici2,*, almina kargacıoğlu1, hazal nurcan ağırman1 1department of biology, faculty of science, trakya university, edirne, 22030, turkey 2department of science education, faculty of education, trakya university, edirne, 22030, turkey *corresponding author. e-mail : nuranekici@yahoo.com abstract. in this study gynoecium, megasporogenesis, megagametogenesis and female gametophyte of gladiolus italicus miller were examined cytologically and histologically by using light microscopy techniques. ovules of g. italicus are of anatropous, bitegmic and crassinucellate type. embryo sac development is of monosporic polygonum type. polar nuclei fuse before fertilization to form a secondary nucleus near the antipodals. the female gametophyte development of g. italicus was investigated for the first time with this study. keywords: gladiolus italicus, iridaceae, embryo sac, polygonum type. introduction iridaceae family is represented by approximately 66 genera and 2245 species in the world (christenhusz and byng 2016; burgt et al. 2019). this family includes ornamentals like gladiolus, belamcanda, iris, crocus, eleutherine, etc. and also plants of commercial value like crocus sativa and lris spp. (venkateswarlu et al.1980). genus gladiolus l. has more than 260 species (goldblatt 1996). 11 gladiolus species are found in various regions of turkey and 6 of them; g. anatolicus (boiss.) stapf, g. attilae kit tan, b. mathew & a. baytop, g. halophilus boiss. & heldr., g. humilis stapf, g. micranthus stapf, g. osmaniyensis sağıroğlu) are endemic (tan et al. 2006; güner et al. 2012; sağıroğlu and akgül 2014). gladiolus italicus is in iucn red list category of turkey. g. italicus is distributed in macaronesia, mediterranean basin to central asia. it is also introduced and naturalized in california. it naturally grows in many parts of turkey (demir and çelikel 2019). it is a monocotyledon with spectacular flowers (tan and edmondson 1984). gladiolus corms are used in the treatment of dysentery and gonorrhea in some countries of africa (nguedia et al. 2004). in turkey, it is known as an aphrodisiac and it is known to have emetic property (baytop 1999). g. italicus and g. atroviolaceus corms are used in ice cream and also in other 92 ciler kartal et al. dairy foods (öztürk and özçelik 1991). the chemical composition of gladiolus plants is studied. demeshko et al. (2020) studied carboxylic acid content of g. hybridus leaves. the chemical composition of the essential oil and the antibacterial, antifungal and antioxidant properties of the essential oil extract of g. italicus are investigated by üçüncü et al. (2016). the seed testa structure of g. italicus is studied with scanning electron microscope by erol et al. (2006). üzen (1999) studied g. italicus morphologically and anatomically. it is also studied karyologically and cytologically. the chromosome numbers of the species are resulted 2n=30, 60 (iran) and 2n=120 (aegean islands and spain) in several populations (perez and pastor 1994; kamari et al. 2001; fakhraei et al. 2011). in addition, chromosome numbers such as 2n = 60, 90, 110-120, 120, ~170, 176 are reported by other researchers (ohri and khoshoo 1985; van raamsdonk and de vries 1989). mensinkai (1939) reported that cytomixis was observed in meiosis in a study of four gladiolus species (g. tristis, g. byzantinus, g. primulinus, and g. dracoce) mitosis and meiosis divisions. the pollen morphology of g. italicus is examined using light and scanning electron microscopy by dönmez and işık (2008). they described the pollen grains of g. italicus as monosulcate, heteropolar, elliptic, spinulose-perforate and tectate-collumellate (dönmez and işık 2008). embryological studies in family iridaceae are rather limited. studies about the development of the embryo sac are reported by davis (1966) in iris japonica and i. tenax. then other studies are done in sisyrinchium striatum and s. californicum by lakshmanan and philip (1971) and crocus sativus and c. thomasii by chichiricco (1987, 1989). the aim of this study is to determine the development of female gametophyte in g. italicus. cytological and embryological features of g. italicus have not been studied yet. this study is also an attempt toward a better understanding of taxonomic relationships between closely related taxa within the iridaceae and are indirectly useful to the efforts to protect this species in vitro. materials and methods in this study, g. italicus plants were collected from höyüklütatar village of edirne a1 (e) in european turkey. they were brought to the botanical garden of trakya university. voucher specimens were placed in the herbarium of trakya university (edtu). ovaries were examined under an olympus sz61 stereomicroscope. for cyto-histological studies, flowers and buds were fixed in carnoy’s fluid (3:1, ethyl alcohol: acetic acid). dehydration process was done with increasing alcohol series for 24 hours (70%, 80%, 90%, 96%, absolute alcohol). then, the material was kept in a mixture of 1 absolute alcohol: 1 basic resin + activator for 24 hours. after being kept in pure resin for 24 hours, the next day, they were embedded in historesin (leica, historesinembedding kit), which was prepared by adding hardener to the basic resin and activator mixture in an appropriate ratio according to manufacturer’s protocols (leica microsystems, nussloch). semi-thin (2 µm) sections taken from the materials embedded in historesin with leica rm2255 rotary microtome and stained with 1% toluidine blue (o’brien et al. 1964). slides were examined with an olympus cx31 microscope and photographed by progress c12 camera. results gynoecium gynoecium of  gladiolus italicus, contains a pistil  with inferior ovary, a long style and a three-lobed, spatulate stigma (figure 1). g. italicus has a trilocular, syncarpous ovary. in the ovary 22-24 ovules are marginal-central placented (figure 2). megasporangium ovules of g. italicus are anatropous, crassinucellate and bitegmic. the outer integument consisted of 5-6 cell layers and the inner integument consisted of 2 cell lines. the micropyle is formed by the inner integument. the inner and outer integuments are fiveto seven-layered around the micropyle (figure 3a). megasporogenesis one of the sub-epidermal cells at the tip of the ovule of g. italicus differentiates to form a megaspore mother cell (mmc). the mmc cell forms deep within the nucellus. it has a large volume and larger nucleus and is easily distinguishable from other cells (figure 3b). as the outer integument become apparent, the first meiotic division begins in the mmc (figure 3c). the volume of the mmc increases during meiosis (figure 3d). a dividing wall is formed between the two nuclei after meiosis i. after a short period, also meiosis ii is completed. a linear megaspore tetrad forms as a result of meiosis of the mmc (figure 3e). 93megasporogenesis and megagametogenesis in gladiolus italicus megagametogenesis after megasporogenesis, atrophy of the three megaspores on the micropylar side occurred. then, the active megaspore on the chalazal side began mitosis. when the active megaspore divided into two nuclei at the end of the first mitosis, they moved towards the opposite poles and a large central vacuole was formed between them (figure 3f). the nuclei in the poles enters in the second mitosis and an embryo sac with 4 nuclei is formed (figure 3g). after the third mitosis, there are eight nuclei in the embryo sac. female gametophyte the embryo sac of g. italicus is of the polygonum type. it has eight nuclei and seven cells. it shows a clear polarization. the mature embryo sac contains an egg apparatus, three antipodal cells and a central cell with two polar nuclei. the polar nuclei fuse before fertilization and form a secondary nucleus near the antipodal cells. antipodal cells antipodal cells are haploid. they have densely stained cytoplasm and evident, large nucleolus (figure 3h). they are surrounded by whole cell wall. their chalazal sides are embedded within the hypostasis. (figure 3i). central cell the central cell is located in the middle of the embryo sac, initially contains a polar nucleus on the micropylar side and a polar nucleus near the antipodal cells on the chalazal side. the nucleus on the micropylar side moves to the side of the polar nucleus, which is located close to the chalazal side (figure 3j). these two polar nuclei fuse before fertilization and they form the secondary nucleus near the antipodal cells. (figure 3k). egg apparatus the egg apparatus is located on the micropylar side of the embryo sac. it consists of two synergid cells and an egg cell. the synergid cells form the filiform apparatus with their walls towards the micropylar side. the filiform apparatus enlarges the wall surface of the synergid cells. this facilitates nutrient and water intake. one of the synergid cells begins to degenerate before fertilizafigure 2. cross section of gladiolus italicus’ ovary (ov, ovule; ow, ovary wall). figure 1. pistil and stamens of gladiolus italicus. (ov, ovary; sg, stigma; st, stamens; sy, style). 94 ciler kartal et al. tion and a vacuole is formed (figure 3l). the egg cell is located between the two synergid cells in the embryo sac (figure 3m). hypostasis in the advanced stages of embryo sac development, tissue differentiation is observed on the chalazal side, in g. italicus. this tissue is named as hypostasis and it differentiates from the tissue in the nucellus between integuments and the chalaza. the hypostasis in the embryo sac of g. italicus consists of cells with thickened walls and it is cup-shaped (figure 3n). discussion in this study, the developmental stages of the embryo sac in g. italicus is presented for the first time by using light microscopy techniques. g. italicus shows the characteristics of the iridaceae family in terms of ovules and embryo sac development (davis 1966; lakshmanan and philip 1971; chichiricco 1987; 1989; zhang et al. 2011). 22-24 ovules are located in the inferior, trilocular ovary in g. italicus. they are marginal-central placented. zhang et al. (2011) reported that iris mandshurica had also inferior, trilocular ovary, but its ovules were axial placented. ovules of g. italicus are anatropous, bitegminous and crassnucellate like iris mandshurica. in g. italicus, 5-6 cell lines formes the outer integument and 2 cell lines formes the inner integument. the micropyle is formed by the inner integument in both g. italicus and iris mandshurica (zhang et al. 2011). similar features are observed in sisyrinchium striatum and s. californicum (lakshmanan and philip 1971), crocus sativus (chichiricco 1987), c. thomasii figure 3. female gametophyte in gladiolus italicus and its developmental stages; 3a, ovule of g. italicus; 3b, crassinucellate ovule of g. italicus; 3c, bitegmic ovule of g. italicus; 3d, prophase i phase of meiosis in megaspore mother cell in g. italicus; 3e, g. italicus’ linear type of megaspore tetrad; 3f, two-nucleate embryo sac in g. italicus; 3g, four-nucleate embryo sac in g. italicus; 3h; antipodal cells of g. italicus; 3i, densely stained cytoplasm and nucleoli in the antipodal cells of g. italicus; 3j, polar nuclei of g. italicus; 3k, secondary nucleus of g. italicus; 3l; synergid cells of g. italicus; 3m, egg apparatus of g. italicus; 3n, hypostase in g. italicus. (a, antipodal cell; c, chalaza; dm, degenerated megaspores (arrows); ea, egg apparatus; ec, egg cell; f, funiculus; fa, filiform apparatus; fm, functional megaspore (arrow); h, hypostase; ii, inner integument; mmc, megaspor mother cell; m, micropyle; n, nucleus; nu, nucleolus; nuc, nucellus; oi, outer integument; pn, polar nucleus; s, synergid; sn, secondary nucleus; v, vacuole). 95megasporogenesis and megagametogenesis in gladiolus italicus (chichiricco 1989). in previous studies, it was reported that both outer and inner integuments were formed by 2 cell lines in sisyrinchium striatum and s. californicum (lakshmanan and philip 1971). the inner integument is formed by 2 cell lines in crocus thomasii (chichiricco g. 1989). the outer integument is formed by 6-8 layers and the inner integument was formed by 4-6 cell lines in iris mandshurica (zhang et al. 2011). these findings showed that g. italicus had characteristics of the iridaceae family in terms of ovule type and development. it is also closer to crocus and sisyrinchium genera in terms of number of cell lines of the inner integument. one of the subepidermal cells in the ovule of g. italicus differentiates from others to form the megaspore mother cell (mmc). the mmc appears below the nucellus. when the outer integument initiated, the first meiosis was taking place. a cell wall is formed between the two nuclei after meiosis i. after a short rest period, meiosis ii is completed. linear megaspore tetrad occurs as a result of meiosis. megasporogenesis is of the polygonum type. in this kind of embryo sac, in the beginning of megagametogenesis, 3 micropylar megaspores are degenerated. the degeneration of the three supernumerary meiotic products is hence a case of developmental programmed cell death (pcd) and it is of interest to investigate its features with the aim of checking resemblance to apoptotic morphological syndrome as described in general and as described for plants (papini et al. 2011). tem observations on the degenerating nonfunctional megaspores in larix leptolepis (sieb. et. zucc.) gordon (pineaceae) showed morphological features that are typical of pcd (cecchi fiordi et al. 2002). then, megasporogenesis and pcd in tillandsia (bromeliaceae) were studied in a comprehensive study by papini et al. (2011). the general shrinkage of the cell protoplast and the condensation of the cytoplasm and particularly of the nucleus observed in the degenerating supernumerary megaspores are signs of pcd (pennell and lamb 1997). the pcd of the supernumerary megaspores in angiosperms is a deletional pcd, since the developmental program leading to the female gametophyte formation and maturation implies their disappearance (papini et al., 2011). the chalazal megaspore becomes functional and mature embryo sac is formed after three sequential mitosis. similar findings have been observed in the previously studied species; sisyrinchium striatum, s. californicum (lakshmanan and philip 1971) crocus sativus (chichiricco 1987), c. thomasii (chichiricco 1989), and iris mandshurica (zhang et al. 2011). a large number of tissue remodeling occurs during the seed development, with some of the cells being eliminated as a result of pcd. synergids die during double fertilization (doronina et al. 2020) vacuolization is one of the morphological patterns accompanying pcd in synergids. in nicotiana tabacum (tian and russell, 1997), cytoplasmic vacuolization, in proboscidea louisianica (mogensen, 1978), penniseturn glaueum (chaubal and reger, 1993), nicotiana tabacum (huang and russel, 1994), helleborus bocconei (bartoli et al., 2017) vacuole rupture were seen in synergid cells. in g. italicus, vacuolization is also occurred in one of the synergids due to early stage of fertilization. in g. italicus, bowl-like hypostasis with thickened walls is seen and it is densely stained. in some plants, although the walls of the hypostasis are thickened due to substances such as cutin, suberin and lignin, in some plants they remain thin walled. they have a secretory cell structure (johri et al. 1992). it is reported that thickened walled hypostasis was seen in crocus sativus (chichiricco 1987) and c. thomasii (chichiricco 1989). in leucojum aestivum (amaryllidaceae), hypostasis cells are thin-walled and have abundant cytoplasm (ekici and dane 2008). there are no reports on hypostasis in sisyrinchium striatum, s. californicum (lakshmanan and philip 1971) and iris mandshurica (zhang et al. 2011). ünal (2011) reported that hypostasis developing from nucellar cells beneath the embryo sac plays a role in preventing embryo growth. they also deliver nutrients from the vascular bundles to the embryo sac. in some taxa they play a role in maintaining the water balance. in the light of these findings, it is seen that g. italicus is close to genus crocus in terms of hypostasis. conclusion in conclusion, the ovule and the development of female gametophyte of g. italicus were studied for the first time and it was seen that the findings obtained from this study were compatible with the previously examined species belonging to the iridaceae family. pcd occurred when functional megaspore formed at the end of megasprogenesis. it has also occurred in the degeneration of synergid cells. g. italicus showed characters of the iridaceae family in terms of female gametophyte development. data gained from this study will also contribute to the general knowledge about the embryological characters used in the taxonomy of iridaceae family. acknowledgement this study was supported by trakya university scientific research projects coordination unit. project number: tubap-2019/27 96 ciler kartal et al. references bartoli g, felici c, castiglione m.r. 2017. female gametophyte and embryo development in helleborus bocconei ten. 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(iridaceae) and its systematic relation. plant syst. evol. 293:43-52. caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 3 2021 firenze university press chromomycin a3 banding and chromosomal mapping of 45s and 5s ribosomal rna genes in bottle gourd ahmet l. tek*, hümeyra yıldız, kamran khan, bilge ş. yıldırım development of a protocol for genetic transformation of malus spp federico martinelli1,*, anna perrone2, abhaya m. dandekar3 cytogenetic and cytological analysis of colombian cape gooseberry genetic material for breeding purposes viviana franco-florez, sara alejandra liberato guío, erika sánchez-betancourt, francy liliana garcía-arias, víctor manuel núñez zarantes* palynological analysis of genus geranium (geraniaceae) and its systematic implications using scanning electron microscopy jun wang1,2,*, qiang ye1, chu wang2, tong zhang2, xusheng shi2, majid khayatnezhad3, abdul shakoor4,5 influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats yuri a. kirillov1, maria a. kozlova1, lyudmila a. makartseva1, igor a. chernov2, evgeniya v. shtemplevskaya1, david a. areshidze1,* the morphological, karyological and phylogenetic analyses of three artemisia l. (asteraceae) species that around the van lake in turkey pelin yilmaz sancar1,*, semsettin civelek1, murat kursat2 molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant jinxin cheng1,*, dingyu hu1, yaran liu2, zetian zhang1, majid khayatnezhad3 genetic variations and interspesific relationships in salvia (lamiaceae) using scot molecular markers songpo liu1, yuxuan wang1, yuwei song1,*, majid khayatnezhad2, amir abbas minaeifar3 development of female gametophyte in gladiolus italicus miller (iridaceae) ciler kartal1, nuran ekici2,*, almina kargacıoğlu1, hazal nurcan ağırman1 colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae) harshita dwivedi*, girjesh kumar statistical evaluation of chromosomes of some lathyrus l. taxa from turkey hasan genç1, bekir yildirim2,*, mikail açar3, tolga çetin4 use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater treatment and water of the kamniška bistrica river (slovenia) peter firbas1, tomaž amon2 the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bo shi1,*, majid khayatnezhad2, abdul shakoor3,4 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(2): 5-13, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1314 caryologia international journal of cytology, cytosystematics and cytogenetics citation: piyaporn saensouk, surapon saensouk, rattanavalee senavongse (2022) cytogenetic studies of six species in family araceae from thailand. caryologia 75(2): 5-13. doi: 10.36253/caryologia-1314 received: may 14, 2021 accepted: may 20, 2022 published: september 21, 2022 copyright: © 2022 piyaporn saensouk, surapon saensouk, rattanavalee senavongse. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. cytogenetic studies of six species in family araceae from thailand piyaporn saensouk1, surapon saensouk2,*, rattanavalee senavongse2 1 plant and invertebrate taxonomy and its applications unit group, department of biology, faculty of science, mahasarakham university, kantharawichai, maha sarakham, 44150, thailand 2 plant and invertebrate taxonomy and its applications unit group, biodiversity program, walairukhavejbotanical research institute, mahasarakham university, kantarawichai, maha sarakham, 44150, thailand *corresponding author. e-mail: surapon.s@msu.ac.th abstract. the chromosome numbers and karyotypes of six species belonging to five genera of the family araceae distributed in natural areas from thailand were analyzed. these species are aglaonema modestum schott ex engl., ag. simplex (blume) blume, amorphophallus serrulatus hett. & a. galloway, arisaema maxwellii hett. & gusman, hapaline benthamiana schott and homalonema griffithii (schott) hook.f. three of them (ag. simplex, am. serrulatus and ar. maxwellii) are not common and endemic species in thailand. the chromosome number and karyotypes of all species were determined as ag. modestum 2n = 40 with karyotype to be 20m + 14sm + 6st, ag. simplex 2n = 42 with karyotype to be 6m + 26sm + 10st, am. serrulatus 2n = 26 with karyotype to be 12m + 6sm + 8st and two satellite chromosomes, ar. maxwellii 2n = 24 with karyotype to be 22m + 2sm and two satellite chromosomes, ha. benthamiana 2n = 26 with karyotype to be 26m and one satellite chromosome and ho. griffithii 2n = 40 with karyotype to be 30m + 10sm and two satellite chromosomes. karyotype analysis indicated that six species of family araceae generally have metacentric, submetacentric and subtelocentric chromosomes. in addition, satellites were observed in am. serrulatus, ar. maxwellii, ha. benthamiana and ho. griffithii. the chromosome numbers from three species of them and karyotypes from five species of them in this study were determined for the first time in this study. these cytogenetic studies of this report can be used for classification of six species in the family araceae. keywords: araceae, chromosome number, first report, karyotype, satellite, thailand. introduction family araceae is the largest primitive monocot family and the most diverse (grayum 1990; boyce et al. 2012; nauheimer et al. 2012). currently, 120 genera and more than 3,800 species are recognized in worldwide. this family is distributed in tropical regions of north and south america, asia and throughout tropical western pacific and eastern australia, tropical africa, temperate eurasia, southern africa, madagascar and seychelles (mayo et 6 piyaporn saensouk, surapon saensouk, rattanavalee senavongse al.1997; boyce et al. 2012). in thailand, 30 genera with about 210 species of family araceae are reported by boyce et al. (2012). the traditionally uses of this family are found to be local food, ornamental plants and medicinal plants (boyce et al. 2012). the status of cytogenetic data is vital for taxonomic decision-making and biosystematic investigations (stace 2000). classical cytological techniques and fundamentals are still an excellent starting point for basic plant cy togenetic studies. the chromosome numbers and chromosome characteristics can be used to identify the genetic diversity and phylogenetic information (jahier 1996; stace 2000; guerra 2008; figueroa and bass 2010). a few years ago, the authors collected specimens of the family araceae in thailand for study diversity and the chromosomes and found that the not common six species in this study – ag. modestum schott ex engl., ag. simplex (blume) blume, am. serrulatus hett. & a. galloway, ar. maxwellii hett. & gusman, ar. maxwellii hett. & gusman, ha. benthamiana schott, and ho. griffithii (schott) hook.f. were reported to use for daily life in the thai communities. ag. modestum schott ex engl. is used as an ornamental and medicinal plant (boyce et al. 2012). ag. simplex (blume) blume is reported conservation status from the iucn red list of threatened species to be a least concern (lc) species (allen 2011). am. serrulatus hett. & a. galloway is an endemic species in thailand (boyce et al. 2012). ar. maxwellii hett. & gusman is reported conservation status in iucn red list to be  vulnerable species (vu) and it is reported to be a rare species from thailand which is presented in the book title threatened plants in thailand (chamchumroon et al. 2017). ha. benthamiana schott is used as traditional food in thailand (boyce et al. 2012). ho. griffithii (schott) hook.f. is mainly distributed in the south of thailand and used as an ornamental plant (boyce et al. 2012). the chromosome numbers and karyotypes of few species from family araceae was studied by few botanists – larsen 1969; okada 1982; okada 2000; chen et al. 2003; eksomtramage et al. 2007; liu et al. 2010; senavongse et al. 2018; saensouk et al. 2019 and senavongse et al. 2020. this study is expected to find more new information of plant chromosome. therefore, the aims of this study are to study the chromosome numbers and karyotypes of the six species of araceae in thailand. materials and methods six species of araceae in thailand, namely ag. modestum schott ex engl. (coll. no. r. senavongse 001/2016), ag. simplex (blume) blume (coll. no. r. senavongse 006/2016), am. serrulatus hett. & a. galloway (coll. no. r. senavongse 031/2016), ar. maxwellii hett. & gusman (coll. no. r. senavongse 036/2016), ha. benthamiana schott (coll. no. r. senavongse 056/2016) and ho. griffithii (schott) hook.f. (coll. no. r. senavongse 061/2016) were collected from the field in thailand (table 1) and voucher specimens were deposited in the mahasarakham university herbarium (msu) all fresh specimens were grown in a nursery at the walai rukhavej botanical research institute, mahasarakham university, maha sarakham province, thailand. the chromosome numbers study follows saensouk et al. (2019) and senavongse et al. (2018, 2020). the nomenclature of the chromosome morphology follows the classification of levan et al. (1964). the classification of the karyotype symmetry degree is proposed by following stebbins (1971). the diploid chromosome numbers of each species in this study are counted from 20 cells. for the arrangement of the karyotypes the following parameters such as average length of the short arm (ls), average length of the long arm (ll), length of each chromosome (lt), average measurement of relative length (rl), chromosome index (ci), standard deviation (sd) of rl and ci from metaphase chromosomes were calculated by methods of saensouk et al. (2019) and senavongse et al. (2018, 2020). result and discussion the chromosome number, chromosome length range, haploid chromosome length, arm ratio, relative length, and karyotype formula were determined from 20 metaphase cells in each species. the diploid chromosome numbers in this study are reported as 24–42 chromosomes (table 1; figure 1). the characteristics of karyotype of examined six species in family araceae from thailand is are given below (table 1; figure 2). the somatic chromosome number of ag. modestum was found to be 2n = 40 and fundamental number (nf) = 80 (figure 1(a)). the karyotype formula was 20m + 14sm + 6st including 10 pairs including metacentric chromosomes, seven pairs of submetacentric chromosomes and three pairs of subtelocentric chromosomes. it has an asymmetrical karyotype due to the karyotype formula ratio (symmetrical karyotype comprise of metacentric and submetacentric chromosome and asymmetrical karyotype comprise of metacentric, submetacentric, subtelocentric and telocentric chromosome). the short arm chromosome length ranged from 1.01±0.01 to 4.72±0.02 μm, the long arm chromosome length ranged from 2.13±0.01 to 5.07±0.03 μm, the 7cytogenetic studies of six species in family araceae from thailand total arm chromosome length ranged from 3.14±0.02 to 9.79±0.05 μm. relative lengths were 2.50–7.79 %. centromeric indexes were 0.53–0.77 (table 2; figure 2(a)). the somatic chromosome number of ag. modestum in this study differs from earlier reported (chen et al. 2003 2n = 60; eksomtramage et al. 2007, 2n = 80). whereas, the somatic chromosome number of this species in this study was corresponding to the previous reported by liu et al. (2010) and they reported the karyotype to be 22m + 18sm, which differs from this study (table 1). table 1. summarizes of the cytogenetic reviews of studies in six species of the araceae family in thailand. species localities 2n karyotype formulas previous recorded 2n karyotype formulas author ag. modestum loei 40 20m+14s+ 6st 60 80 40 22m+18sm chen et al. (2003) eksomtramage et al. (2007) liu et al. (2010) ag. simplex kalasin 42** 6m+26sm+10st 42 larsen (1969) am. serrulatus ubon ratchathani 26*,** 12m+6sm+8st ar. maxwellii changmai 24*,** 22m+2sm ha. benthamiana chaiyaphum 26** 26m 13 26 larsen (1969) ho. griffithii songkhla 40** 30m+10sm 40 okada (1982) okada (2000) * = new report chromosome numbers for the first time. ** = new report karyotype for the first time. figure 1. microphotographs of somatic metaphase plate of ag. modestum (a), ag. simplex (arrow = non chromosome) (b), am. serrulatus (c), ar. maxwellii (d), ha. benthamiana (e) and ho. griffithii (f). scale bars = 10 µm. 8 piyaporn saensouk, surapon saensouk, rattanavalee senavongse the somatic chromosome number of ag. simplex were 2n = 42 and nf = 84 (figure 1(b)). the karyotype formula was 6m + 26sm + 10st including 21 pairs, which comprised three pairs of metacentric chromosomes, 13 pairs of submetacentric chromosomes and five pairs of subtelocentric chromosomes. it has an asymmetrical karyotype due to the karyotype formula ratio. the short arm chromosome length ranged from 0.83±0.00 to 2.97±0.01 μm, the long arm chromosome length ranged from 2.80±0.01 to 7.50±0.03 μm, the total arm chromosome length ranged from 3.63±0.02 to 10.48±0.055 μm. relative lengths were 2.21–6.37 %. centromeric indexes were 0.51–0.75 (table 3; figure 2(b)). the somatic chromosome number of this species in this study was corresponding to the previous reported by larsen (1969). the karyotype of ag.simplex in this study is reported for the first time. the somatic chromosome number of am. serrulatus were found as 2n = 26 and nf = 52 (figure 1(c)). the karyotype formula was asymmetrical karyotype due to figure 2. karyotypes by conventional staining. ag. modestum (a), ag. simplex (b), am. serrulatus (c), ar. maxwellii (d), ha. benthamiana (e) and ho. griffithii (f). arrows in c-f indicate satellite. scale bar = 10µm. 9cytogenetic studies of six species in family araceae from thailand table 2. mean length of short arm chromosome (ls), long arm chromosome (ll), total arm chromosome (lt), relative length (rl), centromeric index (ci) and standard deviation (sd) of rl, ci from 20 metaphases of ag. modestum (2n=40). chromosome pair ls±sd (µm) ll±sd (µm) lt±sd (µm) rl (%) ci chromosome type 1 4.72±0.02 5.07±0.03 9.79±0.05 7.79 0.53 metacentric 2 3.72±0.01 4.96±0.03 8.68±0.04 6.91 0.58 metacentric 3 4.12±0.02 4.39±0.03 8.51±0.04 6.78 0.56 metacentric 4 1.70±0.01 5.92±0.03 7.62±0.04 6.06 0.77 subtelocentric 5 1.96±0.01 4.89±0.03 6.85±0.04 5.45 0.69 submetacentric 6 3.10±0.01 3.59±0.02 6.68±0.04 5.32 0.54 metacentric 7 2.71±0.01 3.84±0.02 6.55±0.04 5.22 0.58 metacentric 8 2.49±0.01 3.89±0.03 6.38±0.03 5.08 0.56 metacentric 9 3.03±0.02 3.32±0.02 6.35±0.03 5.06 0.59 metacentric 10 2.52±0.01 3.64±0.02 6.16±0.03 4.90 0.57 metacentric 11 1.46±0.01 4.68±0.02 6.14±0.03 4.89 0.72 subtelocentric 12 1.78±0.01 4.32±0.02 6.11±0.03 4.86 0.74 subtelocentric 13 1.98±0.01 4.12±0.02 6.10±0.03 4.86 0.59 metacentric 14 2.23±0.01 3.70±0.02 5.94±0.03 4.73 0.62 submetacentric 15 2.29±0.01 3.46±0.02 5.75±0.03 4.58 0.62 submetacentric 16 2.15±0.01 3.09±0.02 5.23±0.03 4.17 0.63 submetacentric 17 2.13±0.01 3.00±0.02 5.13±0.03 4.08 0.61 submetacentric 18 2.10±0.01 2.50±0.01 4.60±0.02 3.66 0.60 submetacentric 19 1.33±0.01 2.59±0.01 3.92±0.02 3.12 0.61 submetacentric 20 1.01±0.01 2.13±0.01 3.14±0.02 2.50 0.59 metacentric table 3. mean length of short arm chromosome (ls), long arm chromosome (ll), total arm chromosome (lt), relative length (rl), centromeric index (ci) and standard deviation (sd) of rl, ci from 20 metaphases of ag. simplex (2n=42). chromosome pair ls±sd (µm) ll±sd (µm) lt±sd (µm) rl (%) ci chromosome type 1 2.97±0.01 7.50±0.03 10.48±0.05 6.37 0.64 submetacentric 2 2.58±0.01 7.41±0.03 9.98±0.04 6.07 0.66 submetacentric 3 3.12±0.01 6.61±0.03 9.73±0.04 5.92 0.65 submetacentric 4 1.86±0.01 7.83±0.03 9.69±0.04 5.89 0.75 subtelocentric 5 2.55±0.01 6.60±0.03 9.16±0.04 5.57 0.69 submetacentric 6 2.52±0.01 6.62±0.03 9.14±0.04 5.56 0.66 submetacentric 7 2.37±0.01 6.56±0.03 8.93±0.04 5.43 0.75 subtelocentric 8 1.74±0.01 7.07±0.03 8.81±0.04 5.36 0.60 submetacentric 9 3.60±0.02 5.17±0.02 8.77±0.04 5.33 0.51 metacentric 10 3.61±0.02 4.83±0.02 8.44±0.04 5.13 0.64 submetacentric 11 2.58±0.01 5.66±0.03 8.23±0.04 5.01 0.55 metacentric 12 3.10±0.01 5.02±0.02 8.12±0.04 4.94 0.70 subtelocentric 13 1.87±0.01 5.76±0.03 7.63±0.03 4.64 0.68 submetacentric 14 2.57±0.01 4.96±0.02 7.53±0.03 4.58 0.63 submetacentric 15 2.46±0.01 4.68±0.02 7.14±0.03 4.34 0.73 subtelocentric 16 1.91±0.01 4.92±0.02 6.83±0.03 4.16 0.75 subtelocentric 17 1.89±0.01 4.29±0.02 6.18±0.03 3.76 0.70 submetacentric 18 2.53±0.01 3.53±0.02 6.07±0.03 3.69 0.52 metacentric 19 2.34±0.01 3.09±0.01 5.43±0.02 3.30 0.69 submetacentric 20 1.53±0.01 3.01±0.01 4.53±0.02 2.76 0.69 submetacentric 21 0.83±0.00 2.80±0.01 3.63±0.02 2.21 0.61 submetacentric 10 piyaporn saensouk, surapon saensouk, rattanavalee senavongse 12m + 6sm + 8st with two visible satellite chromosomes including 13 pairs, which comprised six pairs of metacentric chromosome, three pairs of submetacentric chromosomes and four pairs of subtelocentric chromosomes with two visible satellite chromosomes. the short arm chromosome length ranged from 1.41±0.01 to 4.95±0.02 μm, the long arm chromosome length ranged from 3.75±0.02 to 5.51±0.03 μm, the total arm chromosome length ranged from 5.16±0.03 to 10.46±0.05 μm. relative lengths were 5.45–11.05 %. centromeric indexes were 0.55 0.74 (table 4; figure 2(c)). this study of the chromosome number and karyotype of this endemic species to thailand was never previously reported. the somatic chromosome number of ar. maxwellii, vulnerable species (vu) and a not common species from thailand, is reported here to be 2n = 24 with nf = 48 (figure 1(d)). the karyotype formula was asymmetrical karyotype due to 22m + 2sm with two visible satellite chromosomes including 12 pairs, which comprised 11 pairs of metacentric chromosomes and one pair of submetacentric chromosomes. the short arm chromosome length ranged from 1.28±0.01 to 2.65±0.01 μm, the long arm chromosome length ranged from 1.30±0.01 to 3.00±0.02 μm, the total arm chromosome length ranged from 2.58±0.02 to 5.65±0.04 μm. relative lengths were 5.94–13.00 %. centromeric indexes were 0.50–0.63 table 4. mean length of short arm chromosome (ls), long arm chromosome (ll), total arm chromosome (lt), relative length (rl), centromeric index (ci) and standard deviation (sd) of rl, ci from 20 metaphases of am. serrulatus (2n=26). chromosome pair ls±sd (µm) ll±sd (µm) lt±sd (µm) rl (%) ci chromosome type *1 4.95±0.02 5.51±0.03 10.46±0.05 11.05 0.58 metacentric 2 4.06±0.02 5.29±0.03 9.35±0.04 9.88 0.59 metacentric 3 3.26±0.01 5.55±0.03 8.81±0.04 9.31 0.57 metacentric 4 3.31±0.01 4.65±0.03 7.96±0.04 8.41 0.56 metacentric 5 2.02±0.01 5.91±0.03 7.94±0.04 8.39 0.72 subtelocentric 6 3.19±0.01 4.49±0.03 7.67±0.04 8.11 0.55 metacentric 7 2.21±0.01 5.03±0.03 7.23±0.04 7.64 0.69 submetacentric 8 1.71±0.01 5.09±0.03 6.80±0.04 7.18 0.74 subtelocentric 9 1.88±0.01 4.38±0.02 6.26±0.03 6.61 0.71 subtelocentric 10 2.54±0.01 3.54±0.02 6.09±0.03 6.43 0.59 metacentric 11 1.62±0.01 3.85±0.02 5.48±0.03 5.79 0.68 submetacentric 12 1.54±0.01 3.91±0.02 5.45±0.03 5.76 0.68 submetacentric 13 1.41±0.01 3.75±0.02 5.16±0.03 5.45 0.72 subtelocentric *=satellite chromosome. table 5. mean length of short arm chromosome (ls), long arm chromosome (ll), total arm chromosome (lt), relative length (rl), centromeric index (ci) and standard deviation (sd) of rl, ci from 20 metaphases of ar. maxwellii (2n=24). chromosome pair ls±sd (µm) ll±sd (µm) lt±sd (µm) rl (%) ci chromosome type 1 2.65±0.01 3.00±0.02 5.65±0.04 13.00 0.52 metacentric 2 2.25±0.01 2.44±0.02 4.69±0.03 10.80 0.52 metacentric 3 1.91±0.01 2.36±0.02 4.27±0.03 9.83 0.58 metacentric 4 1.87±0.01 2.37±0.02 4.25±0.03 9.78 0.50 metacentric 5 1.75±0.01 2.05±0.02 3.80±0.03 8.75 0.57 metacentric *6 1.71±0.01 2.03±0.02 3.74±0.03 8.61 0.51 metacentric 7 1.50±0.01 1.71±0.02 3.21±0.03 7.39 0.59 metacentric 8 1.47±0.01 1.56±0.02 3.04±0.03 6.99 0.51 metacentric 9 1.20±0.01 1.66±0.02 2.86±0.03 6.58 0.63 submetacentric 10 1.23±0.01 1.46±0.01 2.69±0.03 6.18 0.53 metacentric 11 1.33±0.01 1.34±0.02 2.68±0.02 6.16 0.53 metacentric 12 1.28±0.01 1.30±0.01 2.58±0.02 5.94 0.56 metacentric *=satellite chromosome. 11cytogenetic studies of six species in family araceae from thailand (table 5; figure 2(d)). the chromosome number and karyotype including the cytological characteristics of this species in this study is reported for the first time. the somatic chromosome number of ha. benthamiana was recognized to be 2n = 26 and nf = 52 (figure 1(e)). the karyotype formula was asymmetrical karyotype due to 26m with one visible satellite chromosomes including 13 pairs, which comprised 13 pairs of metacentric chromosomes. the short arm chromosome length ranged from 1.24±0.01 to 2.20±0.01 μm, the table 6. mean length of short arm chromosome (ls), long arm chromosome (ll), total arm chromosome (lt), relative length (rl), centromeric index (ci) and standard deviation (sd) of rl, ci from 20 metaphases of ha. benthamiana (2n=26). chromosome pair ls±sd (µm) ll±sd (µm) lt±sd (µm) rl (%) ci chromosome type 1 2.20±0.01 2.32±0.02 4.52±0.03 9.74 0.51 metacentric 2 2.11±0.01 2.22±0.02 4.33±0.03 9.34 0.53 metacentric 3 1.99±0.01 2.21±0.02 4.20±0.03 9.06 0.54 metacentric 4 1.97±0.01 2.07±0.02 4.03±0.03 8.70 0.58 metacentric 5 1.73±0.01 1.96±0.02 3.69±0.03 7.96 0.58 metacentric 6 1.66±0.01 1.88±0.02 3.53±0.03 7.62 0.54 metacentric 7 1.59±0.01 1.88±0.02 3.47±0.03 7.48 0.54 metacentric 8 1.67±0.01 1.80±0.02 3.47±0.03 7.48 0.55 metacentric *9 1.52±0.01 1.71±0.02 3.23±0.03 6.96 0.55 metacentric 10 1.46±0.01 1.75±0.02 3.21±0.03 6.93 0.51 metacentric 11 1.48±0.01 1.71±0.02 3.19±0.03 6.87 0.54 metacentric 12 1.28±0.01 1.48±0.02 2.76±0.02 5.95 0.55 metacentric 13 1.24±0.01 1.49±0.02 2.73±0.02 5.89 0.55 metacentric *=satellite chromosome. table 7. mean length of short arm chromosome (ls), long arm chromosome (ll), total arm chromosome (lt), relative length (rl), centromeric index (ci) and standard deviation (sd) of rl, ci from 20 metaphases of ho. griffithii (2n=40). chromosome pair ls±sd (µm) ll±sd (µm) lt±sd (µm) rl (%) ci chromosome type 1 1.98±0.01 2.01±0.02 3.99±0.03 9.05 0.55 metacentric 2 1.46±0.01 1.68±0.02 3.14±0.03 7.13 0.58 metacentric *3 1.26±0.01 1.68±0.02 2.93±0.03 6.65 0.51 metacentric 4 0.60±0.01 1.96±0.02 2.56±0.03 5.81 0.63 submetacentric 5 0.77±0.01 1.87±0.02 2.64±0.03 5.98 0.62 submetacentric 6 0.92±0.01 1.46±0.02 2.38±0.03 5.39 0.61 submetacentric 7 0.95±0.01 1.28±0.02 2.23±0.03 5.06 0.59 metacentric 8 0.73±0.01 1.45±0.02 2.18±0.03 4.94 0.62 submetacentric 9 0.77±0.01 1.39±0.02 2.16±0.02 4.91 0.59 metacentric 10 0.93±0.01 1.20±0.01 2.13±0.02 4.82 0.59 metacentric 11 0.81±0.01 1.25±0.02 2.06±0.02 4.66 0.55 metacentric 12 0.82±0.01 1.23±0.01 2.05±0.02 4.65 0.56 metacentric 13 0.73±0.01 1.33±0.02 2.05±0.02 4.65 0.68 submetacentric 14 0.91±0.01 1.11±0.01 2.02±0.02 4.58 0.53 metacentric 15 0.88±0.01 1.02±0.01 1.89±0.02 4.29 0.58 metacentric 16 0.90±0.01 1.00±0.01 1.89±0.02 4.29 0.53 metacentric 17 0.77±0.01 1.03±0.01 1.80±0.02 4.08 0.55 metacentric 18 0.73±0.01 1.03±0.01 1.76±0.02 3.99 0.55 metacentric 19 0.69±0.01 0.93±0.01 1.62±0.02 3.67 0.58 metacentric 20 0.21±0.00 0.41±0.01 0.62±0.01 1.40 0.59 metacentric *=satellite chromosome. 12 piyaporn saensouk, surapon saensouk, rattanavalee senavongse long arm chromosome length ranged from 1.48±0.02 to 2.32±0.02 μm, the total arm chromosome length ranged from 2.73±0.02 to 4.52±0.03 μm. relative lengths were 5.89–9.74 %. centromeric indexes were 0.51–0.58 (table 6; figure 2(e)). the somatic chromosome number of ha. benthamiana in this study is consistent with larsen (1969) reported 2n = 26. the karyotype studies of this species were reported for the first time. the somatic chromosome number of ho. griffithii was found to be 2n (diploid) = 40 with nf = 80 (figure 1(f)). the karyotype formula was asymmetrical karyotype due to 30m + 10sm with two satellite chromosomes including 20 pairs, which comprised 15 pairs of metacentric chromosomes and five pairs of submetacentric chromosomes (table 7; figure 2(f)). the short arm chromosome length ranged from 0.21±0.00 to 1.98±0.01 μm, the long arm chromosome length ranged from 0.41±0.01 to 2.01±0.02 μm, the total arm chromosome length ranged from 0.62±0.01 to 3.99±0.03 μm. relative lengths were 1.40–9.05 %. centromeric indexes were 0.51–0.68 (table 7; figure 2(f)). the somatic chromosome number of ho. griffithii is consistent with okada (1982) and okada (2000). in addition, the karyotype of this study was investigated for the first time. while, darlington and wylie (1955) reported the chromosome numbers of plants in the family araceae between 2n = 24–140 because it is able to cross-pollinate, and when there are hybrids, the doubling of the chromosome number and resulting plant species have allopolyploidy, and it was found that the polyploidy was related to the evolution of this family (larsen 1969) or environmental factors, such as weather, humidity, light, soil or altitude above sea level in each of the areas. from the literature, it was found that this family has a wide range of chromosome numbers between 2n = 24–140. however, this study found that all five genera with six species had chromosome numbers 2n = 24–42, including three species with symmetry and three species with asymmetry. acknowledgements we are deeply indebted to mahasarakham university for financial support. we are grateful to the walai rukhavej botanical research institute, mahasarakham university, maha sarakham, thailand, for their facilities during the study. in addition, thanks to dr. jolyon dodgson (a native speaker from uk) for language editing and suggestions to improve the manuscript. references allen dj. 2011. aglaonema simplex. the iucn red list of threatened species 2011; 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(poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(4): 121-128, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1270 caryologia international journal of cytology, cytosystematics and cytogenetics citation: marina souza cunha, silvana melo, filipe schitini salgado, cidimar estevam assis, jorge abdala dergam (2021) repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil. caryologia 74(4): 121-128. doi: 10.36253/caryologia-1270 received: april 01, 2021 accepted: november 27, 2021 published: march 08, 2022 copyright: © 2021 marina souza cunha, silvana melo, filipe schitini salgado, cidimar estevam assis, jorge abdala dergam. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil marina souza cunha1,2,*,#, silvana melo1,3,#, filipe schitini salgado1,2, cidimar estevam assis1, jorge abdala dergam1,* 1 departamento de biologia animal, universidade federal de viçosa, viçosa, minas gerais, brazil 2 departamento de biologia geral, universidade federal de viçosa, viçosa, minas gerais, brazil 3 departamento de morfologia, instituto de biociências, universidade estadual paulista, botucatu, são paulo, brazil *corresponding authors. e-mail: marina.cunha@ufv.br; jdergam@gmail.com #m.s. cunha and s. melo should be considered joint first author. abstract. within the neotropical region, the genus oligosarcus represents an interesting assembly of small-sized freshwater predators. the goal of this study was to cytogenetically analyze oligosarcus acutirostris from the espírito santo stream, paraíba do sul river basin. the following cytogenetic techniques were performed: giemsa staining, ag-nor and cbandings, fluorescence in situ hybridization (fish) using 18s and 5s rdna probes, and (ca)15 and (ga)15 microsatellite probes. diploid number was 2n=50 and the karyotypic formula 4m+14sm+18st+14a. ag-nor sites were present on the subtelocentric chromosome pair number 10. c-banding showed a few pericentromeric and conspicuous terminal heterochromatic blocks. the 18s and 5s rdna probes marked chromosome pairs number 10 and number 19, respectively. fish patterns obtained with (ca)15 and (ga)15 probes hybridized pericentromeric and terminal regions in almost all chromosomes, and interstitial regions of some chromosomes. interestingly, microsatellite (ca)15 showed a conspicuous centromeric mark on chromosome pair number 14, which could be an autapomorphy of this species, or it might characterize some species of this genus. the oligosarcus cytogenetic patterns suggest that this genus is prone to fixation of chromosomal rearrangements and may be useful to detect biogeographical subunits within the coastal brazilian basins. keywords: characiformes, cytotaxonomy, coastal river basins, fluorescence in situ hybridization (fish), freshwater fishes. introduction the genus oligosarcus günther, 1864 currently encompasses 22 species adapted to inhabit shallow places with dense vegetation in small tributar122 marina souza cunha et al. ies, river channels, although they are also collected in large rivers (araújo et al. 2005; ribeiro and menezes 2015; fricke et al. 2021). they are distributed throughout most of south america (menezes 1988), and its endemism patterns and biogeographic relevance have been addressed (menezes 1987, 1988; ribeiro and menezes 2015; wendt et al. 2019). eight oligosarcus species have been studied with cytogenetic techniques, showing a conserved diploid number of 2n = 50 (martinez et al. 2004; centofante et al. 2006; rubert and margarido 2007; barros et al. 2015). some species have shown high levels of population chromosome variation (table 1), including 18s rdna amplification (up to 10 chromosomes) (barros et al. 2015) and the presence of odd numbers (i.e. 3, 7, 9) of ribosomal clusters (hattori et al. 2007; usso et al. 2018). the cytogenetic tools have been instrumental on systematic studies for understanding phylogenetic relationships in several animal groups. over the recent years, the increasing use of the molecular cytogenetic techniques have added important insights in studies of cryptic and closely related species (supiwong et al. 2013; yano et al. 2016; utsunomia et al. 2018; conde-saldana et al. 2019; ibagon et al. 2020; salgado et al. 2021), and have been a valuable tool to evidence possible hybridization cases (peres et al. 2012; gavazzoni et al. 2020). within the oligosarcus genus, oligosarcus acutirostris menezes, 1987 is broadly distributed among the rivers belonging to the coastal eastern basins of brazil (between espírito santo and bahia states) (menezes 1987; fricke et al. 2021). the aim of this study was to cytogenetically analyze o. acutirostris from the espírito santo stream, paraíba do sul river basin, with an additional cytogenetic review of the genus oligosarcus. material and methods oligosarcus acutirostris specimens (four males, two females, and one juvenile) were collected in the espírito santo stream, paraibuna river, paraíba do sul river basin (21º41’27” s 43º28’25” w), with collection license sisbio 14975-1 issued to jorge abdala dergam. the specimens were identified (menezes, 1987; ribeiro and menezes, 2015) and deposited in the ichthyological collection of the museu de zoologia joão moojen in the universidade federal de viçosa, minas gerais, brazil (lot number mzufv 4104). the animals were anesthetized and euthanized using 300 mg.l-1 clove oil aqueous solution (lucena et al. 2013) following the universidade federal de viçosa animal welfare committee protocols (authorization 68/2014). mitotic metaphase chromosomes were obtained through air-drying technique (bertollo et al. 1978). chromosomes were stained with giemsa to characterize the diploid number, karyotypic formula and the number of chromosome arms (fundamental number fn). the chromosomes were measured with image-pro plus® software and classified according to the arm ratios proposed by levan et al. (1964) in metacentric, submetacentric, subtelocentric, and acrocentric. the nucleolar organizing regions were detected using silver nitrate impregnation technique (ag-nor) (howell and black 1980), and the heterochromatic regions were evidenced using c-banding (sumner 1972) and dyed with dapi. the fluorescence in situ hybridization (fish) was used to characterize the chromosomal distribution patterns of 18s and 5s ribosomal sites (double-fish), and (ca)15 and (ga)15 microsatellites (single-fish). fish protocols were carried out according to pinkel et al. (1986). the 18s probe was labeled with biotin using the bio-nick translation mix kit (roche applied science) and the signal was detected with avidin-fitc (sigma), whereas the 5s rdna probe was labeled with digoxigenin using the dig-nick translation mix kit (roche applied science) and the signal was detected with antidigoxigenin-rhodamine (roche applied science). the microsatellite repetitive probes (ca)15 and (ga)15 were synthesized and labeled with fluorochrome cy3 on the 5’ end (sigma). digital images were obtained in bx53f olympus microscopes with olympus dp73 and xm10 cameras, for giemsa and fluorescent techniques respectively, both using cellsens imaging software (olympus). results the diploid number of o. acutirostris was 2n = 50, karyotypic formula of 4m + 14sm + 18st + 14a, fn = 86, with no differences between males and females (fig. 1). the ag-nor was located on the short arm of the largest subtelocentric chromosome pair number 10 (box on fig. 1). c-banding evidenced heterochromatic blocks mainly on pericentromeric and terminal regions of the chromosomes, although not all chromosomes showed heterochromatic positive markings (fig. 2). the 18s rdna fish probe marked subtelocentric pair number 10, whereas the 5s rdna probe marked the acrocentric pair number 19 (fig. 2). the microsatellite (ca)15 probe hybridized in pericentromeric and terminal regions of most chromosomes, and in interstitial regions of a few chromosomes, with a conspicuous centromeric mark on pair number 14, observed in both sexes. the (ga)15 probe hybridized in 123cytogenetics of o. acutirostris terminal regions of almost all chromosomes, with a few pericentromeric and interstitial blocks (fig. 2). discussion all oligosarcus species are characterized by a diploid number of 50 chromosomes, which is considered a plesiomorphic trait within the family characidae (kavalco et al. 2005). however, the karyotypic formulae and cytogenetic banding patterns are highly variable (table 1), underlining the relevance of chromosomal inversions and/or translocations in the karyotypic evolution of this group (centofante et al. 2006; rubert and margarido 2007; barros et al. 2015). this condition is a stark contrast with the conserved chromosomal macrostructure observed in other families, such as anostomidae (salgado et al. 2021), and prochilodontidae (voltolin et al. 2013; melo et al. 2017). small amounts of heterochromatin, with few pericentromeric and conspicuous terminal blocks, can be considered a widespread trait of the genus oligosarcus (reviewed in usso et al., 2018). within characidae, closely related genera typically show high levels of interspecific karyotypic variation, such as large amounts of heterochromatin found in deuterodon taeniatus (jenyns, 1842) (cunha et al. 2016), contrasting with the low amounts in deuterodon pedri eigenmann, 1907 (coutinho-sanches and dergam 2015). also, there are cases of intraspecific heterochromatin variation, such as in astyanax lacustris (lütken 1875) (cunha et al. 2019) and astyanax scabripinnis (jenyns, 1842) (santos et al. 2012). among oligosarcus species, ag-nor cistrons have been observed on metacentric, submetacentric, subtelocentric, and acrocentric chromosomes (martinez et al. 2004; rubert and margarido 2007; barros et al. 2015). although the occurrence of a single pair of agnors is common in this genus, up to eight sites have been observed (table 1). in o. acutirostris, coincidental markings of ag-nor and 18s rdna fish probe demonstrates that the nucleolar organizing region is restricted to one chromosome pair. in some other oligosarcus species, discrepancy between these cytogenetic markers indicate that not all ribosomal sites highlighted by the 18s probe are active (table 1). the presence of only one pair of 5s rdna is the most widespread trait observed in oligosarcus spp., showing less variability than the 18s rdna clusters (table 1). figue 1. giemsa-stained karyotype of oligosarcus acutirostris (2n = 4m + 14sm + 18st + 14a, nf = 86). mean values of chromosome arm ratios are in parentheses. the ag-nor on chromosome pair number 10 is shown in the box. 124 marina souza cunha et al. based on non-simultaneous fish patterns, hattori et al. (2007) suggested the existence of synteny between the 18s and 5s rdna cistrons in o. hepsetus, o. pintoi, and o. jenynsii. however, this putative syntenic pattern has not been observed in other studies that applied doublefish (barros et al. 2015; usso et al. 2018; present study). the ribosomal 18s and 5s probes constitute potential phylogenetic markers for populations or species groups in the family characidae (kavalco et al. 2004; coutinhosanches and dergam 2015; piscor et al. 2019). the family characidae has a complex evolutionary history, and the phylogenetic relationship of its members have been assessed using morphological and molecular data (mirande 2010; oliveira et al. 2011; silva et al. 2017; wendt et al. 2019). most small-sized fish of this family, which includes the genus oligosarcus, have complex taxonomic issues. although this is the first study using microsatellite dna probes to characterize an oligosarcus species, the conspicuous mark on chromosome pair number 14 with the (ca)15 probe in o. acutirostris could be an autapomorphy of this species, or it might be a cytotaxonomic marker for some species within this genus. in other fish groups, these probes are distributed mainly in terminal chromosome regions, but additional interstitial markings have been useful as cytotaxonomic markers (supiwong et al. 2013; cunha et al. 2016; salgado et al. 2021), as well as in the identification of sex chromosome systems (cioffi et al. 2011; poltronieri et al. 2014; yano et al. 2016). most of the oligosarcus species are allopatric, just a few are sympatric but not syntopic (ribeiro and menezes 2015). this habitat partitioning together with competitive exclusion may act as geographical or ecological barriers isolating populations, favoring the diversification and speciation of this taxon. classical chromosomal evolutionary models suggest that high rates of chromofigure 2. dapi-stained c-banding and fluorescence in situ hybridization (fish) patterns of oligosarcus acutirostris. double-fish was performed with the probes 18s (pair number 10) and 5s rdna (pair number 19), and single-fish with the repetitive microsatellite probes (ca)15 and (ga)15. a conspicuous centromeric mark on pair number 14 was observed with the (ca)15 probe (indicated by the arrow). 125cytogenetics of o. acutirostris some rearrangement fixation are associated with species subdivided in small populations (king 1987; sites and moritz 1987), but they may also arise when selection favors reduction of crossing-over rates between chromosome regions, favoring chromosome rearrangement fixation and speciation (faria and navarro 2010). we conclude that oligosarcus species are prone to fixation of chromosomal rearrangements and this characteristic may be useful to detect biogeographical subunits within the coastal brazilian basins. table 1. cytogenetic variation in the oligosarcus species regarding the karyotypic formulae and the number of chromosomes marked by the ag-nor, 18s and 5s rdna markers. species locality karyotype ag nor 18s rdna 5s rdna references o. acutirostris espírito santo stream, paraíba do sul basin 4m+14sm+18st+14a 2 2 2 present study o. argenteus doce river basin 6m+12-14sm+16-20st+12-14a 4 8#-10# 2 barros et al. 2015 o. hepsetus grande stream, paraíba do sul basin 6m+12sm+14st+18a 3 4 centofante et al. 2006 o. hepsetus santo antônio stream, paraíba do sul basin 4m+12sm+16st+18a 3 6 centofante et al. 2006 o. hepsetus ipiranga and juquia rivers, paraíba do sul basin 2m+26sm+4st+18a falcão and bertollo 1985 o. hepsetus paraíba do sul river, paraíba do sul basin 2m+16sm+16st+16a 2 2-3 2 hattori et al. 2007 o. hepsetus paraitinga river and jacui stream, paraíba do sul basin 6m+10sm+16st+18a 2 4 4 kavalco et al. 2005 o. jenynsii ipiranga rivers, paraíba do sul basin 6m+22sm+6st+16a falcão and bertollo 1985 o. jenynsii uruguay river, santa catarina state, brazil 2m+24sm+10st+14a 2 2 2 hattori et al. 2007 o. longirostris iguaçu river, upper paraná basin 4m+10sm+16st+20a 2 rubert and margarido 2007 o. longirostris iguaçu river, upper paraná basin 2m+20sm+10st+18a 4 martinez et al. 2004 o. macrolepis turvo river, minas gerais state 8m+20sm+6st+16a falcão and bertollo 1985 o. paranensis keller river, upper paraná basin 2m+26sm+8st+14a 2-6 martinez et al. 2004 o. paranensis tunas river, upper paraná basin 4m+10sm+16st+20a 2-6 rubert and margarido 2007 o. paranensis três bocas stream, tibagi basin 8m+18sm+10st+14a 2-8 7 2 usso et al. 2018 o. paranensis quexada river, ivaí basin 6m+10sm+16st+18a 2-6 9 2 usso et al. 2018 o. pintoi mogi-guaçu river, upper paraná basin 4m+20sm+10st+16a falcão and bertollo 1985 o. pintoi mogi-guaçu river, upper paraná basin 2m+20sm+12st+16a 2 3 3 hattori et al. 2007 o. pintoi tunas river, upper paraná basin 4m+10sm+16st+20a 2-4 rubert and margarido 2007 o. solitarius doce river basin 4m+14-16sm+14-20st+12-18a 2 6# 2 barros et al. 2015 oligosarcus sp. das velhas river, são francisco basin 6m+14sm+18st+12a 4 10# 2 barros et al. 2015 # some chromosomes showed biterminal markings. 126 marina souza cunha et al. acknowledgments the authors wish to thank dr. vivian gemiliano pinto (instituto federal de educação ciência e tecnologia do sudeste de minas gerais campus juiz de fora) for logistical support. the authors also wish to thank “conselho nacional de desenvolvimento científico e tecnológico (cnpq)”, “coordenação de aperfeiçoamento de pessoal de nível superior (capes)”, and “fundação de amparo à pesquisa do estado de minas gerais (fapemig)”. statement of ethics the protocols followed the universidade federal de viçosa animal welfare committee authorization 68/2014. authors’ contributions m.s.c. and s.m. collected the data; m.s.c, s.m., and f.s.s. analyzed the data; all authors contributed to the manuscript writing and approved the final version. references araújo fg, andrade cc, santos rn, santos afgn, santos ln. 2005. spatial and seasonal changes in the diet of oligosarcus hepsetus 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(caprifoliaceae), using scot molecular markers fengzhen chen1, dongmei li2,* , mohsen farshadfar3 the new chromosomal data and karyotypic variations in genus salvia l. (lamiaceae): dysploidy, polyploidy and symmetrical karyotypes halil erhan eroğlu1,*, esra martin2, ahmet kahraman3, elif gezer aslan4 cytogenetic survey of eight ant species from the amazon rainforest luísa antônia campos barros1, gisele amaro teixeira2, paulo castro ferreira1, rodrigo batista lod1, linda inês silveira3, frédéric petitclerc4, jérôme orivel4, hilton jeferson alves cardoso de aguiar1,5,* molecular phylogeny and morphometric analyses in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f. neda atazadeh1,*, masoud sheidai1, farideh attar2, fahimeh koohdar1 a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) melika tabasi1, masoud sheidai1,*, fahimeh koohdar1, darab hassani2 genetic diversity and relationships among glaucium (papaveraceae) species by issr markers: a high value medicinal plant lu feng1,*, fariba noedoost2 morphometric analysis and genetic diversity in rindera (boraginaceae-cynoglosseae) using sequence related amplified polymorphism xixi yao1, haodong liu2,*, maede shahiri tabarestani3 biosystematics, fingerprinting and dna barcoding study of the genus lallemantia based on scot and remap markers fahimeh koohdar*, neda aram, masoud sheidai karyotype analysis in 21 plant families from the qinghai–tibetan plateau and its evolutionary implications ning zhou1,2, ai-gen fu3, guang-yan wang1,2,*, yong-ping yang1,2,* some molecular cytogenetic markers and classical chromosomal features of spilopelia chinensis (scopoli, 1786) and tachybaptus ruficollis (pallas, 1764) in thailand isara patawang1,*, sarawut kaewsri2, sitthisak jantarat3, praween supanuam4, sarun jumrusthanasan2, alongklod tanomtong5 centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates) simona ceraulo, vanessa milioto, francesca dumas* repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil marina souza cunha1,2,*,#, silvana melo1,3,#, filipe schitini salgado1,2, cidimar estevam assis1, jorge abdala dergam1,* karyomorphology of some crocus l. taxa from uşak province in turkey aykut yilmaz*, yudum yeltekin variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l. egizia falistocco1,*,+, gianpiero marconi1,+, lorenzo raggi1, daniele rosellini1, marilena ceccarelli2, emidio albertini1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(2): 141-148, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1091 caryologia international journal of cytology, cytosystematics and cytogenetics citation: özgün tuna-gülören, ferhan korkmaz, meltem erdir, ebru ataşlar (2021) cytotoxic and genotoxic effects of methanol extracts of vegetative parts of some gypsophila l. species using allium cepa assay. caryologia 74(2): 141-148. doi: 10.36253/caryologia-1091 received: september 22, 2020 accepted: july 20, 2021 published: october 08, 2021 copyright: © 2021 özgün tuna-gülören, ferhan korkmaz, meltem erdir, ebru ataşlar. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. cytotoxic and genotoxic effects of methanol extracts of vegetative parts of some gypsophila l. species using allium cepa assay özgün tuna-gülören1, ferhan korkmaz2*, meltem erdir2, ebru ataşlar2 1 eskişehir osmangazi university, graduate school of natural and applied sciences, department of biology, 26040, eskişehir, turkey 2 eskişehir osmangazi university, faculty of science and letters, department of biology, 26040, eskişehir, turkey *corresponding author; e-mail ferhanatahan@gmail.com; ferhanka@ogu.edu.tr abstract. in this study, the cytotoxic and genotoxic effects of gypsophila perfoliata l. var. perfoliata, gypsophila perfoliata l. var. araratica kit tan, gypsophila pilosa hudson and gypsophila osmangaziensis ataşlar & ocak plant extracts have been examined using allium assay. methanol extracts of plants have been prepared in 4 different concentrations (0.625 mg/ml, 1.250 mg/ml, 2.500 mg/ml and 5.000 mg/ml). after the onion roots were treated in these concentrations of plant extracts for 24 hours and 48 hours, mitosis slides were prepared from these root tips. with the data obtained by examining the slides, mitotic index (%) and chromosome aberration (%) values have been calculated. distilled water has been used as the control group. it was found that mitotic index and chromosome aberration values of all species showed significant differences compared to the control group in the extract concentration range of 1.250– 5.000 mg/ml. it has been also determined that the most widely observed chromosome aberrations were disturbed metaphase, sticky metaphase, c-metaphase, disturbed anaphase and anaphase bridge. keywords: gypsophila l., caryophyllaceae, methanol extracts, allium test, mitotic index, chromosome aberrations. introduction gypsophila l., is a genus of caryophyllaceae family, which is found between 30° and 60° latitudes and is represented with nearly 150 species in this region (antkowiak and dyba 2004, schweingruber 2007). their underground parts, containing saponins (4-25%), were used for washing wool and silk, giving halva its fragility, and as fire extinguisher agents, while in medicine they were believed to have expectorant, laxative, and emetic properties (kołodziej et al. 2018). gypsophila which is the third largest genus of the caryophyllaceae in turkey, is represented by nearly 60 genus, 67% of which are endemic. turkey 142 özgün tuna-gülören et al. is located in gypsophila species’ main centers of variation and is one of the most important locations for their gene diversity (barkoudah 1962, bittrich 1993, davis 1967, davis et al.1988, güner et al. 2000, güner et al. 2012). studies on the saponin content of gypsophila species have shown that the ratio of pure saponin can reach up to 25% in some species, for example g. bicolor (freyn. & sint.) grossh. (sezik 1982). furthermore, one of the recent studies related to this subject was conducted by kołodziej et al. (2018). in the study, 7 gypsophila species with a potential use in the pharmaceutical industry for saponin production have been examined. the study shows that those species which were most abundant in saponins were g. acutifolia steven ex spreng., g. pacifica kom., g. scorzonerifolia ser. and g. zhegulensis krasnova. on the other hand, kołodziej et al. (2019) studied antimicrobial and antioxidant activities of the g. paniculata l., g. pacifica, and g. scorzonerifolia. the results of this study showed that gypsophila had valuable bioactive properties and the hexane extracts showed higher antifungal and antibiotic activity. also, kotelnaya et al. (2019) mentioned plant saponins exert cardiotonic, neurotrophic, hypotensive, tonic, hypocholesterolemic and antisclerotic, diuretic, corticotropic, adaptogenic, sedative, antiulcer genic and mild laxative effects on human subjects. natural products are potential anticancer agent sources and these are considered as alternatives to synthetic anticancer drugs which have disadvantages such as toxicity, high costs and negative side effects. it is stated that nearly 3000 plant types produce metabolites that have anticancer activity (hartwell 1971). furthermore, 350 plant types have been defined as potential source of agents against cancer (graham et al. 2000). due to the reason that modern drugs are costly, the demand for natural plant products has increased in clinical applications. usage of plant extracts as traditional drugs in the health area is a practice which is as old as the human history. allium test is an important and well known test system which is used in determining safe concentrations in the therapeutic use of plant extracts. (roy and roy 2019). besides, allium test is recommended as the standard test, especially for cytogenotoxicity in environmental monitoring (fiskesjö 1985). this test has advantages such as being useful, having low costs, and showing good correlation with mammalian test systems. results of allium test are also compatible with test systems composed of prokaryotes and/or eukaryotes (çelik and aslantürk 2007). in this study, the cytotoxic and genotoxic effects of methanol extracts from four gypsophila species, two of which are endemic, were investigated using allium cepa root tip meristem cells. the species that were chosen for this study were: gypsophila perfoliata l. var. perfoliata, gypsophila perfoliata l. var. araratica kit tan (endemic), gypsophila pilosa hudson and gypsophila osmangaziensis ataşlar & ocak (endemic). there are some reports about cytotoxicity of different gypsophila species such as gypsophila bicolor (freyn & sint.) grossh. and gypsophila ruscifolia boiss. (rad et al. 2018). to the best of our knowledge this is the first report on the cy totoxicity and genotoxicity of methanolic extracts of vegetative parts ofall of the studied species and we hope our research will helpshed light on other studies about gypsophlia species and provide data for future studies on medicinal plants. materials and methods plant samples in this study, four gypsophila species were used. plant samples were collected from two different locations. voucher specimens were deposited at the herbarium of the department of biology, eskişehir osmangazi university (oufe). g. perfoliata var. perfoliata and g. perfoliata var. araratica samples were collected from b3 afyon: emirdağ-çifteler junction point, steppe, 1035 m, 39°22´34.27” n and 31°02´15.21” e, 23.08.2009 (ataşlar, oufe 15942, oufe 15943). on the other hand, the samples of g. pilosa and g. osmangaziensis were collected from b3 eskişehir: eskişehir osmangazi university campus area, west sides, open stone areas, 810 m, 39°45´10.6164” n and 30°29´16.9620” e, 19.06.2009 (ataşlar, oufe 15944, oufe 15945). these sites lie in the central anatolian region which is characterized by its continental climate, extreme heat and virtually no rainfall in summers with winters recieving heavy, lasting snows. long term avarage of annual temperature is around 10 °c while humidity is around 65% (apaydin et al. 2011) preparation of plant extracts the extraction procedure of the plant samples was performed according to ataşlar et al. (2019). briefly, fresh vegetative parts of the studied species were treated with 0.8% tween 80, tap water and distilled water and then were dried at room temperature. the dried samples were grinded to obtain the powder form of the studied gypsophila species. ten grams of powdered samples were extracted with 250 ml 80% petroleum ether to remove their oily constituents with soxhlet apparatus. the degreased plant materials were dried overnight and were extracted with 250 ml 70% methanol. at this step, the flasks were mixed for half an hour in the blender 143cytotoxic and genotoxic effects of methanol extracts of vegetative parts of some gypsophila species using allium cepa assay consecutively three times. all of the methanol extracts were combined and filtrated with whatman 1 filter paper. the methanol in the total extract was removed by rotary evaporator. the obtained dry extracts (yield= 6.90%, 5.00%, 8.96% and 7.36%, respectively) were maintained at 4 °c for future use in genotoxicity studies. genotoxicity test genotoxicities of plant extracts used in the study have been determined with allium test. for this purpose, the methanol extracts of g. perfoliata var. perfoliata, g. perfoliata var. araratica, g. pilosa and g. osmangaziensis species were prepared in 4 different concentrations (0.625 mg/ml, 1.250 mg/ml, 2.500 mg/ml and 5.000 mg/ml). distilled water has been used as negative control. for each concentration, 6 onions rooted in distilled water for 24 hours. onion roots were left to interact with extract concentrations at 25 ± 1 ºc for 24-48 hours. at the end of 24 and 48 hours, the root tips were cut and included in the farmer fixative (3: 1 ethyl alcohol: glacial acetic acid) and stored at +4 ºc. fixative residues that could be found on the root tips were removed by washing with distilled water. afterwards, the root tips have been hydrolyzed for 12 minutes in 1 n hcl acid at 60 ºc water bath. after the hydrolysis, the roots were submerged in feulgen dye and chromosomes were stained for 1 hour with the schiff reaction. after this procedure, slides were prepared from the dyed root tips (1-2 mm) by crushing and spreading. (fiskesjö 1985, rank et al. 2002, rank 2003). slides were phtographed using a light microscope, nikon eclips 80i. mitotic index % (mi%). mitotic index (mi%) have been calculated with the following formula (sehgal et al. 2006) and chromosome aberration % (ca%) have been calculated with the other formula (ivanova et al. 2003). mitotic index (mi) (%)=(p+m+a+t)/(total number of cells)×100 chromosome aberration (ca) (%)=(number of abnormal cells)/(number of cells in mitosis)×10 where (p+m+a+t) is the sum of all cells in phase as prophase, metaphase, anaphase and telophase, respectively. statistical analysis the results have been interpreted statistically, using independent sampling t test, one-way variance analysis (anova) and tukey test. results and discussion the values of mi% (mitotic index %) and ca% (chromosome aberration %) of the methanol extracts of gypsophila species used in the study are given in table 1. in the 24-hour treatment, 5.000 mg / ml concentrationss significantly decreased mi% in g. osmangiensis and g. pilosa extracts compared to the control group (p <0.05). mi% values have statistically decreased to a significant extent compared to the control group, at 2.500 mg/ml and 5.000 mg/ml concentrations of g. perfoliata var. perfoliata extract and at 1.250 mg/ml, 2.500 mg/ ml and 5.000 mg/ml concentrationsof g. perfoliata var. araratica extract (p<0.05). as a result of treatment for 48 hours, none of the mi% values relating to g. osmangaziensis extract showed a significant difference compared to the control group. for g. pilosa extract, 0.625 mg/ml, 1.250 mg/ml concentrations have increased mi% compared to the control group, whereas 2.500 mg/ml has reduced it. when mi% values of g. perfoliata var. perfoliata have been considered, it was determined that only 5.000 mg/ml has shown a significant decrease compared to the control group and for g. perfoliata var. araratica extract, it was determined that 2.500 mg/ml and 5.000 mg/ml concentrations have shown a significant decrease compared to the control group (p<0.05). the mitotic index is a cytogenetic parameter that helps measure the proliferation (m phase) of mitotic cells in the cell cycle, and inhibition of the mitotic index is considered as cell death. (öney-birol and gündüz 2019). taking this into consideration, the result of the treatment for 24 hours in the g. osmangaziensis extract which had a concentration of 5.000 mg/ml, mi% decreased to a significant extent, which means that it showed a cytotoxic effect. however, no such effect could be observed from 48 hours treatment in the same concentration for 48 hours. in the case of g. pilosa, in the samples that were treated for 24 hours, the 5.000 mg/ ml concentration decreased mi% and in the samples that were treated for 48 hours, 2.500 mg/ml concentration decreased mi%, meanwhile 1.250 mg/ml and 0.625 mg/ml concentrations increased mi%. when we look at the experiment data of the g. perfoliata var. perfoliata extract samples, it is seen that after 24 hours treatment the 2.500 mg/ml and the 5.000 mg/ml concentrations decreased mi% meanwhile in the samples that were treated for 48 hours only the 5.000 mg/ml concentration decreased mi%. the treatment of samples in g. perfoliata var. araratica for 24 hours showed a decrease in mi% in 1.250 mg/ml, 2.500 mg/ml and 5.000 mg/ml concentrations while treatment for 48 hours only showed a decrease in mi% in 2.500 mg/ml and 5.000 mg/ml 144 özgün tuna-gülören et al. table 1. mitotic index and chromosome aberration types and their frequency induced by gypsophila extracts in root tip cells of allium cepa. d ur at io n plant species concentration of treatment (mg/ml) total number of cells scored number of dividing cells number of abnormal cells mitotic index (%)±sd chromosome aberration types (%) the frequency of total chromosome aberration (ca %)±sd d is tu rb ed m et ap ha se st ic ky m et ap ha se cm et ap ha se d is tu rb ed a na ph as e a na ph as e br id ge “2 4 ho ur s g. osmangaziensis control 12959 740 21 5.76±2.69 43 48 0 9 0 2.65±1.05 0.625 13498 902 29 6.72±1.40 45 38 3 3 11 3.25±085 1.250 12704 966 53 7.69±1.88 43 30 4 17 7 5.50±1.41 b 2.500 12740 739 46 5.81±1.19 54 31 0 13 2 6.16±1.49 b 5.000 11916 364 30 2.90±1.25a 53 43 0 4 0 6.09±4.71 b g. pilosa control 11094 588 0 5.41±1.02 0 0 0 0 0 0.00±0.00 0.625 12763 636 0 5.06±0.75 0 0 0 0 0 0.00±0.00 1.250 12698 648 0 5.18±0.81 0 0 0 0 0 0.00±0.00 2.500 11397 511 30 4.44±1.35 43 27 0 27 3 6.28±2.53 b 5.000 11827 231 28 2.02±1.29 a 39 29 0 32 0 14.15±10.97 b g. perfoliata var. perfoliata control 13001 687 0 5.43±1.47 0 0 0 0 0 0.00±0.00 0.625 12756 905 0 7.11±1.24 0 0 0 0 0 0.00±0.00 1.250 12202 695 0 5.70±0.60 0 0 0 0 0 0.00±0.00 2.500 13271 389 19 2.86±1.22 a 63 27 0 5 5 5.51±5.52 b 5.000 10574 281 16 2.61±1.10 a 13 50 6 31 0 6.09±4.26 b g. perfoliata var. araratica control 10753 849 0 7.83±1.39 0 0 0 0 0 0.00±0.00 0.625 11338 820 0 7.38±1.88 0 0 0 0 0 0.00±0.00 1.250 12230 689 13 5.85±1.68 a 38 31 8 23 0 2.05±1.64 b 2.500 12744 609 23 4.80±0.87 a 48 30 0 13 9 3.88±2.18 b 5.000 11810 337 37 2.92±1.02 a 54 16 3 27 0 10.61±5.39 b 48 h ou rs g. osmangaziensis control 12243 550 1 4.29±2.82 100 0 0 0 0 1.19±2.92 0.625 12628 612 27 4.88±1.47 52 33 0 11 4 3.78±3.23 1.250 12149 520 45 4.28±1.07 20 31 0 49 0 8.60±7.62 b 2.500 12429 558 15 4.53±1.75 20 53 0 27 0 3.19±2.82 5.000 13159 502 13 3.84±1.39 31  69   0  0  0 2.44±2.07 g. pilosa control 12904 661 0 5.22±0.77 0 0 0 0 0 0.00±0.00 0.625 11542 811 0 7.07±0.78 b 0 0 0 0 0 0.00±0.00 1.250 10919 587 0 5.28±1.25 b 0 0 0 0 0 0.00±0.00 2.500 10854 340 15 3.13±1.30 a 40 47 0 13 0 5.11±3.02 b 5.000 11708 238 12 1.97±1.23 61 23 8 8 0 4.48±2.68 b g. perfoliata var. perfoliata control 14266 726 0 5.11±1.22 0 0 0 0 0 0,00±0,00 0.625 12969 827 0 6.39±1.00 0 0 0 0 0 0.00±0.00 1.250 11701 704 0 5.99±0.71 0 0 0 0 0 0.00±0.00 2.500 12947 520 33 4.10±1.99 49 42 0 9 0 5.43±3.70 b 5.000 11895 155 16 1.26±0.70 a 31 38 6 25 0 9.44±6.02 b g. perfoliata var. araratica control 10731 675 0 6.15±1.85 0 0 0 0 0 0.00±000 0.625 12499 732 0 5.94±0.82 0 0 0 0 0 0.00±0.00 1.250 12960 793 22 6.11±0.69 18 59 0 23 0 2.76±1.50 b 2.500 12507 523 29 4.28±0.83 a 48 28 3 21 0 5.56±3.84 b 5.000 12121 362 33 2.94±1.03 a 52 36 0 12 0 10.95±6.51 b means in a column followed by the same superscript letters are significantly different according to their control groups (p<0.05, one-way anova, tukey post hoc test; a: reduction of mi and ca, b: increase in mi and ca). 145cytotoxic and genotoxic effects of methanol extracts of vegetative parts of some gypsophila species using allium cepa assay concentrations. thus, it can be said that at the end of 48 hours a decrease in the cytotoxicity of these plant extracts is observed. when reviewing the literature, it can be seen that caryophyllaceae, which the studied plant species are included, constitute a wide family that has cytotoxic species. cytotoxic activity of g. bicolor and g. ruscifolia’s methanol extracts on mcf-7 (human breast adenocarcinoma), a-549 (non-small cell lung carcinoma) and ago1522 (human fibroblast) cell lines were examined using mtt method and it was determined to be cytotoxic for mcf-7 (human breast adenocarcinoma) cells figure 1. chromosome aberrations in allium cepa root meristem cells after treatment with extracts of gypsophila species. a: disturbed metaphase (g. perfoliata var. araratica-24 hours-5.000 mg/ml); b: sticky metaphase (g. pilosa-24 hours-2.500 mg/ml); c: c-metaphase (g. perfoliata var. araratica-24 hour-5.000 mg/ml); d: disturbed anaphase (g. perfoliata var. perfoliata-48 hours-5.000 mg/ml); e: anaphase bridge (g.osmangaziensis-24 hours-2.500 mg/ml);f: normal metaphase and; g: normal anaphase. 146 özgün tuna-gülören et al. (rad et al. 2018). in another study, it was determined that gypsophila saponins showed a synergistic cytotoxicity in macrophage-like (pma-treated) u937 cells with type i rips saporin and his-tagged saporin (weng et al. 2008). in a study conducted by gevrenova et al. (2014), it was determined that saponins were plant glycosides having one or more sugar chains being covalently linked as a steroid or triterpenoid aglycon or aglycon and it was emphasized that caryophyllaceae was an extremely rich source of triterpene saponin. in this study, it was shown for the first time that extracts of caryophyllaceae species including saponaria officinalis l., gypsophila trichotoma wend. and dianthus sylvestris wulffen, had impact on the vitality of mammalian monocytes/macrophage cell lines and that they induced apoptosis through caspase-3 activation (gevrenova et al. 2014). in the literature research that has been made, no study was found using the allium test in determining the cytotoxicity of plant extracts belonging to gypsophila species. another parameter determined in this studyis genotoxicity. for this purpose, ca% values were calculated. these values are shown in table 1. in the chromosome analysis of the four gypsophila species being studied, aberrations in the form of disturbed metaphase, sticky metaphase, c-metaphase, disturbed anaphase and anaphase bridge were observed. these aberrations are shown in figure 1. similar results were observed in the literature. ždralović and colleagues found that methanol extracts of the plantago lanceolata l. plant also caused chromosome aberrations like sticky metaphase and anaphase bridge in allium cepa chromosomes (ždralović et al. 2019). the deterioration of microtubules frequently causes mitotic aberrations like laggard chromosomes resulted from disturbed anaphase-telophase (amer and ali, 1986). various abnormalities like lagging chromosomes, vagrants, distrubed metaphases and anaphases, and chromosome stickiness can be induced by the inhibition of proteins’ effect on the spindle function (tkalec et al. 2009). and chromosome stickiness, usually of an irreversible type leading to cell death, is definite proof of genotoxicity (khanna, n., & sharma, s. (2013). moreover, vagrant chromosomes and c-metaphases increase the risk for aneuploidy, whereas chromosome bridges indicate the clastogenic effect caused by chromosome breaks (leme and marin-morales 2009). when plant extracts’ genotoxicity was examined, distilled water was used as negative control. in the 24 hours treatment, it was seen that ca% values of g. osmangaziensis extract with concentrations of 1.250 mg/ml and 2.500 mg/ml 5.000 mg/ml increased significantly compared to the control group. for g. pilosa extract, concentrations of 2.500 mg/ml and 5.000 mg/ ml increased ca%. for g. perfoliata var. perfoliata, concentrations of 2.500 mg/ml and 5.000 mg/ml increased ca% significantly compared to the control group and for g. perfoliata var. araratica, concentrations of 1.250 mg/ml, 2.500 mg/ml and 5.000 mg/ml increased ca% significantly compared to the control group (p<0.05). as a result of treatment for 48 hours, it was seen that for g. osmangaziensis extract, only the concentrationof 1.250 mg/ml increased ca% statistically and that for g. pilosa, concentrations of 2.500 mg/ml and 5.000 mg/ ml statistically increased the ca% value. for g. perfoliata var. perfoliata, concentrations of 2.500 mg/ml and 5.000 mg/ml increased ca% values and for g. perfoliata var. araratica, concentrations of 1.250 mg/ml, 2.500 mg/ ml and 5.000 mg/ml increased ca values. according to these results; after 24 hour treatment, only the 1250 mg/ml, 2250 mg/ml and 5000 mg/ml concentrations of g. osmangaziensis extract and after 48 hour treatment, only the 1250 mg/ml concentration of the same extract exhibited a significant increase. for the other 3 plant extracts; a significant difference in ca% increase between the 24 hour and 48 hour treatments could not be observed. thus it can be said that treatment period does not effect genetoxicity to a great extent concentration. in the literature review that has beenmade, no other genotoxicity study was found on these plant extracts. the fact that g. osmangaziensis is a new specie (ataşlar and ocak 2005), emphasizes the importance of data submitted in this study. however, it is considered appropriate for in vitro tests such as this to be evaluated with other test systems. our future studies will aimto support the data in this study related to the species investigated with other in vitro test systems. acknowledgements authors are thankful to dr. mustafa yamaç for his contribution in writing and mrs. demet büyükemir for her technical support during preparation. references amer, s. m., ali, e. m. 1986. cytological effects of pesticides xvii. effect of the insecticide dichlorvos on root-mitosis of vicia faba. cytologia 51(1): 21-25. apaydin, h., anli, a. s., ozturk, f., 2011. evaluation of topographical and geographical effects on some cli147cytotoxic and genotoxic effects of methanol extracts of vegetative parts of some gypsophila species using allium cepa assay matic parameters in the central anatolia region of turkey. int j climatol 31(9): 1264-1279. ataşlar e, ocak a, 2005. gypsophila osmangaziensis (caryophyllaceae), a new species from central anatolia, turkey. ann bot fenn. 42: 57–60. ataşlar e, tuna-gülören ö, filik-i̇şcen c, i̇lhan s. 2019. in vitro antimicrobial and antioxidant activities of four gypsophila l. species plant extracts. fresen environ bull. 28 (3): 1841–1851. antkowiak w, dyba s. 2004. effect of differentiated npk fertilization on saponin content in roots of tall and spread gypsophyll (gypsophila paniculata l. and g. repens l.) in the second and third years of cultivation. prace z zakresu nauk rolniczych. 97: 9–14. barkoudah, yi. 1962. a revision of gypsophila, bolanthus, ankyropetalum and phryna. wentia 9: 1–203. bittrich v. 1993. caryophyllaceae. in: kubitzki, k., rohwer, j. g. and bittrich, v. 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radiofrequency electromagnetic fields on seed germination and root meristematic cells of allium cepa l. mutat res, genet toxicol environ mutagen 672: 76-81. weng a, melzig mf, bachran c, fuchs h. 2008. enhancement of saporin toxicity against u937 cells by gypsophila saponins. j. immunotoxicol. 5(3): 287– 292. ždralović, a., mesic, a., eminović, i., parić a. 2019. cytotoxic and genotoxic activity of plantago major l. extracts. caryologia 72(3): 35-40. caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 1 2021 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 74(4): 3-10, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1064 caryologia international journal of cytology, cytosystematics and cytogenetics citation: kevin i. sánchez, fabio h. takagui, alberto s. fenocchio (2021) cytogenetic analyses in three species of moenkhausia eigenmann, 1903 (characiformes, characidae) from upper paraná river (misiones, argentina). caryologia 74(4): 3-10. doi: 10.36253/caryologia-1064 received: august 26, 2021 accepted: november 27, 2021 published: march 08, 2022 copyright: © 2021 kevin i. sánchez, fabio h. takagui, alberto s. fenocchio. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. cytogenetic analyses in three species of moenkhausia eigenmann, 1903 (characiformes, characidae) from upper paraná river (misiones, argentina) kevin i. sánchez1,*, fabio h. takagui2, alberto s. fenocchio3 1 instituto patagónico para el estudio de los ecosistemas continentales (ipeec-conicet), u9120-acd puerto madryn, chubut, argentina 2 laboratório de citogenética animal, departamento de biologia geral, ccb, universidade estadual de londrina, londrina, paraná, brazil 3 facultad de ciencias exactas, químicas y naturales, universidad nacional de misiones, instituto de biología subtropical (unam-conicet), posadas, misiones, argentina *corresponding author. e-mail: ksanchez@cenpat-conicet.gob.ar abstract. moenkhausia eigenmann, 1903 is one of the most diverse genera within characidae, being an important component of the neotropical fish fauna. three members of this genus were cytogenetically analyzed: m. dichroura kner, 1858, m. intermedia eigenmann, 1908 and m. sanctaefilomenae steindachner, 1907. the three species showed 2n = 50 bi-armed chromosomes (nf = 100) and different karyotype formulas: 22m + 22sm + 6st in m. dichroura, 16m + 28sm + 6st in m. intermedia, and 12m + 32sm + 6st in m. sanctaefilomenae. in addition, supernumerary chromosomes (or b-chromosomes) were detected in m. intermedia and m. sanctaefilomenae. c-positive bands were restricted to pericentromeric regions, secondary constrictions and supernumerary chromosomes. active nucleolus organizer regions (ag-nors) and positive cma3 bands were observed in a single pair of sm chromosomes. pericentromeric dapi positive signals were evidenced on chromosomes of m. sanctaefilomenae only. overall, the three species showed a conservative karyotype macrostructure (diploid number, number of chromosome arms) and variations in microstructure (karyotype formulas, presence/absence of supernumerary chromosomes). we discuss how the observed differences could have been shaped. keywords: neotropical fishes, moenkhausia, supernumerary chromosomes, heterochromatin, ag-nors. 1. introduction the genus moenkhausia comprises 109 species (fricke et al. 2021) and is considered insertae sedis within the family characidae (lima et al. 2003). this genus shows a wide distribution along cis-andean neotropical rivers (lima et al. 2003). its members are characterized by a wide variation in morphological attributes and coloration patterns (carvalho et al. 2014), being 4 kevin i. sánchez, fabio h. takagui, alberto s. fenocchio frequently used as ornamental fishes. phylogenetic relationships within moenkhausia based on morphological (mirande 2010, 2018) and molecular (mariguela et al. 2013) data evidenced their polyphyletic nature, suggesting that this genus could be an artificial grouping. several studies addressed the cytogenetic characterization of members of this genus (portela et al. 1988; foresti et al. 1989; arefjev 1990; alberdi and fenocchio 1997; santos 1999; portela-castro et al. 2001; dantas et al. 2007; hashimoto et al. 2012; scudeler et al. 2015; utsunomia et al. 2016; fernandes and alves 2017; nascimento et al. 2020). however, 10 species have been analyzed so far, mainly from brazilian populations. almost all these populations showed a diploid chromosome number of 50 bi-armed chromosomes (dantas et al. 2007; utsunomia et al. 2016; nascimento et al. 2020). some variations characterized as “cytotypes” were reported in m. gracilima eigenmann 1908 (2n = 48) and m. pittieri eigenmann 1920 (2n = 49) (arefjev 1990; santos 1999), although these observations were not corroborated in subsequent studies. heterochromatic blocks were mainly reported in centromeric and pericentromeric regions, and nucleolus organizer regions (nors) were generally observed on a single chromosome pair (portela et al. 1988; foresti et al. 1989; portela-castro et al. 2001; portela-castro and júlio júnior 2002; dantas et al. 2007; hashimoto et al. 2012; utsunomia et al. 2016; fernandes and alves 2017; nascimento et al. 2020). in addition, supernumerary chromosomes were detected in populations of m. sanctaefilomenae, m. intermedia, m. forestii, and m. oligolepis (foresti et al. 1989; dantas et al. 2007; hashimoto et al. 2012; scudeler et al. 2015; utsunomia et al. 2016; fernandes and alves 2017; nascimento et al. 2020). more recent studies inquired about the molecular composition of this supernumerary chromosomes by means of chromosomal mapping (dantas et al. 2007; scudeler et al. 2015; utsunomia et al. 2016; fernandes and alves 2017; nascimento et al. 2020). in spite of the taxonomic and cy togenetic diversity observed in moenkhausia, the number of analyzed species remains scarce. based on this, the aim of this work was to describe for the first time the karyotypic constitution of argentinean populations m. dichroura, and new populations of m. intermedia and m. sanctaefilomenae. aspects of the chromosomal differentiation between them will also be discussed in an evolutionary context. 2. materials and methods we collected 24 individuals of moenkhausia dichroura kner 1858, 12 individuals of m. intermedia eigenmann 1908, and 12 individuals of m. sanctaefilomenae steindachner 1907 from tributaries of the upper paraná river (misiones province, argentina) (table 1). the specimens were deposited in the collection of grupo de investigación en citogenética animal y monitoreo ambiental (ibs-unam-conicet). mitotic preparations were obtained from kidney cells following the protocol described in moreira-filho and bertollo (1991). c-banding followed sumner (1972), and nors were evidenced by silver nitrate impregnation (ag-nor; howell and black 1980). at and gcrich regions were detected with fluorochromes dapi (4’,6-diamidin-2-phenylindol) and cma3 (chromomycin a3), respectively (schweizer 1980). at least 30 metaphases were analyzed per specimen, and those exhibiting optimal chromosomal morphologies were used in karyotype analysis. chromosomes were classified as metacentrics (m), submetacentrics (sm), subtelocentrics (st) and acrocentrics (a) according to their arm ratios (levan et al. 1964). metacentric, submetacentric and subtelocentric chromosomes were considered as bi-armed, in order to determine the number of chromosome arms (nf). chromosome measures were obtained in karyotype v2 (altınordu et al. 2016) and karyograms were assembled in adobe photoshop®cs6 (san jose, california, usa). table 1. specimens of moenkhausia collected. f: females, m: males, ?: undetermined sex. voucher species stream/locality coordinates sex 2733-47, 2764-68 2758-61 moenkhausia dichroura a° pindapoy grande/garupá/mn/arg. a° mártires/posadas/mn/arg. 27°28’58”s, 55°49’10”w 27°22’50”s, 55°57’14”w 10f, 7m, 3? 1f, 1m, 2? 2751-57 2770, 2773, 2775, 2777, 2779 moenkhausia intermedia a° pindapoy grande/garupá/mn/arg. a° yabebiry/santa ana/mn/arg. 27°29’41”s, 55°49’13”w 27°17’40”s, 55°33’40”w 7? 1f, 1m 3? 2771-72, 2774, 2776, 2778, 2780-86 moenkhausia sanctaefilomenae a° yabebiry/santa ana/mn/arg. 27°17’40”s, 55°33’40”w 8f, 4m 5cytogenetic analyses in three species of moenkhausia from upper paraná river 3. results all three moenkhausia species showed 2n = 50 biarmed chromosomes (nf = 100). sexual differences were not observed. the analysis of karyotype formula revealed subtle differences distinctive of each species (fig. 1): m. dichroura (22m + 22sm + 6st), m. intermedia (16m + 28sm + 6st), and m. sanctaefilomenae (12m + 32sm + 6st). we have not observed differences in karyotype formula among different populations of the same species. in addition to the basic karyotype, moenkhausia intermedia and m. sanctaefilomenae showed a variation from one to three supernumerary microchromosomes (mean = 2 on both species), both in males and females (fig. 1; table 2). silver nitrate staining allowed the identification of one pair of nor-bearing chromosomes in the three species, which showed size heteromorphism. this chromosomes corresponded to pair 16 in m. dichroura, pair 12 in m. intermedia, and pair 13 in m. sanctaefilomenae (fig. 1). heterochromatic c-bands were allocated in centromeric and pericentromeric regions, in the short arms figure 1. giemsa stained karyotypes of moenkhausia species: (a) m. dichroura, (b) m. intermedia and (c) m. sanctaefilomenae. nor-bearing chromosomes of each species are depicted in the boxes. 6 kevin i. sánchez, fabio h. takagui, alberto s. fenocchio of nor-bearing chromosomes, and the supernumerary chromosomes (fig. 2). the ag-nor bands showed correspondence with bright positive signals when stained with cma3, and dark negative bands when stained with dapi (fig. 3). besides, the staining with cma3 made more evident the size heteromorphism evidenced by silver nitrate. bright dapi bands were observed in the pericentromeric region of several chromosomes in m. sanctaefilomenae, matching positive c-bands (fig. 3). 4. discussion diploid number of 50 bi-armed chromosomes are common features of the genus moenkhausia, in agreement with our observations (portela et al. 1988; arefjev 1990; foresti et al. 1989; alberdi and fenocchio 1997; portela-castro et al. 2001; portela-castro and júlio júnior 2002; dantas et al. 2007; hashimoto et al. 2012; scudeler et al. 2015; utsunomia et al. 2016; fernandes and alves 2017; nascimento et al. 2020). however, cytotypes with 2n = 48 and 2n = 49 were described in m. gracilima and m. pittieri, respectively (arefjev 1990; santos 1999). variations reported in karyotype formulas suggests that structural rearrangements could be involved in the karyotypic differentiation in moenkhausia, such as non-robertsonian translocations, inversions and/or translocations (tenório et al. 2013; nascimento et al. 2020). some authors have also postulated that these chromosomic rearrangements could have an important role in the diversification of certain families and orders of neotropical fishes (galetti jr. et al. 2000; silva et al. 2013; takagui et al. 2014; cioffi et al. 2017). the presence of b chromosomes in neotropical fishes has been reported for the first time in prochilodus lineatus (cited as p. scrofa in pauls and bertollo 1983), characiformes being the group with the higher number of species having this special type of chromosomes (carvalho et al. 2008). the presence of supernumerary chromosomes in the genus moenkhausia was reported for the first time by portela et al. (1988), in a population of m. intermedia from mogi-guaçu river (são paulo, brasil). in a later study, a population of this species from paraná river was analyzed, but the authors could not detect supernumerary chromosomes (portela-castro and júlio júnior 2002). thus, our results extends the presence of b-chromosomes in m. intermedia. nearly all analyzed populations of m. sanctaefilomenae have shown supernumerary chromosomes, including our results, even as numerical polymorphisms within populations (foresti et al. 1989; alberdi and fenocchio 1997; portela-castro et al. 2001; dantas et al. 2007; hashimoto et al. 2012; scudeler et al. 2015; utsunomia et al. 2016; fernandes and alves 2017). recent molecular cytogenetic approaches have also revealed an autosomic origin of this elements (scudeler et al. 2015; utsunomia et al. 2016). it has been suggested that numerical polymorphisms of b-chromosomes in m. sanctaefilomenae could represent a genetic diversification process, related to populations restricted to small rivers and tributaries (portela-castro et al. 2001; hashimoto et al. 2012). this can also be attributed to somatic non-disjunction, as suggested in camacho et al. (2000). interestingly, we detected supernumerary chromosomes on specimens of both sexes, contrary to the results of portela-castro et al. (2001), who found their presence only in males. c-banding showed several heterochromatic bands at centromeric and pericentromeric regions in the three species, in concordance with previous studies (foresti et al. 1989; portela-castro et al. 2001; portela-castro and júlio júnior 2002; dantas et al. 2007; hashimoto et al. 2012; fernandes and alves 2017). b-chromosomes detected in m. intermedia and m. sanctaefilomenae exhibited positive c-bands, agreeing partially with studies that demostrated the occurrence of euchromatic and heterochromatic supernumerary chromosomes (foresti table 2. b chromosome counts in metaphase cells of moenkhausia intermedia and m. sanctaefilomenae. f: females, m: males, ?: undetermined sex species voucher sex number of bs n cells with bs1b 2bs 3bs moenkhausia intermedia 2752 ? 2 2 2753 ? 2 6 13 21 2754 ? 6 6 2755 m 3 6 9 2757 ? 3 8 1 12 2773 ? 5 16 21 2775 ? 10 10 2779 f 4 3 7 14 n cells 27 41 27 95 proportion 0.28 0.43 0.28 1 moenkhausia sanctaefilomenae 2771 f 4 4 2776 f 1 6 1 8 2781 f 24 2 1 27 2782 m 2 13 1 16 2783 f 4 4 2785 m 15 15 2786 f 4 14 18 n cells 46 43 3 92 proportion 0.5 0.47 0.03 1 7cytogenetic analyses in three species of moenkhausia from upper paraná river et al. 1989; hashimoto et al. 2012; utsunomia et al. 2016; fernandes and alves 2017). moenkhausia intermedia has been characterized by ag-nors in a single chromosome pair, in agreement with our results (portela et al. 1988; portela-castro and júlio júnior 2002; dantas et al. 2007). on the contrary, simple and multiple nors have been described in m. sanctaefilomenae (foresti et al. 1989; portela-castro and júlio júnior 2002; dantas et al. 2007; hashimoto et al. 2012; fernandes and alves 2017). ag-nors were not described in m. dichroura, this study being the first report. some populations of m. sanctaefilomenae analyzed previously exhibited active nors on supernumerary chromosomes (foresti et al. 1989; hashimoto et al. 2012). this has lead to the suggestion that these elements are not completely inert, being able to contribute to cellular functions (hashimoto et al. 2012; utsunomia et al. 2016). in addition, it has been hypothesized that b chromosomes had a relevant role in the evolutionary history of this species (portela-castro et al. 2001). we have not observed agnor bands in any supernumerary chromosome. fluorochromes that stain preferentially gc base repetitions were employed as an additional method to detect nucleolar organizers independently of their activfigure 2. c-banded chromosomes of (a) moenkhausia dichroura, (b) m. intermedia and (c) m. sanctaefilomenae. 8 kevin i. sánchez, fabio h. takagui, alberto s. fenocchio ity (amemiya and gold 1986). ag-nor bearing chromosomes of m. intermedia and m. sanctaefilomenae showed positive cma3 signals on secondary constrictions, according to previous observations (portela-castro and júlio júnior 2002). moenkhausia dichroura exhibited a similar pattern. the observation of pericentromeric dapi+ blocks restricted only to m. sanctaefilomenae could indicate a prevalence of at-rich regions in these species. pericentromeric dapi+ heterochromatic blocks were also detected in other neotropical fish species such as astyanax argyrimarginatus (tenório et al. 2013), bryconamericus aff. iheringii (da silva et al. 2014), and hollandichthys multifasciatus (balen et al. 2013). this fact could be an exception since it has been suggested that bright dapi+ regions are not common in fishes, negative bands coincident with cma3+ sites being more frequently observed (souza et al. 2008). supernumerary chromosomes were not stained by the fluorochromes, neither in m. intermedia nor m. sanctaefilomenae, preventing us to make inferences about their molecular composition. the species of moenkhausia analyzed here showed a conservative macrostructure of bi-armed chromosomes, similar c-band patterns and simple nors systems. however, species-specific differences were evidenced regarding composition of chromosome types (m, sm and st), position of ag-nors, and dapi banding patterns. overall, m. dichroura and m. intermedia showed more similarities between them in comparison to m. sanctaefilomenae, supporting phylogenetic hypotheses that grouped m. dichroura and m. intermedia in a branch separated from m. sanctaefilomenae (mariguela et al. 2013; 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international journal of cytology, cytosystematics and cytogenetics volume 74, issue 4 2021 firenze university press cytogenetic analyses in three species of moenkhausia eigenmann, 1903 (characiformes, characidae) from upper paraná river (misiones, argentina) kevin i. sánchez1,*, fabio h. takagui2, alberto s. fenocchio3 genetic variations and interspesific relationships in lonicera l. (caprifoliaceae), using scot molecular markers fengzhen chen1, dongmei li2,* , mohsen farshadfar3 the new chromosomal data and karyotypic variations in genus salvia l. (lamiaceae): dysploidy, polyploidy and symmetrical karyotypes halil erhan eroğlu1,*, esra martin2, ahmet kahraman3, elif gezer aslan4 cytogenetic survey of eight ant species from the amazon rainforest luísa antônia campos barros1, gisele amaro teixeira2, paulo castro ferreira1, rodrigo batista lod1, linda inês silveira3, frédéric petitclerc4, jérôme orivel4, hilton jeferson alves cardoso de aguiar1,5,* molecular phylogeny and morphometric analyses in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f. neda atazadeh1,*, masoud sheidai1, farideh attar2, fahimeh koohdar1 a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) melika tabasi1, masoud sheidai1,*, fahimeh koohdar1, darab hassani2 genetic diversity and relationships among glaucium (papaveraceae) species by issr markers: a high value medicinal plant lu feng1,*, fariba noedoost2 morphometric analysis and genetic diversity in rindera (boraginaceae-cynoglosseae) using sequence related amplified polymorphism xixi yao1, haodong liu2,*, maede shahiri tabarestani3 biosystematics, fingerprinting and dna barcoding study of the genus lallemantia based on scot and remap markers fahimeh koohdar*, neda aram, masoud sheidai karyotype analysis in 21 plant families from the qinghai–tibetan plateau and its evolutionary implications ning zhou1,2, ai-gen fu3, guang-yan wang1,2,*, yong-ping yang1,2,* some molecular cytogenetic markers and classical chromosomal features of spilopelia chinensis (scopoli, 1786) and tachybaptus ruficollis (pallas, 1764) in thailand isara patawang1,*, sarawut kaewsri2, sitthisak jantarat3, praween supanuam4, sarun jumrusthanasan2, alongklod tanomtong5 centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates) simona ceraulo, vanessa milioto, francesca dumas* repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil marina souza cunha1,2,*,#, silvana melo1,3,#, filipe schitini salgado1,2, cidimar estevam assis1, jorge abdala dergam1,* karyomorphology of some crocus l. taxa from uşak province in turkey aykut yilmaz*, yudum yeltekin variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l. egizia falistocco1,*,+, gianpiero marconi1,+, lorenzo raggi1, daniele rosellini1, marilena ceccarelli2, emidio albertini1 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(2): 73-80, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-135 citation: s. mohsenzadeh, m. sheidai, f. koohdar (2020) populations genetic study of the medicinal species plantago afra l. (plantaginaceae). caryologia 73(2): 73-80. doi: 10.13128/caryologia-135 received: april 26, 2019 accepted: february 23, 2020 published: july 31, 2020 copyright: © 2020 s. mohsenzadeh, m. sheidai, f. koohdar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. populations genetic study of the medicinal species plantago afra l. (plantaginaceae) saeed mohsenzadeh*, masoud sheidai, fahimeh koohdar faculty of life sciences & biotechnology, shahid beheshti university, tehran, iran *corresponding author. e-mail: s_mohsenzadeh@yahoo.com abstract. plantago afra (plantaginaceae) is the most medicinally important species in genus plantago and it is native to the western mediterranean region, west asia and north africa, and cultivated extensively in asia and europe for seed husk known as black psyllium. we have no data on the population genetic structure of this species in the world. therefore a population genetic and morphological investigation was performed through light on genetic and morphological variability in this taxa. we used issr molecular markers for population genetic investigation. genetic diversity analyses revealed a moderate genetic variability within plantago afra, while pcoa showed some degree of genetic admixture among populations. amova produced significant genetic difference among populations. the mantel test showed a positive significant correlation between the genetic and geographic distance of the studied populations. structure analysis showed that there are different genetic groups in the studied populations. morphometric analysis showed that one population differed in seed color and mean stem diameter. the same population contained specific allele combinations and differed genetically from the rest of the studied populations. therefore, we considered it as a new variety within plantago afra. keywords: plantago afra, issr, pcoa, structure analysis. introduction genus plantago l. is the largest genus of the plantaginaceae family which contains more than 200 annual and perennial herbs and subshrubs with a worldwide distribution (rahn 1996; rønsted et al. 2002). most species of the genus plantago are small, with rosette leaves, ovoid and cylindrical spikes that contain tiny flowers. plantago species have been used in both conventional and traditional systems of medicine throughout asia, europe, and north america (sarihan et al. 2005; goncalves and romano 2016). moreover, few species like p. afra l. and p. ovata forssk. are highly valued in the nutraceutical, pharmaceutical and cosmetic industries the polysaccharides obtained from husks in these species can improve intestinal performance, obesity, high cholesterol, colon cancer, constipation and diabetes (goncalves and romano 2016). 74 saeed mohsenzadeh, masoud sheidai, fahimeh koohdar p. afra (syn. p. psyllium l.) is native to the western mediterranean region, west asia, and north africa. it is an annual herb with well-developed stems (grow up to 40 cm long), leaves narrow-linear and opposite or whorl covered sparsely with short, hard and glandular hairs (kazmi 1974). this medicinal plant species grows in different regions of the country and forms several local populations. we have no information on genetic diversity and available gene pools in this species. population genetic studies is a proper approach to investigate geographical populations within a single species and to identify divergent plant populations, both in genetic content as well as morphological differentiation (sheidai et al. 2016a,b, 2018). population genetic analyses provide valuable data on the rate of genetic divergence, genetic variability within/ between populations, self-pollination versus outcrossing, gene flow and inbreeding. also, data regarding morphological differentiation among populations, together with data on genetic diversity, are vital to support population management and conservation strategies (zanella et al. 2011; sheidai et al. 2016a). different molecular markers have been used in population genetic studies such as neutral multi-locus markers (rapd, rflp, issr, ssr, srap, aflp, etc.) and gene sequence data (cp-dna, nuclear its, ets, etc.) (ferreira et al. 2013; mosaferi et al. 2015; sheidai et al. 2013, 2016a,b, 2018). in the present work, we carried out population genetic analyses of p. afra by using inter-simple sequence repeats (issr) markers as they are reproducible, and cheap in cost (sheidai et al. 2013; 2016 a,b; safaei et al. 2016). morphological analyses of these populations were also performed to study if genetic divergence in populations is accompanied by morphological differentiation. material & methods plant materials for the present study, 88 plant accessions were collected from 10 geographical populations in two provinces of fars and bushehr, that are located in south of iran. details of localities are provided in table 1. the voucher specimens are deposited in the herbarium of shahid beheshti university (hsbu). identification of p. afra was done by using different references (patzak & rechinger 1965; kazmi 1974; sell et al. 2010). dna extraction and pcr details total dna was extracted from 40 mg of leaf tissue by using ctab-activated charcoal protocol (križman et al. 2006). quality of extracted dna was examined by running on 0.8% agarose gel. each issr amplification was performed in a 25μl volume containing 20 ng of genomic dna, 10 mm trishcl buffer at ph 8, 50 mm kcl, 1.5 mm mgcl2, 0.2 μm of a single primer, 0.2 mm of each dntp and 3 u of taq dna polymerase (bioron germany). issr analyses the issr primers employed were (ga)9a, (ga)9t, ubc 807, ubc 811, ubc 810, ubc 834, cag(ga)7, (ca)7ac, (ca)7at and (ca)7gt commercialized by the university of british columbia. amplification reactions were done in a techne thermocycler (germany) with the following program: 5 min for initial denaturation step at 94 °c, 1 min at 94 °c, 45s at 55 °c, 2 min at 72 °c and a final run of 10 min at 72°c. the amplification products were visualized by running on 1% agarose gel, followed table 1. the studied populations, their localities and voucher numbers. pop. locality longitude latitude altitude (m) voucher no. 1 fars, kazeroun, taleghanei mountain 51°40’13” 29°38’29” 970 hsbu-2018410 2 fars, kazeroun, aboali village 51°42’2” 29°31’32” 838 hsbu-2018411 3 fars, kazeroun, mountains around baladeh village 51°56’48” 29°17’22” 781 hsbu-2018412 4 fars, farashband, nougin village 52°0’46” 29°10’16” 740 hsbu-2018413 5 fars, bishapour 51°35’21” 29°44’43” 840 hsbu-2018414 6 fars, mountains around ghaemieh 51°25’21” 29°50’26” 928 hsbu-2018415 7 fars, mountains around noorabad 51°35’1” 29°58’51” 1080 hsbu-2018416 8 fars, konartakhteh 51°24’40” 29°31’45” 512 hsbu-2018417 9 bushehr, dalaki 51°17’10” 29°25’13” 83 hsbu-2018418 10 bushehr, chahkhani village 51°6’27” 29°11’42” 20 hsbu-2018419 75populations genetic study of plantago afra l. by the ethidium bromide staining. the fragment size was estimated by employing a 100 bp molecular size ladder (fermentas, germany). data analyses morphological analysis morphological characters studied are stem diameter, peduncle length, internode length, leaf length and width, spike length, seed color. we used ward clustering (minimum spherical cluster method) based on euclidean distance after 100 times bootstrapping for grouping of the accessions. data analysis performed by using past ver. 2.17 software (hammer et al. 2012). molecular analysis we used the issr bands as binary characters and coded them accordingly (absence = 0, presence = 1,). the number of common bands versus private bands was determined. genetic diversity parameters such as the percentage of allelic polymorphism, diversity (he), allele diversity, nei’s gene and shannon information index (i) were determined (weising et al. 2005). we used genalex 6.4 for these analyses (peakall and smouse 2006). nei’s genetic distance (weising et al. 2005) was determined among the studied populations followed by neighbor joining (nj). amova test with 1000 permutations performed for examining the genetic difference of the studied populations (peakall and smouse 2006). dca (detrended correspondence analysis) was performed for estimating the distribution of loci in the genome. pcoa (principal coordinate analysis) analysis was performed to group the plant specimens according to issr data. the mantel test (podani 2000) was implemented to study the association between genetic distance and geographical distance of the studied populations. data analyses were performed by using genalex 6.4 (peakall and smouse 2006) and past ver. 2.17 (hammer et al. 2012). bayesia n model-based structur e a na lysis (pritchard et al. 2000) was utilized to examine the genetic structure of the studied populations. for this analysis data were scored as dominant markers and analysis developed the method advised by falush et al. (2007). the structure harvester website (dent and von holdt 2012) to perform the evanno method (evanno et al. 2005) was used for the optimal value of k in the studied populations. the selection of the most likely number of clusters (k) was performed by calculating an ad hoc statistic δk based on the rate of change in the log probability of data between successive k values, as defined by evanno et al. (2005). results morphometry the ward tree (fig. 1), of the selected studied populations based on morphological features, separated plants of population 1, from the others. this population differed in stem diameter (fig. 2a,b) and seed color (fig. 2c,d) from other populations. the mean stem diameter was 4mm in population 1, while it ranged from 1mm up to 3mm in other populations. similarly, the seed color was dark brown in population 1, while it was light brown in the other studied populations. issr analyses we obtained 31 issr bands (loci) in total (table 2). the highest mean number of bands occurred in populations 1 and 3 (31 and 28 bands, respectively). some of the populations had private bands while, few common bands occurred in the studied population. these are shared alleles among these populations. the studied p. afar populations varied in genetic variability (table 3), for example, population 1 had the highest degree of genetic polymorphism (58.97%), while the population 10 showed the lowest degree (5.13%). the highest value of new gene diversity was observed in population 1 (0.15) followed by populations 3-5 (>0.10). however, the studied populations had almost a similar value for the mean effective alleles. figure 1. ward tree of morphological data of the selected populations of p. afra. 76 saeed mohsenzadeh, masoud sheidai, fahimeh koohdar dca detrended correspondence analysis plot of issr alleles (fig. 3) revealed that the loci studied are distributed in the genome and are not closely linked as they are scattered in this plot. therefore, issr loci studied are suitable molecular markers for genetic variability investigation in p. afar. the discriminating power of the issr loci is presented in table 4. we presented only the loci with at least 0.70 gst value/ or above 1 nm indicating their migration and shared value. the mean gst value 0.62 for all issr loci, indicates that these molecular markers have a good discriminating power and can be used in date plantago figure 2. a: stem of population 1; b: stem of other populations; c: dark brown seed in population 1; d: light brown seed in other populations. figure 3. dca plot of issr alleles of p. afra. figure 4. pcoa plot of the studied populations based on issr data. table 2. issr bands in p. afar populations studied. population pop1 pop2 pop3 pop4 pop5 pop6 pop7 pop8 pop9 pop10 no. bands 31 26 28 24 23 17 14 17 16 14 no. bands freq. >= 5% 31 26 28 24 23 17 14 17 16 14 no. private bands 3 0 1 1 1 0 0 0 0 0 no. lcomm bands (<=25%) 4 2 2 1 1 0 0 0 1 1 no. lcomm bands (<=50%) 13 9 10 5 6 2 1 1 2 2 77populations genetic study of plantago afra l. cultivar genetic fingerprinting. the studied p. afar populations almost showed a high degree of genetic similarity (mean = 0.84) (table 4). the highest degree of genetic distance (0.32) occurred between populations 3 and 9, while the lowest degree (0.03) between populations 9 and 10. pcoa grouping of the p. afar populations based on issr data (fig. 4), placed the studied specimens in 4 major groups. individuals of populations 9 and 10 were intermixed and formed the first major group at the left top part of the pcoa plot. individuals from three populations 1-3, are also close to each other and comprised the second major group, at the right top corner of this plot. similarly, plants in populations 6-8 and 4 and 5 comprised the next two major groups, which are placed in lower parts of the pcoa plot. these results indicate that a limited degree of genetic admixture has occurred in some of the studied populations. nj tree obtained (fig. 5), revealed the genetic affinity between p. afar populations. the four genetic groups are well separated in distinct clusters. as also indicated before, the populations 6-8 formed a distinct cluster, in which populations 6 and 7 showed closer genetic similarity. the populations 9 and 10 formed the second genetic figure 5. nj tree of the studied populations based on issr data. figure 6. structure plot of the studied p. afra populations. table 3. genetic diversity parameters in the studied populations (n = number of samples, na= number different alleles, ne = number of effective alleles, i= shannon’s information index, he gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism). pop na ne i he uhe %p pop1 1.385 1.236 0.241 0.151 0.160 58.97% pop2 1.000 1.120 0.124 0.077 0.081 33.33% pop3 1.154 1.216 0.202 0.131 0.139 43.59% pop4 0.974 1.179 0.168 0.109 0.114 35.90% pop5 0.897 1.180 0.160 0.106 0.116 30.77% pop6 0.692 1.176 0.143 0.097 0.105 25.64% pop7 0.538 1.089 0.084 0.054 0.058 17.95% pop8 0.641 1.128 0.111 0.075 0.079 20.51% pop9 0.513 1.031 0.035 0.021 0.022 10.26% pop10 0.410 1.035 0.029 0.020 0.021 5.13% table 4. discriminating power of issr loci studied in p. afar populations. locus sample size ht hs gst nm* locus2 88 0.0333 0.0306 0.0823 5.5745 locus4 88 0.4011 0.0982 0.7949 0.1290 locus7 88 0.4790 0.0982 0.7949 0.1290 locus11 88 0.1378 0.1267 0.0808 5.6895 locus12 88 0.0215 0.0205 0.0440 10.8533 locus13 88 0.0333 0.0306 0.0823 5.5745 locus15 88 0.0233 0.0208 0.1075 4.1492 locus17 88 0.0233 0.0208 0.1075 4.1492 locus24 88 0.5000 0.1022 0.7956 0.1284 locus25 88 0.0360 0.0300 0.1682 2.4719 locus26 88 0.4434 0.0536 0.8790 0.0688 locus28 88 0.3200 0.0000 1.0000 0.0000 locus30 88 0.4592 0.0929 0.7977 0.1268 locus32 88 0.4899 0.0531 0.8915 0.0608 locus34 88 0.1238 0.1071 0.1343 3.2217 locus35 88 0.2314 0.1685 0.2716 1.3407 locus36 88 0.0604 0.0487 0.1933 2.0870 locus36 88 0.0730 0.0664 0.0899 5.0614 mean 88 0.2266 0.0840 0.6293 0.2945 st. dev 0.0335 0.0041 * nm = estimate of gene flow from gst or gcs. e.g., nm = 0.5(1 gst)/gst; see mcdermott and mcdonald, ann. rev. phytopathol. 31: 353-373 (1993). ht = totale diversity, hs = diversity due to population. 78 saeed mohsenzadeh, masoud sheidai, fahimeh koohdar group and were positioned in a separate cluster. the same holds true for the populations 4 and 5. similarly, populations 1-3 comprised the last genetic group, with populations 2 and 3 showing closer genetic similarity. amova produced significant genetic difference among the studied populations (p = 0.001). it reveals that 61% of total genetic difference occurred due to among populations difference, while 39% was due to within populations genetic variability. similarly, pairwise amova among populations (table 6), revealed that most of the populations differed significantly in their genetic content. structure analysis revealed more detailed information on the genetic structure of the studied p. afra populations (fig. 6). the populations 2-3, and the populations 9-10 are genetically similar due to a high degree of shared common/ancestral alleles (similarly colored segments). population 1 and 4, contained distinct allele combinations and differed in color segments. the mantel test performed between the genetic and geographical distance of the studied populations produced a significant positive correlation (p = 0.0002). therefore, with an increase in the population geographical distance, they become genetically more diverged. this is called isolation by distance (ibd). this also indicates that gene flow may occur among the neighboring populations only, which is in agreement with low degree of nm and genetic admixture obtained. identification of new variety in plantago afar based on both morphological and genetic results, we consider population 1 as a new variety for plantago afra. seed color and diameter of the stem are among important morphological characters that can be used in the taxonomy of the genus at the bellow species rank. discussion p. afra is medicinally important plant and producing data on its genetic affinity, genetic structure and variability can be used in conservation and probably future breeding programs. in the process of populations divergence, they may form new taxonomic entity bellow the species rank. this may be, ecotype, variety or subspecies (sheidai et al. 2013, 2014). the species delimitation in complex groups and in cases where the species have morphological overlap is a tedious and difficult task. therefore, using multiple approaches is suggested to determine the species boundaries (carstens et al. 2013). in the recent years, combined approaches of morphological and molecular studies have been used to delimit the species and subtable 5. nei genetic distance versus genetic identity of p. afar populations. nei’s genetic identity (above diagonal) and genetic distance (below diagonal). pop id 1 2 3 4 5 6 7 8 9 10 1 **** 0.87 0.81 0.77 0.76 0.82 0.80 0.76 0.78 0.76 2 0.13 **** 0.92 0.83 0.84 0.78 0.73 0.83 0.76 0.75 3 0.20 0.08 **** 0.84 0.84 0.78 0.74 0.83 0.72 0.74 4 0.25 0.17 0.16 **** 0.89 0.90 0.80 0.85 0.77 0.78 5 0.26 0.17 0.16 0.11 **** 0.89 0.81 0.86 0.75 0.78 6 0.19 0.23 0.24 0.10 0.11 **** 0.95 0.91 0.85 0.85 7 0.21 0.31 0.30 0.21 0.20 0.04 **** 0.92 0.842 0.84 8 0.26 0.17 0.18 0.16 0.15 0.08 0.08 **** 0.87 0.87 9 0.23 0.26 0.32 0.25 0.27 0.15 0.17 0.13 **** 0.96 10 0.26 0.28 0.30 0.24 0.23 0.15 0.16 0.13 0.03 **** table 6. pair-wise amova among p. afar populations. phipt values below diagonal. probability, p(rand >= data) based on 999 permutations is shown above diagonal. pop1 pop2 pop3 pop4 pop5 pop6 pop7 pop8 pop9 pop10 0.000 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 pop1 0.431 0.000 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 pop2 0.459 0.263 0.000 0.001 0.001 0.001 0.001 0.002 0.001 0.001 pop3 0.567 0.547 0.475 0.000 0.001 0.001 0.001 0.001 0.001 0.001 pop4 0.555 0.559 0.452 0.459 0.000 0.005 0.002 0.001 0.001 0.001 pop5 0.506 0.615 0.542 0.392 0.477 0.000 0.004 0.001 0.001 0.001 pop6 0.590 0.732 0.653 0.623 .658 0.245 0.000 0.001 0.001 0.001 pop7 0.596 0.589 0.526 0.534 0.558 0.360 0.463 0.000 0.001 0.001 pop8 0.679 0.756 0.714 0.724 0.783 0.660 0.750 0.692 0.000 0.001 pop9 0.698 0.775 0.715 0.731 0.764 0.682 0.750 0.701 0.575 0.000 pop10 79populations genetic study of plantago afra l. species (seif et al. 2014; mosaferi et al. 2015; sheidai et al. 2016a). this multiple approaches seems to be an efficient method in p. afra as wolff & morgan-richards (1988) concluded that pcr-generated polymorphic markers like rapd and inter-ssr are useful tools to study populations of the two subspecies of p. major and to group the plants into the two subspecies. population genetic study produces important information on the genetic structure of plants, the stratification versus gene flow among the species populations, genetic divergence of the populations, etc. (sheidai et al. 2014). the issr-pcr marker technique is also efficient for genetic characterization even at the varietal level of a species. for example, charters et al. (1996) distinguished 20 cultivars of brassica napus using issr markers, similarly, seif et al. (2012) used combined analysis of morphological and issr-rapd molecular markers in 13 populations of cirsium arvense to recognize new varieties within this species. meyers and liston (2008) recognized 4 varieties of p. ovata in the new and old world by using sequence data of its and morphological characters included corolla lobe length/width ratio, trichome length to length of bracts, color midrib on corolla lobes and bracts. the occurrence of ibd in the studied populations indicates that the neighboring populations are genetically more alike than distantly placed populations. therefore, the reason for the genetic similarity of population 2 with 3, and populations 9 and 10 together, and population 6 with 7 and 8 revealed by structure plot is probably their geographical vicinity, followed by their pollination system and the distribution of their seeds by the wind, which can bring about a frequent gene flow among these populations. genetic study revealed that there are different genetic groups in the studied populations. morphological study of the selected studied population showed that we have two different groups based on morphological features. therefore, we suggest the existence of new taxa within this species. taxonomy plantago afra l. var. kazerunensis sheidai var. nov. iran. fars prov.: kazerun, 970 m, saeed mohsenzadeh, 10 april 2018, 2018410 (hsbu). description: plants annual, ca. 20 cm tall; all parts covered with short, hard and glandular hairs, stems, erect, highly branched usually of basal, diameter ca. 4 mm; internodes 3–3.5 cm; leaves opposite 3–3.5 long up to 1mm broad, linear-lanceolate or linear, margins entire; spikes ovate, 8–10 mm; peduncle 3–3.5 cm; fertile bracts 3-5 mm long, covered with glandular hairs, narrow-ovate to ovate, lower bracts in the upper part produced into a long, narrow acuminate part; sepals 3-3.5 mm long narrow-ovate covered with similar hairs as on the bracts; corolla tube up to 4 mm long, rugulose, lobes 2 mm long, narrow-ovate, acute; seeds 2, dark brown, narrow-elliptic, shining, 3 mm long. distribution and habitat: plantago afra var. kazerunensis was found at only one locality in kazerun, in the west fars province, iran. dozens of individuals were found at the type locality in the hillside of taleghanei mountain to 970 m above sea level. this area is the southern iranoturanian phytogeographic region (takhtajan 1986), which is characterized by mean temperatures of 44˚c (in the hottest month) and 3˚c (in the coldest month) and a mean annual precipitation of 260 mm. iucn red list category: plantago afra var. kazerunensis has not yet been evaluated using iucn red list criteria (iucn 2010), although it is abundant at the collection site and produces many seeds. for now, its conservation status is estimated as data deficient (dd). key to the varieties of plantago afra 1stem diameter up to 3 mm, seed color light brown plantago afra var. afra stem diameter, usually more above, about 4mm, seed color dark brown plantago afra var. kazerunensis references carstens bc, pelletier ta, reid nm, satler jd. 2013. how to fail at species delimitation. mol ecol. 22:4369-4383. charters ym, robertson a, wilkinson mj, ramsay g. 1996. pcr analysis of oilseed rape cultivars (brassica napus l. ssp. oleifera) using 5’-anchored simple sequence repeat (ssr) primers. theor appl genet. 92:442-447. dent ea, vonholdt bm. 2012. structure harvester: a website and program for visualizing structure output and implementing the evanno method. conserv genet resour. 4(2):359–361. evanno g, regnaut s, goudet j. 2005. detecting the number of clusters of individuals using the software structure: a simulation study. mol ecol. 14:26112620. falush d, stephens m, pritchard jk. 2007. inference of population structure using multilocus genotype data: 80 saeed mohsenzadeh, masoud sheidai, fahimeh koohdar dominant markers and null alleles. mol ecol notes. 7:574-578. ferreira v, matos m, correia s, martins n, gonçalves s, romano a, pinto-carnide p. 2013. genetic diversity of two endemic and endangered plantago species. biochem syst ecol. 51:37-44. goncalves s, romano a. 2016. the medicinal potential of plants from the genus plantago (plantaginaceae) ind crops prod. 83:213–226. hammer ø, harper dat, ryan pd. 2012. past: paleontological statistics software package for education and data analysis. palaeontol electron. 4:9. iucn. 2010. the iucn red list of the threatened species, version 2010.4. available from: http://www.iucnredlist.org kazmi ma.1974. plantaginaceae. in: flora of pakistan. 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(plantaginaceae) saeed mohsenzadeh*, masoud sheidai, fahimeh koohdar a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding timir baran jha1,*, partha sarathi saha2, sumita jha2 chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india harminder singh2, jaswant singh1,*, puneet kumar2, vijay kumar singhal1, bhupendra singh kholia2, lalit mohan tewari3 population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies raheleh tabaripour1,*, masoud sheidai1, seyed mehdi talebi2, zahra noormohammadi3 induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions girjesh kumar, shefali singh* karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(2): 37-44, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1206 caryologia international journal of cytology, cytosystematics and cytogenetics citation: teresa garnatje,a, jaume pellicer,a, joan vallès, nathan hall, curtis hansen, leslie goertzen (2021) first genome size assessments for marshallia and balduina (asteraceae, helenieae) reveal significant cytotype diversity. caryologia 74(2): 37-44. doi: 10.36253/ caryologia-1206 received: february 02, 2021 accepted: july 14, 2021 published: october 08, 2021 copyright: © 2021 teresa garnatje,a, jaume pellicer,a, joan vallès, nathan hall, curtis hansen, leslie goertzen. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid tg: 0000-0001-6295-6217 jp: 0000-0001-7632-9775 jv: 0000-0002-1309-3942 first genome size assessments for marshallia and balduina (asteraceae, helenieae) reveal significant cytotype diversity teresa garnatje1,a, jaume pellicer1,2,a,*, joan vallès3, nathan hall4, curtis hansen4, leslie goertzen4 1 institut botànic de barcelona (ibb, csic-ajuntament de barcelona). passeig del migdia s.n. 08038 barcelona, catalonia, spain 2 royal botanic gardens, kew, richmond, tw9 3ab, united kingdom 3 laboratori de botànica unitat associada al csic. facultat de farmàcia i ciències de l’alimentació, institut de recerca de la biodiversitat irbio, universitat de barcelona. av. joan xxiii 27-31, 08028 barcelona 4 department of biological sciences and auburn university museum of natural history, auburn university, auburn, al 36849, u.s.a. *corresponding author. e-mail: jaume.pellicer@ibb.csic.es a both authors contributed equally abstract. the genus marshallia is made up by seven to ten species of perennial herbs growing mainly in open habitats, whereas the genus balduina is represented by three sympatric species; two perennial herbs and one annual, growing in open pine forest habitats. both genera belong to the family asteraceae, tribe helenieae, and are endemic to the southeast united states, in north america. cytogenetic studies concerning these two genera are scarce and genome size data is lacking for both. the main goals of this study were to (i) generate novel insights into the evolution of the genome size and (ii), contribute to filling existing gaps on our knowledge of the asteraceae family from this point of view. nuclear dna contents range from 11.42 pg/2c in marshallia trinervia to 31.58 pg/2c in marshallia mohrii. the combination of genome size with chromosome data (and inferred cytotypes) suggests the existence of multiple cytotypes, and provides interesting insights into the potential impact of polyploidy in the evolution of these genera in general, and the shaping of genome size diversity, in particular. keywords: barbara’s buttons, chromosome counts, compositae, nuclear dna content, karyology, polyploidy. introduction the genus marshallia schreb. (asteraceae: helenieae), commonly known as barbara’s buttons, is endemic to the southeast united states of america (hansen and goertzen 2014). this small genus is made up of seven (baldwin 2009; watson 2006) to ten species (weakley 2020) of perennial herbs, which grow mainly in open habitats such as pine forests and roadsides, although 38 teresa garnatje et al. some species show preference for wet habitats as bogs, shoals or stream sides. morphologically, the genus is characterized by possessing discoid inf lorescence heads of deeply lobed, rotate corollas that are colored either white or pink. some of its morphological features are shared with other groups of asteraceae (baldwin 2009). this author placed the genus within subtribe marshalliinae, closely related to gaillardiinae (which includes balduina nutt., gaillardia foug., and helenium l.) in the tribe helenieae, but its sister group has not yet been clearly established (baldwin and wessa 2000). although species of marshallia can be difficult to distinguish from each other based on morphological characters, a more recent study carried out by hansen and goertzen (2014) revealed that nuclear ribosomal its sequences serve as an acceptable dna barcode marker in the genus, with sufficient nucleotide differences to discriminate amongst most species. the genus balduina nutt. is endemic to the southeast united states, and it is represented by just three sympatric species, two perennial herbs and one annual (keener 2006). parker and jones (1975) putatively related this genus to the tribe helenieae based on an analysis of flavonoid and sesquiterpene lactone composition. genome size (gs, usually estimated as the 2c-value), refers to the total amount of dna in an unreplicated somatic nucleus (i.e. holoploid genome size, greilhuber et al. 2005). this parameter is considered as a biodiversity trait given the 2,400-fold variation encountered among land plants (pellicer et al. 2018), with representatives having some of the largest eukaryotic genomes so far reported (c. 300 gbp/2c) in contrasting lineages such as the monocots and pteridophytes (hidalgo et al. 2017). certainly, the relevance of this parameter in the evolution of plants is without doubt and further supported by the multiple correlations reported between gs and several ecological, life cycle and karyological attributes (e.g. bennett and leitch 2005; beaulieu et al. 2008; knight and ackerly 2002; pellicer et al. 2010a; pustahija et al. 2013; pellicer et al. 2014). genome size diversity and evolution studies in the asteraceae have been examined by several authors (e.g. vallès et al. 2013, vitales et al. 2019). however, achieving a comprehensive understanding of gs evolution in a family as large as the asteraceae (c. 25.000 species) is challenging. in fact, only about 6% of the extant taxonomic diversity at the species level in this family has been studied from this point of view (vitales et al. 2019). despite the gaps in our knowledge, those studies have evidenced a relative high diversity of gs across species, ranging about 139-fold, mostly driven by the ubiquitous nature of polyploidy across the family. indeed, the lack of correlation between gs and chromosome number among diploids suggests that chromosomal rearrangements have a relatively minor impact on the overall dna content at the family level (vitales et al. 2019). although some species of marshallia have recently been the subject of studies of nuclear gene regulation in non-model systems (melton et al. 2019), and also of conservation biology (knapp et al. 2020), cytogenetic studies concerning marshallia or balduina are very scarce and mostly restricted to chromosome counts. so far gs data are entirely absent for both genera according to the plant c-values database (https://cvalues.science.kew.org, pellicer and leitch 2020) as well as the family-specific asteraceae genome size database (https://www.asteraceaegenomesize.com, vitales et al. 2019). for that reason, the main goal of this study was to provide new gs and chromosome data for most species of these genera, aiming at (i) generating novel insights into the evolution of this parameter and (ii) contributing to filling existing gaps on our knowledge of asteraceae genome size evolution. materials and methods plant material the species and populations studied as well as their origin and herbarium vouchers (deposited in the john d. freeman herbarium (aua), of the auburn university museum of natural history, auburn, alabama, usa) are shown in table 1. nuclear dna content assessments genome sizes of the target species were estimated using flow cytometry. pisum sativum l. ‘express long’ (2c = 8.37 pg, marie and brown 1993) was used as an internal standard. young, healthy leaf tissue (about 25 mm2) from each species was placed in a plastic petri dish and chopped in 1,200 µl of lb01 lysis buffer (doležel et al. 1989) with a razor blade. the suspension of nuclei was filtered through a nylon mesh with a pore size of 70 µm and stained for 20 min with 36 µl of propidium iodide (60 µg/ml; sigma-aldrich química) and supplemented with 100 µg/ml ribonuclease a (boehringer). for each individual, two replicates were prepared and processed on the f low cytometer. measurements were made at the centres científics i tecnològics de la universitat de barcelona using an epics xl flow cytometer (coulter corporation, hialeah, fla.). the instrument was set up with the standard configuration: excitation of 39first genome size assessments for marshallia and balduina (asteraceae, helenieae) reveal significant cytotype diversity the sample was done using a standard 488 nm air-cooled argon-ion laser at 15 mw power. acquisition was automatically stopped at 8,000 nuclei. the half-peak coefficient of variation was calculated for both target plant material and the internal standard. chromosome counts root-tip meristems were obtained from achenes germinated on wet filter paper in petri dishes at room temperature. seedlings were pretreated with 0.05% aqueous colchicine at room temperature for 2.5 h. material was fixed in absolute ethanol and glacial acetic acid (3:1) for 2 h at room temperature and stored in the fixative at 4°c. samples were hydrolyzed in 1 n hcl for 5 min at 60°c, stained with 1% aqueous aceto-orcein for 4h, and squashed on slides in 45% acetic acid-glycerol (9:1). the best metaphase plates were photographed with a digital camera (axiocam mrc5 zeiss) mounted on a zeiss axioplan microscope, and images were analyzed with axio vision ac software version 4.2. phylogenetic tree and data mapping in order to plot and visualize gs data from a phylogenetic perspective, genbank its sequences from hansen and goertzen (2014) and an outgroup (helianthus annuus l.) were downloaded using geneious prime 2020.1.2 (https://www.geneious.com), and aligned with clustal omega (sievers et al. 2011). a maximum likelihood tree was then constructed using the default settings and 10,000 bootstrap, as implemented in geneious. genome size data were plotted on the tree using the plottree.wbars function implemented in phytools package (revell 2012), and c-value scatterplots were carried out using ggplot2 package (wickham 2016), both available in r (r core team 2019). results the results obtained for gs, complemented with chromosome numbers in some of the accessions are shown in table 2. illustrative chromosome pictures and the distribution of gss from a phylogenetic perspective in marshallia are depicted in figure 1. in all investigated accessions, flow histograms with coefficients of variation below 3.5 were obtained, illustrating the high quality of the results obtained. as highlighted above, these two genera have never been studied from this perspective, and therefore, our results represent the first estimates for all of these species. discussion the combination of gss with actual chromosome data (plus inferred cytotypes) provides interesting insights into the potential impact of polyploidy in the evolution of both marshallia and balduina. semple and watanabe (2009) attributed to the tribe helenieae s. str., to which the two genera considered here belong, a secondarily derived base number of x = 19. however, all counts reported here as well as those previously recorded in the literature (see below) correspond to a primary base number of x = 9, one of the most frequent in the family asteraceae. table 1. marshallia and balduina species studied including population code and origin. species code voucher (in herbarium aua) balduina uniflora nutt. b1 live material from au davis arboretum marshallia caespitosa nutt. ex dc. var. caespitosa m26 watson 12-01, pottawatamie co., ok m. graminifolia (walt.) small m1 hansen 4951, covington co., al m. graminifolia (walt.) small m39 hansen 5814, jackson co., ms m. graminifolia (walt.) small m40 hansen 5814, beauregard par., la m. mohrii baedle and f.e.boynton m20 hansen 5055, bibb co., al m. mohrii baedle and f.e.boynton m21 hansen 5056, bibb co., al m. obovata (walt.) baedle and f.e.boynton m3 hansen 4956, macon co., al m. obovata (walt.) beadle and f.e.boynton m22 hansen 5471, macon co., al m. obovata (walt.) beadle and f.e.boynton m34 hansen 5786, bullock co., al m. ramosa beadle and f.e.boynton m19 hansen 5054, ben hill co., ga m. ramosa beadle and f.e.boynton m38 hansen 5795, washington co., fl m. trinervia (walt.) trel. m2 hansen 4954, lee co., al 40 teresa garnatje et al. genome size and chromosome diversity in marshallia nuclear dna contents varied 2.76-fold in marshallia, ranging from 11.42 pg/2c in marshallia trinervia (walt.) trel. to 31.58 pg/2c in the population m21 of marshallia mohrii beadle and f.e. boynton (see table 2). the large gs found in the latter, is further supported by the fact that this particular accession is a hexaploid, as confirmed by our chromosome counts (2n = 56, figure 1a). furthermore, a likely hybrid origin of this species, table 2. marshallia and balduina species studied including genome size measurements and chromosome counts. species code n1 2c (pg) 2c (mbp)2 1cx (pg) hpcv plant hpcv standard 2n 2n3 balduina uniflora nutt. 2 12.96±0.00 12675 6.48 2.44±0.37 3.03±0.16 18* 72 m. caespitosa nutt. ex dc. m26 1 22.83 22328 5.70 1.87 2.85 36* 18,36 m. graminifolia (walt.) small m1 1 12.74 12460 6.37 2.50±0.19 2.82±0.05 18 18 m. graminifolia (walt.) small m39 1 12.72 12440 6.36 3.23±0.32 3.81±0.06 18* 18 m. graminifolia (walt.) small m40 5 12.89±0.27 12606 6.45 2.42±0.48 3.01±0.37 18* 18 m. mohrii baedle and f.e.boynton m20 1 16.70 16333 5.56 0.67±0.02 3.31±0.10 27* 36 m. mohrii baedle and f.e.boynton m21 4 31.58±0.97 30885 5.26 1.27±0.95 3.08±0.28 54 36 m. obovata (walt.) baedle and f.e.boynton m3 3 13.60±0.51 13300 6.80 2.39±0.35 2.75±0.22 18 18 m. obovata (walt.) beadle and f.e.boynton m22 1 13.73 13428 6.86 2.95±1.60 3.71±0.07 18 18 m. obovata (walt.) beadle and f.e.boynton m34 1 13.92 13614 6.96 2.07±0.06 2.39±0.26 18* 18 m. ramosa beadle and f.e.boynton m19 1 16.77 16401 5.59 1.25±0.32 2.20±0.13 27* 18 m. ramosa beadle and f.e.boynton m19 2 23.92±0.12 23394 5.98 2.51±0.41 3.27±0.90 36* 18 m. ramosa beadle and f.e.boynton m38 2 13.37±0.19 13076 6,68 3.02±0.29 3.44±0.25 18* 18 m. trinervia (walt.) trel. m2 3 11.42±0.06 11169 5.71 3.21±0.26 3.30±0.26 18* 18 1 n = numer of individuals. 2 1 pg = 978 mbp (doležel et al. 2003). 3 cromosome counts database (rice et al. 2015). * chromosome numbers inferred from nuclear dna contents. figure 1. a. illustrative chromosome counts in marshallia: (top) marshallia obovata (walt.) baedle and f.e. boynton (m3, 2n = 18). (bottom) marshallia mohrii (m21, 2n = 56). scale bars are 10 μm. b. phylogenetic mapping of genome size data (2c-values) based on its sequences from hansen and goertzen (2014). inferred ploidy levels based on nuclear dna contents are indicated. 41first genome size assessments for marshallia and balduina (asteraceae, helenieae) reveal significant cytotype diversity with subsequent introgression has been suggested in the past. for example, watson et al. (1991) and more recently hansen and goertzen (2014) suggested an allopolyploid origin of this species, hypothesizing that m. trinervia (2n = 18) could be one of the parents, which is supported by the very close phylogenetic relationship among both species (hansen and goertzen 2014). other species possibly involved in the origin of m. mohrii could be either m. caespitosa or m. ramosa, given their relatively close phylogenetic relationship with this species (hansen and goertzen 2014). considering the federally endangered status of this imperiled species, further research into its apparent cytotype diversity is warranted. of the two investigated accessions of m. mohrii, the specimen belonging to population m20 had a gs of 16.70 pg/2c. compared to other confirmed diploid accessions in this study, such as m. obovata (2c = 12.89 pg) or m. graminifolia (2c = 13.60 pg), its nuclear dna content is larger than would have been expected for a diploid accession. several mechanisms could be invoked to provide an explanation for this gs, such as activation of amplification of repetitive dna and/or polyploidy. based on the value obtained for the hexaploid population of this species (m21, 31.58 pg/2c), a triploid cytotype could have, in theory, a similar gs as that found in population m20 (as inferred in figure 1). however, to avoid excessive speculation, further studies would be needed to confirm this point including an actual chromosome count, and thus discard the existence of bursts of dna amplification as the main driver of such genomic expansion. concerning m. caespitosa, only one individual was analyzed in the present study (22.83 pg/2c, table 2). from this value, a tetraploid cytotype can be also inferred (figure 1), if compared with the results in chromosomally-confirmed diploid taxa. certainly, both diploid (2n = 18) and tetraploid (2n = 36) levels are known in the species (watson and estes 1990), which makes our inference more feasible. marshallia ramosa, the other possible genome donor of m. mohrii, is highly variable in morphology in the field, and also in gs (table 2). in the present study, observed nuclear dna content is compatible with three ploidy levels (2x, 3x and 4x; figure 1), although only 2n = 18 has been previously reported for this species (watson and estes 1990). our results thus support a scenario where hybridization and introgression might have taken place, influencing changes in gs through the likely existence of multiple ploidy levels. in contrast to the above-mentioned cytogenetic variability, data from m. graminifolia and m. obovata, suggest overall intraspecific gs stability, with values ranging only 1.02 and 1.01-fold among accessions, respectively. our results confirmed that both species are diploid (table 2, figure 1), as previously reported by watson and estes (1990), and therefore the small intraspecific differences in gs among them could have arisen through chromosomal reorganizations, as previously found in other asteraceae (e.g. anacyclus; vitales et al. 2020). is genome size diversity mostly driven by polyploidy in marshallia? the nuclear dna content estimates and somatic chromosome numbers from this study set up a scenario where polyploidy has played a significant and ongoing role in the in the evolution of marshallia, influencing gs in particular. genome sizes of around 12-13 pg/2c for the diploid level (i.e. 2n = 18), 23-24 pg/2c for the tetraploid (putatively corresponding to 2n = 36), and 31-32 for the hexaploid level (corresponding to 2n = 54) were confidently inferred (figure 1). in addition, two populations presented nuclear dna amounts around 16-17 pg/2c, suggesting the existence of triploid representatives in the genus. if our overall ploidy inferences hold true, this would indicate that while m. obovata and m. graminifolia clades are essentially diploid, the clade including m. mohrii (3x and 6x), m. ramosa (2x, 3x, 4x) and m. caespitosa (4x) is cytogenetically highly diverse in a somewhat lineage-specific manner (figure 1, hansen and goertzen 2014). polyploidy and whole genome duplications have been shown to have a direct impact on the gs, especially since it involves, at least, a duplication of the overall dna content (pellicer et al. 2018). however, genomic restructuring after polyploid formation can result in elimination of specific dna sequences, leading to a loss of linearity in the accumulation of dna, the so-called genome downsizing (leitch and bennet 2004). this phenomenon can be seen in marshallia, where a reduction of the holoploid nuclear dna content with increasing ploidy levels was observed, which was more patent at higher ploidy levels (figure 2a). for example, bearing in mind that 2c-values of about 12-13 pg were found in diploid accession, nuclear dna contents of ca. 18-20 pg would be expected in 3x, 24-26 pg in 4x, and 36-40 pg (6x) would be expected under the assumption of proportional genome expansion. however, the observed results are lower in each case (table 2, figure 2a). the impact of such reduction in marshallia, is further illustrated by the fact that monoploid c-values (i.e. 1cx) are lower in polyploids with respect to their diploid counterparts (figure 2b). as stated, genome downsizing in polyploids is a very common phenomenon in plants, and the asteraceae family is no exception. among other mechanisms, chromosome rearrangements after polyploidy, particularly 42 teresa garnatje et al. for relatively old genome duplication events, can influence this process (e.g. leitch and bennet 2004, pellicer et al. 2010b, and references therein). in the genus artemisia, even between closely related species, contrasting gs dynamics have been reported (pellicer et al. 2013), involving changes in the number and distribution of repetitive dnas, such as ribosomal dna loci, which could influence changes in gs. however, in some other cases, genome size additivity has been also described, suggesting a more recent origin of such polyploids (e.g. pellicer et al. 2010a). in other groups, both gs increases and decreases have been observed (e.g. nicotiana, leitch et al. 2008). a similar scenario was reported in the genera hieracium and centaurea (bancheva and greilhuber, 2006; chrtek et al. 2009), where multiploid taxa revealed both genome downsizing or upsizing in each genus. the mechanisms undepinning changes on gs in polyploids yet remain to be fully understood, but autopoliploidy and introgression could play a relevant role in determining the gs of the resulting polyploid. genus balduina: nuclear dna content evidences a potential unknown diploid cytotype available chromosome numbers compiled in the ccdb (rice et al. 2015) for the genus indicate the presence of tetraploids in the species balduina atropurpurea harper. and balduina angustifolia (pursh) robinson (2n = 4x; parker and jones 1975), and octoploids in balduina uniflora nutt. (2n = 8x = 72). unfortunately, for our accession of b. uniflora we have only been able to estimate the gs and an actual chromosome count is thus, missing. certainly, its nuclear dna content falls within the range of gs for diploids encountered in the closely figure 2. a. scatter plot of observed 2c-values in marshallia grouped by ploidy level. the dotted line represents the projection of expected 2c-values given a proportional increase of gs with ascending ploidy levels (note that the prediction is based on average 2c-values of diploid taxa). b. scatter plot of observed 1cx-values in marshallia grouped by ploidy level, which illustrate the reduction of the monoploid genome in ascending ploidy levels. 43first genome size assessments for marshallia and balduina (asteraceae, helenieae) reveal significant cytotype diversity related genus marshallia (table 2), suggesting that this accession could likely represent a diploid population. if this assumption holds true, then this finding would represent a new ploidy level report in the species, meaning a baseline level for chromosome evolution of the genus, which subsequently underwent several rounds of polyploidy. in any case, further chromosome research will be necessary to confirm this point and discard any other potential taxonomic issues. conclusions we have performed the first gs assessments in the genera marshallia and balduina, complemented with chromosomes counts and chromosome number inferences based on nuclear dna content. the significant, ongoing role of polyploidy and hybridization in these genera has been discussed. in order to confirm some patterns deduced from the data, further research focused on chromosome counts should be carried out in all species lacking this information, complemented with gs in the remaining species of both genera. in balduina angustifolia, the only annual species in the genus (keener 2006), this research could be particularly interesting to test whether it shows a reduced gs associated with the faster life cycle than in perennials, as reported in many annual taxa (pellicer et al. 2014, and references therein). acknowledgements we thank miquel veny and patrick thompson for technical assistance with cultivation of plants, and ricard álvarez, jaume comas, chary gonzález and sonia ruíz for technical help in flow cytometric measurements. this work was supported by the catalan government under grant 2017sgr1116. j.p. benefited from a ramón y cajal fellowship supported by the ministerio de ciencia y tecnología (ryc-2017-2274). references baldwin bg. 2009. heliantheae alliance. in: funk va, susanna a, stuessy tf, bayer rj (eds.) systematics, evolution, and biogeography of compositae. iapt, vienna. baldwin bg, wessa bl. 2000. phylogenetic placement of pelucha and new subtribes in helenieae sensu stricto (compositae). systematic botany. 25: 522-538. bancheva s, greilhuber j. 2006. genome size in bulgarian centaurea s.l. 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analysis of marshallia (asteraceae). systematic botany 15: 403-414. watson le, elisens wj, estes jr. 1991. electrophoretic and cytogenetic evidence for allopolyploid origin of marshallia mohrii (asteraceae). american journal of botany 78: 408-416. weakley as. 2020. flora of the southeastern united states. university of north carolina herbarium, north carolina botanical garden. wickham h. 2016. ggplot2: elegant graphics for data analysis. springer-verlag new york. isbn 978-3-31924277-4, https://ggplot2.tidyverse.org. caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 1 2021 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 73(4): 45-54, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-1024 caryologia international journal of cytology, cytosystematics and cytogenetics citation: v. p. sobhakumari (2020) exploration of diversity and distribution of cytotypes of saccharum spontaneum, a wild species of sugarcane, in india. caryologia 73(4): 45-54. doi: 10.13128/ caryologia-1024 received: july 20, 2020 accepted: december 12, 2020 published: may 19, 2021 copyright: © 2020 v. p. sobhakumari. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. exploration of diversity and distribution of cytotypes of saccharum spontaneum, a wild species of sugarcane, in india v. p. sobhakumari crop improvement division, icar-sugarcane breeding institute, coimbatore, tamil nadu, india e-mail: vpsobhakumari@rediffmail.com abstract. the present investigation was undertaken to examine the geographic distributions of cytotypes of saccharum spontaneum l., wild species of sugarcane, in india. new chromosome determinations has been done for 524 accessions collected mainly from four ecological zones, west, east, north and north-east, of the country. a detailed evaluation of cytogeographic pattern of s. spontaneum has been done with these clones along with the clones in which chromosome data is already available. twenty six cytotypes ranging from 2n=40 (8x) to 2n=112 (14x) has been identified in s. spontaneum from india. gangetic valley of sub himalayan region and deltaic region of south-east zone can be considered as regions of cytogeographic interest with largest concentration of different chromosome numbers. north-east region of the country appears to have had a higher evolutionary activity in this species which is evidenced by the occurrence of multiple cytotypes and sympatric growth with other species and genera. the overall cytogeographic pattern of s. spontaneum includes the incidence of mixed polyploidy, aneuploidy, sympatric with different ploidy levels and disjunct distribution of some cytotypes indicate that this species likely to have had multiple independent origin in different parts of india. keywords: saccharum spontaneum, chromosome number, mitosis, india, cytogeography, polyploidy, cytotype diversity. introduction the genus saccharum comprises of six species viz., saccharum officinarum l., saccharum barberi jesweit, saccharum sinense roxb. amend. jeswiet and saccharum edule that are cultivated and saccharum robustum brandes and jesweit ex grassl and saccharum spontaneum l. that are wild. among these species s. spontaneum was subjected to detailed studies in india due to its wide distribution and high variability in morphology and chromosome number and most importantly its contribution towards the genetic improvement of cultivated sugarcane by conferring resistance to major diseases, providing vigor and hardiness for increased abiotic stress tolerance (cold and drought), increased tillering and improved ratoonability. 46 v. p. sobhakumari the commercial success of the early interspecific hybrids involving s. spontaneum generated interest in the collection and utilization of wild sugarcane germplasm. india is one of the major centers of diversity for s. spontaneum. it has a wider distribution throughout the country, from sub – himalayan region to peninsular india. the sugarcane germplasm collection from india dates back to 1912. during that time dr. c.a. barber collected s. spontaneum clone coimbatore which later become the male parent of the first sugarcane variety, co 205. further collections of s. spontaneum were made by sir venkataraman and dr. janakiammal. later the ‘spontaneum expedition scheme (ses)’ sponsored by the indian central sugarcane committee was operated during 1948-1957, with the objective of collecting the wild saccharum germplasm from the distributional areas in the country and outside. collection efforts were revived during 1980s and six more explorations were conducted to north eastern states of india during 19811990. further exploration for collection of saccharum germplasm was conducted under the national agricultural technology project on plant biodiversity (natppb) during 1999-2004. under this programme explorations were conducted in arunachal pradesh, mizoram, orissa, andra pradesh, karnataka, tamil nadu, kerala and andaman and nicobar islands (nair, 2013). explorations are being continued thereafter under the institute programmes and the states of india like tripura (2005), meghalaya (2006), gujarat (2007), rajasthan (2008), himachal pradesh (2009), uttarakhand (2009), west bengal (2010), nagaland and manipur (2011), maharashtra (2015), punjab and har yana (2016), jharkhand (2017), west bengal and sikkim (2018 and assam (2019) were explored during the subsequent years. ). the explorations covered all states of the country and the cytogenetic studies revealed that the s. spontaneum germplasm collected represent the whole range of cytotype diversity present in the respective state. extensive cytological studies have been conducted in s. spontaneum accessions available in germplasm collection at icar-sugarcane breeding institute, coimbatore, india. natural occurrence of around 31 cytotypes in s. spontaneum ranging from 2n=40-128 was established from these studies of accessions from in and out of the country (janakiammal 1939; panje and babu 1960; mehra and sood 1974; sreenivasan 1975; kandaswamy et al. 1983; sreenivan and sreenicasan 1984, 1994; praneetha and nair, 2005; sobhakumari and mallika 2007; sobhakumari 2009; sobhakumari 2013 and sobhakumari and stanly 2017 survey on the geographical distribution of different cytotypes of s. spontaneum, which were identified till 1960, has been conducted by panje and babu (1960) and they could substantiate the proposal of parthasarathy and rao (1946) that the indian sub-continent (including nepal, bangladesh, pakistan and sri lanka) has mostly low numbers ranging 2n=40-80. after this survey the geographic distribution of the cytotypes of s. spontaneum has not been analyzed critically. a detailed knowledge of the geographic distribution of ploidy variation within the species comprising of polyploid complexes is critical to our understanding of the history and evolution of such complexes. in this study an elaborative cytological analysis of accessions of s. spontaneum from different states of india is used to resolve its cytogeographic pattern in the country. in particular, the study will address the following questions. (1) what is the ploidy variation of s. spontaneum across its distribution range (based on the representative samples collected from different states of india), (2) is this variation geographically/ecologically structured (3) where is the center of ploidy level diversity (4) how many cytotypes are available for this species in india and its role in the evolution of euploids and aneuploids in the same species. materials and methods the materials included in the study are collections about 524 in number, from different states of the country which were made during 2001-2017. the details of the number of clones used in the study from different states of the country are given in table 1. most of these clones were documented in s. spontaneum catalogues (sreenivasan et al. 2001, nair et al. 2013). the clones were numbered based on the following method. ind represents the country of origin (india), next number (0117) represents the year of collection and the last number represents the accession number. from the place of origin the materials were collected as clumps/ suckers and were established at germplasm fields of icar-sugarcane breeding institute, coimbatore after proper quarantine. for cytological studies small parts of clumps were planted in pots to collect root tips. for mitotic studies the root tips were pretreated with saturated solution of alpha bromo naphthalene at 4°c for 2h. then the washed materials were fixed in 3:1 (alcohol: acetic acid) fixative overnight. washed root tips were hydrolyzed in 1n hcl and stained in 1% acetocarmine. a minimum of 10 well spread metaphase plates were used for chromosome count and photographed in carton 402t microsystem. the cytologically analyzed 524 accessions of s. spontaneum include 478 new determinations of chromosome number from the clones collected under institute project 47exploration of diversity and distribution of cytotypes of saccharum spontaneum, a wild species of sugarcane, in india (2001-2017), 19 clones collected from arunachal pradesh during 1990 (ind 90 clones) and 27 clones which were collected under ses programme and r collections done by dr janakiammal. the clones recently analyzed (478) were mostly covered the west zone, east zone, north zone and north-east zone of india. in order to cover the whole country the earlier cytological reports of s. spontaneum were also considered for cytogeographic survey. for this purpose the clones studied by panje and babu (1960), sreenivasan (1975), sreevivasan and sreevivasan (1984 and 1994), praneetha and nair (2005), sobhakumari and mallika (2007), sobhakumari (2009), sobhakumari (2013) and sobhakumari and stanly (2017) were included. all the chromosome number data have been pooled together and a superimposed map of on the cytogenetic distribution of s. spontaneum in india has been resolved. to avoid confusion the chromosome numbers of all the clones are referred by their 2n numbers, although most of the earlier determinations have been made on pollen mother cells (meiosis). to avoid hinder during discussion the authorship references are not given each time when a cytotype is mentioned. results and discussion among the saccharum species, the wild s. spontaneum was subjected to detailed studies in india due to its wide distribution, extensive variability in morphology and chromosome number, and most importantly its use in genetic improvement of cultivated sugarcane. its somatic chromosome number as on now known extends from 2n=40-128 with basic chromosome number x=8. in the present study we examined the geographic distributions of cytotypes of s. spontaneum in different parts of india. for the present study india has divided into 6 geographic zones viz., (1) south zone, (2) west zone, (3) east zone, (4) north zone, (5) north-east zone and (6) central zone. the cytotype of 524 accessions were determined based on somatic chromosome count which were mainly distributed in four zones viz. (1) west zone, (2) east zone, (3) north zone and (4) north-east zone. few clones were studied from peninsular india and also from andaman islands. west zone this zone includes rajasthan, gujarat and maharashtra. three cytotypes, 2n=64, 72 and 80 were common in these states. of 34 s. spontaneum clones collected from gujarat, 25 were studied cytologically. majority of the clones were 2n=80 (80%) and many of them were morphologically dwarf. surprisingly only one clone, ind 07-1486, showed 2n=64 chromosomes. with two clones of 2n=72 two aneuploids of 2n=74 and 76, one each, were also present in gujarat. though rajasthan is a nearby state, its collection was having only one clone, ind 08-1502, with 2n=80. other than this 2n=64 and 2n=72 cytotypes were also present in low frequencies. this state reports only cytotypes with multiples of eight, i.e., 8x, 9x and 10x. in maharashtra out of 41 clones collected, 39 clones were cytologically analyzed. six cytotypes, 2n=60, 62, 64, 66, 72 and 80 were identified from this collection. majority of the clones were with 2n=64 (77%). other two euploids present in the collection were 2n=72 (9x) and 2n=80 (10x). few aneuploids like 2n=60, 62 and 66 were also present with polyploids that of multiples of eight. as the range of somatic chromosome number reported in s. spontaneum is 2n= 40-128, from west zone of india intermediate numbers of this range were reported. majority of the clones were 2n=62, 72 and 80. as far as the distribution of the cytotypes concerned a specific segregation could observe in the states of this zone. in gujarat majority of the clones were with 2n=80 (80%). this was an unusual occurrence while compare to other table 1. details of s. spontaneum clones cytologically analyzed. clone year of collection state no. of clones studied ind 01 2001 orissa 3 ind 02 2002 andhra pradesh 6 ind 03 2003 andaman 24 ind 04 2004 mizoram 39 ind 05 2005 tripura 13 ind 06 2006 meghalaya 16 ind 07 2007 gujarat 25 ind 08 2008 rajasthan 11 ind 09 2009 himachal pradesh & uttaranchal 45 ind 10 2010 west bengal 26 ind 11 2011 nagaland & manipur 91 ind 15 2015 maharashtra 39 ind 16 2016 punjab & haryana 88 ind 17 2017 jharkhand 52 ind 90 1990 arunachal pradesh 19 ses,ind81 & r collections 1954, 1981, 1937 madhya pradesh, bihar and ap 27 48 v. p. sobhakumari states of the country where mixed ploidy was observed. this finding contrast with the report of panje and babu (1960) where they have identified 2n=80 chromosome forms restrictedly in nepal, assam and the western ghats of india. the nearest state rajasthan was having only one clone with 2n=80 cytotype and also absent in aneuploids. this indicates that 2n=64, 72 and 80 cytotypes are cytologically stable with normal meiosis and normal chromosome segregation. east zone this zone covers the states bihar, jharkhand, west bengal and odisha. the clones that studied from bihar were collected during 1950 (ses) and 1981 (ind 81). most of them were 2n=64 cytotypes and one clone each of 2n=56 and 2n= 90 cytotypes were also there. jharkhand collections were done recently in 2017. from this collection 52 clones were subjected to cytological analysis. cytotype range identified in this collection was 2n=40-72. among this, lower cytotypes i.e., 2n=54 and 56 were in higher frequency. the reported lowest chromosome type of s. spontaneum has identified in jharkhand collection, i.e., 2n=40 (ind 17-1852). a total of 8 cytotypes were identified from this state in which aneuploids were lesser in number while compared to euploids. in west bengal collection (ind 10) only four cytotypes were identified, i.e., 2n=60, 64, 70, 72. out of 26 clones analyzed cytologically it was found that majority of them were with 2n=60 (73%). the euploids (multiples of 8) like 2n=64 and 72 were present only in low frequency. from odisha three cytotypes were identified as 2n=52, 64, 112. ind 01-1157 (2n=112) from odisha is the s. spontaneum clone with highest number reported from the present study. from the four states of east zone of india, the lowest chromosome number, 2n=40, and the highest chromosome number, 2n=112 were reported. these extreme types were only in low frequency, i.e., one in each. west bengal was showing a peculiar cytotype, i.e., 2n= 60 in high frequency. this cytotype was identified from other states also, but in low frequency. in bihar the lowest number was 2n=56 and highest number was 2n=90. as very few clones were studied from this state at present it is not sensible to conclude the position of s. spontaneum cytotypes existing in this state. there are chances for having the intermediate chromosome numbers from natural hybridization. to substantiate this view earlier reports revealed that from bihar 14 cytotypes were identified by analyzing more number of accessions of s. spontaneum (sreenivasan and sreenivasan, 1994). jharkhand was having low cytotypes like 2n= 40, 56, 64, 72 (euploids) and this may be the cause of the existence of other aneuploids in this region due to intraspecific natural hybridization. panje and babu (1960) reported the possibility of existence of cytotypes with different range of chromosome numbers in the region where low cytotypes are abundant. odisha was having the highest chromosome number 2n=112 and lowest chromosome number 2n=52. in this study only few clones from odisha has been included and the picture of chromosome survey in this state is not adequate to come to the conclusion about its cytogeographic pattern. this will be clearer in the later part of the study where all previous reports on cytological analysis of s. spontaneum from the same area were considered. north zone in this zone s. spontaneum has been collected from four states namely himachal pradesh, uttaranchal, punjab and haryana. during 2009 a combined collection has been done from himachal pradesh and uttaranchal. all the collected clones of s. spontaneum, 45 cloones, were subjected to cytological analysis. majority (71%) of them comes under the cytotype category 2n=54 and 2n=56. more number of clones (6 clones) were identified from here with 2n=40. other cytotypes present in himachal pradesh and uttaranchal were 2n=60, 64 and 72. surprisingly only one clone was with 2n=64 though it is considered as the most prevailing cytotype in india. during 2016 a combined collection has been made from punjab and haryana. high chromosomal diversity has been revealed by studying 88 accessions collected from these states. in this region twelve cytotypes were identified such as 2n=40, 48, 50, 52, 54, 56, 60, 64, 70, 72, 74 and 76. as in himachal pradesh and uttaranchal the lower cytotypes 2n=54 and 56 were more in punjab and haryana also. it covered around 60% of the whole collection. all the other cytotypes were present only in less than 8%. very clear demarcation in the chromosome number of s. spontaneum has been shown by north zone of india from other zones because of the high frequency of low chromosome number types like 2n=40, 54 and 56. 2n=64 cytotype was in less frequency in this zone. in punjab and haryana among 12 cytotypes 2n=40, 48, 56, 64, and 72 were euploids (multiples of basic chromosome number 8) with chromosome constitution 5x, 6x, 7x, 8x and 9x respectively. others were aneuploids and may be originated from intraspecific hybridization among the different ploidy cytotypes at the place of origin itself. though in punjab and haryana six clones of 2n=64 were available, only one clone was with 2n=64 in himachal 49exploration of diversity and distribution of cytotypes of saccharum spontaneum, a wild species of sugarcane, in india pradesh and uttaranchal collection. it has been reported that inter and intraspecific natural hybridization are responsible for the existence of extensive euploidy and aneuploidy in s. spontaneum (janaki ammal, 1936; janaki ammal and singh, 1936; raghavan, 1953; kandasami. 1961a; bremer, 1961a; kandaswamy and rao, 1963; sreenivasan and jagthesan, 1973). analysis on the evolutionary origin of different cytotypes of punjab and haryana collection revealed its independent as well as multiple origins (data not published). north-east zone in this zone the cytological analysis has been done for s. spontaneum clones collected from the states sikkim, meghalaya, tripura, mizoram, manipur, nagaland and arunachal pradesh. in sikkim only one cytotype, i.e., 2n=64 was identified. from meghalaya 16 clones were studied and four cytotypes, 2n=60, 64, 70 and 80 were identified. of this 2n=64 (8x) was showing majority (59%) and next to it was 2n=80 (10x). the other two aneuploids would have been derived as a result of intraspecific hybridization of euploids. these aneuploids, 2n=60 and 2n=70, were in 12% and 16% respectively. in 2005 collections of were made from tripura. thirteen clones were cytologically analyzed and chromosome number has been determined by root tip mitosis. it was found that majority of the clones were with 2n=64 (31%). the other cytotypes available in tripura were 2n=80 (23%), 2n=72 (23%). these clones were with chromosome numbers that were the multiples of 8. two types of aneuploids identified from tripura were 2n=60 and 52. they were less in percentage, 15% and 8% respectively. mizoram collections were made during 2004. thirty nine clones were cytologically analyzed from this collection and 12 cytotypes were identified. they were 2n= 56, 58, 60, 62, 64, 70, 72, 76, 78, 80, 88 and 90. while considering eight as the basic chromosome number of s. spontaneum, 2n=56, 64, 72, 80 and 88 were polyploids with chromosome constitution 7x, 8x, 9x, 10x and 11x respectively. 2n=64 (26%) and 2n=80 (28%) cytotypes were in majority in this state and next to this was 2n=56 (13%). all the other cytotypes were in less frequency and it was found that all together the nine cytotypes covered 33% of the total clones studied. combined collection was made from manipur and nagaland during 2011 and cytological analysis has been done in 91 clones of s. spontaneum. nine cytotypes, 2n=54, 56, 58, 60, 64, 70, 72, 74, and 80 were identified from these states. majority of the collection (62%) was with 2n=64 and next to it was 2n=80 (22%). all other cytotypes were in low frequency. nineteen clones were cytologically analyzed from arunachal pradesh and six cytotypes were identified as 2n=54, 56, 58, 62, 64 and 90. majority of the collections were with 2n=64. all other cytotypes present in this collection were in less number. from arunachal pradesh one clone, ind 90-755, was with 2n=90 which is a rare cy totype with high chromosome number occurred in india. from the result of cytological studies conducted in s. spontaneum clones from the states of north east region of india make us to recall the statement of dr. c.a. barber that “one of the keys which can unlock the question of ancestral sugarcane forms is concealed in north india”. in the present study 179 clones of s. spontaneum from different states of north east zone were cytologically analyzed. these clones have been collected from diverse habitats and different altitudes. fourteen cytotypes including 2n=52, 54, 56, 58, 60, 62, 64, 70, 72, 74, 76, 80, 86 and 90 were identified from this study. this revealed that this region is showing high ploidy diversity for its cytotypes. this high genetic variability is due to its high compatibility between the groups and even with other related genera and species. though we could see variable numbers with euploids of 7x, 8x, 9x, 10x and many intermediate aneuploids it was interesting to note that the lowest chromosome numbers in this species, 2n=40 and 48, were absent in this region. in earlier report the cytotype 2n=40 has been reported from sikkim and arunachal pradesh (sreenivasan and sreenivasan, 1994). they observed that irrespective of the climatic condition prevailing in the distributional area, all clones with 2n=40 were short, saturated, less cane forming with very narrow leaves due to reduction of lamina to midrib. contradictory to this 2n=80 cytotypes in most of its morphological characteristics it resembles s. barberi. in the present study determination of chromosome numbers from the recent collections have revealed new cytotypes. earlier reports of the occurrence of low chromosome types in sikkim and arunachal pradesh, occurrence of other related species and genera and existence of natural hybrids with different chromosome numbers in the north east region provides further evidence for the evolutionary significance of this zone. due to the existence of all members of “saccharum complex”, overlapping of flowering time, and high compatibility between the species makes this area much evolutionary significant as far as sugarcane is concerned. during 2003 collection of s. spontaneum has been done in andaman islands and the cytological analysis showed that only 2n=64 (8x) and 72 (9x) were available here. the essential first step when gaining insight into 50 v. p. sobhakumari the evolution of polyploid is cytogeography, the study of cytotype diversity and its past and predicted future distribution patterns. knowledge of cytotype distribution pattern usually reveals phenomena such as environmental segregation or productive isolation of cytotypes (rejlova et al., 2019). while superimposed the map of figure 1 which explained the state wise chromosome numbers of recently collected s. spontaneum clones from different zones of india with the earlier reports on the same aspect, we are getting a comprehensible picture of the geographic distribution of different cytotypes of s. spontaneum throughout indian sub-continent. in figure 2 the merged map with distribution details of so far reported cytotypes of s. spontaneum has been given. in figure 3 the distribution pattern of 26 cytotypes of s. spontaneum in six geographical zones of india has been specified. the earlier report says that in the case of s. spontaneum wherever the low cytotypes occur there is a certain concentration of other chromosome numbers also (panje and babu, 1960). the cytotype distribution pattern of north and north-east zones substantiate this statement by having maximum number of cytotypes that in the country reported. although there are many incidents of polyploid coexistence in nature, the minority cytotype exclusion hypothesis predicts that mixed figure 1. (a) – (t) somatic chromosomes in different cytotypes of s. spontaneum a) ind 16-1812 (2n=40), b) ind 16-1792 (2n=48), c) ind 11-1606 (2n=54), d) ind 11-1604 (2n=56), e) ind 11-1614 (2n=58), f ) ind 10-1574 (2n=60), g) ind 89-754 (2n=62), h) ind 11-1610 (2n=64), i) ind 01-1156 (2n=52), j) ind 17-1862 (2n=66), k) ind 17-1866 (2n=70), l) ind 15-1741 (2n=72), m) ind 10-1585 (2n=74), n) ind 09-1552 (2n=76), o) ind 07-1457 (2n=80), p) ind 08-1494 (2n=64), q) ind 03-1312 (2n=64), r) ind 04-7353 (2n=86), s) ind 90-775 (2n=90), t) ind 01-1157 (2n=112). 51exploration of diversity and distribution of cytotypes of saccharum spontaneum, a wild species of sugarcane, in india cytotype population will eventually lose one cytotype (levin, 1975). this may be the reason for absence of 2n=40 and 2n=48 in majority of the states of north east zone even though around 17 cytotypes were available in this zone. a wide ranging track could be marked to incorporate all lowest chromosome number cytotypes (2n=40, 48, 54 and 56). these cytotypes could be identified from two areas which covers the entire northern sub himalayan plain of ganga and yamuna river and this extended to the south east coast which encircled the deltas of mahanadhi, the godavari and krishna. this identified area is coincides with the geographic pattern explained by panje and babu (1960). while considering the geographic pattern of different cytotypes of s. spontaneum in india the above mentioned track is of special interest figure 2. distribution of s. spontaneum cytotypes (from 2001-2017 collection) in different states of india. figure 3. superimposed map showing distribution of s. spontaneum cytotypes (so far reported) in different states of india. 52 v. p. sobhakumari because it is found to have as many as 18 out of 26 cytotypes reported from india. the cytotype of 2n=64 has the widest distribution in india (figure 3). this may be due to its better competitive ability compare to other cytotypes. the lower part of the peninsular india which consists of tamil nadu and kerala was having more number of 2n=64 types than in north india. in kerala only euploids like 8x, 9x and 10x were present and no intermediate chromosome numbers. given the geographical separation and habitat similarity among cytotypes, mixed-ploidy populations may be transitional and subject to the forces of minority cytotype exclusion which lead to pure-ploidy populations (castro et al, 2018). in most of the states with 2n=64 types, its aneuploids, 2n=60, 62, and 66, were also observed in low concentration. mating between intraspecific cytotypes which are at the levels of ploidy often produce offsprings with odd number of genomes or imbalanced ploidy having lower fitness than those produced by individuals of same cytotype. generally, the odd polyploids show meiotic abnormalities and consequent decreased viability of gametes leading to little success in existence (rani et al., 2015). in contrast to this in west bengal relatively higher concentration of (73%) 2n=60 cytotype was observed which seems to be distinct from other states. 2n=54 cytotype groups are more confined to the north zone of the country. its relative concentration is more in himachal pradesh, uttaranchal, punjab and haryana. this is the only chromosome number so far reported from jammu and kashmir (ses 352, 2n=54). though many cytotypes inhabit the neighboring zones around gangetic plain, the central zone of india was having only few cytotypes of s. spontaneum which consist of 2n=64 and its aneuploids. chromosome number less than 64 was not reported from here. 2n=80 cytotype has been identified from all parts of the country except in north zone states. in many states it was in india chromosome number s ta tes 4 0 4 8 5 0 5 1 5 2 5 4 5 6 5 8 6 0 6 1 6 2 6 3 6 4 6 6 6 8 7 0 7 2 7 4 7 6 7 8 8 0 8 6 8 8 9 0 9 6 1 1 2 south zone k e ra la k a rna ta ka t a mil n a du a ndhra p ra des h c e n t r a l z o n e m a dhya p ra des h west zone r a ja s tha n g uja ra t m a ha ra s htra north zone j a mmu k a s hmir h p & u tta ra ncha l p unja b & h a rya na u tta r p ra des h east zone b iha r j ha rkhand w e s t b e nga l o dis ha north east zone s ikkim m e gha la ya t ripura m iz ora m n a ga la nd & m a nipur a s s a m & a runa chalp ra des h figure 4. distribution pattern of s. spontaneum cytotypes in different geographic zones of india. 53exploration of diversity and distribution of cytotypes of saccharum spontaneum, a wild species of sugarcane, in india low frequency whereas in gujarat 80% of the s. spontaneum clones were with 2n=80. this disproves the prior assumption that this cytotype was restricted assam and western ghats. the world collection of sugarcane germplasm maintained by icar-sugarcane breeding institute, coimbatore, india is the largest germplasm collection at present. a large assembly of s. spontaneum representing the entire range of availability collected from its distributional areas of the country is currently available in the institute. these accessions were used for the present study and it revealed that s. spontaneum is rich in genetic variability and are compatible group for inter and intraspecific hybridization. polyploid series from lowest chromosome number 2n=5x=40 to the highest of 2n=14x=112 in this species has been revealed from this study. natural hybridization between the cytotypes with multiples of 8 (x=8) resulted in other cytotypes and also aneuploids in its distributional areas. a total of 26 cytotypes were identified from india. in north and northeast india the evolutionary mechanism are highly active in this species and it is found that the cyto-morphological variability favor the accumulation of adaptability characters, especially to biotic and abiotic stresses. in sugarcane, compared to most of the other commercial crops, the information on genetic variability and geographic distribution pattern of the wild species, s. spontaneum, is available and it can be contributed to the development of superior clones with desirable characters. acknowledgements the author wishes to thank dr. bakshi ram, director and dr. g. hemaprabha, head, crop improvement division, icar-sugarcane breeding institute, coimbatore for the support and facilities provided for the work. she also thank dr. karthikeyan, principal scientist, icar-sbi, for providing the s. spontaneum clones in time to time for the study and mrs remadevi, mr. selvamuthu and dr. harunipriya for their technical support. references bremer g. 1961. problems in the breeding and cytology of sugarcane. 11. the sugarcane breeding from a cytological view-point. euphytica 10: 121-133. castro m, castro s, figueiredo a, husband b, loureiro j.  2018. complex cytogeographical patterns reveal a dynamic tetraploid–octoploid contact zone. aob plants 10: ply012; doi: 10.1093/aobpla/ply012 janaki ammal ek, singh tsn. 1936. preliminary note on a new saccharum x sorghum hybrid. indian j agri sci 6: 1105-1106. janaki ammal ek. 1936. cytogenetical analysis of some saccharum spontaneum l. 1. chromosome studies in some indian forms. indian j agri sci 6: 1-8. janakiammal ek. 1939. triplopolyploidyin saccharum spontaneum l. curr sci. 8:74-76. kandasami pa, sreenivasan tv, ramana rao tc, palanichami k, natarajan bv., alexander, k. c., et al. 1983. catalogue on sugarcane genetic resources i. saccharum spontaneum l. sugarcane breeding institute (icar), coimbatore. kandasami pa. 1961a. interspecific and intergeneric hybrids of saccharum spontaneum l. 1. functioning of gametes. cytologia 26(2): 117-123. kandaswamy pa, rao kks. 1963. artificially synthesized forms as an induction of the probable origin of certain naturally occurring forms of saccharum spontaneum l. indian j. sugarcane res. developm. 8: 25-31. levin da. 1975. minority cytotype exclusion in local plant populations. taxon 24: 35–43. mehra pn, sood op. 1974. floating chromosomal populations in saccharum spontaneum l. cytologia. 39: 681–696. nair nv, 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nettle. plos one 14(7): e0218389. https://doi.org/ 10.1371/journal.pone.0218389. sobhakumari vp, stanly ma. 2017. high ploidy diversity in saccharum spontaneum population of north east region of india. j. sugarcane res. 7: 52-59. 54 v. p. sobhakumari sobhakumari vp, mallika s. 2007. a cytological survey of saccharum officinarum and s. spontaneum clones. the nucleus 50: 27-32. sobhakumari vp. 2009. chromosome survey of wild and cultivated species of of saccharum. the nucleus 52: 17-23. sobhakumari vp. 2013. new dterminations of somatic chromosome number in cultivated and wild species of saccharum. caryologia 66: 268-274. sreenivasan tv, amalraj va , jebadhas aw. 2001a. catalogue on sugarcane genetic resources v s.spontaneum (part-2). sugarcane breeding institute, coimbatore. sreenivasan tv, sreenivasan j. 1984. cytology of saccharum complex from new guinea, indonesia and india. caryologia 37: 351-357. sreenivasan tv, jagathesan d. 1973. cytogenetic studies in interspecific hybrids of saccharum spontaneum l. the nucleus 16: 44-48. sreenivasan tv, sreenivasan j. 1994. chromosome numbers of saccharum and related grasses. sugarcane. 1: 16-22. sreenivasan tv. 1975. cytogenetical studies in saccharum spontaneum. proc. indian. acad. sci. 81: 131–144. caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 75-81, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-138 citation: i. petrescu, i. sarac, e. bonciu, e. madosa, c.a. rosculete, m. butnariu (2020) study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.). caryologia 73(1): 75-81. doi: 10.13128/caryologia-138 received: january 9, 2019 accepted: february 23, 2020 published: may 8, 2020 copyright: © 2020 i. petrescu, i. sarac, e. bonciu, e. madosa, c.a. rosculete, m. butnariu. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 1 banat’s university of agricultural science and veterinary medicine “regele mihai i al româniei” timisoara, romania 2 university of craiova, faculty of agronomy, craiova, romania *corresponding author. e-mail: catalin_rosculete@yahoo.com abstract. the cytogenetic study on the meristematic tissues of basil (ocimum basilicum l.) aimed to evaluate some cytotoxic effects induced by two heavy metals (cadmium cd and zinc zn) applied in three different concentrations: 10, 50 and 100 ppm. cytogenetic tests reveal a decrease of the mitotic index and the occurrence of various chromosomal aberrations following heavy metal treatments. the cell division was significantly affected, especially in the case of cd treatment, which showed the highest degree of toxicity in all variants compared to control variant. instead, zn has a lower degree of toxicity but only at concentrations of 50 ppm and 100 ppm. types of chromosomal aberrations were relatively varied, being randomly distributed and concentration dependent, for both cd and zn. were observed cells with large nucleus and disorganized-looking; interphases with pyknotic nucleus; cells with laggard chromosomes, pyknotic and sticky chromosomes, as well as cells with telophase bridge. the results reveal that cd (at all tested concentrations) and zn in concentrations higher than 10 ppm exhibit significant cytotoxic potential to ocimum basilicum l. as a result of the effects reported in cell divisions of the meristematic tissues. we can also appreciate that the ocimum basilicum l. species could be used as a test plant to determine the degree of soil pollution with heavy metals. keywords. basil, cadmium, chromosomal aberrations, mitodepresive, zinc. introduction basil (ocimum basilicum l.) is an herbaceous, annual and aromatic plant belonging to the lamiaceae family. basil is currently grown in many other parts of the world. due to medical and culinary properties, as well as the spiritual and symbolic connotations that have been in the culture of the romanian people since ancient times, basil is one of the most appreciated aromatic plants in romania, along with rosemary, mint and sage. the basil is used as a seasonal functional food, being used both in tea and fresh salads, due to its health benefits. functional foods have an important contribution to improving the quality of life (butnariu and caunii, 2013). 76 irina petrescu et al. basil is a plant used not only in the food industry but also in the pharmaceutical and perfume industry. for example, from a pharmaceutical point of view, the basil may be used as a nutritional supplement or therapeutic drug to protect against aspirin-induced gastric ulcers, a common problem resulting from the use of aspirin (abd el-ghffar et al., 2018). heavy metals are identifiable components in the environment, occurring in significant concentrations and under natural conditions. in the 21st century, the metaliferic loading of air, water, soils, and consequently of plants, animals and the human body became an urgent concern for nature pollution. the aim of this study was to determine how the basil (ocimum basilicum l.) responded to increasing cd and zn concentrations in terms of changes in cellular activity and especially in chromosomes structure. the vegetal meristematic tissues that are used for testing the effects of chemicals on chromosomes should be easy to obtain and less expensive and from this point of view, the basil can be suitable. materials and methods plant material dry seeds of ocimum basilicum l. belong to the genovese variety was placed in glass petri dishes on filter paper. three treatment variants with 4 replicates were performed for each of the heavy metals experienced (cd and zn). solutions for the treatment of seeds have been obtained by dissolving the respective amounts of heavy metals in distilled water. equal volumes of the different concentrations of cadmium nitrate cd(no3)2 and zinc nitrate zn(no3)2 solutions (10, 50 and 100 ppm), respectively were administered while the control was treated with distilled water. these concentrations have been established taking into account that basil is an aromatic and medicinal herbaceous plant that reacts easily to any stressful environmental factor, being easily contaminated with heavy metals during growth. the seeds (in the amount of 100 seeds per every variant) were germinated in climatic chamber (model binder kbf 720, binder manufacturer, usa), at 22°c. after 72 hours, the basil roots that grew to a length of 1-1.5 cm were cut and processed for microscopic preparation. microscopic preparations the biological material were fixed with a mixture of absolute ethyl alcohol and glacial acetic acid in a volume ratio of 3:1 for 24 hours at 6°c in the refrigerator, followed by hydrolysis with 1 n hydrochloric acid for 5 minutes at room temperature. the stage of the meristematic roots staining was performed using the feulgenrossenbeck method (baik et al. 2017; rosculete ca et al. 2019). colouring was achieved in a basic fuchsine solution, in concentration of 10%. the microscopic slides were prepared using the squash technique (asita okorie et al. 2017). five slides for each variant were analysed for calculating the mitotic index and the chromosomal aberration frequency. the same slides used to calculate the mitotic index were studied to identify the chromosomal aberration. all slides were examined using a kruss microscope with digital camera (kruss manufacturer hamburg, germany). statistical analyses statistical analysis was done using ms excel 2007. the data obtained were analysed to determine the effects of cd and zn treatments on the mitotic activity to ocimum basilicum l. the mean and standard error (se) were calculated for the mitotic index (mi) and differences between treatment means were compared using the lsd-test at probability level of 0.05% (botu and botu, 1997) after anova analysis. the mitotic index (mi) was calculated according to balog (1982): total number of cells in division mi (%) = × 100 total number of analysed cells the index of the chromosomal aberrations (ca) and the percentage of germination (g) were also calculated: total number of aberrant cells ca (%) = × 100 total number of cells in division germinated seed g (%) = × 100 total seed results the heavy metals have differently influenced seed germination and root length to ocimum basilicum l. as can be seen in table 1. the inhibitory effect on germination is evident to the highest concentration of heavy 77study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) metals (v4/100 ppm) at which the germination percentage was 23.33% for cd and 70.00% for zn. also the concentration of 50 ppm, both to cd and zn, inhibited the germination of basil seeds in the proportion of 56.66% (cd) and 74.11% respectively (zn). the highest germination percentage was recorded to v2/zn/10 ppm variant (93.33%, percentage equal to that of the untreated control). as for the increase in length of the roots, the highest values were recorded in the variants with the lowest concentrations of heavy metals: v2/zn/10 ppm (1.28 ± 0.13 cm) respectively v2/cd/10 ppm (1.16 ± 0.19 cm). the most powerful inhibitory effect was found in the v4/cd/100 ppm variant, where the average length of the roots was 0.21 ± 0.04 cm, compared to the control (2.26 ± 0.37). table 2 presents the results of the effects of cd and zn on the mitotic index and the cell division phases to ocimum basilicum l. a significant reduction (p=0.05) of the mitotic index compared to the control was observed in all treatment variants. mitotic index value decreased with the increase concentration of heavy metal solutions. thus, the intensity of mitotic activity was decreasing in order of treatment with cd to zn treatment. the higher mitodepresive effect was found in the treatment of cd at the concentration of 100 ppm, when mi was 9.23%, i.e. 79.8% lower mitotic activity compared to control variant. however, in all variants treated with cd, there was a significant decrease in the mitotic index compared to the control (10.26% v3/50 ppm and 12.64% v2/10 ppm). in case of the zn-treated variants, the decrease in the mitotic index was also correlated with the increase in the concentration of heavy metal, but the strongest mitodepresive effect compared to the control was found only at the concentration of 100 ppm (v4 17.29% and 50 ppm (v3 29.14%). in low concentrations (10 ppm), zn did not negatively influence the values of the mitotic index as compared to control variant. from point of view of the cell distribution on mitotic phases, the highest percentage was registered by prophase, followed by telophase, metaphase and anaphases in all the analysed variants, including the control. frequency of cells in prophase ranged from 73.4785.61% for cd-treated variants and 75.54-84.11% for zn-treated variants. the frequency of cells in metaphase ranged from 7.33% (v4/zn/100 ppm) to 10.29% (v2/ cd/10 ppm). on the other hand, the frequency of cells in anaphase stage ranged from 1.58% (v4/cd/100 ppm) to 4.82% (v3/zn/50 ppm). the smallest values of the mitotic index of telophase compared to control were recorded at the highest concentrations of heavy metals: 3.70% (v4/cd/100 ppm) and 6.54% (v4/zn/100 ppm) respectively. heav y metals tested induced a high number of mitotic aberrations when compared with control. the increase of mitotic aberrations was dependent on the increasing treatment concentrations (table 3). the types of chromosomal aberrations identified in meristematic table 1. influence of cadmium and zinc on the seeds germination and root length to ocimum basilicum l. variants germination (%) root length (x ± se) (cm) v1 (control) 93.33 2.26±0.37 v2/cd/10 ppm 80.00 1.16±0.19 v3/cd/50 ppm 56.66 0.26±0.04 v4/cd/100 ppm 23.31 0.21±0.04 v2/zn/10 ppm 93.33 1.28±0.13 v3/zn/50 ppm 74.11 1.18±0.19 v4/zn/100 ppm 70.00 0.65±0.09 table 2. mitotic index (%) and the cell division phases (%) to ocimum basilicum l. treated with different concentrations of cadmium and zinc nitrate. variants tcn mi ± se % mip % mim % mia % mit % v1 (control) 500 45.82±0.68 75.35 6.35 1.93 16.37 v2/cd/10 ppm 500 12.64±0.42* 73.47 10.29 1.86 14.38 v3/cd/50 ppm 500 10.26±0.35* 78.36 9.74 1.64 10.26 v4/cd/100 ppm 500 9.23±0.34* 85.61 9.11 1.58 3.70 v2/zn/10 ppm 500 43.61±0.63 76.28 7.46 2.01 14.25 v3/zn/50 ppm 500 29.14±0.60* 75.54 9.22 4.82 10.42 v4/zn/100 ppm 500 17.29±0.58* 84.11 7.33 2.02 6.54 tcn = total cells number; mi = mitotic index; mip = mitotic index of prophase; mim = mitotic index of metaphase; mia = mitotic index of anaphase; mit = mitotic index of telophase; se = standard error; * significant at level 5% (p=0.05). 78 irina petrescu et al. cells of ocimum basilicum l. were interphases with pyknotic nucleus; pyknotic and sticky chromosomes, cells with laggard chromosomes, as well as cells with telophase bridge. the most common types of chromosomal aberrations were stickiness and pyknosis while the least frequent were bridges. compared with the control variant, total chromosomal aberration rate recorded insignificant values for all variant exposed to zn, from 4.58% (v2/ zn/10 ppm) to 14.51% (v4/zn/100 ppm). on the other hand, in all variants exposed to cd treatment total chromosomal aberration recorded significantly positive values from 19.51% (v2/cd/10 ppm) to 39.70% (v4/cd/100 ppm) respectively. discussion the contamination of soil and water by heavy metals is a major environmental problem. in this regard it presents an ecotoxicology risk for food chains because of strongly toxic properties of these elements for all human beings (lassoued et al. 2014; bonciu et al. 2018; coroian et al. 2017; puia et al. 2019). understanding the phenomenon of bioaccumulation of heavy metals in living substance is of extremely complex. this contamination can have very long-term effects (bilal et al. 2014; lassoued et al. 2014; bonciu et al. 2018). heavy metal pollution is one of the most serious problems of industrialization, who affects significantly soil and biodiversity and its impact continues to increase (bae et al. 2016), due to these metals’ non-biodegradability and high toxicity (chul kong, 2013). generally, heavy metals are dangerous because they tend to bioaccumulate and can cause altered physiological and metabolic processes to plants or disturbing the metabolism of essential elements (petrescu et al. 2015; sarac et al. 2015; wójcik and tukiendorf 2014; butnariu 2012; mohanpuria et al. 2007; dong et al. 2006). in our experiment, the seed germination of ocimum basilicum l. was heavily affected by the concentration of heavy metals, especially by cd, which exhibited the highest degree of toxicity. instead, zinc recorded a lower degree of toxicity and only at concentrations of 50 ppm and 100 ppm. the inhibition of seed germination with increasing concentration of cd has been found also in other plants: vigna radiata (maheswari et al. 2017); triticum aestivum (guilherme et al. 2015); suaeda salsa (liu et al. 2012); spartiana alterniflora (mrozek and funicelli, 1982), etc. we can appreciate that the highest toxicity in basil seed germination as well as the increase in length of meristematic roots was induced by cd at the concentration of 100 ppm. in other authors’ opinion, at low concentrations cd is not toxic to plants, but at higher concentrations it is toxic and preferentially accumulates in the meristematic and elongation root zones (xu et al. 2009; karcz and kurtyka, 2007). the heavy metals can disturb the nucleolar cycle. indirect immunof luorescence detects nucleolar material and their movement into the cytoplasm following heavy metal stress (liu et al. 2016). the length roots of ocimum basilicum l. was influenced differently from one heavy metal to another and from one concentration to the other, the most powertable 3. type and percentage of mitotic aberrations induced by cadmium and zinc on the meristematic roots to ocimum basilicum l. variants mitotic aberrations (%) total aberrations (%)pn pc l s b v1 (control) 0 0 0 1.05 0 1.05 v2/cd/10 ppm 4.03 5.27 3.89 5.09 1.23 19.51* v3/cd/50 ppm 6.21 8.63 4.03 7.26 1.58 27.71* v4/cd/100 ppm 8.15 12.86 6.23 10.04 2.42 39.70* v2/zn/10 ppm 0 1.04 1.53 2.01 0 4.58 v3/zn/50 ppm 3.05 1.01 2.03 2.34 0.89 9.32 v4/zn/100 ppm 2.84 2.63 3.82 4.01 1.21 14.51 pn = pyknotic nucleus; pc = pyknotic chromosomes; l = laggards; s = stickiness; b = bridges; * significant at level 5% (p=0.05). a b c d figure 1. some chromosomal aberrations identified in meristematic cells of ocimum basilicum l. exposed to cd and zn: pyknosis (a); sticky metaphase whit laggards chromosomes (b); bridges (c); disturbed telophase whit pyknotic chromosomes (d). 79study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) ful inhibitory effect being found at the highest cd concentrations. results suggest that cd is highly toxic and can affect the metabolism of meristematic roots. similar results have been reported by gharebaghi et al. (2017) to two basil species (ocimum basilicum l. and ocimum basilicum var. purpurescens). cytogenetic tests on ocimum basilicum l. show a decrease of the mitotic index following heavy metal treatments. the mitodepresive effect of cadmium was obvious even at the lowest concentration (10 ppm). the other authors results showed that cd causes irregularities in mitotic activity to pisum sativum (fusconi et al. 2007) and allium sativum (xu et al. 2009) and can induce increased frequency of the chromosomal aberrations to allium cepa (1.5 times more than in control group), while mitotic index was significantly decreased (evseeva et al. 2001). to allium cepa, cd affected the spindle and decreased anaphase and telophase stages while the metaphase stage was increased. in the presence of certain external stimuli, the cellular progress can be blocked in one of the phases of the cell cycle or cell division, and their action is called mitoinhibition. mitogens act to overcome intracellular braking mechanisms that block cell cycle progression, and their action is called mitostimulatory. any deviation from the orderly and directed progression of the cell cycle, and respectively, of mitosis and cytokinesis, is reflected in a state of cytotoxicity and genotoxicity (bonciu et al. 2018; rosculete e et al. 2019) and some chromosomes variation (bouziane et al. 2019). some research shows that the electron energy loss spectroscopy (eels) and electron spectroscopic imaging (esi) are good methods for identifying sites of localization of heavy metals at the sub-cellular level in cell organelles, cytoplasm or cell walls and clarifying the process involved in their uptake, transport and deposition or detoxification in plant cells (liu and kottke 2003, 2004). the results of previous investigations indicate that heavy metals including cd and zn at excessive concentration can disturb cell division process and induce ca comprising c-mitosis and lagging chromosomes, anaphase bridges, and chromosome stickiness in the root tips of a. cepa (liu et al. 1995). during mitosis, metal ions can interfere with the proper positioning of nucleolar organizing regions on chromosomes. under metal stress, an obviously toxic phenomenon appears in nucleoli of root tips of a. cepa (bonciu et al. 2018). the results of this study highlight the strong cytotoxic effect of cd to ocimum basilicum l. even at low concentrations of 10 and 50 ppm. the most common types of chromosomal aberrations were stickiness; sticky chromosomes can lead, in opinion of some authors, to cell death (singh, 2015; karaismailoğlu, 2017). in most cases the percentages of abnormal mitotic phases were seen to increase with increasing concentration, this result being recorded in other studies also (samanta and bandyopadhyay, 2012; verma et al. 2016; șuțan et al. 2018). the cytotoxicity effect of zn occurred only at concentrations higher than 10 ppm. in low concentrations, zn did not negatively influence the values of the mitotic index, the percentage of chromosome aberrations being insignificant. of the two heav y metals tested, cd showed the highest degree of cy totoxicity and inhibits normal growth to ocimum basilicum l. the results also suggest that basil may be used for bio-greening of soil, since it absorbs the heavy metals and synthesizes them in the cells. besides, the decontamination of soils polluted with heavy metals through phytoremediation is one of the cheapest and simplest methods, and from this point of view, the cultivation of basil involves very low costs. references abd el-ghffar ea,  al-sayed e, shehata sm, eldahshan oa,  efferth t. 2018. the protective role of  ocimum basilicum  l. 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regul. 44(1):71-80. caryologia international journal of cytology, cytosystematics and cytogenetics volume 73, issue 1 2020 firenze university press karyotypic investigation concerning five bromus species from several populations in iran sara sadeghian, ahmad hatami, mehrnaz riasat high genetic diversity and presence of genetic structure characterise the endemics ruta corsica and ruta lamarmorae (rutaceae) marilena meloni1, caterina angela dettori2, andrea reid3, gianluigi bacchetta2,4,*, laetitia hugot5, elena conti1 cytogenetic effects of c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. and mathematical analysis cihangir alaca1, ali özdemir1, bahattın bozdağ2, canan özdemir2,* clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 74(3): 45-51, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1122 caryologia international journal of cytology, cytosystematics and cytogenetics citation: yuri a. kirillov, maria a. kozlova, lyudmila a. makartseva, igor a. chernov, evgeniya v. shtemplevskaya, david a. areshidze (2021) influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats. caryologia 74(3): 45-51. doi: 10.36253/caryologia-1122 received: october 26, 2020 accepted: june 01, 2021 published: december 21, 2021 copyright: © 2021 yuri a. kirillov, maria a. kozlova, lyudmila a. makartseva, igor a. chernov, evgeniya v. shtemplevskaya, david a. areshidze. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats yuri a. kirillov1, maria a. kozlova1, lyudmila a. makartseva1, igor a. chernov2, evgeniya v. shtemplevskaya1, david a. areshidze1,* 1 federal state budgetary scientific institution «research institute of human morphology» 2 fsbei he tyumen state medical university of the ministry of health of russia *corresponding author. e-mail: labcelpat@mail.ru abstract. the features of the diurnal dynamics of the area of rat hepatocyte nuclei and their ploidy were studied under conditions of a standart (fixed) light regime and constant illumination, as well as under chronic exposure to alcohol in the mentioned light regimes. it has been shown that exposure to alcohol and exposure to constant illumination separately lead to a change in the amplitude-phase characteristics of the circadian rhythm of the nucleus area, while the combined effect of these factors leads to a complete destruction of the rhythm, which indicates a violation of adaptation processes. an increase in the average ploidy of hepatocyte nuclei in chronic alcohol intoxication is also shown, while in animals kept under constant illumination without drinking alcohol, the values of this parameter decrease, which indicates a successful course of the adaptation process. the conducted research indicates that the results of karyometric and ploidometric analysis characterize the degree of influence of alcohol intoxication and changes in the light regime on the liver of rats, reflecting the rate of efficiency of adaptation to these factors. key words: caryometry, ploidy, hepatocyte, circadian, morphometry. introduction the liver is the main organ of metabolism of various exogenous and endogenous chemical compounds, while the main functionally active liver cells (hepatocytes) are among the first to be exposed to these factors. damage and death of these cells renders to the disability of the liver to perform its functions (aizava et al., 2020). one of the mechanisms for maintaining the structural and functional integrity of the liver is cellular regeneration, which occurs due to mitotic and amitotic division of hepatocytes (gilgenkrantz et al., 2018; clemens et al., 2019). mass death of hepatocytes by necrosis and/or apoptosis activates the processes that trigger the entry of “resting” hepatocytes into the cell-division cycle to restore the original cell mass and maintain the cellular homeostasis 46 yuri a. kirillov et al. of the organ. the liver always has a reserve of hepatocytes with polyploid nuclei, constantly ready to divide. thus, an increase in the level of ploidy of hepatocytes is one of the main compensatory-adaptive reactions of the liver, ensuring the preservation of function of the organ. it is known that in mammals in the prenatal and at the early stages of postnatal ontogenesis, diploid hepatocytes prevail, then their polyploidization occurs, and the proportion of polyploid hepatocytes can reach 80% of the total number of cells. in addition, it was found that ploidy of hepatocytes increases with aging, after hepatectomy, under the influence of a number of unfavorable factors, but at the same time ploidy decreases, for example, in hepatocellular carcinomas (duncan, 2013; zhang et al., 2019; donne et al., 2020). another, very important and informative approach to determining the functional state of the liver, as well as diagnosing various kinds of diseases of this organ is karyometry. karyometric analysis is used to assess the intensity of dystrophic, inflammatory, reparative processes in chronic viral hepatitis, liver fibrosis, hepatocellular carcinoma, etc (el-sokkary et al., 2005;esperandim et al., 2010; makovsky p et al., 2018). thus, the approaches of the study of the liver in different morphofunctional states can be associated with the karyometric and ploidometric assessment of hepatocytes. the data obtained using these methods will make it possible to assess the morphofunctional state of the liver more accurately and, in accordance with this, to solve the problems of prognosis. rhythmicity of functioning is peculiar for living systems at every level of organization. biosystems have rhythms with different periodicity, however, diurnal, or circadian rhythms (cr) are the most significant for mammals (gillette, 2013; mckenna et al., 2018). circadian system of mammals includes central circadian rhy thm generators (suprachiasmatic nuclei of the hypothalamus (scn), pineal gland), which are connected with peripheral pacemakers – morphological structures in organs and tissues. it is endogenous and is determined genetically (genes per1, per2, cry, etc.), however, it has significant plasticity and can be modulated by the action of external zeitgebers (time givers), the most important of which is light (tahara et al., 2017). separate biorhythms of physiological processes in various systems form a strongly coordinated ensemble, the chronostructure of the organism. the presence of a rhythmic structure of biological processes ensures the necessary order of their course, coherence, maintenance of the functioning of systems of organism at an optimal level (roennenberg et al., 2016). exposure to endogenous or exogenous desynchronizing factors leads to disorganization of circadian rhythmicity (roennenberg et al., 2017). in the case of prolonged or regular exposure to desynchronizing factors, for example, constant lighting at night, desynchronosis develops, which is a pathological condition characterized by a mismatch of rhythms in phase, the loss of their mutual synchronization or their destruction (beauvalet et al., 2017; walker et al., 2020). one of the organs, the normal rhythm of the functioning of which is very important for maintaining homeostasis, is the liver (trefts et al., 2017). in the regulation and realization of plastic and energy metabolism, the coordination of rhythmic processes in the liver with the rhythms of processes in other organs and systems of the organism plays a fundamental role. moreover, most of these processes demonstrate the daily rhythm (tahara et al., 2016). in most cases, the rhythm of metabolic processes arises and is maintained due to dynamic interactions between the molecular clock of the organism and external zeitgebers, such as, for example, light (main cr synchronizer) and nutrition (secondary synchronizer) (stubblefield et al., 2016; ding et al., 2018). the disruption of circadian rhythmicity in the form of a shift in biorhythms or desynchronosis in the liver entails the development of pathological conditions and diseases such as cholestasis, fatty hepatosis, impaired biotransformation of toxic and medicinal substances, hepatitis, cirrhosis and liver tumors (tong et al., 2013; debruyne et al., 2014). an indicator of functional changes in hepatocytes is the modification of their morphological structure, which has a wide range of variations, from subtle ultrastructural transformations to cell death. (li et al., 2020). the linear dimensions of hepatocytes and their nuclei, nuclear-cytoplasmic ratio, and ploidy of hepatocytes are the significant parameters for assessing the morphofunctional state of the liver (junatas et al., 2016). the significant reason of desorganization of biorhythms in the modern world is the disturbance of natural light regime, known as light pollution. due to a number of social reasons (prolonged interaction with digital technique, overtime and shift work, transmeridian flights, etc.), a person is currently exposed to abundant exposure to artificial lighting in the dark, which leads to a shift in the circadian rhythms of the organism, or to the development of desynchronosis (lunn et al., 2017). another factor that influences cr is alcohol consumption. in a study of the effect of alcohol on rhythms in mammals, two areas of interest are distinguished. the first one considers the chrono-effecter action of alcohol, i.e. how the effects of alcohol change depending on the 47influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats time of day in which it was consumed. the second area of interest is chronergic, with wider approach, exploring mainly the effect of alcohol on biorhythms of other parameters of organism (wasielewski et al., 2001). alcohol abuse and alcoholic disease are associated with widespread disturbances in cr (rosenwasser, 2015; davis et al., 2018). it is shown that disturbances in circadian homeostasis make liver and intestines more susceptible to alcohol toxicity. studies in human alcoholics have shown altered expression of circadian genes. anyway, alcohol has a significant chronotoxic effect, which causes desynchronosis (huang et al., 2010; filiano et al., 2013; martínez-salvador j. et al., 2018.) we considered it important to study the daily dynamics of the area of hepatocyte nuclei and their ploidy under normal light conditions and under constant illumination, as well as in combination of these conditions with experimental chronic alcohol intoxication. matherials and methods animals the study was conducted on 160 male wistar rats at age of 6 months, weighing 300±20 g. animals were taken from the stolbovaya nursery (the “stolbovaya” affiliate of the fsbis “scientific center for biomedical technologies of the federal medical and biological agency). design of experiment rats were divided into 4 equal groups. the experiment lasted 3 weeks for every group. 1st group (control): animals of the first group served as control. the individuals were housed in plastic cages with free access to water under the conditions of a fixed light regime “light-dark” (10:14 hours). 2nd group: animals of the second group were kept under the same conditions, but instead of water, a 15% ethanol ad libitum solution was offered daily as a drink. 3rd group: animals of the third group were kept under the same conditions as the animals of the first group, except for the light regime, which represented constant lighting (“light-light”). 4th group: animals of the fourth group were kept under conditions of constant lighting and got 15% ethanol ad libitum solution as a drink instead of water. the criterion for the selection of rats in the 2 and 4 group, along with the absence of visible deviations in the state and behavior, was the initial preference for a 15% solution of ethyl alcohol to a tap water. for this, a preliminary experiment was carried out for 3 days in individual cages with free access to both liquids. euthanasia was carried out three weeks after the start of the experiment in a carbon dioxide chamber equipped with a device for the upper gas supply (100% co2) at 9.00, 15.00, 21.00 and 3.00. the chamber volume was filled with gas at a rate of 20% per minute to avoid dyspnea and pain in animals. after sacrifice, the liver was removed for morphological examination. all animal experiments were performed in according to the compliance with ec directive 86/609/eec and with the russian law regulating experiments on animals. the liver was fixed in 10% neutral buffered formalin with further passage through alcohols of increasing concentration (50°, 60°, 70°, 80°, and 96°) and xylol, followed by pouring into histomix histological medium (biovitrum, russia). when conducting studies of organs embedded in paraffin, serial sections with a thickness of 5-6 μm were prepared. histological sections were made on the rotor microtome mps-2 (ussr). hematoxylineosin staining was carried out according to the standard technique. stained sections were put in a biomount mounting medium (biovitrum, russia). microscopy of histological preparations was performed using a nikon eclipse 80i digital microscope with use of a nikon di-fi digital camera (japan). for microscopy, eyepieces ×10, ×15, lenses ×4, ×10, ×20, ×40, ×100 were used. from each studied preparation, 10 digital images of randomly selected visual fields were taken at a magnification of ×400, ×1000, with the use of which karyometry were subsequently carried out, the daily dynamics of the nucleus was determined, estimated by their area. in morphometric studies, the imagej program (usa) with the appropriate plug-ins was used to determine the crosssectional area of hepatocyte nuclei (broeke et al., 2015). the measurements were carried out in micrometers after preliminary geometric calibration on an object-micrometer scale digitized with the same magnification. for ploidometry, paraffin sections were stained with methylene-green pyronin g, with following processing of sections with rna-ase. the hepatocyte ploidy was calculated in units of ploidy relative to the optical density of the staining results of diploid nuclei of small lymphocytes. micromorphometry of only mononuclear interphase hepatocytes without signs of pathological changes was carried out. methods of statistical processing the obtained data were analyzed using the graphpad prism 6.0 program by calculating average values, 48 yuri a. kirillov et al. standard deviation, and arithmetic mean error. the numerical rows characterizing the diurnal fluctuations of the studied physiological rhythms of animals were subjected to mathematical processing, on the basis of which group chronograms were drawn. we studied the form of chronograms and calculated daily average values. statistical differences in studied parameters were determined using t-student test. a p value <0.05 was considered statistically significant. for the statistical estimation of the amplitude and acrophase of crs, cosinor analysis was performed, which is an international, recognized method for the unified study of biological rhythms using the cosinorellipse2006-1.1 program. the presence of a reliable circadian rhythm was determined, as well as its acrophase and amplitude. acrophase is a measure of the peak time of the total rhythmic variability over a 24-hour period. the amplitude corresponds to half the total rhythmic variability in the cycle. acrophase is expressed in hours; amplitude values are expressed in the same units as the studied variables (cornelissen, 2014). results considering the results of karyometry, we found that the cross-sectional area of hepatocyte nuclei of rats of the first three groups, which amounted to 41.79±8.13 μm2, 42.65±4.80 μm2, and 42.72±5.63 μm2, respectively, did not differ significantly from each other, but the significant decrease in this parameter up to 35.50±3.01 μm2 in hepatocytes of animals of the fourth group was found. the daily rhythm of the cross-sectional area of the hepatocyte nuclei of rats of 2-4 groups significantly differed from the control (fig. 1). in particular, the maximum of area of nuclei in control group is noted at 15:00 with acute decrease to minimum at evening and nighttime – 21:00 and 3:00. in the second group the rhythm is less pronounced, a maximum at 15:00 is noted. in the third group, the maximum values are noted at 9:00 with a gradual decrease to a minimum at 3:00. in the fourth group, daily fluctuations in the area of hepatocyte nuclei are unreliable. the results of the cosinor analysis of diurnal changes in the area of the nucleus indicate the presence of a reliable cr of this process in the first three groups and its destruction in the fourth group. therewith, acrophases of rhythms in groups 1 and 3 are noted in the daytime at 1221 and 1136, with an amplitude of 10.03 μm2 and 4.60 μm2, respectively, and the acrophase of the rhythm in the second group shifts by 1802 with an amplitude of 3.37 μm2 (fig. 2). considering the results of measuring the ploidy of mononuclear hepatocytes, it was found that the studied samples contain diploid, tetraploid and octoploid cells. the average ploidy of the studied hepatocytes is 4.47±2.12n in the first group, 5.02±2.18n in the second, 4.04±2.16n in the third, and 5.18±2.14n in the fourth. analysis of ploidy distribution of hepatocyte nuclei revealed significant intergroup differences (table 1). in particular, in groups in which animals were exposed to chronic alcohol intoxication, the number of diploid hepatocytes significantly decreases, but at the same time, the proportion of octoploid cells in the second group significantly increases, as well as and the proportion of tetraploid cells in the fourth group. 30 35 40 45 50 55 i group ii group iii group iv group 9 hours 15 hours 21 hours 3 hours figure 1. daily rhythm of the cross-sectional area of hepatocyte nuclei of rats. figure 2. results of cosinor analysis of circadian rhythmicity of area of nuclei of hepatocytes of rats. 49influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats discussion and conclusion the conducted study allowed us to establish that both the violation of the light regime and the effect of ethanol, individually and jointly, have a significant effect on the studied parameters. the alcoholic intoxication at fixed light regime causes the decrease in proportion of diploid cells with a simultaneous increase in the proportion of octaploid cells. changing of the normal light regime to constant light leads to a change in the nature of the ploidy distribution of hepatocytes. the increase in proportion of diploid hepatocytes indicates a successful course of adaptation processes in the organ, apparently due to the division of cells of higher ploidy, the proportion of which has decreased (nagy et al., 2001; yelchaninov et al., 2011; lazzeri et al., 2019). the alcoholic intoxication in conditions of constant lighting lead to decrease in size of nuclei and increase in proportion of tetraploid hepatocytes. the increase in general ploidy in groups 2 and 4 (i.e. in those where animals were exposed to alcohol) occurs due to tetraand octaploid nuclei, which, according to a number of authors, indicates the development of hepatocyte hypertrophy against the background of an increase in nuclear ploidy (miyaoka et al., 2012; zhou et al., 2016). it has been suggested that the polyploid state functions as a growth suppressor, limiting the proliferation of most of cells and causing compensatory-adaptive reactions in the form of diploid cell hypertrophy. in turn, the nature of the circadian rhythm of the size of the cell nuclei indicates that constant illumination and ethanol, acting separately, cam use a rearrangement of the cr, but the combined action of these parameters leads to the destruction of the circadian rhythm, which indicates a disruption of adaptation processes in animals of this group (maruani et al., 2018; matkarimov, 2020) so, the conducted study testifies that the results of caryometric and ploidometric studies characterize the degree of influence of alcohol intoxication and changes in the light regime on the liver of rats, representing the degree of effectiveness of adaptation to these factors. figure 3. liver of rat of control group, methylene-green pyronin g, ×400. figure 4. liver of rat of iv group, methylene-green pyronin g, ×400. table 1. distribution of hepatocyte nuclei in rat liver depending on ploidy. group ploidy of nuclei of hepatocytes 2n, % 4n, % 8n, % 1st group (n=40) 23.98±3.51 52.1±4.60 23.23±2.20 2nd group (n=40) 14.15±2.02 *** 53.47±5.18 32.38±3.21*** 3rd group (n=40) 34.0±4.81 *** 45.9±3.95 *** 20.1±1.89*** 4th group (n=40) 13.70±2.84 *** 79.6±5.18 *** 6.70±0.81*** note: hereinafter: *(p≤0.05); **(p≤0.005); ***(p≤0.0005) statistical significance of differences in comparison with the control group. 50 yuri a. kirillov et al. acknowledgements financial support for this study was carried out by research institute of human morphology. compliance with ethics guidelines all the experimental protocols were performed in accordance to ethical guidelines approved by the research and ethics committee of scientific center for biology of cells and applied biotechnology of the moscow state regional university, moscow, russian federation prior 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issue 3 2021 firenze university press chromomycin a3 banding and chromosomal mapping of 45s and 5s ribosomal rna genes in bottle gourd ahmet l. tek*, hümeyra yıldız, kamran khan, bilge ş. yıldırım development of a protocol for genetic transformation of malus spp federico martinelli1,*, anna perrone2, abhaya m. dandekar3 cytogenetic and cytological analysis of colombian cape gooseberry genetic material for breeding purposes viviana franco-florez, sara alejandra liberato guío, erika sánchez-betancourt, francy liliana garcía-arias, víctor manuel núñez zarantes* palynological analysis of genus geranium (geraniaceae) and its systematic implications using scanning electron microscopy jun wang1,2,*, qiang ye1, chu wang2, tong zhang2, xusheng shi2, majid khayatnezhad3, abdul shakoor4,5 influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats yuri a. kirillov1, maria a. kozlova1, lyudmila a. makartseva1, igor a. chernov2, evgeniya v. shtemplevskaya1, david a. areshidze1,* the morphological, karyological and phylogenetic analyses of three artemisia l. (asteraceae) species that around the van lake in turkey pelin yilmaz sancar1,*, semsettin civelek1, murat kursat2 molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant jinxin cheng1,*, dingyu hu1, yaran liu2, zetian zhang1, majid khayatnezhad3 genetic variations and interspesific relationships in salvia (lamiaceae) using scot molecular markers songpo liu1, yuxuan wang1, yuwei song1,*, majid khayatnezhad2, amir abbas minaeifar3 development of female gametophyte in gladiolus italicus miller (iridaceae) ciler kartal1, nuran ekici2,*, almina kargacıoğlu1, hazal nurcan ağırman1 colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae) harshita dwivedi*, girjesh kumar statistical evaluation of chromosomes of some lathyrus l. taxa from turkey hasan genç1, bekir yildirim2,*, mikail açar3, tolga çetin4 use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater treatment and water of the kamniška bistrica river (slovenia) peter firbas1, tomaž amon2 the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bo shi1,*, majid khayatnezhad2, abdul shakoor3,4 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(2): 103-109, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1230 caryologia international journal of cytology, cytosystematics and cytogenetics citation: sazada siddqiui, saad abdurahamn muhammad al amri, huda ahmed al ghamdy, wadha saad saeed alqahtani, sarah mohammed alquyr, habab merghani yassin (2021) impact of bisphenol a on seed germination, radicle length and cytogenetic alterations in pisum sativum l.. caryologia 74(2): 103-109. doi: 10.36253/caryologia-1230 received: march 02, 2021 accepted: june 11, 2021 published: october 08, 2021 copyright: © 2021 sazada siddqiui, saad abdurahamn muhammad al amri, huda ahmed al ghamdy, wadha saad saeed alqahtani, sarah mohammed alquyr, habab merghani yassin. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid ss: 0000-0001-5448-7617 impact of bisphenol a on seed germination, radicle length and cytogenetic alterations in pisum sativum l. sazada siddiqiui*, saad abdurahamn muhammad al amri, huda ahmed al ghamdy, wadha saad saeed alqahtani, sarah mohammed alquyr, habab merghani yassin department of biology, college of science, king khalid university, abha 61413, saudi arabia *corresponding author. e-mail: sasdeky@kku.edu.sa abstract. bisphenol a (bpa) is a global transpiring pollutant and an endocrine disruptor present in the environment which has a substantial harmful effect on plants. in the present study, its effects on seed germination, radicle length and cytogenetic alterations were investigated in p. sativum root tip cells. p. sativum seeds were germinated after treating with various concentrations of bpa (2 mg/l, 5 mg/l, 10 mg/l, 15 mg/l, 20 mg/l and 25 mg/l) at 24±1°c for 72 hours and the cytogenetic variations were assessed. the investigation showed that bpa reduced the percentage of seed germination, mitotic index, radicle length (at higher concentrations) and instigated a rise in chromosomal anomalies in a dose-related manner. in total, there is an enhanced occurrence of c-mitosis, stickiness, bridges, fragments and laggards in the bpa treated root tip cells of p. sativum seeds. keywords: bpa, seed germination, mitotic index, chromosomal anomalies, pisum sativum l. introduction bisphenol a (bpa, 2,2-bis-(4hydroxyphenyl) propane) is an important transpiring pollutant (clarke and smith 2011). bpa is an abundantly mass-produced industrial chemical widely used in the manufacture of various domestic and daily use items like baby feeding plastic bottles, protecting coverings, packing of drinks, food items and in the linings of metal cans used for storing beverages and food products. globally every year bpa is manufactured industrially in huge quantities approximately 0.0037 billion metric tonnes (mihaich et al. 2009). it is constantly released in marine environment by municipal, agriculture and industrial effluents (gatidou et al. 2007; pothitou and voutsa 2008; fu and kawamura 2010). with leaching of bpa by plastics and containers used for keeping food, drinks and beverages, human beings are exposed to it by consuming food and drinks stored in 104 sazada siddiqiui et al. these containers (huang et al. 2012) and it poses a risk for the health of all human beings (le et al. 2008; wagner and oehlmann 2009; cooper et al. 2011). human beings are also at risk by eating fish found in aquatic waters polluted by bpa (mita et al. 2011). agrarian soils usually get polluted by biosolids containing bpa found in sewage sludge (gatidou et al. 2007; stasinakis et al. 2008). through extensive research work on bpa, it has been found that it is an endocrine disruptor (staples et al. 1998; le et al. 2008; clarke and smith 2011). small organisms living in soils and plants could come in contact with soils polluted by bpa (yamamoto et al. 2001; staples et al. 2010). moreover, not many studies have analyzed the toxicologic effects of bpa in plants which absorbs and accumulates it (ferrara et al. 2006). though, it has been established that plants can form bpa-glycosides by metabolizing bpa (noureddin et al. 2004), clastogenic as well as phytotoxic influence of bpa were defined (ferrara et al. 2006). due to the impact of bpa on the pollen of kiwifruit in a dose-related manner, there is a substantial inhibition of tube development and its elongation (speranza et al. 2011). lately, the mitotic and chromosomal anomalies were found in cells of root meristem of allium cepa l treated with 50, 100, 150 and 200 mg/l bpa concentration for five days (jadhav et al. 2012). bpa treatment with 0.044-0.44 mm concentration inhibited the segregation of chromosomes, obstructed the cytokinesis completion, disrupted mitotic mt arrays and interphase and stimulated microtubules creation in p. sativum (adamakis et al. 2013). moreover, bpa treatment influences leaf blade differentiation in arabidopsis thaliana significantly (pan et al. 2013) and in bpa treated seedlings of soybean, it reduced the photosynthetic constraints and growth indexes (qiu et al. 2013). in animals, bisphenol a has revealed to put forth xenoestrogenic action (wang et al. 2021). however, the influence of bpa on plants are not clearly understood. though bpa is consumed regularly and disposed, it may persist in the soil and can potentially cause detrimental effects on the plants. further, there is not sufficient studies available in the literature about its genotoxic effects on plants (palani and panneerselvam 2007). in the present study, we have evaluated the adverse effects of bpa on seed  germination, radicle length, mitotic index and chromosomal anomalies in cells of p. sativum root tips. material and methods purchase of bpa and seeds from a seed shop in saudi arabia, pea seeds (p. sativum variety arkil, 2n = 14) were bought. through sigma–aldrich merck (darmstadt, germany) bisphenol a (bpa) (bpa, 2,2-bis-(4hydroxyphenyl) propane is procured from bayouni trading co. ltd., jeddah, saudi arabia. bisphenol a, cas number is 80-05-7. its melting point is 158-159 °c and its solubility in water at 25ºc is 123–300 mg/l. the molecular weight of bpa is 228.29 and its chemical formula is c16h18o2. seed treatment with bpa for 5 minutes, seeds were sterilized in 0.1% hgcl2 solution and they were washed in distilled water 2-3 times. thirty seeds were soaked in bpa solutions of each concentrations (2 mg/l, 5 mg/ l, 10 mg/l, 15 mg/l, 20 mg/l and 25 mg/l) for 3 hours. for control group, a group of thirty seeds was soaked in distilled water. seeds were repeatedly shaken for sufficient air supply. thirty sterilized seeds were then spread over three whatman filter papers, grade one and then kept in petri-dishes (150 mm x 15 mm diameter). for more readings, these petri dishes were kept in a biological oxygen demand incubator (bod) at a temperature of 24±20c. as per the procedure defined by rank (2003), root elongation toxicity and seed germination tests were performed. radicle length were measured and germination of seeds were recorded, each day on an interval of 24 hours for 3 consecutive days. in similar settings, this test was done thrice. toxicity was stated as compared with control, the difference of germination of seeds and root elongation. cytotoxicity and genotoxicity evaluations to assess the cytotoxicity and genotoxicity evaluations caused by bpa in p. sativum plant, the root tips of germinated seeds were used as a source of mitotic cells. the root tips were washed in water. in a blend of ethanol and acetic acid (3:1–v/v, merck), roots in length 2 cm were fixed (approximately 2 days). staining of fixed roots were done with schiff ’s reagent, as defined by feulgen and rossenbeck (mello and vidal 1978) and the slides were made by applying the meristematic region as per the protocol stated by siddiqui et al. (2007). by documenting the variations in the meristematic cells mitotic index (mi), cytotoxicity was evaluated. by means of scoring various kinds of chromosomal anomalies (cas), genotoxicity was assessed. each slide was observed and coded blind. by using light microscope under oil immersion, chromosomal anomalies and mitotic index in metaphase and anaphase plates were examined. at least 250 cells were scored from every single slide and mitotic index was computed. chromosomal anomalies such as sticky chromosome, 105impact of bisphenol a on seed germination, radicle length and cytogenetic alterations in pisum sativum l. c-mitosis, laggards, bridges and fragments were examined in at least 150 metaphase and anaphase plates for each slide and stated in percentage. statistical analysis by using graph pad software (san diego, ca, usa), statistical analysis (anova with dunnett’s multiplecomparison test) having significance at p<0.05 was carried out. data were exhibited in the form of mean ± standard error (se). results effect of bpa treatment on seed germination at 24 h interval, in control group 77.33% of seeds germinated which increased to 85% and 99% at 48 h and 72 h respectively (table 1). in seeds treated with lower concentration of bpa (2 and 5 mg/l), percentage of seed germination decreased (p<0.001 and p<0.05) at 24 h. similarly, a significant decrease was observed at 48 h (p>0.05) and 72 h (p<0.01 and p<0.001) compared to control. in all time periods, on and above a concentration of 10 mg/l treatment with bpa caused a very significant decrease in germination percentage of seed in a dose-related manner, as compared to control. lowest percentage of seed germination was reported at 20 mg/l (65% at 72 h) and at 25 mg/l (50.22% at 24 h, 60% at 48 h) in bpa treated seeds. effect of bpa treatment on radicle length in control group, the radicle length increased with increase in time which was 4.0 ± 0.05 at 72 h (fig. 1). at 24 h interval, significant decrease in radicle length was observed in seeds exposed to bpa in a dose-related manner. furthermore, there was no statistically significant difference reported from 2 mg/l to 25 mg/l bpa treatment at 48 h as compared to control. in 2 mg/l, 10 mg/l and 15 mg/l bpa treated seeds no statistically significant difference was noticed but in 5 mg/l and 20 mg/l significant decrease in radicle length was reported and in 25 mg/l very significant decrease was observed at 72 h. in bpa treated seeds lowest root length was recorded in 20 mg/l at 48 h (0.85 ± 0.04) and in 25 mg/l at 24 h (0.35 ± 0.02) and at 72 h (1.5 ± 0.33). maximum root length was recorded in 2 mg/l (0.77 ± 0.04) at 24 h, 10 mg/l (1.5 ± 0.7) at 48 h and at 2 mg/l (3.0 ± 0.03) at 72 h in bpa treated seeds. effect of bpa treatment on mitotic index the control presented a mitotic index of 17.78 ± 5.66 (fig. 2). however, further increase in bpa concentration caused a decline in the mitotic index in a dose-related manner. as compared to control, at a lesser concentration of bpa (2 and 5 mg/l), the mitotic index was nonsignificantly lower. when compared with control, in seeds treated with 10 mg/l bpa, the mitotic index was significantly less (p<0.05), in 15 mg/l the mitotic index was found to be very significantly lower (p <0.01) and in seeds treated with 20 and 25 mg/l bpa, the mitotic index was highly significantly lower (p<0.001). in seeds treated with 25 mg/l bpa, the lowest mitotic index (5.45 ± 2.05) was determined. table 1. germination rates of p. sativum treated with different concentrations of bpa. concentrations of bpa seed germination (%) 24 h 48 h 72 h 00.00 77.33 ± 0.33 85.0 ± 0.88 99.0 ± 3.11 2 mg/l 72.77 ± 0.88a 84.0 ± 0.68 88.0 ± 2.09b 5 mg/l 74.33 ± 0.77c 78.0 ± 3.20 82.2 ± 0.33a 10 mg/l 66.66 ± 0.15a 70.0 ± 1.15a 75.0 ± 0.88a 15 mg/l 61.22 ± 0.03a 68.0 ± 1.15a 70.0 ± 1.33a 20 mg/l 55.33 ± 0.66a 61.0 ± 0.88a 65.0 ± 0.77a 25 mg/l 50.22 ± 0.42a 60.0 ± 0.55a 65.6 ± 0.66a ap<0.001 compared to control; bp<0.01 compared to control; cp<0.05 compared to control. data are mean of three replicates ± sem; 00.00 = control group. figure 1. effect of different concentrations of bpa on the radicle length of p. sativum. ap<0.001 compared to control; bp<0.01 compared to control; cp<0.05 compared to control. yp≤0.001v/s 15, 20, 25 mg/l; xp≤0.01 v/s 25 mg /l; pp≤0.05 v/s 20 mg/l. data are mean of three replicates ± sem; 0.0 = control group. 106 sazada siddiqiui et al. effect of bpa treatment on chromosomal anomalies. as shown in table 2 and fig. 3 treatment with bpa caused numerous mitotic anomalies in p. sativum. in control, the occurrence of abnormal metaphase-anaphase plates was 00 ± 00. in the present study, in case of root tips of p. sativum enhanced occurrence of chromosomal anomalies such as sticky chromosomes, c-mitosis, laggards, bridges and fragments were observed in various doses of bpa treatment (table 2, fig. 3). treatment with bpa resulted in a dose-related increase in the percentage of root tip cells with abnormal metaphase-anaphase plates. in lower concentration (2 mg/l of bpa treatment), minimum chromosomal anomalies such as fragments (0.42 ± 0.01), c-mitosis (0.52 ± 0.01), sticky chromosomes (0.61 ± 0.01), laggards (0.83 ± 0.06) and bridges (0.91 ± 0.02) were found which were non-significant (p>0.05) when compared with control. highest percentage of bridges (10.72 ± 2.2), c-mitosis (8.1 ± 2.15), fragments (6.78 ± 0.56), sticky chromosomes (6.1 ± 0.77) and laggards (6.01 ± 2.56) were found in 25 mg/l bpa treated root tip cells. st ick y chromosomes were high ly signif ica nt (p<0.001) in 5 to 25 mg/l, c–mitosis was found to be significant (p<0.05) at 25 mg/l, laggards were found to be significant (p<0.05) at 20 mg/l, bridges were found to be very significant (p<0.01) at 10 mg/l and highly significant (p<0.001) at 15 to 25 mg/l (p<0.01) and fragments were found to be very significant (p<0.01) at 15 mg/l and highly significant (p<0.001) at 20 to 25 mg/l when compared with control. discussion the outcome of the present study revealed that bpa inhibits and delays the germination of seeds, mitotic index, radicle length and chromosomal anomalies in figure 2. effect of different concentrations of bpa on the mitotic index in root tip cells of p. sativum. ap<0.001 compared to control; bp< 0.01 compared to control; cp< 0.05 compared to control. yp≤0.001v/s 15, 20, 25 mg/l; xp≤0.01 v/s 25 mg /l; data are mean of three replicates ± sem; 0.0 = control group. table 2. chromosomal anomalies in metaphase–anaphase plates in root tip cells of p. sativum treated with different concentrations of bpa. anomalies in 150 plates concentrations of bpa 00.00 2 mg/l 5 mg/l 10 mg/l 15 mg/l 20 mg/l 25 mg/l sticky chromosome (%) 00 ± 00 0.61 ± 0.01 2.78± 0.09a 4.99 ± 0.90a 4.25± 0.04a 7.80 ± 0.44a 6.10 ± 0.77a c-mitosis (%) 00 ± 00 0.52 ± 0.01 3.15 ± 1.12 2.25 ± 1.20 5.70 ± 1.13 6.70 ± 3.20 8.10 ± 2.15c laggards (%) 00 ± 00 0.83 ± 0.06 1.32 ± 0.91 2.45 ± 1.01 6.75 ± 2.05 8.15 ±3.25c 6.01 ± 2.56 bridges (%) 00 ± 00 0.91 ± 0.02 2.25 ± 1.00 4.23 ± 1.20b 5.78 ± 0.09a 7.62 ± 1.50a 10.72 ± 2.2a fragments (%) 00 ± 00 0.42 ± 0.01 0.71 ± 0.45 1.75 ± 0.76 2.91 ± 0.66b 4.62 ± 0.78a 6.78 ± 0.56a ap<0.001 compared to control; bp<0.01 compared to control; cp<0.05 compared to control. data are mean of three replicates ± sem; 0.0 = control group. figure 3. chromosomal anomalies induced by bpa in p. sativum root tip cells. (a) sticky chromosome, (b) c-mitosis, (c) laggards, (d) bridge at anaphase (e) fragment. 107impact of bisphenol a on seed germination, radicle length and cytogenetic alterations in pisum sativum l. seeds of p. sativum in a dose-related manner. it was shown in our experimental outcome that there is a substantial concentration-effect of bpa on the germination of seeds, mitotic index, radicle length and chromosomal anomalies in seedlings of p. sativum (table 1, 2 and fig. 1-3). seed germination is inhibited by bpa (zhiyong et al. 2013; pan et al. 2013; dokyung et al. 2018)). similar findings have been found by the present study that bpa delays and inhibits the germination of p. sativum seeds. seed germination is affected by various causes for example light, temperature of incubation, humidity and oxygen level (isabelle et al. 2000). eunkyoo et al. (2004) proved that an essential helix-loop-helix transcription feature pif3-like 5 (pil5) protein was a significant adverse regulator of phytochrome-mediated germination of seeds. it is known that etiolated seedlings generally have higher quantities of phytochromes a (hanumappa et al.1999), therefore, the possible functioning of bpa on phytochromes in seeds germination phase is interesting. in the present test, it was revealed that bpa showed inhibitory effects on root length in p. sativum treated with different doses. this may be caused by the noxious influence of bpa in root tips mitotic cell division (adamakis et al. 2013; 2016; amer 2017; dokyung et al. 2018. in p. sativum the root tip mitotic index is directly associated with decrease in root length. the same influence of bpa on mitotic index was recorded (pan et al. 2013; jadhav et al. 2012). primary roots elongation is facilitated by relating hormonal signal paths and a variety of enzymes for example phospholipase d, auxin and phosphatidic acid (ohashi et al. 2003; li et al. 2006; saini et al. 2013). though, the paths comprised in the molecular process related to the elongation of roots altered by bpa is not known. the decrease in the quantity of mitotic cells in root tips treated with bpa may be because of its mode of action on the progress of cell cycle. synthesis of dna may be inhibited by bpa (adamakis et al. 2019; ozge et al. 2019) or in g2 stage of cell cycle, bpa could also obstruct the cells and thus blocking them to enter into mitosis. moreover, bpa might affect enzymes for dnarepair, by altering the structure of proteins present in the enzymes or in mitotic cells, by decreasing the formation of enzymes at transcription phase that could induce chromosomal anomalies (ozge et al. 2019; nasir et al. 2018). p. sativum seeds treated with bpa showed numerous chromosomal anomalies in root tips mitotic cells for example c-mitosis, bridges, laggards, fragments and sticky chromosomes. the occurrence of chromosomal anomalies increases with increase in bpa concentration. in cell division, spindle fiber arrangement and its movement are a mechanism reliant on atp (can et al. 2005; nasir et al. 2018; adamakis et al. 2019). because of decreased synthesis and obtainability of atp, arrangement of spindle fiber in root tips treated with bpa, cells might get influenced, and it could disturb the chromosomal organization at metaphase plate and chromosomal migration to opposite poles in anaphase. the irregularity in spindles formation and segregation of chromosomes in mitosis, will cause chromosomal anomalies like laggards, bridges and sticky chromosomes. in bpa treated root tips, c-mitosis is generally linked to spindle defects (shahin and el-amoodi, 1991). since earlier studies have shown that bpa is a strong inhibitor of spindle microtubule organization (george et al. 2008; adamakis et al. 2013; xin et al. 2014; adamakis et al. 2016) which may explain high incidence of c-mitosis in bpa group.  the bridges found in the cells of bpa treated root tips are possibly produced by breaking and merging of chromosome bridges which got enhanced with treatment by bpa. chromosome bridges may be formed because of stickiness of chromosomes and consequent collapse of freed anaphase separation or because of an uneven translocation or chromosome segment inversion (gomurgen 2000; siddiqui 2012; siddiqui and al-rumman 2020 a and b). moreover, chromosome fragments may get formed because reactive oxygen species can induce double strand breaks in dna. conclusion conclusively, the outcome of this investigation revealed that bpa has substantial repressing effects on seed germination and enhances chromosomal anomalies in p. sativum root tip cells. as found in the present study and on the basis of the occurrence of numerous types of chromosomal abnormalities, it is rational to presume that bpa might reveal genotoxic effect on plants at elevated concentrations. moreover, a greater insight into the 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growth, photosynthesis and chlorophyll fluorescence in above-ground organs of soybean seedlings. chemosphere. 90(3):1274–80. caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 2 2021 firenze university press chromosomal analysis of eight cultivars in three species of cultivated yam (dioscorea l.) species in nigeria joshua matthew1,2,*, julius oloaye faluyi1 mutagenic and cytotoxic activity of insecticide napoleon 4ec in allium cepa and ames test dilek akyil assessment of antimitotic and programmed cell death stimulation potentials of galium sinaicum (delile ex decne) boiss. at the cellular level shaimaa s. sobieh1,*, dina m. fahmy2 first genome size assessments for marshallia and balduina (asteraceae, helenieae) reveal significant cytotype diversity teresa garnatje1,a, jaume pellicer1,2,a,*, joan vallès3, nathan hall4, curtis hansen4, leslie goertzen4 comparative study and genetic diversity in salvia (lamiaceae) using rapd molecular markers ruonan zheng1,*, shuhua zhao2, majid khayatnezhad3, sayed afzal shah4 identification of regions of constitutive heterochromatin and sites of ribosomal dna (rdna) in rhogeessa hussoni (genoways & baker, 1996) (chiroptera; mammalia; vespertilionidae) adriano silva dos santos1, karina de cassia faria2 analysis of cma-dapi bands and preparation of fluorescent karyotypes in thirty indian cultivars of lens culinaris timir baran jha1,*, biplab kumar bhowmick2, partha roy1 toxic and genotoxic effects of aqueous extracts of polygonum weyrichii fr. schmidt on the allium test taken as an example maria v. smirnova*, anna v. korovkina comparative cytogenetic analysis between species of auchenipterus and entomocorus (siluriformes, auchenipteridae) amanda de souza machado, samantha kowalski, leonardo marcel paiz, vladimir pavan margarido, daniel rodrigues blanco, paulo cesar venere, sandra mariotto, liano centofante, orlando moreira-filho, roberto laridondo lui* impact of bisphenol a on seed germination, radicle length and cytogenetic alterations in pisum sativum l. sazada siddqiui*, saad abdurahamn muhammad al amri, huda ahmed al ghamdy, wadha saad saeed alqahtani, sarah mohammed alquyr, habab merghani yassin first report on nucleolar organizer regions (nors) polymorphism and constitutive heterochromatin of moonlight gourami, trichopodus microlepis (perciformes, osphronemidae) patcharaporn chaiyasan1, sumalee phimphan2, teamjun sarasan1, sippakorn juntaree3, alongklod tanomtong1, sitthisak pinmongkhonkul4, weerayuth supiwong3,* morphological method and molecular marker determine genetic diversity and population structure in allochrusa kun zhu1,*, lijie liu1, shanshan li1, bo li1, majid khayatnezhad2, abdul shakoor3,4 genetic diversity and comparative study of genomic dna extraction protocols in tamarix l. species xiao cheng1,*, xiaoling hong1, majid khayatnezhad2, fazal ullah3,4 cytotoxic and genotoxic effects of methanol extracts of vegetative parts of some gypsophila l. species using allium cepa assay özgün tuna-gülören1, ferhan korkmaz2*, meltem erdir2, ebru ataşlar2 molecular techniques in the assessment of genetic relationships between populations of consolida (ranunculaceae) jing ma1, wenyan fan1,*, shujun jiang1, xiling yang1, wenshuai li1, di zhou1, amir abbas minaeifar2,* caryologia. international journal of cytology, cytosystematics and cytogenetics 74(4): 69-76, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1380 caryologia international journal of cytology, cytosystematics and cytogenetics citation: xixi yao, haodong liu, maede shahiri tabarestani (2021) morphometric analysis and genetic diversity in rindera (boraginaceae-cynoglosseae) using sequence related amplified polymorphism. caryologia 74(4): 69-76. doi: 10.36253/caryologia-1380 received: august 24, 2021 accepted: december 17, 2021 published: march 08, 2022 copyright: © 2021 xixi yao, haodong liu, maede shahiri tabarestani. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. morphometric analysis and genetic diversity in rindera (boraginaceae-cynoglosseae) using sequence related amplified polymorphism xixi yao1, haodong liu2,*, maede shahiri tabarestani3 1 college of agriculture and animal husbandry, qinghai university, xining,qinghai, 810016, china 2 gansu polytechnic college of animal husbandry & engineering, wuwei, gansu, 733006, china 3 assistant professor, department of agriculture, payame noor university, tehran, iran *corresponding author. e-mail: mindkeeper@126.com; chunou41@gmail.com abstract. the genus rindera comprises about 20–25 species distributed in central eastern europe to central asia. ninety-five individuals related to six rindera were collected in 9 provinces. a total of 147 (number of total loci) (ntl) dna bands were produced through polymerase chain reaction amplifications (pcr) amplification of six rindera species. these bands were produced with the combinations of 10 selective primers. the total number of amplified fragments ranged from 8 to 22. ). the predicted unbiased heterozygosity (h) varied between 0.15 (rindera media) and 0.30 (rindera regia). high shannon’s information index was detected in rindera regia. the genetic similarities between six species are estimated from 0.73 to 0.95. clustering results showed two major clusters. according to the srap (sequence-related amplified polymorphism) markers analysis, rindera regia and rindera media had the lowest similarity. this study also detected a significant signature of isolation by distance (mantel test results). present results showed that sequence-related amplified polymorphism have the potential to identify and decipher genetic affinity in rindera species. current results have implications in biodiversity and conservation programs. keywords: sequence-related amplified polymorphism, population structure, gene flow, network, genetic admixture, rindera. introduction: sequence-related amplified polymorphism (srap) is pcr –based marker system. it is one of the efficient and simple marker systems to study gene mapping and gene tagging in plant species (li and quiros 2001; guo, et al. 2021; cheng, et al. 2021), and srap are potential markers to assess plant systematics and genetic diversity studies (robarts and wolfe 2014). these past studies showed that molecular markers, including srap markers, are efficient to investigate genetic diversity analyses and phylogenetic relationship among paracaryum species in boraginaceae family. the family boraginaceae 70 xixi yao, haodong liu, maede shahiri tabarestani s.str consists of approximately 131 genera and 2,500 species, mainly distributed in dry, cliffy and sunny habitats of eurasia, the mediterranean region and the western north america (binzet and akcin 2009). they are mainly annual, bi-annual or perennial herbs and shrubs, some trees and a few lianes, distributed throughout the temperate and subtropical regions of the world (retief and vanwyk 1997), with a high distribution in iran (willis 1973). given the negative impact of biodiversity threats and over exploitation of rindera plant species in iran, it is necessary to conduct genetic diversity studies on rindera species. genetic diversity based studies pave our understanding to develop conservation strategies (esfandani-bozchaloyi et al. 2017). subfamily cynoglossoideae weigend., is the largest subfamily having about 900 species and 50 genera. recent molecular studies have shown that a wide range of the previously recognized tribes places into this subfamily (chacón et al. 2016). the subtribe cynogolossinae dumort. (tribe cynoglosseae w.d.j.koch) is entirely restricted to the old world, with a center of diversity in western asia and the mediterranean (chacón et al. 2016). the genus rindera pallas (1771: 486), comprises about 20–25 species distributed in central eastern europe to central asia (bigazzi et al. 2006). this taxon is closely related to paracaryum boissier (1849: 128) and mattiastrum brand (1915: 150), nested in cynoglossum linnaeus (1753: 134) s.str. (weigend et al. 2013, weigend et al. 2016). all species of rindera are perennial and linked to the dry and continental climate of the steppe and semidesertic belts (bigazzi et al. 2006). rindera is represented by 6 species in iran, 4 of which rindera albida (wettst.) kusn.; rindera bungei (boiss.) gürke; rindera regia kusn., rindera media (turrill) riedl. are endemic (khatamsaz 2001). rindera is characterized by tubular corollas, stamens usually inserted at the throat of the corolla, with a style mostly exserted from the corolla, and usually eglochidiate large mericarpids with a broad, membranous wing (bigazzi et al. 2006). rindera species are widely known as “yünlü gelin” and used as an anti-inflammatory agent in anatolian folk medicine (altundag and ozturk 2001). r. lanata is used to alleviate joint pains in iranian folk medicine (mosaddegh et al. 2012). in order to develop conservation strategies and proper utilization of plant genetic resources, it is important to characterize plant species based on genetic studies (kharazian et al. 2015), particularly this approach will serve better to understand genotypes of the geographically differentiated genus, such as echium l. and onosma (boraginaceae) (maria et al. 2007; dana et al. 2007). the present study investigated the molecular variation of six species in iran. objectives of the study were; a) to estimate genetic diversity; b) to evaluate population relationships using ward approaches. current results have implications in breeding and conservation programs. materials and methods: plants collection ninety-five (95) individuals were sampled. six rindera species in west azerbaijan, mazandaran, hamadan, kurdistan, esfahan, semnan, khorasan and razavi khorasan provinces of iran were selected and sampled during july-august 2018-2020 (table 1). morphometric and srap analyses on 95 plant accessions were carried out. five to twelve samples from each population belonging to six different species were selected based on other eco-geographic characteristics. samples were stored at -20 °c till further use. detailed information about locations of samples and geographical distribution table 1. list of the investigated taxa including origin of voucher specimens. all material is collected by majid khayatneshad. taxa locality latitude longitude altitude(m) rindera albida (wettst.) kusn. kurdestan, sanandaj hamedan, 20km s of nahavand 37°07’48” 49°54’04” 165 rindera bungei (boiss.) gürke razavi khorasan, kashmar, kuhsorkh district 37°07’08” 49°54’11” 159 rindera lanata (lam.) bunge kurdestan, sanandaj esfahan, ardestan on road to taleghan 38°52’93” 47°25’92” 1133 rindera cyclodonta bunge bojnord, ghorkhod protected area semnan, 20km nw of shahrud 38°52’93” 47°25’92” 1139 rindera regia kusn v mazandaran, 40 km tonekabon to janat abad mazandaran, nowshahr 35°50’36” 51°24’28” 2383 rindera media (turrill) riedl n west-azarbaijan, urumieh, silvana 35°42’29” 52°20’51” 2421 71morphometric analysis and genetic diversity in rindera using sequence related amplified polymorphism of species are mentioned (table 1 and fig 1). morphological studies each species was subjected to morphometric analysis and twelve samples per species were processed. qualitative (3) and quantitative (4) morphological characters were studied. data were transformed before calculation. different morphological characters of flowers, leaves, and seeds were studied. ordination analyses were conducted while using euclidean distance (podani 2000). sequence-related amplified polymorphism method: fresh leaves were used randomly from one to twelve plants. these were dried with silica gel powder. genomic dna was extracted while following previous protocol (esfandani-bozchaloyi et al. 2019). srap assay was performed as described previously (li and quiros 2001). ten srap in different primer combinations were used (table 2). a 25μl volume containing 10 mm of trishcl buffer at ph 8; 50 mm of kcl; 1.5 mm of mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of single primer; 20 ng of genomic dna and 3 u of taq dna polymerase (bioron, germany) were subjected to pcr reactions. the overall reaction volume consisted of 25 μl. this pcr reaction was carried out in techne thermocycler (germany). the following cycles and programs were observed. the initial denaturation step was performed for 5 minutes at 94°c. the initial denaturation step was followed by 40 cycles for 1 minute at 94°c; 1 minute at 52-57°c, and 2 minutes at 72°c. the reaction was completed by a final extension step of 7-10 min at 72°c. staining was performed with the aid of ethidium bromide. dna bands/fragments were compared against a 100 bp molecular size ladder (fermentas, germany). data analyses: upgma (unweighted paired group using average) ordination method was implemnented to assess morphological characters. anova (analysis of variance) was conducted to assess morphological differences among species. principal component analysis (pca) was implemented to identify variable morphological characters in rindera species. multivariate statistical analyses i.e., pc analysis, were performed in past software version 2.17 (hammer et al. 2001). molecular analyses sequence-related amplified polymorphism (srap) bands were recorded. presence and absence of bands were scored present (1) and absent (0), respectively. total loci (ntl) and the number of polymorphism loci (npl) for each primer were calculated. furthermore, the polymorphic ratio was assessed based on npl/ntl values. polymorphism information content was calculated as previously suggested by roldan-ruiz et al. (2000). resolving power for individual marker system was calculated as: rp = σib. ib (band informativeness) was estimated while figure 1. provinces and collection sites of rindera species. table 2. srap primer information and results. primer name ntl npl p pic rp em1-me1 13 12 92.31% 0.44 43.77 em2-me2 12 12 100.00% 0.66 36.77 em1-me4 18 17 94.4% 0.43 40.46 em2-me4 15 15 100.00% 0.49 33.76 em2-me5 8 8 100.00% 0.44 50.99 em3-me4 10 10 100.00% 0.41 32.24 em3-me1 24 19 79.00% 0.30 26.55 em4-me1 11 11 100.00% 0.44 44.23 em5-me1 16 16 100.00% 0.47 38.55 em5-me2 22 22 100.00% 0.35 29.65 mean 16 15 94.00% 0.48 37.55 total 147 133 359.85 abbreviations: ntl = number of total loci; npl = number of polymorphic loci; p = polymorphic ratio; pic = polymorphic information content; rp = resolving power. 72 xixi yao, haodong liu, maede shahiri tabarestani following equation: proposed as: ib= 1 [2 x (0.5-p)]. in the equation, p indicates the presence of bands (prevost and wilkinson, 1999). pairwise genetic similarity between species was evaluated to reveal genetic affinity between species (jaccard, 1908). unbiased expected heterozygosity and shannon information index were calculated in genalex 6.4 software (peakall and smouse, 2006). gene flow was conducted in popgene software, version 1.32 (yeh et al. 1999). analysis of molecular variance test was conducted in genalex (peakall and smouse 2006). mantet test was performed with 5000 permutations in past, version 2.17 (hammer et al. 2001). the comparison of genetic divergence or genetic distances, estimated by pairwise fst and related statistics, with geographical distances by mantel test is one of the most popular approaches to evaluate spatial processes driving population structure. the mantel test, as originally formulated in 1967, zm = gij × dij where gij and dij are, respectively, the genetic and geographic distances between populations i and j, considering populations. because zmis given by the sum of products distances its value depends on how many populations are studied, as well as the magnitude of their distances. the zm-value can be compared with a null distribution, and mantel originally proposed to test it by the standard normal deviate (snd), given by snd =zm/ var(zm)1/2 (mantel 1967). these analyses were done by past ver. 2.17 (hammer et al. 2012), darwin ver. 5 (2012) software. results mophometery the anova findings showed substantial differences (p<0.01) between the species in terms of quantitative morphological characteristics. principal component analysis results explained 55% cumulative variation. the first pca axis explained 40% of the total variation. the highest correlation (> 0.7) was shown by morphological characters such as calyx length, calyx width, corolla length, corolla color. the morphological characters of rindera species are shown in ward tree (fig. 2). each species formed separate groups based on morphological characters. the morphometric analysis showed clear difference among rindera species and separated each groups. in rindera albida and r. bungei nutlets are 8–14 mm, two-winged; outer wing 3 mm broad, margin undulate, inner 2 mm broad, incurved, margin cristatedentate, glochids entirely absent, while in r. lanata, r. cyclodonta nutlets are 15.8–23 mm, smooth, wing with smooth or undulate often blue margin, without glochids. species identification and genetic diversity ten (10) suitable primer combinations (pcs), out of 25 pcs were screened in this research. figure 3 illustrates the banding pattern of em3-me4, em1-me4, em5-me2 and em1-me1 primer by the srap marker profile. one hundered and thirty three (133) amplified polymorphic bands (number of polymorphic loci) were produced. these bands (fragments) had different range i.e. 150bp to 3000 bp. maximum and minimum numbers of polymorphic bands were 22 and 8 for em5-me2 and 8 em2me5, respectively. each primer produced 15 polymorphic bands on average. the pic ranged from 0.30 (em3-me1) to 0.66 (em2-me2) for the 10 srap primers, with an average of 0.48 per primer. rp of the primers ranged figure 2. morphological characters analysis of rindera species by ward. figure 3. electrophoresis gel of studied ecotypes from dna fragments produced by srap profile; 1,7,14,20: rindera albida ; 2, 8,15,21: rindera bungei ; 3,9, 16, 22: rindera lanata; 4, 10, 17, 23: r. cyclodonta;; 5, 11, 18, 24: rindera regia and 6, 12-13, 19, 25-26: rindera media; l = ladder 100 bp. 73morphometric analysis and genetic diversity in rindera using sequence related amplified polymorphism from 26.55 (em3-me1) to 50.99 (em2-me5) with an average of 37.55 per primer (fig. 3, table 3). the calculated genetic parameters of rindera species are shown (table 3). the unbiased heterozygosity (h) varied between 0.15 (rindera media) and 0.30 (rindera regia) with a mean of 0.23. shannon’s information index (i) was maximum in rindera regia (0.37), where as we recorded minimum shannon’s information index in rindera media (0.18). the observed number of alleles (na) ranged from 0.299 in rindera albida to 1.155 in rindera cyclodonta. the significant number of alleles (ne) ranged from 1.016 (rindera lanata) to 1.440 (rindera regia). analysis of molecular variance results in significant genetic difference (p = 0.01) among rindera species. the majority of genetic variation occurred among species. amova findings revealed that 82% of the total variation was between species and comparatively less genetic variation was recorded at the species level (table 4). genetic difference between rindera species was highlighted by genetic statistics (nei’s gst), as evident by significant p values i.e. nei’s gst (0.66, p = 0.01) and d_est values (0.122, p = 0.01) .mantel test after 5000 permutations produced significant correlation between genetic distance and geographical distance in these populations (r = 0.77, p = 0.001). therefore, the populations that are geographitable 3. genetic diversity parameters. sp n na ne i he uhe p rindera lanata 8.000 0.333 1.016 0.192 0.17 0.22 48.23% r. cyclodonta 12.000 1.155 1.190 0.271 0.184 0.192 55.91% r. regia 5.000 0.358 1.440 0.374 0.30 0.29 66.50% r. albida 6.000 0.299 1.029 0.231 0.18 0.23 44.38% r. bungei 5.000 0.462 1.095 0.288 0.25 0.22 62.05% r. media 5.000 0.358 1.117 0.18 0.15 0.12 34.30% abbreviations: n = number of samples, na= number of different alleles; ne = number of effective alleles, i= shannon’s information index, he = genetic diversity, uhe = unbiased gene diversity, p = percentage of polymorphism, populations. table 4. molecular variance analysis. source df ss ms est. var. % φpt among pops 30 1501.364 92.789 16.154 82% 82% within pops 100 334.443 3.88 2.888 18% total 130 1955.807 20.060 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance; φpt: proportion of the total genetic variance among individuals within an accession, (p < 0.001). figure 4. dendrograms of rindera species. 74 xixi yao, haodong liu, maede shahiri tabarestani cally more distant have less amount of gene flow, and we have isolation by distance (ibd) in the rindera. the constructed dendrogram highlighted two major clusters (fig. 4). group a consisted of 3 species rindera lanata; r. cyclodonta and rindera regia .two subclusters were in the b group: three species of rindera bungei, rindera albida and rindera media. we detected strong correlation between geographical and genetic distances (r = 0.22, p=0.0002) and gene flow (nm) score of 0.356 was reported among species. detailed information about genetic distances and genetic identity (nei’s) are described (supplementary table). the findings suggested that there was the highest degree of genetic similarity (0.95) between rindera lanata and r. cyclodonta. on the contrary to this, rindera regia and rindera media (0.73) had lowest genetic resemblance. discussion in the present study, we used morphological and molecular (srap) data to evaluate species relationships in rindera species. morphological analyses of rindera species showed that quantitative indicators (anova test results) and qualitative characteristics are well differentiated from each other. pca analysis suggests that morphological characters such as corolla color, nutlet shape, nutlet length, stamens position, nutlet margin, nutlet disc have the potentials to identify and delimitate rindera species. principal component analysis results suggests the utilization of morphological characters to identify and delimitate rindera species. morphological characters including nutlet shape, nutlet length, stamens position, nutlet margin play key role in plant systematics and taxonomy. our work also highlighted the significance of morphological characters and molecular data to identify and study species genetic diversity. in general, genetic relationships obtained from srap data coincides with morphometric results. this is in accordance with the parameters of amova and genetic diversity results. srap molecular markers detected clear genetic difference among species. these results indicate that srap have potentials to study plant systematics and taxonomy in rindera members. genetic diversity studies are conducted through appropriate selection of primers and indexes including polymorphic information content (pic) and marker index (mi) are important indexes to fathom genetic variation in species (sivaprakash et al. 2004). common logic suggests that different makers have different abilities to assess genetic diversity, and usually, genetic diversity is linked with polymorphism (sivaprakash et al. 2004). in this research, we reported pic values of srap primers from 0.30 to 0.66, with a mean value of 0.48. pic values indeed show low and high genetic diversity among genotypes. values are ranging from zero to 0.25 show low genetic diversity; in contrast to this, 0.25 to 0.50 highlight mid-level of genetic diversity. in addition to this, values higher than 0.5 are associated with high genetic diversity (tams et al. 2005). present results highlighted the efficiency of srap markers to estimate genetic diversity in rindera species. in our study, srap markers detected average percentage of polymorphism (94%). current research results also described average pic values of srap makers (0.48) and average rp (resolving power) values i.e. 37.55 of srap markers. these current reported values are higher than other reported markers on rindera species (maria et al. 2007; dana et al. 2007). in the recent study, low gene flow (nm) was detected among rindera species. despite the presence of limited gene flow in rindera species, two distinct ecotypes were reported previously. these ecotypes were formed due to reproductive isolation caused by altitude gradient and different niches (moein et al., 2019). the present study also depicted a significant correlation between genetic and geographical distances. our findings revealed that isolation by distance (ibd) existed between rindera species (mantet test results). several mechanisms, such as isolation, local adaptation, and genetic drift, shape the species or population differentiation (frichot et al. 2013; de kort et al. 2014; zhang et al. 2021; zheng et al. 2021; guo et al. 2021). the magnitude of variability among na, ne, h, and i indices demonstrated a high level of genetic diversity among rindera species. dendrogram and principal component analysis results showed clear difference among rindera species . this shows the high utilization of the srap technique to identify salvia species. our results have implications for conservation and breeding programs. furthermore, it may identify suitable ecotypes for forage and pasture. conclusions the present study investigated the molecular variation of six species. molecular and morphometric analysis confirmed morphological and genetical difference between rindera species. this was first attempt to assess genetic diversity through sequence-related amplified polymorphism and morphometrics analysis in iran. current study reported two major clusters. these two major groups were separated on the basis of genetic and morphological characters. the genetic similarities between six species was estimated from 0.73 to 75morphometric analysis and genetic diversity in rindera using sequence related amplified polymorphism 0.95. srap (sequence-related amplified polymorphism) markers analysis, showed that rindera regia and rindera media had the lowest similarity. current study also reported correlation between genetic and geographical distances. this clearly indicated isolation mechanism envloved in the ecology of rindera species. present results indicated the potential of sequence-related amplified polymorphism to assess genetic diversity and genetic affinitiy among rindera species. current results have implications in biodiversity and conservation programs. besides this, present results could pave the way for selecting suitable ecotypes for 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issue 4 2021 firenze university press cytogenetic analyses in three species of moenkhausia eigenmann, 1903 (characiformes, characidae) from upper paraná river (misiones, argentina) kevin i. sánchez1,*, fabio h. takagui2, alberto s. fenocchio3 genetic variations and interspesific relationships in lonicera l. (caprifoliaceae), using scot molecular markers fengzhen chen1, dongmei li2,* , mohsen farshadfar3 the new chromosomal data and karyotypic variations in genus salvia l. (lamiaceae): dysploidy, polyploidy and symmetrical karyotypes halil erhan eroğlu1,*, esra martin2, ahmet kahraman3, elif gezer aslan4 cytogenetic survey of eight ant species from the amazon rainforest luísa antônia campos barros1, gisele amaro teixeira2, paulo castro ferreira1, rodrigo batista lod1, linda inês silveira3, frédéric petitclerc4, jérôme orivel4, hilton jeferson alves cardoso de aguiar1,5,* molecular phylogeny and morphometric analyses in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f. neda atazadeh1,*, masoud sheidai1, farideh attar2, fahimeh koohdar1 a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) melika tabasi1, masoud sheidai1,*, fahimeh koohdar1, darab hassani2 genetic diversity and relationships among glaucium (papaveraceae) species by issr markers: a high value medicinal plant lu feng1,*, fariba noedoost2 morphometric analysis and genetic diversity in rindera (boraginaceae-cynoglosseae) using sequence related amplified polymorphism xixi yao1, haodong liu2,*, maede shahiri tabarestani3 biosystematics, fingerprinting and dna barcoding study of the genus lallemantia based on scot and remap markers fahimeh koohdar*, neda aram, masoud sheidai karyotype analysis in 21 plant families from the qinghai–tibetan plateau and its evolutionary implications ning zhou1,2, ai-gen fu3, guang-yan wang1,2,*, yong-ping yang1,2,* some molecular cytogenetic markers and classical chromosomal features of spilopelia chinensis (scopoli, 1786) and tachybaptus ruficollis (pallas, 1764) in thailand isara patawang1,*, sarawut kaewsri2, sitthisak jantarat3, praween supanuam4, sarun jumrusthanasan2, alongklod tanomtong5 centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates) simona ceraulo, vanessa milioto, francesca dumas* repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil marina souza cunha1,2,*,#, silvana melo1,3,#, filipe schitini salgado1,2, cidimar estevam assis1, jorge abdala dergam1,* karyomorphology of some crocus l. taxa from uşak province in turkey aykut yilmaz*, yudum yeltekin variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l. egizia falistocco1,*,+, gianpiero marconi1,+, lorenzo raggi1, daniele rosellini1, marilena ceccarelli2, emidio albertini1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(3): 31-43, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1109 caryologia international journal of cytology, cytosystematics and cytogenetics citation: jun wang, qiang ye, chu wang, tong zhang, xusheng shi, majid khayatnezhad, abdul shakoor (2021) palynological analysis of genus geranium (geraniaceae) and its systematic implications using scanning electron microscopy. caryologia 74(3): 31-43. doi: 10.36253/caryologia-1109 received: october 06, 2020 accepted: july 21, 2021 published: december 21, 2021 copyright: © 2021 jun wang, qiang ye, chu wang, tong zhang, xusheng shi, majid khayatnezhad, abdul shakoor. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. palynological analysis of genus geranium (geraniaceae) and its systematic implications using scanning electron microscopy jun wang1,2,*, qiang ye1, chu wang2, tong zhang2, xusheng shi2, majid khayatnezhad3, abdul shakoor4,5 1school of management, harbin institute of technology; heilongjiang provice, china 2beijing jinghang computation and communication research institute; beijing, china 3department of environmental sciences and engineering, ardabil branch, islamic azad university, ardabil, iran 4college of environment and planning, henan university, kaifeng, 475004, henan, china 5key laboratory of geospatial technology for the middle and lower yellow river regions, ministry of education, kaifeng 475004, henan, china *corresponding authors. e-mail: jxwxuwei@163.com; abdul_shakoor954@yahoo.com abstract. pollen morphology of 23 species belonging to geranium have been studied in details, which represent eight sections of two subgenera i.e., g. sect. dissecta, geranium, and tuberosa of subgen. geranium, divaricata, lucida, ruberta and trilopha of subgen. robertium. these plant species were collected from different phytogeographical regions of iran. the palynological investigation was done using scanning electron microscopy (sem) techniques. different palyno-morphological features have been observed, and the closely related species were distinguished. we used different multivariate statistical methods to reveal the species relationships. ward clustering analyses have been done to check out the relationship among the species. the shapes of pollen grains were monad, radially symmetric, isopolar, apertures were tricolporate, and of spheroid, prolate-spheroid or sub-prolate classes. three pollen types were recognized on the basis of differences in exine sculpturing pattern: reticulate-clavate, striaterugulate, reticulum cristatum with clavae. observed differences were not of diagnostic importance in subgenera and sections level. the main objective of this study is to find distinguish pollen characters in the species of the genus geranium and to elucidate their systematics importance. keywords: geranium, pollen morphology, systematics, phytogeographical regions. research highlights – pollen grains of the studied species were investigated by light and scanning electron microscope – using scanning electron microscopy techniques for the easy and quick identification of plant species – using micro-morphological (sem) characters for the identification and species delimitations. 32 jun wang et al. introduction the geranium l., is the largest genus of geraniaceae and contains 325 species. geranium species are distributed worldwide except in lowland tropical areas (aedo 2017). most of the species belong to subg. geranium (aedo 2001). traditionally the genus geranium was classified into three subgenera: 1subg. geranium, 2subg. erodioidea (picard) yeo, and 3subg. robertium (picard) rouy. according to yeo (1984), the subg. robertium primary diagnostic characteristics are carpel projection of fruit discharge. this subgenus has thirty species, and these are distributed in eight sections. the subgenus. geranium, is the largest subgenus containing 300 species. these 300 species were in at least ten sections. the genus is characterized by a seed-ejection fruit type in which the explosive recurvature of the awn carries the mericarp upwards in an arc, throwing the seed out (marcussen and meseguer 2017). the family geraniaceae juss., is a worldwide family and comprising additionally five genera: hypeocharis j. remy (subfam. hypseocharitoideae), erodium l’herit ex aiton., geranium l., monsonia l., pelargonium l’herit. ex aiton and california being successive sisters to geranium l. (subfam. geranioideae) (fiz et al. 2008; weng et al. 2014). these genera have a moderate number of species than geranium and had a more narrow distribution. t he cha rac ter ist ic f ive-c a r pel led sch i zoc a r p geranioideae fruit with a prominent central rostrum has a great taxonomic value in this genus. the geranium shows four different fruit discharge methods: carpelprojection type, erodium-type, seed-ejection type, and the inoperative type (marcussen and meseguer 2017). these methods have been considered in the delimitation of major groups within the geranium. marcussen and meseguer (2017) pointed that phylogenetic relationships within geranium and the evolution of the different seed discharge methods are still unknown. the new classification has shifted some of the studied taxa from subg. robertium to subg. geranium, while a third subgenus (erodioidea) has been reduced to synonymy under subg. geranium (marcussen and meseguer 2017). the geraniaceae is a eurypalynous family (baser et al. 2016). the pollen morphological features of the family have a great importance in the discrimination at the generic level (perveen and gaiser 1999). the pollen grains are usually isopolar with radial symmetry; generally have oblate-spheroidal shape, rarely sub-oblate; three-lobed, tri-colporate, rarely colpate and the colpi are short; the sexine is thicker than nexine, and the tectum is reticulate with densely baculate or gemmate muri or striate (perveen and gaiser 1999). various workers described the pollen morphology of different species of geraniaceae, species delimitation and species relationships in the genus geranium l. since bortenschlager’s (1967) preliminary study on the pollen morphology of the geraniaceae, the pollen morphology of monsonia (verhoeven and venter 1986), sarcocaulon (verhoeven and venter 1988), and erodium (verhoeven andventer 1987) of the southern african geraniaceae has been investigated. erodium pollen grains of the middle east (ei-oqlah 1983) and northwestern europe have been studied (stafford and blackmore 1991). stafford andblackmore (1991) reviewed the pollen grains of geranium species in north-western europe. pollen morphology of 35 taxa of the genus geranium in asia has been observed through light and scanning electron microscopy (sem) by park and kim (1997). they found high variation in pollen grains size and exine sculpture (muri, lumina, excrescences distribution) among the examined taxa. also, palynological characters were useful to establish the phylogeny within the genus. however, they are not helpful characters for separation at subgenera and sections. perveen and gaiser (1999) studied the pollen morphology of pakistani geraniaceae taxa and based on exine ornamentations and apertural type. they divided the pollen grains into three groups i.e., erodium cicutariumgeranium himalayense, and monsonia senegalensis-types. they observed that the pollen grains were mostly oblatespheroidal, rarely prolate-spheroidal or spheroidal, and often sub-oblate in the examined taxa. shehata (2008) analyzed pollen grains of the geraniaceae family from eg ypt. the results showed that pollen morphological characters, i.e., pollen size, pollen shape, and aperture, were taxonomically significant at the generic level and up to some extent at the specific level. ilcim et al. (2008) analyzed the morphological and palynological properties of g. tuberosum. palynological investigation of 13 species of genus geranium in turkey by deniz et al. (2013), did not reflect significant differences between the sections, except some differences in pollen grains of the subgenus geranium. according to deniz et al. (2013), all examined species’ pollen grains were large, and exine ornamentation was reticulate-clavate; and the apertures were tri-colporate. in iran, the only study on pollen morphology of five geranium species was assessed by keshavarzi et al. (2016). there is no comprehensive palynological investigation of geranium species in iran. the present study aimed to use the pollen grains morphological features as a source of diagnostic characters to distinguish different iranian geranium species. 33palynological analysis of genus geranium and its systematic implications using scanning electron microscopy material and methods totally 60 populations were collected and studied from 23 taxa of geranium from different habitats in iran to explore the pollen features (table 1). ten individuals of each location were studied and examined for three qualitative and eight quantitative features (table 2). for scanning electron microscopy (sem) examinations, pollen grains were not acetolyzed according to the method of erdtman (1960). the pollens were suspended in a drop of water for a while and then directly transferred to a metallic stubby a fine pipette, and double-sided cello tape was used. and then the pollens were sputtered in a chamber coated with gold (sputter coater baltec, scdoos). coating with gold by the physical vapor deposition method (pvd) was restricted to 100 å. the sem examination was carried out on a tescan microscope. pollen sculptures were described according to previous terminology (hesse et al. 2009). the evaluated and measured characters of the species studied of geranium are summarized in tables ii. to detect significant differences in the studied characters among the various studied species, an analysis of variance (anova) was performed. for multivariate analysis, the mean of the quantitative characters was used. principal components analysis (pca) was performed among the species to determine palynological characters useful for separating the species. in order to group the species, cluster analysis using ward (minimum spherical variance) methods and pca ordination plot were performed by past software (hammer et al. 2001), using euclidean and taxonomic distance among the species was calculated (podani 2000). the qualitative traits were coded as binary or multistate. variables were systematized for multivariate statistical analysis. average taxonomic distances and squared euclidean distances were applied as dissimilarity coefficients in the pollen data cluster analysis. image tool version 3.0 (http://ddsdx.uthscsa.edu/dig/itdesc.html) was used to carry out the required measurements. results infrageneric variation we showed that g. purpureum, g. robertianum and g. sylvaticum possess type i pollen grains. sub-genus geranium had type ii pollens. species, i.e., g. biuncinatum ,g. mascatense and g. trilophum belonging to trilopha section .showed the type iii of pollen grains. factor analysis shows three factors explained more than 77% of the total observed variation in studied pollen grains. the first factor revealed equatorial length, pollen shape importance. aperture condition, colpus length, and exine thickness illustrated more than 44% of the observed variation in the second factor. principal component analysis (pca) based on pollen grain qualitative and quantitative traits confirms the cluster analysis results by ward’s method. general pollen grain features the majority of geranium species depicted prolatespheroidal pollen types. for instance, g. columbinum, g. collinum, g. sylvaticum, g. pratense, g. dissectum, g. linearilobum, g. tuberosum, g. kotschyi, g. platypetalum, g. gracile, g. ibericum, g. purpureum, g. pyrenaicum, g. robertianum, g. divaricatum, g. lucidum, and g. molle had prolate-spheroidal shape (figures. 2 a1, b1, c1, d1; 3 e1, f1, g1; 4 m1, o1; 5 p1, q1; 6t1, w1, y1, z1; 7 x1, u1). spheroidal pollen types were observed in g. rotundifolium, g. pusillum, g. albanum (figs. 4 l1, 3 h1, 4 n1). g. biuncinatum, g. mascatense, g. trilophum species had sub-prolate pollen morphology (figures. 5 r1, s1; figures. 7, u4-u6). we found that g. lucidum and g. robertianum had spheroidal-prolate pollen grains. while g. rotundifolium had spheroidal pollen shape (table ii, figures 4 l1-l2). our statistical and microscopy analyses depicted that g. platypetalum had the largest (table ii, figure. 6 z1, z2) and g. pusillum possessed smallest pollen grains. (table ii, figures 3 h1, h2).our observations revealed that pollen grains were generally prolate-spheroidal except g. pusillum, g. rotundifolium, and g. albanum (figures 3h1-h2, figures 4 l1-l2; n1-n2).mean polar axis length varied from 38.55 μm (g. pusillum) to 104.88 μm (g. platypetalum), while the mean of the equatorial axis length varied from 37.55 μm (g. pusillum) to 105μm (g. platypetalum). the main colpus length varied from 12 μm (g. molle) to 58 μm (g. trilophum). p/e ratio differed from 0.89 μm (g. albanum) to 1.5 μm (g. trilophum). the main features of the investigated pollen grains are summarized in table ii. the basic ornamentation of the exine surface in the geranium was reticulate-clavate, striate-rugulate, and reticulum cristatum with clavae. on the basis of differences in exine sculpturing pattern, the following 3 types are recognized: reticulate-clavate, striate-rugulate, reticulum cristatum with clavae. most of the specimens belong to reticulum cristatum with clavae pattern. type i: geranium robertianumtype (“reticulateclavate”) species: geranium robertianum (figure 5, p3; figure 4, o3; figure 3, e2) pollen class: tricolporate 34 jun wang et al. table 1. list of geranium species in iran their localities and voucher numbers. no section sp. locality 1 dissecta yeo g. dissectum l. guilan, lahijan east azerbaijan, kaleybar, cheshme ali akbar east azerbaijan, kaleybar, shoj-abad tehran, damavand khorassan, kashmar-darvaneh 2 geranium g. columbinum l. guilan, siahkal, ezbaram; guilan, langerud, chaff; guilan, bandar-e anzali; tehran, damavand; khorassan, kashmar-darvaneh 3 g. rotundifolium l. tehran, tuchal mazandaran, kandovan-siahbisheh east azerbaijan, kaleybar, shoj-abad 4 g. collinum stephan ex willdenow mazandaran, tonekabon-jannat rudbar hamedan, 20km s of nahavand 5 g. sylvaticum l. east azerbaijan, kaleybar, cheshme ali akbar 6 g. pratense l. east azerbaijan, kaleybar, shoj-abad mazandaran, 40 km tonekabon to janat abad 7 tuberosa (boiss.) reiche g. platypetalum fisch. & c. a. mey. east azerbaijan, kaleybar razavi khorasan, kashmar tehran, damavand 8 g. ibericum cav. tehran, damavand kordestan, sanandaj khorassan, kashmar-darvaneh 9 g. gracile ledeb. ex nordm. mazandaran, noshahr, kheyrud kenar forest kerman, lalehzar, baghabad 10 g. linearilobum dc. tehran, firuz kuh mazandaran, 40 km tonekabon to janat abad 11 g. kotschyi boiss. alborz, karajqazvin tehran, desin 12 g. tuberosum l. east azerbaijan, kaleybar cheshme ali akbar, tehran, tuchal 13 batrachioidea w.d.j. koch g. molle l. east azerbaijan, kaleybar, shojabad east azerbaijan, kaleybar, cheshme ali akbar hamedan, 20km s of nahavand 14 g. pyrenaicum burm. f. east azerbaijan, kaleybar, roadside east azerbaijan, kaleybar, cheshme ali akbar east azerbaijan, kaleybar, shojabad east azerbaijan, babak fort tehran, damavand 15 g. pusillum l. east azerbaijan, kaleybar, roadside east azerbaijan, kaleybar cheshme ali akbar east azerbaijan, kaleybar, shojabad hamedan, 20km s of nahavand tehran, damavand 16 ruberta dumort. g. purpureum vill. east azerbaijan, kaleybar, cheshme ali akbar guilan, gole rodbar guilan, gole rodbar, roadside guilan, jirandeh khorassan, kashmar-darvaneh kerman, lalehzar, baghabad 17 g. robertianum l. guilan,gole rodbar esfahan, 50 km delijan tehran, damavand 18 divaricata rouy g. albanum m. bieb. guilan, sangar, roadside guilan, lahijan guilan, jirandeh mazandaran, siah bisheh to chalus golestan, ramian 35palynological analysis of genus geranium and its systematic implications using scanning electron microscopy no section sp. locality esfahan, 50 km delijan khorasan, birjand tehran, darakeh hamedan, 20km s of nahavand 19 g. divaricatum ehrh. east azerbaijan, kaleybar tehran, darband khorasan, birjand tehran, darakeh kerman, lalehzar, baghabad 20 lucida r. knuth g. lucidum l. east azerbaijan, kaleybar cheshme ali akbar 21 trilopha yeo g. trilophum boiss. hormozgan, amani village, kushk-e nar rural hamedan, 20km s of nahavand 22 g. mascatense boiss. hormozgan, qeshm, bakho mountain 23 g. biuncinatum kokwaro khuzestan, shushtarmasjed soleyman table. 2. evaluated characters of pollen grains in geranium species studied (values m ± sd μm). mmean value; sdstandard deviation. aperture: at the same level =2, protruding = 1; pollen shape: prolate-spheroidal =1, spheroidal= 2, subprolate=3; exine ornamentation type: reticulate-clavate=1, striate-rugulose =2, reticulum cristatum with clavae= 3 taxon po la r ax is le ng th (µ m ) ex in e th ic kn es s (µ m ) eq ua to ri al a xi s le ng th ( µm ) p/ e ra tio a po co lp iu m le ng th ( µm ) m es oc ol pi um le ng th ( µm ) c ol pu s le ng th (µ m ) po re d ia m et er (µ m ) a pe rt ur e po lle n sh ap e ex in e or na m en ta tio n ty pe g. dissectum 55.67±2.1 4.5 55.87±3.3 1.05±0.05 ±0.03 23.67 35.66 36.44 6.87 1 1 3 g. columbinum 62.76±3.4 5.88 62.66±2.9 1.00±0.03 35.87 52.54 22 15 1 1 3 g. rotundifolium 66.44±3.8 5.87 65.54±2.3 1.01±0.02 37.22 35.77 25 8.5 1 2 3 g. collinum 86.5±2.8 5.34 77.32±4.8 1.11±0.04 48.55 34.33 55 12 1 1 3 g. platypetalum 103.32±4.1 4.45 101±2.7 1.01±0.05 68.87 54.77 33 24 1 1 3 g. sylvaticum 78.44±2.9 5.87 75.55±1.4 1.04±0.04 54.22 42.32 25 7 2 1 1 g. pretense 75.55±3.7 5.88 66.77±2.4 1.06±0.02 55.77 60.22 32 7 2 1 3 g. ibericum 81.44±3.9 4.34 78.33±3.6 1.03±0.05 47.22 54.55 28 13 2 1 3 g. gracile 76.44±2.4 5.45 74.56±2.9 1.02±0.03 37.55 53.77 27 16 1 1 3 g. linearilobum 71.88±2.8 5.65 61.55±4.2 1.13±0.06 38.43 38.34 39 14 1 1 3 g. kotschyi 63.66±4.1 5.8 59.55±4.5 1.06±0.03 42.66 35.77 39 11 1 1 3 g. tuberosum 81.55±2.8 5.3 75.66±2.2 1.08±0.02 48.77 46.44 33 12 1 1 3 g. molle 49.44±1.6 4.55 47.34±1.3 1.04±0.04 31 35 13 10 1 1 3 g. pyrenaicum 64.22±2.8 4.3 59.34±2.2 1.08±0.05 33 30 23 4 2 1 3 g. pusillum 39.65±1.4 3.7 38.22±5.3 1.02±0.03 26-27 24 15.66 7.87 1 2 3 g. purpureum 58.27±1.9 4.2 54.55±2.5 1.1±0.04 37.85 29.01 27.68 7.4 2 1 1 g. robertianum 61.76±4.1 4.7 51.54±1.8 1.02±0.02 30 22-28 21-22 7-8 2 1 1 g. albanum 63.44±2.5 5.45 66.34±2.6 0.95±0.03 38.76 39.44 26.33 14 1 2 3 g. divaricatum 59.57±3.7 4.55 51.66±3.9 1.13±0.01 33.66 35.33 32 9 1 1 3 g. lucidum 58.55±5.2 4.76 49.65±3.3 1.09±0.04 33.77 25.44 32 6 1 1 3 g. mascatense 64.66±6.1 5.88 60.33±2.9 1.1±0.05 23.77 47.55 50 14 1 3 2 g. trilophum 65.44±2.6 5.34 54.45±2.5 1.2±0.03 21.45 43.55 56 9 1 3 2 g. biuncinatum 63.25±5.1 4.35 63.76±7.9 1.1±0.03 22.65 45.98 59 12 1 3 2 36 jun wang et al. shape: prolate-spheroidal apertures: ectoaperture-colpi small ornamentation: tectum coarsely reticulate with clavae measurements: polar length (p) (56-75 μm), equatorial diameter (e) (55-78 μm), colpi (20-25μm) in diameter. mesocolpium (20-40 μm). apocolpium (33-59 μm). exine 4.9-5.9 μm species included: g. robertianum, g. purpureum, g. sylvaticum type ii: geranium molle -type (“reticulum cristatum with clavae”) species: g. molle (figures 2, a3, b3, c3, d3; figures 3, f3, g3, h3; figures 4, l3, m3, n3; figure 5, q3; figures 6, t3, w3, y3, z3; figures 7, x3, u3 ) pollen class: tricolporate shape: prolatespheroidal apertures: ectoaperture colpus short (approx. 1/5 to 1/8 of polar axis) ornamentation: tectum reticulum cristatum with clavae measurements: polar length (p) (38-105 μm) , equatorial diameter (e) (38-105μm), colpi (10-58μm) in diameter. mesocolpium (22-67μm). apocolpium (22-69μm) . exine 3.7-5.8 μm species included: g.gracile, g. ibericum, g. pyrenaicum, g. divaricatum, g. lucidum, and g. molle, g. pusillum ; g. columbinum, g. collinum, g. pratense, g. dissectum, g. linearilobum, g. tuberosum,g. kotschyi, g. platypetalum pollen type iii: erodium-type (“striate-rugulose”) (figures. 5, r3, s3; figures. 7, u4-u6) pollen class: tricolporate shape: subprolate apertures: ectoaperture-colpi large ornamentation: tectum striate-rugulose measurements: polar length (p) (68-69 μm) , equatorial diameter (e) (50-64μm), colpi (53-59μm) in diameter. mesocolpium (40-49μm). apocolpium (20-25 μm). exine 5.5-5.9 μm species included: g. mascatense, g. trilophum, g. biuncinatum discussion geranium species are relatively challenging to study due to the overlapping of morphological characters (aedo and pando 2017; ji et al. 2020; wang et al. 2020; sun et al. 2021). therefore, an attempt was carried out to investigate pollen grains of geranium species by scanning electron microscopy. our approach also included the usage of principal component analysis to verify our findings. the current study showed that geranium species have eurypalynous pollens morphology. present results corroborate with a previous study conducted on the geraniaceae family (baser et al. 2016). most of the species showed prolate-spheroidal pollen types. this pattern of pollen type is previously reported in the geraniance family (baser et al. 2016). we observed monad, isopolar, radial symmetry, tricolporate, and short linear colpi pollen grains in geranium species. similar pollen types have been reported in iran while working on five geranium species (keshavarzi et al. 2016). our result recorded spheroidal-prolate pollen grains in g. lucidum and g. robertianum species. however, previously oblatespheroidal pollen grains were reported in g. lucidum, g. robertianum (perveen and gaisar 1999). we argue that such a difference in pollens is due to sampling site and habitat (brodschneider et al. 2019). annual or biennial species, i.e., g. lucidum, g. pusillum, g. molle, g. dissectum, g. rotundifolium showed the smaller pollen grains (from 39 to 66 μm). g. collinum, g. sylvaticum, g. pratense, g. platypetalum, g. gracile, and g. ibericum had larger (63 to 103 μm) pollen grains. such variation in pollen grains has been described in the past (aedo 2001; aedo et al. 2007; jurgens et al. 2012; hao et al. 2020). figure 1. ward clustering of geranium species based on observed pollen data. 37palynological analysis of genus geranium and its systematic implications using scanning electron microscopy clustering results revealed pollen grain type iii in section trilopha. henceforth, type iii pollen is the diagnostic feature to identify trilopha section. deniz et al. (2013) observed the exine ornamentation to be reticulate-clavate in geranium taxa. while in the present study, exine ornamentations were reticulateclavate, striate-rugulose, reticulum cristatum with clavae. most of the specimens belong to reticulum cristatum with clavae pattern. the pollen of all species of geranium sect. trilopha is of the erodium-type (aedo et al. 2016), which is congruent with the present findings. the striate ornamentation is considered a diagnostic feature to differentiate trilopha section. this distinguishing feature may support the close relationship between g. sect. figure 2. pollen micrographs of geranium species: a1-a3: g. collinum, b1-b3: g. columbinum, c1-c3: g. pratense, d1-d3: g. dissectum; a1, b1,c1, d1 – equatorial view; a2, b2, c2, d2 polar view; a3, b3, c3, d3 -exine sculpture. 38 jun wang et al. polyantha and g. sect. trilopha. the scanning electron microscopy (sem) technique has a crucial role in observing the minute biological structure (ullah et al. 2019). several taxonomic and plant systematics questions are also addressed through the application of sem. for instance, seed morphology in caryophyllaceae was studied by scanning electron microscopy to identify caryophyllaceae species correctly (ullah et al. 2019; zou et al. 2019). besides this, detailed plant morphology, anatomy, and pollens in spergula fallax and spergula arvensis revealed the significance of sem to resolve taxonomic complexity (ullah et al. 2018). authors developed identification key and distinguishing features of spergula fallax and spergula arvensis species figure 3. pollen micrographs of geranium species: e1-e3: g. sylvaticum, f1-f3: g. molle, g1-g3: g. pyrenaicum, h1-h3: g. pusillum; e1, f1, g1, h1 – equatorial view; e2, f2, g2, h2 polar view; e3, f3, g3, h3 -exine sculpture. 39palynological analysis of genus geranium and its systematic implications using scanning electron microscopy (ullah et al. 2018). this clearly shows the utilization of new techniques to solve complex questions in plant systematics and biology (pathan et al. 2010; ullah et al. 2018; ullah et al. 2019). cluster revealed groupings of g. uncinatum, g. mascatense, g. trilophum into the trilopha section. similar results at the section level was reported. phylogenetic relationship based on chloroplast (rbcl, trnl-trnf) and nuclear (its) dna sequences highlighted the grouping of g. uncinatum, g. mascatense into the trilopha section (marcussen and meseguer 2017). the phylogenetic tree proved the geraniaceae family as a monophyletic. figure 4. pollen micrographs of geranium species: l1-l3: g. rotundifollium, m1-m3: g. lucidum, n1-n3: g. albanum, o1-o3: g. purpureum ; l1, m1, n1, o1 – equatorial view; l2, m2, n2, o2 polar view; l3, m3, n3, o3 -exine sculpture. 40 jun wang et al. the discrepancy in the phylogenetic results obtained by different molecular markers may be the reason for unresolved species relationship in the genus geranium. evolutionary relationships between geranium, erodium, and are unclear because of the low support for the alternative topologies inferred from both markers (trnl–f and rbcl) figure 5. pollen micrographs of geranium species: p1-p3: g. robertianum, q1-q3: g. divaricatum, r1-r3: g. trilophum, s1-s3: g. mascatense; p1,q1, r1, s1 – equatorial view; p2,q2, r2, s2 polar view; p3,q3, r3, s3 -exine sculpture. 41palynological analysis of genus geranium and its systematic implications using scanning electron microscopy (fiz et al. 2008). inter-simple sequence repeat (issr) markers separated geranium species (esfandani–bozchaloyi et al. 2018). the present study showed that pollen features are not capable of distinguishing different sections in geranium. two subgenera elements were mixed. nonetheless, we found and linked the diagnostic character (pollen type iii) of the trilopha section. future studies may incorporate phylogenetic data and chemotaxonomy methods to decipher genus and species level evolutionary relationship in geranium. linking the morphological and phylogenetic traits may further aid our apprehension in plant evolution and systematics (cohen 2011). in this research, we studied different palynological characteristics of the genus geranium. various characters were crucial for the taxonomic identification of the figure 6. pollen micrographs of geranium species: t1-t3: g. kotschyi, w1-w3: g. linearilobum, y1-y3: g. tuberosum, z1-z3: g. platypetalum; t1,w1,y1, z1 – equatorial view; t2,w2,y2, z2 polar view; t3,w3,y3, z3 -exine sculpture. 42 jun wang et al. species delimitations. this study could serve better to understand the pollen morphology of geranium species in iran. the pollen characters studied here were useful for the taxonomic identification of the species of the genus. acknowledgements we would also thank editor for his comments to improve the quality of the manuscript. references aedo c, barbera p, buira a. 2016. taxonomic revision of geranium sect. trilopha (geraniaceae). syst bot. 41:354–377. aedo c, garcia m, alarcon m, aldasoro j, navarro c. 2007. taxonomic revision of geranium subsect. mediterranea (geraniaceae). syst bot. 32:93–128. aedo c, pando f. 2017. a distribution and taxonomic reference dataset of geranium in the new world. scientific data. 4:170049. aedo c. 2001. taxonomic revision of geranium sect. brasiliensia (geraniaceae). syst bot. 26:205–215. aedo c. 2017. taxonomic revision of geranium sect. ruberta and unguiculata (geraniaceae). ann missouri bot gard. 102:409–465. baser b, firat m, aziret a. 2016. the pollen morphology of pelargonium endlicherianum and pelargonium quercetorum (geraniaceae) in turkey. phytokeys. 75:153-162. bortenschlager s. 1967. vorlaufige mitteilungen zur pollen morphologie in der geraniaceen und ihre systematische bedeutung. grana palynol. 7:400–468. figure 7. pollen micrographs of geranium species:x1-x3: g. ibericum, u1-u3: g. tuberosum, u4-u6: g. biuncinatum. x1,u1, u4 – equatorial view; 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(asteraceae) species that around the van lake in turkey pelin yilmaz sancar1,*, semsettin civelek1, murat kursat2 molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant jinxin cheng1,*, dingyu hu1, yaran liu2, zetian zhang1, majid khayatnezhad3 genetic variations and interspesific relationships in salvia (lamiaceae) using scot molecular markers songpo liu1, yuxuan wang1, yuwei song1,*, majid khayatnezhad2, amir abbas minaeifar3 development of female gametophyte in gladiolus italicus miller (iridaceae) ciler kartal1, nuran ekici2,*, almina kargacıoğlu1, hazal nurcan ağırman1 colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae) harshita dwivedi*, girjesh kumar statistical evaluation of chromosomes of some lathyrus l. taxa from turkey hasan genç1, bekir yildirim2,*, mikail açar3, tolga çetin4 use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater treatment and water of the kamniška bistrica river (slovenia) peter firbas1, tomaž amon2 the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bo shi1,*, majid khayatnezhad2, abdul shakoor3,4 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 115-123, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-136 citation: h. ahmad ganaie, md. n. ali, b.a. ganai (2020) melissa officinalis: a potent herb against ems induced mutagenicity in mice. caryologia 73(1): 115-123. doi: 10.13128/caryologia-136 received: january 9, 2019 accepted: february 23, 2020 published: may 8, 2020 copyright: © 2020 h. ahmad ganaie, md. n. ali, b.a. ganai. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 1 cytogenetics and molecular biology research laboratory, centre of research for development (cord), university of kashmir, srinagar-190 006, j & k, india 2 phytochemistry research laboratory, centre of research for development (cord), university of kashmir, srinagar-190 006, j & k, india *corresponding author. e-mail: hilalganie@hotmail.com abstract. melissa officinalis (l) is used traditionally for different medical purposes such as tonic, antispasmodic, carminative, diaphoretic, surgical dressing for wounds, sedative hypnotic, strengthening the memory and relief of stress induced headache. the methanolic extractof melissa officinalis (mo-me) was investigated for antimutagenic activity. the extraction was done by soxhlet extraction method and the extract was evaluated for antimutagenic assay against ems induced mice by micronucleus and chromosomal aberration assay. briefly, mice were treated with methanolic extract of melissa officinalis (mo-me) (100, 200 300 & 400 mg/kgbw) for 15 days. without the doses of ems, no and mutagenic effects were observed in blood and bone marrow samples of the mice. micronucleus and chromosomal aberration test revealed the protective effects of mo-me when administered at high doses.the reduction profiles in the mn induction of methanolic extract of melissa officinalis at the concentration (100, 200, 300 and 400 mg/kgbw) with ems were estimated as 14.5%, 28.0%, 47.7% and 81.5% respectively. the methanolic extract of melissa officinalis exhibited no cytotoxic and mutagenic effects but only have antimutagenic effects, an effect that can be attributed the presence of majorital compounds, and the antimutagenic property of mo-me is an indication of its medicinal relevance. keywords. melissa officinalis, gc-ms, ems, mice, micronucleus test, chromosomal aberration, antimutagenicity. introduction medicinal plants with antioxidant and antimicrobial properties are gaining a lot of attention as these properties are commonly assumed to play an important role in preventing diseases caused by oxidative stress, such as cancer, coronary arteriosclerosis and the ageing processes (haraguchi et al. 2009). derived forms of medicinal plants (extracts, syrups, etc.) have been the basis of medical therapy for centuries. traditionally used in the treatment of several human disorders, their pharmacological and therapeutic properties are attributed to various chemical constituents isolated from their crude extracts (pereira et al. 2009, kwak and ju, 2015; liu et al. 2015). notwith116 hilal ahmad ganaie, md. niamat ali, bashir a ganai standing, their correct use requires the manipulation of plants selected for their efficacy and safety, based either on folk tradition or scientific validation (tovart, 2009). the use of herbal infusions to cure various disorders is very common in folk medicine especially to those who live in upper reaches of kashmir himalayas (dutt et al. 2015). although the diversity of plant species in kashmir himalayas is a potential source of biologically active compounds, the effects on human health and genetic material are often unknown. there are indications that the protective action on genetic material can lead, not only to its repair, but also the preservation of its integrity (berhow et al. 2000; fernandes and vargas, 2003; souza et al. 2004). not all are harmless, some even presenting toxic and mutagenic substances in their phytochemical composition (bresolin and vargas, 1993; sa-ferreira and vargas, 1999). interest in such popular usage has recently gained strength, through recent knowledge that chemicals, such as proteases and antioxidants may prevent or reduce the development of cancer by blocking genetic damage (hernandez-ceruelos et al. 2002). melissa officinalis belongs to lamiaceae family, a large group of medicinal plants. m. officinalis is native to southern europe and northern africa; although, over the last several centuries it has been successfully cultivated all over the world. today it can be found growing wildly throughout north america, europe, asia, and in the mediterranean. the leaves of m. officinalis have been used in folk medicine especially in turkey and iran, for the treatment of some disease (sadraei et al., 2003). also, the leaves of m. officinalis are often used as herbal teas. m. officinalis contains some phenolic and flavonoid compounds such as rosmarinic acid (herodez et al., 2003). the phenolic contents in plants have some antioxidant properties (chen et al., 2001). essential oils and extracts of this plant have been reported to have antiviral (schnitzler et al., 2008), antimicrobial and antioxidant properties (dastmalchi et al., 2008). as little has been done on the antimutagenicity of melissa officinalis, therefore, the purpose of this study was to determine the antimutagenic activities of methanolic extract of melissa officinalis. material and methods collection and air drying of plant material aerial parts of m. officinalis were collected from bandzoo area of pulwama from the garden of iiim, srinagar and from skuast-k in the month july, 2013. the plant was identified at the centre of biodiversity and plant taxonomy, department of botany, university of kashmir, srinagar, j&k and a voucher specimen (jkash/cbt/227 dated 08. 08. 2014) was deposited there. the parts were allowed to dry under shade (30 °c) for 8-10 days. preparation of extracts after shade drying, the aerial parts were macerated to fine powder, 1 kg of leaves were extracted successively with hexane for defatenning and methanol for 16 h using soxhlet apparatus. the extracts were filtered through a buchner funnel using whatman no. 1 filter paper, and all the extracts were concentrated to dryness under vacuum using a heidolph rotary evaporator, yielding hexane, and methanol crude extracts of 65 and 48g respectively. all the extracts were stored at 4°c in air tight glass bottles before use. gc-ms analysis gc-ms analysis was carried out with gcms-qp2010 plus, shimadzu, japan fitted with programmable head space auto sampler and auto injector. the capillary column used was db-1/rtx-ms (30 metre) with helium as a carrier gas, at a flow rate of 3 ml/min with 1 µl injection volume. samples were analysed with the column held initially at 100°c for 2 min after injection, then increased to 170°c with 10°c/min heating ramp without hold and increased to 215°c with 5°c/min heating ramp for 8 min. then the final temperature was increased to 240°c with 10°c/min heating ramp for 15 min. the injections were performed in split mode (30: 1) at 250°c. detector and injector temperatures were 260°c and 250°c, respectively. pressure was established as 76.2 kpa and the sample was run for 70 min. temperature and nominal initial flow for flame ionization detector (fid) were set as 230 °c and 3.1 ml/min, correspondingly. ms parameters were as follows: scan range (m/z): 40-650 atomic mass units (amu) under the electron impact (ei) ionization (70 ev). the constituent compounds were determined by comparing their retention times and mass weights with those of authentic samples obtained by gc and as well as the mass spectra from the wiley libraries and national institute of standards and technology (nist) database. experimental animals both sex of albino mice, balb/c strain useful for research in cancer and immunology, age of 6 weeks, weighing 25-35 g were obtained from the indian institute of inte117melissa officinalis: a potent herb against ems induced mutagenicity in mice grative medicine (iim), canal road jammu-india, kept in plastic cages in an animal room under controlled conditions of temperature (22 ± 2°c), humidity (55 ± 10%), 12 h light/dark cycles and access to food and water. they were randomized at the beginning of the experiment.the study design was approved by the institutional animal ethical committee, and the experiments undertaken in accordance with the ethical principles of the cpcsea norms. treatment protocol the mice were divided into 8 groups, with 5 animals per group. ethyl methane sulfonate (ems, sigmaaldrich) was used to induce mutations. just before use, the ems was diluted in 0.9% nacl. the exposure route was by gavage(1/4thof ld50 of ems; 117.5 mg/kgbw). evaluation of either dna damage or protection by the methanolic extracts of melissa officinalis was according to protocol developed by azevedo et al. (2003), with the some adaptations. the mice in group 1 received only distilled water (10 ml/kg bw. per day by gavage) for 2 weeks and acted as negative control (table 1). mice in group 2 were exposed to ems (1/4thof ld 50) for 24 h and this group acted as positive control. group 3 and 4 were given different doses (100 & 400 mg/kgbw) of the extract to see the cytotoxic and mutagenic potential of m. officinalis and served as positive control of plant extracts. group 5, 6, 7 and 8 were treated with dose of 100, 200, 300 and 400 mg/kgbw respectively for 15 days after treatment with ems. the mice were killed by cervical dislocation on 16thday for evaluation of micronucleus and chromosomal aberrations. the micronucleus test the method of macgregor et al. (1987) was used for micronucleus test. mice were sacrificed by cervical dislocation. slides were prepared with blood collected from the jugular vein. the slides were air-dried, fixed in absolute methanol, stained in 10% giemsa and then coded for blind analysis. one thousand polychromatic erythrocytes (pce) were analysed per mouse. the proportion of pce and normochromatic erythrocytes (nce) in 1000 erythrocytes/group was calculated, to detect possible cytotoxic effects. the slides were scored blindly, using a light microscope with a 45x and 65x objectives. photography was done using 100x immersion objective. chromosomal aberration mice were injected intraperitoneal with 0.5 ml of 0.06% colchicine and two hours later, were sacrificed by cervical dislocation. both the femurs were fleshed out from the muscles and kept in hbss (hank ’s balanced salt solution). the femurs were then rinsed with 3 ml 0.056% kcl solution in a centrifuge tube. the tube was then incubated at 37oc for 20 minutes. after incubation, centrifugation at 800 rpm for 4 minutes was carried out. supernatant was discarded and fresh carnoy’s fixative was added (3:1 methanol: acetic acid). the process of centrifugation was repeated three times. then slides were prepared, stained with 4% giemsa, air dried and studied under compound microscope. statistical analysis variable normality was assessed using the kolmogorov-smirnov test. micronucleus testing and chromosomal aberration involved multiple pair-wise comparison between experimental groups and positive and negative controls, with the mann whitney u test at a significance level of <0.05. lower the mann whitney statistic value and z score value, higher the difference. table 1. grouping, dose (distilled water, ems and ab-me in concentrations of 100, 200, 300 and 400 mg/kg bw) and duration of experiment. group dose purpose of group duration group 1 distilled water negative control 15 days group 2 1/4th ld50 ems positive control ems 24 h group 3 mo-me 100 mg/kg bw positive control melissa officinalis 24 h group 4 mo-me 400 mg/kg bw positive control melissa officinalis 24 h group 5 mo-me 100 mg/kg bw + ems treated group 15 days group 6 mo-me 200 mg/kg bw + ems treated group 15 days group 7 mo-me 300 mg/kg bw + ems treated group 15 days group 8 mo-me 400 mg/kg bw + ems treated group 15 days mo-me = methanolic extract of melissa officinalis. 118 hilal ahmad ganaie, md. niamat ali, bashir a ganai results gc-ms analysis in order to find out the phytocomponents of melissa officinalis, the methanolic extract was subjected to gc-ms analysis. the active principals present in the methanolic fraction of melissa officinalis along with their retention time (rt), molecular formula, molecular weight (mw) and peak area (%) are presented in table 2. the chromatograms of methanolic extract of melissa officinalis (mo-me) showed three major peaks (fig. 1): 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4hpyran-4-one (51.62%), 5-(hydroxymethyl)-2-furan carboxaldehyde (29.46%), hexadecanoic acid, methyl ester (8.24%), constituting 89.32% of the total peak area. the minor fractions of mo-me include octadecanoic acid (3.26%), stigmast-5-en-3-ol (1.44%), tetradecanoic acid (1.43%), 2, 4cresotaldehyde (1.17), comprising 7.30% of the total peak area. the phytoconstituents identified in the methanolic fraction of melissa officinalis along with their retention time (rt), molecular formula, molecular weight (mw) and peak area (%) are presented in table 3.6. micronucleus test according to mn testing of mouse blood cells the low frequencies of micronucleated cells presumes the too little effects of methanolic extract of melissa officinalis (mo-me) 100 and 400mg/kg (table 3, fig. 2), thereby indicating the virtual absence of mutagenic or cytotoxic effects. in other words, no statistically significant difference in the frequency of mn polychromatic erythrocytes (pce) or the ratio of pce to normochromatic erythrocytes (nce), between the negative control and the groups that ingested extracts could be detected. when evaluating antimutagenicity inmo-me, a significant decrease in the frequency of ems-induced mnpce was observed only in mice that had received 100, 200, 300 and 400 mg/kg ofmo-me (p = 0.05 –mann whitney u test). in the present study, the methanolic extract of m. officinalis showed antimutagenic activities by reducing the % age of micronuclei with increase in the dose of the extract (fig. 3). table 2. phytocomponents identified in the methanolic extract of melissa officinalis (mo-me) by gc-ms. s. no. compound rt % area mf mw 1 2,4-cresotaldehyde 18.52 1.17 c8h8o2 136 2 d-allose 19.41 0.56 c6h12o6 180 3 dodecanoic acid 21.32 0.58 c12h24o2 200 4 tetradecanoic acid 25.79 1.43 c14h28o2 228 5 2,6,10-trimethyl,14-ethylene-14-pentadecne 27.37 0.49 c20h38 278 6 hexadecanoic acid, methyl ester 29.13 8.24 c38h68o8 652 7 5(hydroxymethyl)-2-furan carboxaldehyde 30.07 29.46 c6h6o3 126 8 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4h-pyran-4-one 33.47 51.62 c6h8o4 144 9 octadecanoic acid 33.76 3.26 c18h36o2 284 10 malonic acid, 3-hexyl tridecyl ester 34.05 0.38 c22h42o4 370 11 9-octadecenoic acid 34.22 0.12 c18h34o2 282 12 9,12-octadecadienoic acid 34.77 0.50 c18h32o2 280 13 1,2-benzenedicarboxylic acid 40.36 0.34 c24h38o4 390 14 tochopherol 51.97 0.41 c29h50o2 430 15 stigmast-5-en-3-ol 57.19 1.44 c29h50o 414 figure 1. gc-ms chromatogram of methanolic extract of melissa officinalis. 119melissa officinalis: a potent herb against ems induced mutagenicity in mice chromosomal aberrations the chromosomal aberrations induced by ethyl methanesulphonate (ems 117.5 mg / kg body weight; positive control) were significant (p<0.05) to that of the control group. the frequency of breaks per cell in the ems treated group at 24 h was 0.12 ± 0.010, which was significantly higher when compared to that of total number of breaks per cell in the control group (0.014 ± 0.002) (p<0.05). our results also showed that in the mome and ems combined treatment group, the frequency of chromosomal aberrations was significantly lower in comparison to those observed for the ems only treated group at 24 h. the methanolic extract of melissa officinalis reduced the chromosomal aberrations by 38.1%, 60.1%, 74.5% and 91.5% at 100, 200, 300 and 400 mg/ kgbw (table 4, fig. 4). the chromosomal aberrations induced by ems are mainly chromatid breaks, exchanges, gaps, fragments and rings (fig. 5). discussion from ancient times, medicinal plants are being used as remedies for various diseases in human. in today’s industrialized society, the use of medicinal plants has been traced to the extraction and development of several drugs as they were used traditionally in folk medicine (shrikumar and ravi, 2007). medicinal plants have potent phytoconstituents which are important source of compounds and are responsible for the therapeutic properties (jeeva et al., 2011; florence et al., 2014; sumathiet al., 2014, ganaie et al, 2016; 2017). these phytoconstituents endow them with medicinal properties. many plants possess antioxidant properties because of the presence of phenolic compounds (brown and ricetable 3. frequency profile of micronuclei induced alone by ethyl methanesulphonate and melissa officinalis methanolic extract and their simultaneous exposure for different doses to evaluate antimutagenicity in mus musculus. treatment total no. of cells analysed per mice no. of cells with micronuclei % age of mn % reduction group 1 negative control (distilled water) 1000 2.35 ± 0.12 group 2 positive control (ems) 1000 7.23 ± 0.89 group 3 mome 100 mg/kgbw 1000 2.28 ± 0.10 group 4 mome 400 mg/kgbw 1000 2.27 ± 0.09 group 5 mome 100 mg/kg + ems 1000 6.52 ± 0.70 90.1 14.5 group 6 mo-me 200 mg/kg + ems 1000 5.86 ± 0.58 81.0 28.0* group 7 mo-me 300 mg/kg + ems 1000 4.90 ± 0.50 67.7 47.7** group 8 mo-me 400 mg/kg + ems 1000 3.25 ± 0.43 44.9 81.5*** nc: negative control (distilled water), pc: positive control [ethyl methane sulfonate (ems) 117.5 mg/kgbw; dose is 1/4th ld50], mome: melissa officinalis methanolic extract. values with different asterisks (*p < 0.05: significant, **p < 0.01: highly significant, ***p < 0.001: extremely significant) differ significantly from the positive control (mann-whitney u test). figure 2. graph showing number of micronucleated cells in different groups of mice treated with ems alone, ems + mo-me and mo-me alone in different concentrations. figure 3. bar diagram showing percentage reduction in ems treated micronuclei with increase in concentration of melissa officinalis methanolic extract (mo-me). 120 hilal ahmad ganaie, md. niamat ali, bashir a ganai evans, 1998; krings and berger, 2001). these phenolic compounds possess biological properties such as antiapoptosis, anti-aging, anti-carcinogen, anti-inflammation, anti-atherosclerosis, cardiovascular protection and improvement of endothelial function, as well as inhibition of angiogenesis and cell proliferation activities (han et al., 2007). tannins bind to proline rich protein and interfere with protein synthesis. flavonoids are hydroxylated phenolic substances known to be synthesized by plants in response to microbial infection and they have been found to be antimicrobial substances against wide array of microorganisms in vitro. the activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell wall (marjorie, 1999). they are also effective antioxidant and show strong anticancer activities (salah et al., 1995; delrio et al., 1997). previous reports show that the essential oil of m. officinalis is composed of some important compounds like (e)-caryophyllene and caryophyllene oxide in addition to major constituents such as citronellal, neral and geranial (sorensen, 2000; van de berg et al., 1997). literature reveals that the essential oil of m. officinalis subsp. officinalis contains significant amounts of citral and/or citronellal, whereas m. officinalis subsp. altissima contains only traces (van de berg et al., 1997). van de berg et al. (1997) identified b-caryophyllene, germacrene-d, sabinene, and b-pinene as the main components in leaf oils of m. officinalis subsp. altissima. marnewick et al. (2000) found that the aqueous extracts of fermented and unfermented rooibos tea table 4. frequency profile of chromosomal aberrations induced by ethyl methanesulphonate and melissa officinalis methanolic extract separately and by their combination for different doses to evaluate the antimutagenicity in mus musculus. treatments chromosomal aberrations concentration (mg/kgbw) no. of cells rings fragments exchange breaks gaps total aberrations %age of aberrations % reduction distilled water 500 1 3 7 11 2.2 ems 117.5 mg/kgbw 500 4 18 14 62 31 129 25.8 mo-me alone100 mg/kgbw 500 1 3 7 11 2.2 mo-me alone400 mg/kgbw 500 1 3 6 10 2.0 mo-me 100 mg/kgbw + ems 500 3 13 10 44 14 84 16.8 38.1* a-me 200 mg/kgbw + ems 500 2 9 7 32 8 58 11.6 60.1* mo-me 300 mg/kgbw + ems 500 1 7 5 22 6 41 8.2 74.5** mo-me 400 mg/kgbw + ems 500 4 3 10 4 21 4.2 91.5*** nc: negative control (distilled water), pc: positive control [ethyl methane sulfonate (ems) 117.5 mg/kgbw; dose is 1/4th ld50], mome: melissa officinalis methanolic extract. values with different asterisks (*p < 0.05: significant, **p < 0.01: highly significant, ***p < 0.001: extremely significant) differ significantly from the positive control (mann-whitney u test). figure 5. photomicrograph showing metaphase chromosome preparation from bone marrow. a) normal set of chromosomes, b) exchanges (arrow), c) ring (arrow), d) fragment (arrow), e) micronuclei (arrow). figure 4. bar diagram showing percentage reduction in chromosomal aberrations (ca) induced by ems following post-treatment with methanolic extract of melissa officinalis (mo-me). 121melissa officinalis: a potent herb against ems induced mutagenicity in mice (aspalathus linearis) and honey-bush tea (cyclopia intermedia) possess antimutagenic activity against 2-acetylaminofluorine and aflatoxin b1. vitamin c and e also significantly reduced the ca frequency in mouse bone marrow cells against rifampicin, an anti-tuberculosis drug, (aly and donia, 2002). according to kaur et al. (2010), the phytoconstituents from terminalia arjuna suppressed the mutagenic effect of the aromatic amine, i.e., 2-aminof luorene (2-af). the observed activity caused the inhibition of the metabolic activation of pro-mutagens. hong et al. (2011) found that the extracts of acanthopanax divaricatus were able to rapidly eliminate the mutagenic compounds from the cells before they induce the dna damage. in a similar study, nardemir et al. (2015) observed that the methanol extracts of the lichens have antimutagenic effects against sodium azide.in another study, prakash et al. (2014) found that the different extracts of dioscorea pentaphylla significantly inhibited the effects of methyl methanesulphonate (mms) induced mutagenicity. they also found that the methanolic extract was highly mutagenic in comparison to petroleum ether and chloroform. entezari et al. (2014) compared the antimutagenic and anticancer activities of echinophora platyloba dc on acute promyelocytic leukemia cancer cells and found that the methanolic extract of this plant prevented the reverted mutations and the hindrance was 93.4% in antimutagenic test. akinboro et al. (2014) utilised the leaves of myristica fragrans (houtt.) for antimutagenic activity against benzo[a]pyrene and cyclophosphamide induced mutagenicity in salmonella typhimurium and mus musculus and found that the aqueous extract significantly suppressed more than 50 % of the mutations in all the tested concentrations. sarac, (2015) utilised an edible wild plant, tragopogon longirostis for the evaluation of antioxidant, mutagenic and antimutagenic properties and found that the ethanolic extract of its leaves exhibited antimutagenic properties at 2.5, 0.25, and 0.025 mg/plate concentrations. habibi et al. (2014) found that the ethanolic extract of origanum vulgare reduced the frequency of mn pcr from 10.52 ± 1.07 for cp to 2.17 ± 0.6 for the synergic test of cp and the ethanolic extract. conclusion based on the above results it can be concluded that the methanolic extractof melissa officinalis possess some important phytoconstituents which possess antimutagenic activity. the results of the present study clearly showed that the methanolic extract of melissa officinalis had an antimutagenic and anticlastogenic potential against the mutagenic activity of ethyl methane sulphonate in mice. our results suggest that there may be several ways through which m. of ficinalis extract can work against ems. selection of m. officinalis for the present study on the basis of its folklore usage seems to be justified as it is scientifically proved to have much potentiality. the extracts from such plants could be seen as a good source of useful drugs.however, further studies are needed in other test 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abnormalities in vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 74(1): 63-73, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1014 caryologia international journal of cytology, cytosystematics and cytogenetics citation: i. santra, t. halder, b. ghosh (2021) somatic and gametic chromosomal characterization with fluorescence banding of giloy (tinospora cordifolia): a berberine synthesizing important medicinal plant of india. caryologia 74(1): 63-73. doi: 10.36253/caryologia-1014 received: july 06, 2020 accepted: april 26, 2021 published: july 20, 2021 copyright: © 2021 i. santra, t. halder, b. ghosh. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid bg: 0000-0002-4396-2088 somatic and gametic chromosomal characterization with fluorescence banding of giloy (tinospora cordifolia): a berberine synthesizing important medicinal plant of india indranil santra, tarun halder, biswajit ghosh* plant biotechnology laboratory, post graduate department of botany, ramakrishna mission vivekananda centenary college, rahara, kolkata 700118, india *corresponding author. e-mail: ghosh_b2000@yahoo.co.in abstract. giloy [tinospora cordifolia (willd.) miers.] has been a potential medicinal plant since ancient times; even today, it has great economical values; however, it still receives less attention in cytogenetic study. detailed baseline data of chromosomes in mitotic and meiotic cell division remain very important for genetical characterization and evaluation of the reproductive potentiality of a species. cytogenetic characterization with the aid of a chromosome banding technique is beneficial in searching chromosomal landmarks and constructing an accurate karyotype, which is completely lacking in the genus tinospora. hence, this is the first attempt of the detailed karyological study with fluorescence banding after enzymatic degradation of the cell wall. chromosomes of t. cordifolia are small (1.8–2.4 µm) and 2n=26 having a symmetric karyotype. the secondary constriction of two submetacentric pairs has dapi negative /cma positive bands. the meiosis studies of male flowers show the presence of 13 bivalents having secondary associations among themselves. meiotic abnormalities such as precocious movement (7.56%) and laggard (2.83%) were recorded, and the pollen viability was estimated to be 47.17%. the berberine content produced in the stem of t. cordifolia has been quantitatively measured by high-performance liquid chromatography and found to be 0.424±0.02% on dry weight (dw) basis. in this study, precise karyological profiling by differential banding has been constructed and linked with the medicinal quality of the genotype, thus considered to be beneficial in the selection, cultivation, management, and improvement program of this species. keywords: tinospora cordifolia, fluorescence banding, dapi, cma, karyotype, meiosis, berberine. introduction an important medicinal plant tinospora cordifolia (willd.) miers. commonly known as “giloy,” “guduchi,” or “amrita,” belonging to the menispermaceae family. it is widely distributed ranging from the himalayas to the southern part of india and also found in southeast asian as well as african countries (lade et al. 2018). it is a large deciduous climber with aerial root, 64 indranil santra, tarun halder, biswajit ghosh fleshy stem, heart-shaped leaves, and unisexual flowers. although different parts of the plants such as shoot, leaf, root, fruit, and seed have utility in herbal medicine, but maximum activities are found in stems (bala et al. 2015). this plant has extensive use in the oldest ayurveda system as anticancer, antiulcer, anti-inflammatory, hypoglycemia, antiarthritic, and hepatoprotective potential (srinivasan et al. 2008). this ayurvedic plant is highly recommended in the covid-19 outbreak, as it strengthens and rejuvenates the immune system to fight the global pandemic (prasad et al. 2020). the herbal drugs of this plant exhibit potential immunomodulatory effects to overcome immunosuppression as well as anticancerous activities on human breast cancer and prostate cancer (sachan et al. 2019; deepa et al. 2019). in recent studies, the molecular basis of the anti-inflammatory and antioxidant properties of t. cordifolia has been validated (reddi and tetali 2019). besides, this species is also used in many ayurvedic pharmaceutical industries to produce a cure against chikungunya and dengue (mittal and sharma 2017). tinospora cordifolia contains several alkaloids, including berberine, palmatine, tembetarine, magnof lorine, choline, tinosporin, isocolumbin, tetrahydropalmatine, etc. (singh et al. 2003). berberine is an important isoquinoline alkaloid found in a surplus amount in the stem of t. cordifolia and reported to have a wide range of pharmaceutical activities, including antimalarial, antipyretic, anti-inflammatory, antimicrobial, antidiabetic, antitumor, etc. (srinivasan et al. 2008). among different berberine-containing plants, t. cordifolia covers a wide region in india spreading from kumaon mountains to kanyakumari and mostly found in the wild habitats. because of the distribution and availability, this plant has become the major source of berberine in india (panchabhai et al. 2008). the significant market growth of medicinal plants and herbal drugs in the global context over the last few decades has conveyed the message of consumer’s faith in natural drugs over synthetic ones (ravi and bharadvaja 2019). as stated by the national medicinal plant board (nmpb), india, among 960 species of traded medicinal plants, the consumption of 178 species is estimated to be more than 100 metric tons annually (ved and goraya 2007). the enormous medicinal values of t. cordifolia in traditional medicine elicit the estimated annual demand from 2000 to 5000 metric tons with annual growth registered at 9.1% according to the nmpb, india, in 2012 (abhijeet and mokat 2018). owing to its increased demand, t. cordifolia is placed on the priority list of the nmpb to cultivate in the agro-climatic zones of rajasthan, uttar pradesh, and madhya pradesh in india (mridula et al. 2017). to enhance the production growth, mass multiplication and commercial-level cultivation of this plant have been prioritized. before practicing any commercial-level cultivation for mass production, identification, selection, and characterization of medicinal plants are indispensable (nyarumbu et al. 2019). in this context, basic genetic information by chromosome analysis is essential for the elementary genetical characterization of species to introduce them into plant breeding and crop improvement programs (arroyo martinez et al. 2017). other than conventional breeding, whole-genome duplication or artificial polyploidy induction of medicinal plants is now a flourishing approach to increase the secondary metabolite production of therapeutic value. to step forward in this modern biotechnological research and crop improvement program, accurate knowledge of the chromosomal profile is unconditional. despite the enormous economic importance of medicinal plants, genotype information explored by karyotype analysis is still not sufficient, which may help breeders in identification, selection, and efficient crop management (peruzzi and eroğlu 2013). karyotype analysis and meiotic behavior provide a cytogenetic framework of a plant species, which is subsequently used in the study of genomics, taxonomy, evolution, and reproductive biology (she 2016; kaur and singhal 2019). however, classical karyotype analysis with only chromosomal measurement by orcein-based staining lacks significant markers for individual chromosome identification (she 2016). therefore, to tackle this challenge, karyotype analysis through differential chromosome banding with giemsa and fluorochrome dyes is beneficial (levin 2002). chromomycin a3 (cma) and 4’,6-diamidino-2-phenylindole (dapi) show preferential binding to the gcand at-rich dna sequences, respectively, and give us the access to identify different types of heterochromatin and also serve as a chromosomal marker for karyotype analysis in plants (barros e silva and guerra 2010). in contrast, analysis of the meiotic chromosomes enlightens the genomic behavior during gamete formation and generates the idea about reproductive performance, which helps the breeders in productive manipulation of plants of economic interest (souza et al. 2015, guidini et al. 2017). very restricted information is available considering the genetic identity through the analysis of chromosomal characteristics and karyotype of t. cordifolia. almost the entire study was limited to the numerical value of the chromosomes, while details of structural features of the chromosomes and their measurement remain unresolved (table 1). therefore, karyomorphological analysis with accurate chromosomal landmarks is becoming essential for the prospect of this species. meanwhile, with the identification of a particular genotype of medicinal plants by analyzing chromosomal 65somatic and gametic chromosomal characterization with fluorescence banding of giloy metrics corroborated with the differential banding characters, it is also necessary to assess their medicinal efficacy prior to any crop improvement and conservation program. quantity and quality of the active principle in a medicinal plant species are a dynamic array, which can differ with the natural genetic variation of the species (kroymann 2011). therefore, assessment of the natural products present in the medicinal plants must link with the genetic characters. hence, quantification of the berberine content in t. cordifolia is an essential part to evaluate their medicinal quality. high-performance liquid chromatography (hplc) is considered a powerful analytical technique for the separation of natural products from a complex matrix to analyze and quantify them in a reliable and reproducible way. although the berberine content has been quantified with hplc in this species earlier, quality assessment linked with the particular genetic characteristic received less attention. therefore, in this study, for the first time, attempt has been made to analyze the precise chromosome characteristic to construct the karyotype by differential fluorochrome banding. the meiotic behavior of the reproductive cells of t. cordifolia has also been analyzed. moreover, the berberine content of the genotype has been quantified to evaluate its medicinal efficiency. the outcome of this study may help in the selection and domestication of the species for future biotechnological and agricultural programs to improve from an economic perspective. material and methods somatic chromosome preparation the growing roots of t. cordifolia were collected from the medicinal plant garden. chromosomes were prepared following santra et al. (2020) with minor modifications. roots were pretreated with 4 mm 8-hydroxyquinoline solution at 16 °c for 5 h and then fixed in acetic acid and methanol solution (1:3) overnight. digestion of the cell wall was performed with an enzyme mixture containing 1% cellulase (onozuka-rs, sigma), 0.5% pectolyase (sigma), and 0.75% macerozyme (himedia) in a sodium citrate buffer (ph 4.6) at 37 °c for 60 min. after washing with the same buffer twice, the root tip was broken down into small pieces on a clean slide with the addition of freshly prepared fixative. the slide was air-dried for at least 24 h before staining. chromosome staining chromosomes were stained with 2% giemsa solution in phosphate buffer solution with a ratio of 1:15 (ph 6.8) followed by rinsing with distilled water and analysis under microscope. prior to fluorescent staining, slides were destained with 70% methanol for 15 min and airdried. slides were preincubated in mcilvaine buffer (ph 7.0) supplemented with 5 mm mgcl2, followed by staining with 0.25 mg ml-1 cma for 20 min in dark. after a short rinse in the same buffer, slides were mounted with 50% glycerol containing 5 mm mgcl2 and kept in 4 °c for 48 h before further analysis. after preincubation in mcilvaine buffer (ph 7.0), chromosomes were stained with 0.5 µg ml-1 dapi solution for 20 min in the dark followed by a rinse with the buffer and mounted with 50% glycerol. chromosomes were analyzed under the fluorescent microscope zeiss axio scope a1 equipped with cma and dapi specific filter cassette. photomicrographs were taken with an axiocam icc 5 and zen application suite. individual chromosomes were measured with axiovision 4.9.1 and categorized based on the arm ratio following levan et al. (1964). table 1. previous chromosome counts in tinospora cordifolia. chromosomes in sporophytic (2n) or gametophytic (n) chromosome number karyotype* reference n 12 joshi (1934), joshi and rao (1935) 2n 24 nanda (1962) n 12 sanjappa (1978) n 13 abraham (1942) 2n 26 sharma and bhattacharyya (1955) 2n 26 sharma and sharma (1957) n 13 sarkar et al. 1980 2n 22 asymmetric jain and prasad (2014) 2n 26 mathew (1958) *reports of the karyotype are based on the online available resources. 66 indranil santra, tarun halder, biswajit ghosh study of meiotic behavior and pollen viability tinospora cordifolia is a dioecious  creeper with unisexual flowers. male buds of the appropriate size were taken and fixed in the carnoy’s fixative (acetic acid: ethanol: 1:3 v/v) at 4 °c until use. anthers were then removed and squashed with 2% acetocarmine stain. pollen viability assessment through acetocarmine staining was performed according to haque and ghosh (2017). the pollen viability was estimated after an analysis of more than 1000 pollens. to stain with dapi, anther was squashed with 45% acetic acid, and then, cover glass was removed by freezing the slides at –80 °c temperature for 10 min. slides were then stained with 0.5 µg ml–1 dapi solution and mounted with 50% glycerol in mcilvaine’s buffer (ph 7.0). photomicrographs were taken with an axiocam icc 5 and zen application suite equipped with bright field and dapi-specific filter cassette. quantification of berberine content preparation of standard a stock solution (1.0 mg ml–1) of berberine (sigma-aldrich) was prepared with hplc-grade methanol freshly. preparation of sample the stem was dried at room temperature, and fine powder was prepared in a mechanical grinder. hplcgrade methanol was dissolved with 20 mg of powder and was sonicated for 45 min at 40 °c. after centrifugation at 5000 rpm for 5 min, samples were filtered through 0.22 mm teflon-coated membrane and aliquoted. the analysis was performed with three replicas. hplc conditions the analytical hplc experiments were performed with the waters 1525 binary hplc pump and the waters 2489 uv–vis. detector. the separation was carried out with reverse-phase c-18 column (5 µm particle size, 4.6×250 mm) using potassium dihydrogen phosphate buffer (solvent a) with ph 3.2 adjusted by orthophosphoric acid and acetonitrile (solvent b) with different solvent scales (0 min 90:10 v/v, 18 min 5:95 v/v, and 20 min 90:10 v/v); the flow rate was 1.0 ml min–1 under gradient condition. injected sample volume was 20 µl with 20 min run times. berberine was detected in a uv detector at 266 nm. results karyomorphological studies in this analysis with t. cordifolia, it has been found that they possess 2n=26 chromosomes in somatic cells (figure 1a-c). chromosomes are small in length and range between 1.8 and 2.4 µm. individual chromosome size has been mentioned in table 2. detailed karyotype analysis revealed that 11 pairs of chromosomes have median to nearly median primary constriction, whereas two pairs have submedian primary constriction. hence, the karyotype formula is 22m+4sm (figure 1i). two pairs of submetacentric chromosomes are also associated with secondary constrictions in the long arm (figure 1a, i). the position of all secondary constrictions is intercalary. the karyotype is symmetric and falls into 1a category of stebbins’s (1971) classification. staining with cma reveals that two pairs of chromosomes show bright cma positive banding in their secondary constrictions (figure 1b). whereas dapi stained the metaphase chromosomes uniformly, no dapi positive band has been found (figure 1e). instead, dapi negative bands have been detected in the secondary constrictions colocalized with the cma positive bands (figure 1f–h). however, when stained with dapi at the prometaphase stage, some of the less condensed chromosomes reveal dapi positive signals in their centromeric and pericentromeric regions (figure 1d). meiosis studies meiosis in the male flowers of t. cordifolia revealed 13 bivalents in metaphase-i confirming n=13 chromosomes in the studied material. chiasma formation in diplotene and diakinesis stages has been found to be normal, and the frequency is 10.83±1.16 per pollen mother cell (figure 2a). metaphase-i in some pollen mother cell, when stained with acetocarmine and dapi, revealed 13 perfect bivalents (figure 2b, c). in addition to the usual bivalents, secondary association between the chromosomes causes the formation of trivalent and tetravalent and multivalent configurations frequently (figure 2d, e). in addition to the secondary associations, several other meiotic abnormalities such as chromosome stickiness, laggard chromosome and precocious movement have been recorded. chromosome stickiness has been observed in 21.66% and 17.86 % of total metaphasei and metaphase ii respectively (figure 2f, g, l). in anaphase-i, separation of the chromosomes in most of the plates was regular; however, chromosomes showed stickiness between themselves in each pole (figure 2h-j). in addition, 2.83% of total anaphase-i stage having laggard 67somatic and gametic chromosomal characterization with fl uorescence banding of giloy figure 1. somatic metaphase chromosomes of tinospora cordifolia showing 2n=26 chromosomes; (a) enzymatic maceration of the cell wall and giemsa staining of chromosomes (arrows indicate secondary constrictions); (b) metaphase plate stained with cma, showing 4 positive bands at the secondary constriction region (marked with arrows); (c) karyogram representation of giemsa stained chromosomes; (d) prometaphase stained with dapi; (e) metaphase plate stained with dapi; (f-h) same metaphase plate stained with giemsa, cma and dapi respectively. th e magnifi ed chromosomes having a secondary constriction showing cma positive and dapi negative bands; (i) ideogram representation of the chromosomes in metaphase. scale bars=5µm. 68 indranil santra, tarun halder, biswajit ghosh chromosomes during the separation (figure 2i). precocious movement found in 7.56% of total metaphase-i resulted in early separation of some bivalents (figure 2k). staining of pollens with acetocarmine shows bold red colors for the viable pollens and weak or colorless for the nonviable pollens. the estimated pollen viability was 47.17% (figure 2m). estimation of berberine through hplc in hplc, chromatogram of standard berberine peak was obtained at 10.706 min (figure 3a), and a peak at 10.773 min was obtained from the methanolic extract of t. cordifolia (figure 3b). in the case of our plant, the berberine content was found to be 0.424±0.02% on dry weight (dw) basis. discussion chromosomes of the menispermaceae family are small and have basic chromosome number, x=12 and 13. only a very few genera of the menispermaceae family have been considered for cytological assessment, and the same is also true for the genus tinospora. mitotic cell division and mitotic index have been recorded earlier in t. cordifolia that revealed different growth responses of the plant, based on the changing eco-climate (shervani and mishra 2020). previous chromosome investigations revealed that the species have three cytotypes, 2n=22, 24, and 26 (table 1). in the present report, chromosome analysis performed on t. cordifolia having diploid somatic chromosome number 2n=26 (figure 1a) based on basic number x=13 agrees with the study by sharma and bhattachary ya (1955), sharma and sharma (1957), and mathew (1958). as per the online available resources, analysis of chromosomal characters and their measurements have not been done before in this cytotype (2n=26). karyological measurement has been reported for the cytotype having 2n=22 chromosomes (jain and prasad 2014). mathew (1958) stated the range of chromosome size as small (2–3 µm). individual chromosome measurement has not been mentioned. further centromeric position of chromosomes could not be revealed. nonetheless, these parameters are very essential for chromosome characterization. in plants with small chromosomes, it remains a challenge to prepare high-quality chromosomes spread by a conventional squashing method, where the details of chromosomal attributes can be resolved (yamamoto et al. 2019). this may be a reason for the limited studies on cytogenetic of the family menispermaceae despite being very important and economic. therefore, enzymatic maceration of the cell wall becomes a reliable technique that removes the wall and helps spread the chromosomes on a cytoplasm-free background, so chromosomal details can be studied more rigorously. the chromosomes of t. cordifolia are small and categorized into metacentric and submetacentric (figure 1a, c). in the cytotype with 2n=22, the presence of a subtelocentric pair has been reported, which is absent in the present investigation (jain and prasad 2014). the two pairs of submetacentric chromosomes are also associated with the secondary constriction, which is intercalary in table 2. chromosome parameters in tinospora cordifolia. chromosome number s (µm) l (µm) total (µm) arm ratio chromosome type* dapi/cma bands 1 1.07±0.06 1.34±0.01 2.42±0.06 1.25 m 2 0.9±0.13 1.51±0.11 2.41±0.01 1.67 sm dapi negative/cma positive 3 1.05±0.07 1.26±0.07 2.31±0 1.20 m 4 0.76±0.04 1.44±0.07 2.21±0.04 1.89 sm dapi negative/cma positive 5 0.95±0.07 1.21±0.09 2.15±0.02 1.27 m 6 0.85±0.04 1.26±0.14 2.11±0.05 1.48 m 7 0.96±0.03 1.15±0.02 2.11±0.01 1.19 m 8 0.83±0.09 1.27±0.09 2.1±0 1.53 m 9 0.94±0.09 1.09±0.08 2.03±0.01 1.15 m 10 0.84±0.01 1.17±0.01 2.02±0.01 1.39 m 11 0.8±0.08 1.13±0.06 1.93±0.03 1.41 m 12 0.85±0.03 1.03±0.01 1.88±0.02 1.21 m 13 0.9±0 0.92±0.02 1.82±0.02 1.02 m *m = metacentric, sm= submetacentric. 69somatic and gametic chromosomal characterization with fl uorescence banding of giloy position. intercalary secondary constriction originated through chromosome breakage and inversion events that hold several evolutionary implications in species formation, studied in different plant genera such as richardia and melilotus (schlarbaum et al. 1984; siljakyakovlev et al. 2017). figure 2. meiosis and pollen viability of tinospora cordifolia; (a) diplotene stage with chiasma; (b-c) metaphase-i, showing n=13 bivalents (stained with acetocarmine and dapi respectively); (d-e) secondary associations between chromosomes in metaphase-i showing bivalents trivalents, tetravalents and multivalents confi guration (arrows indicate secondary association); (f-g) chromosome stickiness in metaphasei; (h) separation of chromosomes in anaphase-i; (i) laggard in anaphase-i; (j) stickiness between the chromosomes aft er anaphasic separation; (k) precocious movement in metaphase-i; (l) chromosome stickiness in metaphase-ii; (m) viable pollens and non-viable pollen stained with acetocarmine. scale bars=10µm. 70 indranil santra, tarun halder, biswajit ghosh th e cma positive bands colocalized with the nucleolar organizer region (nor) associated heterochromatin part can readily be observed in diff erent plant species (guerra 2000; guidini et al. 2017). in the present study, the four intense cma positive bands signifying the presence of gc-rich content in the secondary constriction (figure 1b). in contrast, the uniformity of dapi stain all over the chromosomes and the absence of any particular dapi positive bands in metaphase stage indicate a lack of suffi cient at repeats (figure 1c). th e at stretches of the dna are required to generate distinct fl uorescent signals, as dapi is predominantly specifi c to the at-rich region (bhowmick et al. 2016). however, dapi negative band associated with cma positive bands signifi es gc-rich dna contents. noticeably, dapi positive signals can be visible in the less condensed chromosome of prometaphase, where any higher condensation in metaphase failed to generate the dapi positive signal (figure 1d). perhaps, the loose condensation of the prometaphase provides a better resolution for dapi than over condensed metaphase chromosomes. th e karyological data along with their fl uorescent banding is found to be reproducible for this genotype. th e meiosis studies show the presence of 13 bivalents in metaphase i which is in agreement with the studies by abraham (1942) and mathew (1958). similar to somatic chromosomes, meiosis studies on this genus are also very limited despite the signifi cance of meiotic behavior in reproductive events. failure in successful meiosis during gamete formation can lead to pollen sterility and reduction in reproductive performance (shin et al. 2021). along with the regular meiotic behavior, various abnormalities in pollen mother cells have been observed in the present study and are mainly categorized into two classes. th e fi rst one is chromosome stickiness that may be a consequence of secondary association in metaphases-i. secondary association is a result of the residual attraction between distantly related chromosomes owing to their structural rearrangements such as duplication, interchanges, or stickiness and has been reported across diff erent plant genera (bala and gupta 2011). data from diff erent plant families indicate that the presence of the secondary association between bivalents evidences the occurrence of polyploidy or interspecifi c hybridization events as an extent of the genome evolution of plant species (heilborn 1936; bala and gupta 2011). polyploidy and intergeneric hybridization have appeared in diff erent genera, namely cocculus, menispermum, and even in tinospora of the family menispermaceae (wang et al. 2004; lian et al. 2019). however, due to the less attention to the meiotic study of this family, the naturally occurred secondary association between chromosomes was never mentioned before. in the artifi cial colchitetraploid species of t. cordifolia, the occurrence of different chromosomal association such as quadrivalents, trivalents, bivalents, and univalents in the metaphase-i of meiosis is similar to that mentioned in the present study, which also explains the link between the appearance of secondary association and polyploidization (th akur et al. 2020). chromosome stickiness is a common phenomenon in plants where the secondary association is involved between the chromosomes (bala and gupta 2011). in addition to these, other common meiotic irregularities such as laggard chromosome and precocious separation result from abnormal spindle activity (kumar and singh 2003). in the present study, the percentage of the sterile pollens is higher than that of the viable pollens, suggesting that the meiotic abnormalities signifi cantly aff ect the microsporogenesis process, which later on decides the fate of sexual reproduction. together, cytogenetic assessment aided with the fl uorescence banding and analysis of pollen infertility is utilized as a potent tool in the identifi cation of stable genotypes that are further used in the breeding programs (samatadze et al. 2020). in the previous studies, berberine content has been detected through chromatographic separation in the extract of t. cordifolia (srinivasan et al. 2008; satija et al. 2020). srinivasan et al. (2008) also reported the figure 3. hplc chromatogram of berberine. (a) standard. (b) stem of tinospora cordifolia. 71somatic and gametic chromosomal characterization with fluorescence banding of giloy quantitative variation of the berberine content in different samples of t. cordifolia studied through hplc technique. therefore genetic characterization together with quantification of active compounds is essential that relates a genotype with the medicinal efficacy. best of our knowledge, genetic assessment along with the measurement of berberine content in t. cordifolia is remaining very poor. hence, in the present study, the amount of berberine has been measured after details cytogenetical characterization of the plant. the stem of t. cordifolia contains 0.424±0.02% (dw basis) of berberine that is more or less resembles the report of srinivasan et al. (2008). moreover, the above study would be beneficial for the correct assessment of a genotype and reproductive performance linked with their medicinal quality which again is significant for any further quality improvement programs. acknowledgements a l l t h e a u t h o r s a c k n o w l e d g e s w a m i kamalasthananda, principal, ramakrishna mission vivekananda centenary college, rahara, kolkata, india for the facilities provided for this study. further, the authors are thankful regional cum facilitation centre (eastern region), nmpb, jadavpur university, kolkata, india for the financial assistance. references abhijeet r, mokat d. 2018. on vegetative propagation through stem cuttings in medicinally lucrative tinospora species. journal of pharmacognosy and phytochemistry. 7(2), 2313–2318. abraham a. 1942. chromosome number in tinospora. current science. 11:282. arroyo martínez ha, arzate fernández am, barba gonzález, r, piña escutia jl. 2017. karyotype determination of three tigridia species (asparagales, iridaceae). caryologia. 70(3):211–215. bala m, pratap k, verma pk, singh b, padwad y. 2015. validation of ethnomedicinal potential of tinospora cordifolia for anticancer and immunomodulatory activities and quantification of bioactive molecules by hptlc. journal of ethnopharmacology. 175:131– 137. bala s, gupta rc. 2011. effect of secondary associations on meiosis, pollen fertility and pollen size in cape gooseberry (physalis peruviana l.). chromosome botany. 6(2):25–28. barros e silva ae, guerra m. 2010. the meaning of dapi bands observed after c-banding and fish procedures. biotechnic & histochemistry. 85(2):115–125. bhowmick bk, yamamoto m, jha s. 2016. chromosomal localization of 45s rdna, sex-specific c values, and heterochromatin distribution in coccinia grandis (l.) voigt. protoplasma. 253(1):201–209. deepa b, babaji hv, hosmani jv, alamir awh, mushtaq s, raj at, patil s. 2019. effect of tinospora cordifolia-derived phytocomponents on cancer: a systematic review. applied sciences. 9(23):5147. https://doi. org/10.3390/app9235147. guerra m. 2000. patterns of heterochromatin distribution in plant chromosomes. genetics and molecular biology. 23(4):1029–1041. guidini cc, pinto-maglio caf, lombello ra. 2017. karyotype, rdna localization and meiotic behavior of hovenia dulcis thunb. 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cells of allium sativum. caryologia 75(1): 99-107. doi: 10.36253/ caryologia-1307 received: may 6, 2021 accepted: december 17, 2021 published: july 6, 2022 copyright: © 2022 guadalupe velázquezvázquez, beatriz pérez-armendáriz, verónica rodríguez soria, anabella handal-silva, luis daniel ortega. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid gvv: 0000-0001-9879-0968 bpa: 0000-0002-4956-2480 vrs: 0000-0002-2287-8338 ahs: 0000-0002-6915-5655 ldo: 0000-0003-4672-8809 genotoxicity and cytotoxicity of sambucus canadensis ethanol extract in meristem cells of allium sativum guadalupe velázquez-vázquez1, beatriz pérez-armendáriz1, verónica rodríguez soria1, anabella handal-silva2, luis daniel ortega1,* 1 decanato de ciencias biológicas. facultad de biotecnología. universidad popular autónoma del estado de puebla. 21 sur 1103 col. santiago. 72160. puebla, méxico 2 departamento de biología y toxicología de la reproducción. instituto de ciencias, benemérita universidad autónoma de puebla. 72570 puebla, méxico *corresponding author. e-mail: : luisdaniel.ortega@upaep.mx abstract. sambucus canadensis is used in traditional medicine mainly in indigenous communities as an anti-inflammatory, antiviral, to treat cough, fever and other ailments, however, its use must be validated on scientific bases. the aim of this study was to evaluate the genotoxic and cytotoxic effect of the ethanol extract of sambucus canadensis in meristem cells of allium sativum with 5 treatments at concentrations of 125, 250, 500, 1000 and 1500 mg/l. two thousand cells were counted per treatment; the mitotic index (mi) and nuclear abnormalities (na) were evaluated. data were analyzed using variance analysis (anova) and chi square (x2) (p < 0.05). root growth was found to be inhibited based on the concentration with statistically significant differences (p < 0.05). as the dose and exposure time of the ethanol extract increased, the mi decreased. the na increased at the highest concentrations of 500, 1000 and 1500 mg/l and these differences were statistically significant compared to the control (p = 0.001). with the results obtained, it can be shown that the species has antiproliferative effects and genotoxic activity on the allium sativum cell cycle, which can be extrapolated to other types of eukaryotic cells. therefore, despite being a plant with health benefits, moderate use and low concentrations are recommended to avoid harmful effects. keywords: traditional medicine, chromosomal aberrations, biomodel, allium sativum, genotoxicity, elderberry, plant extract. introduction medicinal plants are used by rural and urban populations in the treatment of numerous diseases (chabán et al. 2019; trap et al. 2020). in various parts of the world, they are the only source of medical care, mainly due to economic and geographical factors, customs and traditions (marcotullio et al. 2018; tedesco et al. 2017; ullah et al. 2013). according to the world health organization (who), about 80% of the world’s population uses herbal remedies as primary health care. the use of plants to treat dis100 guadalupe velázquez-vázquez et al. eases is still based on empirical knowledge, although they have been considered low risk compared to other synthetic drugs (de smet 2007; monroy et al. 2005, newman and cragg 2016). the scientific information available for most medicinal plants is still insufficient to guarantee their safe and efficient use (moore et al. 2020; pastori et al. 2013). various studies have indicated the importance of evaluating their safety (ganjhu et al. 2015; huang et al. 2015; neira et al. 2018; palatini and komarnytsky 2019; soliman 2010; sousa et al. 2011, vazirian et al. 2018) due to possible risks associated with their components, which may be potentially toxic, mutagenic, carcinogenic or teratogenic (abdelmigid 2013; bratu et al. 2012; prasansuklab et al. 2020). among the medicinal plants with high curative potential is sambucus canadensis (l.) bolli. a native of mexico belonging to the adoxaceae family, it is commonly known as elderberry. this species has been used medicinally in indigenous communities as a bactericide, anti-inflammatory, against flu, cough, dysentery, fever, as well as in uses related to rituals for pregnant women (álvarez-quiroz et al. 2017; lee and finn 2007; sánchez-gonzález et al. 2008; wu et al. 2004). its antimicrobial, antiviral, antioxidant and chemopreventive activities, among others (sidor et al. 2014; tedesco et al. 2017; thole et al. 2006) have been associated with the components present in the species such as triterpenes, tannins and various types of flavonoids such as anthocyanins (abdelmigid 2013; ozgen 2010; vujosevic et al. 2004), however, the presence of these compounds could also cause harmful effects such as nausea, vomiting and diarrhea, such as in the case of sambucus nigra species, whose consumption in pregnant and lactating women, as well as in children and teenagers under 18 years of age, should be avoided (ema/hmpc european medicines agency 2012). information on toxicology, cy totoxicity and genotoxicity of sambucus canadensis leaves is limited (knudsen and kaack 2015; lee and finn 2007; schmitzer et al. 2012). it is important therefore to carry out studies to evaluate chromosomal damage and alterations of the mitotic cycle. according to hister et al. (2017); nefic et al. (2013); pinho et al. (2010); souza et al. (2010); tedesco et al. (2015), the allium sp. biomodel is a widely used, efficient, fast, low-cost method, with extrapolatable results since animal and plant chromosomes have similar structures. the aim of this study was to evaluate the cytotoxic and genotoxic effect of sambucus canadensis on meristem cells of allium sativum. materials and methods plant material the sambucus canadensis (l.) bolli. plant was collected in san miguel eloxochitlan in the sierra negra zone of puebla, mexico, coordinates 18°30’32”n and 96°55’22”w. the plant material was identified using taxonomic techniques and one specimen was deposited in the arboretum of the university of puebla botanic garden (jb-buap) with the id: 83771. preparation of sambucus canadensis extract the leaves of sambucus canadensis (l.) bolli. were used. the extract was obtained by macerating 750g the dry leaves of seven plants with 4l of 96% ethanol with double filtering. the extracts were vacuum filtered with whatman no. 4 paper, the supernatant was concentrated on a buchi® rotary vapor under reduced pressure at 35 ± 15 °c and the ethanol extract evaporated in vacuo. later, different concentrations of the extract were made, specifically, 125 mg/l, 250 mg/l, 500 mg/l, 1000 mg/l and 1500 mg/l. phytochemical tests were carried out for the qualitative identification of the different metabolite groups, each test was performed in triplicate (carvajal et al. 2009; patil and bhise 2015). fourier-transform midinfrared  spectroscopy (ftir) from 4000 to 600 cm-1 was used to obtain information about the functional groups present in the plant using a bruker spectrometer at a resolution of 4 cm-1. the analyses were done in the high technology service center (cesat-upaep). allium sativum bioassay meristem cells from the roots of a. sativum were used to evaluate the nuclear abnormalities (na) and the mitotic index (mi) in the concentrations (125, 250, 500, 1000, 1500 mg/l); water was used as a control. five repetitions were performed on each concentration with bulbs of uniform size (3 cm in diameter). the control bulbs were kept in water. the other bulbs were transferred to the different concentrations for 120 hours. at the end of exposure, the length of the roots and stem were measured and examined to detect visible morphological anomalies: changes in consistency and root color and the presence of hooks or twists in the roots as a sign of general toxicity (çelik and aslantürk 2010). subsequently, the meristem zone of the garlic roots (2 mm) was cut and placed on a slide, hydrolyzed in 1n hcl for 10 minutes, 101genotoxicity and cytotoxicity of sambucus canadensis ethanol extract in meristem cells of allium sativum then washed with distilled water, and stained with acetic orcein. the slides were fixed with the squash method sealing the edges with resin. the samples were analyzed with a leica dm1000 led fluorescence optical microscope with a jenoptik progres c10 digital camera. some 2000 meristem cells were counted for each treatment. in the stages of mitosis (interphase, prophase, metaphase, anaphase and telophase), the cellular alterations were counted: chromosomal breakage, bridges, lagging chromosomes, strays, among others. the values obtained were used to calculate the mitotic indices (mi) and the percentage of cellular alterations (ca) with the following formulae: mi = numberofcells ∈ mitosis ÷ totalcells × 100 ca = nunberofcellswithabnormalchromosomes ÷ totalcells × 100 the results of the number of roots and length of roots and stem, and the mitotic index were analyzed with the anova (bonciu et al., 2018). the differences were evaluated with dunnett’s post hoc test. the nuclear abnormalities were evaluated with the chi-squared test (x2) using the minitab 8.1 statistics program. values of p < 0.05 were considered significant differences. results the phytochemical tests of the sambucus canadensis extract revealed the presence of alkaloids, flavonoids, saponins and tannins. the infrared spectrum tests (ftir) on the extract showed different frequencies of stretching and bending; the stretching frequencies of the o-h bond at 3350 cm-1 is associated with phenol groups; the involvement in hydrogen bonding produces a widening of the band. the c-h bond stretching vibrations corresponding to methyl and methylene groups appear in the 3000–2850 cm-1 range and the bands in the fingerprint region are due to the bending vibrations at 1386 cm-1 for methyl and 716 cm-1 for ethyl: the stretching vibrations of the carbonyl bond, c=o, appears in the 1750–1680 cm-1 range related to the presence of flavonoids. similarly, a conjugated double bond c=c of the aromatic rings appears in the 1600–1450 cm-1 range, characteristic of the basic structure of flavonoids (figure 1). the length of allium sativum roots and stem at the 1000 and 1500 mg/l concentrations were 7.66 mm and 0.72 mm, respectively, showing statistically significant differences compared to the control (p = 0.000). there were no significant differences with the other treatments (p > 0.05). the stem length at a concentration of 1500 mg/l (2.36 mm) showed a statistically significant difference compared to the stem length of the control group (p = 0.000). it can be seen that the average length and number of roots decreased depending on the concentration (table 1). in terms of morphology, at concentrations of 125 and 250 mg/l no differences were observed compared to the control, however, at concentrations of 500 and 1000 mg/l the roots appeared yellow, at 1500 mg/l the sparse roots were brown and stiff. likewise, effects on the stem such as twisting and color change were observed, mainly at concentrations of 1000 and 1500 mg/l. regarding the results of the mitotic index, table 2 shows a decrease in the mi as concentration increases; the number of dividing cells (prophase, metaphase, anaphase and telophase) differed between concentrations, however, at 125, 250 and 500 mg/l there were no significant differences compared to the control (p > 0.05). at a concentration of 1500 mg/l a statistically significant difference was observed in the mitotic index compared to the control. the nuclear abnormalities in allium sativum are shown in table 3. concentrations of 500, 1000 and 1500 mg/l of the ethanol extract had the highest number, concentrations of 125 and 250 mg/l a lesser amount. the number of cells in mitosis with anomalies was related to the increase in concentration. figure 2 shows abnormalities such as breakage, chromosome loss, bridges, chromosomes with inactivated centromere, among others. figure 1. ftir spectra of sambucus canadensis in the range of 4000 to 600 cm-1. 102 guadalupe velázquez-vázquez et al. discussion the test with allium spp. is a suitable biomodel for identifying the cytotoxic and genotoxic effects of different plants (bagatini et al. 2007; lubini et al. 2008; trapp et al. 2020). the study of raw extracts is important since traditional medicine uses part of the plant structure (leaves, stem, root) or the whole plant, without separating its components. furthermore, it has been shown that different bioactive compounds act synergistically (tallarida 2011) and that a combination of compounds exhibits a greater effect than individual compounds, suggesting that the effects of some plants are the result of the interaction of their components (lamy et al. 2018). in this study, the qualitative analyses (phytochemical tests; ftir) of the plant showed the presence of alkaloids, tannins, saponins and flavonoids. the spectroscopy used provides important information about functional groups as well as being an accessible and useful technique in the chemical and structural analysis of plants (günzler and gremlich 2002; herediaguerrero et al. 2014). the functional group associated with the signals reported in the evaluated spectra (ftir) is flavonoids, which present inhibitory activity against diverse fungi and bacteria species. the metabolites reported (alkaloids, tannins, saponins, flavonoids) showed antimicrobial, antioxidant and antiviral activtable 1. number and length of roots, and stem length of a. sativum exposed to the ethanol extract of sambucus canadensis. treatment mg/l number of roots (x) (δ) length of roots (x) (δ) stems length (x) (δ) 125 13.2 ± 2.66 15.82 ± 724 25.52 ± 17.54 250 9.84 ± 4.94 11.47 ± 6.12 18.76 ± 10.81 500 11.20 ± 6.81 19.76 ± ±12.95 20.8 ± 12.62 1000 7.36 ± 3.68 8.66 ± 10.32* 17.6 ± 8.43 1500 0.92 ± 0.90 * 0.72 ± 0.42 * 2.36 ± 0.46* control 13.4 ± 7.96 25.84 ± 19.79 36.3 ± 44.25 values are mean ± s.e, one way anova (*) are not significantly different p <0.05. table 2. allium sativum merismatic cell numbers in the different cell cycle phases, and index mitotic extract of sambucus canadensis. treatment mg/l interphase prophase metaphase anaphase telophase cells in division mitotic index (%) control 3831 1988 81 59 41 2169 36,1 125 4020 1840 66 39 35 1980 33 250 4356 1571 29 23 21 1644 27,4 500 4306 1580 58 34 32 1704 14,3 1000 4558 1330 36 15 21 1402* 12,0 1500 5398 556 17 13 16 602* 5,03 *p <0.05 in one way anova. table 3. cellular abnormalities observed in allium sativum exposed to the ethanolic extract of sambucus canadensis. treatments mg/l control 125 250 500 1000 1500 number of cells in division 2169 1980 1644 1704 1442 602 bridges 1 4 37 25 chromosome fragments 2 4 5 binucleate 2 3 24 99 40 30 chromosome lagging and disoriented 2 1 1 7 7 4 sticky chromosome 1 1 5 2 vagrant chromosome 2 3 4 trinucleated 10 5 20 total cells aberrations 4a 5a 28b 123b 101b 90b cells aberration (%) 0,2 0,3 1,7* 7,2* 7,0b 15,0b *the chi-square test. significant difference p <0.05. 103genotoxicity and cytotoxicity of sambucus canadensis ethanol extract in meristem cells of allium sativum ity, among others. flavonoids in particular exhibit important pharmacological activities, in addition to being eff ective in chemoprevention and chemotherapy (paduch et al. 2007; perveen 2018). th e evaluation of the s. canadensis extract with the allium sativum test allowed us to determine the eff ects on root and stem growth as well as morphology; the highest concentrations,1000 and 1500 mg/l, signifi cantly inhibited root and stem growth compared to the control. th e mitotic index (mi) decreased signifi cantly as the concentration of sambucus canadensis increased, matching the results reported for other species of sambucus sp. (tedesco et al. 2017; th ole 2006). other authors have reported that plant extracts such as p. leiocarpa and p. myriantha (lubini et al. 2008), campomanesia xanthocarpa (pastori et al. 2013), vernonanthura polyanthes (almeida et al. 2020), amaranthus spinosus (prajitha and th oppil, 2016), achyrocline satureioides (fachinetto et al. 2007), luehea divaricata (frescura et al. 2012) caused a reduction in the mitotic index when increasing the concentration, which may be an indication of antiproliferative activity such as that reported by bagatini et al. (2009), knoll et al. (2006). th e results obtained in this study may be associated with the plant components; in this sense, the fl avonoids found in the ftir analysis may inhibit or stimulate the cellular cycle. tedesco et al. (2017) found that sambucus australis has fl avonoids such as rutin, kaempferol and quercetin, among others, to which diff erent pharmacological eff ects have been attributed, including antiproliferative and anticancer action. one study developed by lee and finn, (2007) reported that sambucus canadensis presents a high quantity of anthocyanins and polyphenols which have a potent antioxidant eff ect, perhaps also explaining the inhibition of cellular division in allium sativum. in the same way, the phenolic components of the species have been associated with a more potent anticancer activity than sambucus nigra (th ole et al. 2006). figure 2. allium sativum cells exposed to the ethanolic extract of sambucus canadensis a) sticky chromosome b) bridges c) sticky and lagging chromosome with bridges d) lagging and sticky chromosome e) abnormal anaphase f ) lagging chromosome g) vagrant chromosome and lagging chromosome, h) bridge and vagrant chromosome, i) bridges. 104 guadalupe velázquez-vázquez et al. the main chromosomal aberrations found in this study include the formation of bridges, which, according to türkoğlu (2007), are produced due to the fusion of chromosomes or chromatids as a result of chromosomal stickiness or due to unequal translocation. lagging chromosomes moving to both sides of the poles without being fused by the spindle apparatus can also induce bridges. another aberration found in the results of this investigation were sticky chromosomes formed by the free movement of chromosomes, which can produce chromosomal breakage and may lead to the loss of genetic material (dutta et al. 2018). stray chromosomes advance ahead of the chromosome group towards the poles resulting in an unequal distribution of chromosomes in daughter cells (sondhi et al. 2018). similarly, fachinetto and tedesco (2009) attribute various chromosomal anomalies, bridges, binucleated cells, among others, to the components of the plants. along these lines, bagatini et al. (2009); toloza et al. (2006), indicate that the genotoxic and antiproliferative activity presented by some plant extracts are the result of the interactions of their different chemical components. in this regard, amado et al. (2020) reported that the smilax brasiliensis extract and the rutin and quercetin fractions, which have also been found in the species sambucus sp. cause genotoxic effects. it has been suggested that, if extracts cause damage to plant cell chromosomes, they may also be potentially harmful for mammalian cell chromosomes (feretti et al. 2007). according to the results, no abnormalities in the a. sativum root were found at low concentrations of 125 mg/l and 250 mg/l. however, in concentrations of 500, 1000 and 1500 mg/l, a considerable number of alterations, such as bridges, chromosome breaks and strays were found, suggesting that the extract presents a genotoxic effect at high concentrations. the results match those reported by bratu (2012) which indicate that at low concentrations the species sambucus nigra presents no mutagenic effects. according to ifeoluwa et al. (2013) and sabini et al. (2011), some plants induce cytotoxicity but not mutagenic effects. generally speaking, the frequency of aberrations increases significantly as the concentration increases, suggesting that sambucus canadensis leaf extract presents clastogenic effects, which agrees with the reports of bidau et al. (2004); çelik and aslantürk (2010) and mattana et al. (2014) on the effects of herbal plants. sambucus canadensis is a plant that should be used with caution not only because it is used for curing diseases but also because it is used by pregnant women (velazquez-vázquez et al. 2019) and could be harmful to their health. the use of plants before and after pregnancy may cause conditions from vomiting, infection and gastrointestinal problems to placental retention, uterine hypotonia, cervical tear, miscarriage, uterine bleeding and others. since few studies exist on the safety and efficacy of the use of herbal plants during pregnancy, a situation which exposes both the mother and the fetus, it is recommended that they are not used during pregnancy unless such use is supported by scientific studies which validate their safety (ahmed et al. 2017; frawley et al. 2015; hall et al. 2011; illamola et al. 2020; nergard et al. 2015). conclusion the results of this study suggest that the ethanol extract from sambucus canadensis induces antiproliferative effects. it was also found that in concentrations higher than 500 mg/l the extract affects root growth, cellular division and chromosomal changes in the cells of allium sativum. it is important to know the effects of plants that are used as the primary source of medical care in order to contribute to the regulation of their use and consumption as an important measure for protecting human health. references abdelmigid hm. 2013. new insights into toxity and drug testing. chapter 5. 89-253. http://dx.doi. org/10.5772/54858 ahmed s, hasan mm, mahmood za. 2017. antiurolithiatic plants of family fabaceae: a memoir of mechanism of action, therapeutic spectrum, formulations with doses. j. pharmacogn. phytochem. 6(3): 592596. alvarez-quiroz v, caso-barrera l, aliphat-fernández m, galmiche-tejeda a. 2017. plantas medicinales con propiedades frías y calientes en la cultura zoque de ayapa, tabasco, méxico. bol latinoam caribe plant med aromat 16 (4): 428 – 454. amado pa, castro ah f, zanuncio vss, stein vc, da silva db, dos santos lima lar. 2020. assessment of allelopathic, cytotoxic, genotoxic and antigenotoxic potential of smilax brasiliensis sprengel leaves. ecotoxicol environ saf 192: 110310 bagatini md, silva acf, tedesco sb. 2007. uso do sistema teste de allium cepa como bioindicador de genotoxicidade de infusões de plantas medicinais. rev bras farmacogn. 17:444-447. bagatini md, vasconcelos tg, laughinghouse iv hd, martins af, tedesco sb. 2009. biomonitoring hos105genotoxicity and cytotoxicity of sambucus canadensis ethanol extract in meristem cells of allium sativum pital effluents by the allium cepa test. bull environ contam toxicol 82:590–592. bidau ag, amat m, yajia da, marti ag, riglos a. 2004. evaluation of the genotoxicity of aqueous extracts of ilex paraguariensis st. hil. 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(mallow) is the genus within the malvaceae juss. family, which includes twentyfive-forty. species and several hybrids. this genus contains herbaceous annual, biennial, and perennial species that are native to regions of africa, asia, and europe. malva species contain a lot of mucilage, malvin, flavonoids, terpenoids, polysaccharides, and vitamin. no detailed random amplified polymorphic dna (rapd) studies were conducted to study malva genetic diversity. therefore, we collected and analyzed seven species from seven provinces of iran regions. overall, eighty-five plant specimens were collected. we showed significant differences in quantitative morphological characters in plant species. malva verticillata l. depicted unbiased expected heterozygosity (uhe) in the range of 0.053. shannon information was high (0.67) in malva parviflora l. malva vericillata showed the lowest value, 0.083. the observed number of alleles (na) ranged from 1.16 to 2.33 in malva vericillata and malva parviflora. the effective number of alleles (ne) was in the range of 1.078-1.922 for malva vericillata and malva parviflora.gene flow (nm) was relatively low (0.63) in malva. the mantel test showed correlation (r = 0.76, p=0.0001) between genetic and geographical distances. we reported high genetic diversity, which clearly shows the malva species can adapt to changing environments since high genetic diversity is linked to species adaptability. present results highlighted the utility of rapd markers and morphometry methods to investigate genetic diversity in malva species.our aims were 1) to assess genetic diversity among malva species 2) is there a correlation between species genetic and geographical distance? 3) genetic structure of populations and taxa. keywords: population structure, gene flow, random amplified polymorphic dna (rapd), malva species, network. introduction the use of medicinal plants can be influenced by the economic condition, the high cost of medicines and the difficult access to public consultations. in addition to that, there is a difficulty of access by residents in rural areas to health care units located in urban areas. moreover, the increase the trend for considering traditional knowledge that supports using natural 78 yinan liu, jiaqing wang, hongling kang resources as an alternative to synthetic drugs (battisti et al., 2013). malvaceae juss. (‘the mallows’) is a botanical family with a rich diversity of species for textile, medicinal, and ornamental purposes. it consists of 4465 species and about 245 genera (tate et al., 2005) and mallows present a cosmopolitan distribution, but with a high number of species in the tropics. the principle economic use of malvaceae plants is as a source of natural fibers, the family providing perhaps the worlds three most important fiber crops plants of the family are also used for food, beverages, timber, in traditional medicine and in horticulture (la duke and dobley, 1995; erbano et al. 2015; frankham 2005; ellegren and galtier 2016; turchetto et al. 2016). many researches have been published on the ecology, taxonomy, genetic, cytology, chemotaxonomy, physiology, seed germination and economic uses of family malvaceae such as (el-rjoob and omari 2009) in ecology; in taxonomy (tate et al., 2005), in chemotaxonomy (blunden et al., 2001; gomez et al. 2005; cires et al. 2013, esfandani-bozchaloyi et al. 2018a, 2018b, 2018c, 2018d) and in genetic researches (baum et al., 2004) studied the pollen. the malva genus has 25-40 species and it can be considered as an annual and/or biannual herb. flowers with an epicalyx and 8-15 reticulated mericarps are the typical one (fryxell, 1988; dellagreca, et al., 2009). in medicine, mallow species are used in the treatment of respiratory, urinary, and digestive problems as they have high bactericidal, antiulcerogenic, anti-inflammatory, hepatoprotective, and antidiabetic activities (pandey et al, 2012). the malva genus is morphologically very diverse, but some species are hardly distinguishable based on morphological features (escobar et al., 2009). several studies have been conducted to clarify the taxonomic affiliation of malva species using different features, such as molecular data (nuclear ribosomal dna (rdna), internal transcribed spacer (its) region, intron–exon splice junction (isj), and inter simple sequence repeat polymerase chain reaction (issr) markers) (celka, et al., 2010), differentiation of seed and seed coat structure (el naggar, 2001), morphology of pollen grains (el naggar, 2004), epidermal structures and stem hairs (akçin, and özbucak, 2006), and plant morphological traits (michael et al., 2009). the variability in mallow species is due, at least in part, to hybridization. natural crossings between malva pusilla sm. and malva neglecta wallr., malva alcea l., and malva moschata l. as well as malva sylvestris l. and malva neglecta were found in europe. ray (1995) stated that hybridization or polyploidy is probably a factor in the evolution of these species, but this aspect has not been investigated so far. the taxonomy and systematics of the malva genus are still unclear and very complicated. taxonomic doubts have appeared because of the high level of homoplasty in morphological traits that are usually used as diagnostic features (escobar garcía, et al.,2009). based on the flower structure, dalby (1968) divided the malva genus into two sections: bismalva (with malva alcea, malva excisa rchb., and malva moschata) and malva (malva neglecta, malva pusilla, malva sylvestris, and malva verticillata) a different classification based on its molecular markers as well as fruit morphology and seed structure was reported by ray (1995), and two groups were distinguished: malvoid and lavateroid. a similar division was proposed by escobar garcia et al. (2009) based on five its molecular markers (matk plus trnk, ndhf, trnl-trnf, and psba-trnh). these genetic relationships and the classification of malva species were also confirmed by celka et al. (2010) and lo bianco et al. (2017) based on its and issr molecular markers along with seed image analysis. genetic diversity studies are usually tapped due to molecular markers. molecular markers are an excellent method to disentangle phylogenetic association between species and population. among molecular methods or markers, rapd (random amplified polymorphic dna) are sensitive to detect variability among individuals of species. rapd method is cost-effective and can work with limited sample quantities. in addition to this, rapd can amplify and target genomic regions with potential and several markers (esfandani-bozchaloyi et al. 2017). taxonomical systematics studies were conducted in the past to identify the malva species. according to the best of our knowledge, there is no existing rapd data on genetic diversity investigations in iran. we studied seventy samples. our aims were 1) to assess genetic diversity among malva species 2) is there a correlation between species and geographical distance? 3) genetic structure of populations and taxa 4) are the malva species able to exchange genes? materials and methods plant materials seven malva species were collected from different regions of iran (table 1). these species were studied via morphological and molecular methods. eighty-five plant samples (nine-fifteen per plant species) were examined for morphometry purposes (figure 1). the random amplified polymorphic dna analysis method was limited to eighty-five samples. we focused on the following species malva neglecta wallr., malva pusilla sm., malva 79random amplified polymorphic dna profiling in detecting genetic variation in malva l. species table 1. list of the investigated taxa including origin of voucher specimens. taxa locality latitude longitude malva neglecta wallr. west azerbaijan, kaleybar 38°5’46.4604” 46°16’23” malva parviflora l. hormozgan, bandar abbas 27°33’12” 56°44’16” malva pusilla sm. khuzestan, behbahan 30°17’01” 50°54’10” malva sylvestris l. esfahan, ardestan on road to taleghan 32°15’44” 51°16’33” malva verticillata l. kerman, hamun-e jaz murian 27°10’13” 58°33’19” malva nicaeensis all. mazandaran, 40 km tonekabon to janat abad 35°10’16” 51°55’18” malva aegyptia l. golestan, gorgan 35°13’19” 52°10’31” figure 1. presence of species in different regions of iran. 80 yinan liu, jiaqing wang, hongling kang sylvestris l., malva verticillata l., malva nicaeensis all., malva aegyptia l. and malva parviflora l. according to previous references, all the species were identified (escobar garcía, et al., 2009; ray, 1995 ). morphometry in total thirty-eight morphological (ten qualitative, twenty-eight quantitative) characters were studied .̀ five to ten plant specimens were randomly studied or morphological analyses. data were transformed (mean= 0, variance = 1) prior to ordination . euclidean distance was implemented to cluster and ordinate plant species (podani 2000). random amplified polymorphic dna we extracted dna from fresh leaves. leaves were dried. dna extraction was carried out according to the previous protocol (esfandani-bozchaloyi et al. 2019). dna quality was checked on an agarose gel to confirm the purity. we amplified the dna with the aid of rapd primers (operon technology, alameda, canada). these primers belonged to opa, opb, opc, opd sets. we selected those primers (10) which could show clear bands and polymorphism (table 2). overall, the polymerase chain reaction contained 25μl volume. this 25 volume had ten mm tris-hcl buffer, 500 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp; 0.2 μm of a single primer; 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). we observed the following cycles and conditions for the amplification. five minutes initial denaturation step was carried out at 94°c after this forty cycles of 1 minute at 94°c were observed. then 1-minute cycle was at 52-57°c followed by two minutes at 72°c. in the end, the final extension step was performed for seven to ten minutes at 72°c. we confirmed the amplification steps while observing amplified products on a gel. each band size was confirmed according to 100 base pair molecular ladder/standard (fermentas, germany). data analyses we used an unweighted pair group method with arithmetic mean (upgma) and ward methods. ordination methods such as multidimensional scaling and principa l coordinate ana lysis were a lso performed (podani 2000). the morphological difference among species and population was assessed through analysis of variance (anova). pca analysis (podani 2000) was done to find the variation in plant population morphological traits. multivariate and all the necessary calculations were done in the past software, 2.17 (hammer et al. 2001). to assess genetic diversity, we encoded rapd bands as present and absent. numbers 1 and 0 were used to show the presence and absence of bands. it is essential to know the polymorphism information content and marker index (mi) of primers because these parameters ser ve to obser ve polymorphic loci in genotypes (ismail et al. 2019). marker index was calculated according to the previous protocol (heikrujam et al. 2015). other parameters such as the number of polymorphic bands (npb) and effective multiplex ratio (emr) were assessed. gene divertable 2. rapd primers and other parameters. note: tnb the number of total bands, npb: the number of polymorphic bands, ppb (%): the percentage of polymorphic bands, pi: polymorphism index, emr, effective multiplex ratio; mi, marker index; pic, polymorphism information content for each of cbdp primers. primer name primer sequence (5’-3’) tnb npb ppb pic pi emr mi opa-05 5’-aggggtcttg-3’ 13 12 92.31% 0.54 8.21 10.23 4.55 opa-06 5’-ggtccctgac-3’ 17 17 100.00% 0.47 7.32 11.55 4.18 opb-01 5’-gtttcgctcc-3’ 11 9 96.89% 0.43 6.56 9.34 7.17 opb-02 5’-tgatccctgg-3’ 13 12 95.81% 0.34 4.21 6.60 5.59 opc-04 5’-ccgcatctac-3’ 12 12 100.00% 0.47 3.37 9.55 3.25 opd-02 5’-ggacccaacc-3’ 11 11 100.00% 0.56 4.86 11.88 3.45 opd-03 5’-gtcgccgtca-3’ 9 7 84.99% 0.43 3.51 8.43 3.85 opd-05 5’-tgagcggaca-3’ 15 13 93.84% 0.66 4.66 11.33 4.67 opd-08 5’-gtgtgcccca-3’ 12 11 94.91% 0.48 5.21 12.50 5.65 opd-11 5’-agcgccattg-3’ 14 13 95.74% 0.67 5.66 9.57 5.37 mean 12.7 11.7 95.88% 0.55 5.5 9.4 4.8 total 127 117 81random amplified polymorphic dna profiling in detecting genetic variation in malva l. species sity associated characteristics of plant samples were calculated. these characteristics include nei ’s gene diversity (h), shannon information index (i), number of effective alleles (ne), and percentage of polymorphism (p% = number of polymorphic loci/number of total loci) (shen et al. 2017). unbiased expected heterozygosity (uhe), and heterozygosity were assessed in genalex 6.4 software (peakall and smouse 2006). neighbor-joining (nj) and networking were studied to fathom genetic distance plant populations (huson and br yant 2006; freeland et al. 2011). the mantel test was carried out to find the correlation between genetic and geographical distances (podani 2000). as we were interested in knowing the genetic structure and diversity, we also investigated the genetic difference between populations through amova (analysis of molecular variance) in genalex 6.4 (peakall and smouse 2006). furthermore, gene flow (nm) was estimated through genetic statistics (gst) in popgene ver. 1.32 (yeh et al. 1999). we also did structure analysis to detect an optimum number of groups. for this purpose, the evanno test was conducted (evanno et al. 2005). first data were scored as dominant markers (issr) so we used from structure analysis for estimate the parameters that related to gene flow among studied population. burn-in = 10000, and 10 runs were performed for relationship between genetic structure and distance of geographical. ma ximum likelihood method and bayesian information criterion (bic) was studied by structure analysis (falush et al. 2007; evanno et al. 2005; meirmans 2012). results morphometry significant anova results (p <0.01) showed differences in quantitative morphological characters in plant species. principal component results explained 67% variation. first component of pca demonstrated 49% of the total variation. leaf morphology and traits such as calyx length, calyx width positively correlated with corolla length, corolla color (>0.7). the second and third components explained floral characters such as corolla apex, seed length and number of segment stem leaves. unweighted pair group method with arithmetic mean (upgma) and principal coordinate analysis (pcoa) plots showed symmetrical results (figure 2, figure 3). generally, plant specimens belonging to different species were separated from each other due to differences in morphology. morphological characters divided malva species into two groups, as evident in the upgma tree (figure 2). populations belonging to malva aegyptia were in the first group. on the other hand, the second group consisted of two sub-groups. malva pusilla and malva verticillata formed the first sub-group. malva neglecta, malva sylvestris, malva parvif lora, malva nicaeensis formed the second sub-group. these groups and subgroups were formed due to morphological differences among the individuals of malva. our pcoa results also confirmed the application of morphological characters in separating and clustering the species in separate groups (figure 3). identical results were also reported in the upgma tree (figure 2). species identification and genetic diversity the primers, i.e., opc-04, opb-01, opa-05 and opd-11 could amplify plant (malva species) dna (figure 4). 119 polymorphic bands were generated and amplified. amplified products ranged from 100 to 3000 bp. we recorded the highest polymorphic bands for opa-06. opd-03 had the lowest polymorphic bands. the average polymorphic bands ranged to 11.9 for each primer. the polymorphic information content (pic) had figure 2. upgma clusters of morphological characters revealing species delimitation in malva species. 82 yinan liu, jiaqing wang, hongling kang values in the range of 0.34 (opb-02) to 0.67 (opd011). primers had 0.55 average polymorphic information content values. marker index (mi) values were 3.25 (opc-04) to 7.17 (opb-01), with an average of 4.8 per primer. effective multiplex ratio (emr) values are useful to distinguish genotypes. in our study, we reported 6.60 (opb02) to 12.50 (opd-08) emr values. emr values averaged 9.4 per primer (table 2). all the necessary genetic features calculated of seven malva species are shown (table 3). malva verticillata depicted unbiased expected heterozygosity (uhe) in the range of 0.053. shannon information was high (0.67) in malva parviflora. malva verticillata showed the lowest value, 0.083. the observed figure 3. pcoa plot morphological characters revealing species delimitation in malva species. figure 4. gel electrophoresis image of dna fragments of malva species. l = ladder 100 bp. arrows show polymorphic bands.1,8,15,22: malva neglecta 2,9,16,23: malva parviflora 3,10,17,24: malva pusilla. 4,11,18,25: malva sylvestris 5,12,19,26: malva verticillata 6,13,20,27: malva nicaeensis7,14,21,28: malva aegyptia. 83random amplified polymorphic dna profiling in detecting genetic variation in malva l. species number of alleles (na) ranged from 1.16 to 2.33 in malva verticillata and malva parviflora. the effective number of alleles (ne) was in the range of 1.078-1.922 for malva veritcillata and malva parviflora. gene flow (nm) was relatively low (0.63) in malva. analysis of molecular variance (amova) test highlighted genetic differences among malva species (p = 0.001). amova showed that 50% of genetic variation was among the species. relative less variation (12%) was reported within the species (table 4). genetic similarity and dissimilarity assessed through genetic statistics (gst) showed significant differences i.e., (0.567, p = 0.001) and d_est values (0.876, p = 0.001). the neighbor-joining tree and mds plot of malva populations based on rapd data produced similar results therefore only neighbor-joining tree is presented and discussed (fig. 5). nj net tree revealed that the seven species are well differentiated on the genetic grounds. in both upgma and nj trees, samples of the malva aegyptia were placed far from each other. malva pusilla was placed close to malva verticillata , and far from malva aegyptia . in both analyses, malva nicaeensis showed closer affinity with malva sylvestris, malva parviflora. genetic distance of the two subsp. was estimated to be 1.66 by kimura 2p distance. gene flow (nm) was relatively low (0.63) in malva species. genetic identity and phylogenetic distance in the rindera members are mentioned (table 5). malva verticillata and malva nicaeensis were genetically closely related (0.856) to each other. malva nicaeensis and malva aegyptia were dissimilar due to low (0.694) genetic similarity. the mantel test showed correlation (r = 0.76, p=0.0001) between genetic and geographical distances. the evanno test showed δk =6 (figure 6). figure 6, showed the genetic details of the malva species. according to structure analysis malva pusilla and malva aegyptia were closely related to common alleles (figure 6). the rest of the malva species are genetically differentiated due to different allelic structures (figure 6). the neighbor-joining plot also showed the same result. limited gene flow results were supported by k-means and structure analyses too. we could not identify substantial gene flow among the malva species. this result is in agreement with grouping we obtained with neighborjoining (figure 5), as these populations were placed close to each other. as evidenced by structure plot based on admixture model, these shared alleles comprise table 3. genetic diversity variables of malva (n = number of samples, na= number of different alleles, ne = number of effective alleles, i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism in populations). taxon n na ne i he uhe %p malva neglecta 10.000 1.500 1.311 0.279 0.267 0.187 50.00% malva parviflora 9.000 2.333 1.922 0.670 0.333 0.417 83.33% malva pusilla 12.000 1.500 1.441 0.330 0.233 0.233 50.00% malva sylvestris 13.000 1.333 1.232 0.196 0.200 0.133 33.33% malva verticillata 10.000 1.167 1.078 0.083 0.150 0.053 16.67% malva nicaeensis 15.000 1.200 1.462 0.337 0.290 0.240 50.00% malva aegyptia 15.000 1.433 1.196 0.150 0.183 0.090 19.67% table 4. analysis of molecular variance (amova) of the studied species. source df ss ms est. var % among regions 5 42.297 12.648 0.337 20% among pops 15 96.827 8.802 0.774 50% among indiv 59 64.383 2.130 0.363 18% within indiv 65 14.500 0.204 0.204 12% total 133 215.007 1.678 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance. figure 5. integer nj net tree produced while using rapd data. table 5. the nei genetic similarity (gs) estimates using rapd markers. pop1 pop2 pop3 pop4 pop5 pop6 pop7 1.000 pop1 0.766 1.000 pop2 0.760 0.764 1.000 pop3 0.750 0.730 0.827 1.000 pop4 0.774 0.797 0.762 0.794 1.000 pop5 0.733 0.770 0.727 0.707 0.856 1.000 pop6 0.679 0.722 0.750 0.704 0.719 0.698 1.000 pop7 84 yinan liu, jiaqing wang, hongling kang very limited part of the genomes in these populations and all these results are in agreement in showing high degree of genetic stratification within malva populations. discussion the malva is a relatively complex taxonomic group, and several morphological characters make it difficult to identify and classify malva species (ray 1995; escobar garcía, et al., 2009). given the complexity, it is necessary to explore other methods that could complement the traditional taxonomical approach (erbano et al. 2015). advent and developments in molecular techniques have enabled plant taxonomists to utilize molecular protocols to study plant groups (erbano et al. 2015; abeshu & zewdu 2020.; amar et al 2021; beltran et al. 2021). we examined genetic diversity in malva by morphological and molecular methods (das et al 2021; gutierrezpacheco et al 2021; hindersah et al 2021; jordaan & rooyen et al 2021). we mainly used rapd markers to investigate genetic diversity and genetic affinity in malva. our clustering and ordination techniques showed similar patterns. morphometry results clearly showed the utilization or significance of morphological characters in malva species. pcoa plot results also confirmed the application of morphological characters to separate malva species. the present study also highlighted that morphological characters such as corolla color, leaf shape, leaf length, stamens position, leaf margin and corolla lenght could delimit the malva group. the malva species highlighted morphological differences. we argue that such a dissimilarity was due to differences in quantitative and qualitative traits. in our study, morphology and genetic diversity in seven taxa of malva species are given in detail for the first time. the aim of the present study was to find diagnostic features to separate species of malva in iran. morphological characters are considered as an useful tool for the identification of the species, as indicated previously ray (1995). malvaceous germplasm has been variously investigated by different molecular marker techniques but the earlier studies either focused on the comparison of the malvaceae with other families in the order malvales or to explore the genetic relationships and diversity within and among population and limited number of species in the same genus. very little attention has been given to the analysis at interspecific and intergeneric levels. la duke and dobley (1995) has the only worth mentioning work in this regard. their results showed that, the genetic relationships and diversity within and between 12 malvaceous species belonging to five genera are investigated by using the amplified fragment length polymorphism (aflp). shaheen et al., (2009) with used aflp (amplified fragment length polymorphism) marker to explore phenetic relationships and diversity within and between 13 malvaceae species belonging to 5 different genera. their primary objective of the study was to evaluate the taxonomic potential, usefulness and applicability of aflp marker system to reconstruct genetic relationships at interspecific and intergeneric level in malvaceae. two primer pairs produced a total of 73 bands, of which 70 were polymorphic. according to celka et al (2010) two categories of dna markers were used to determine genetic relationships among eight malva taxa. a maximum parsimony analysis validated the division of the genus malva into the sections bismalva and malva. the species classified into those sections formed separate clusters. malva moschata was a distinctive species in the section bismalva, as confirmed by previous genetic research based on its and cpdna sequence analyses. the applied markers figure 6. structure plot of rapd data in malva populations studied. 1. malva neglecta; 2. malva parviflora; 3. malva pusilla; 4. malva sylvestris; 5. malva verticillata; 6. malva nicaeensis; 7. malva aegyptia. 85random amplified polymorphic dna profiling in detecting genetic variation in malva l. species revealed a very high level of genetic identity between malva alcea and malva excisa and enabled molecular identification of m. alcea var. fastigiata. jedrzejczyk and rewers (2020) applied flow cytometry and inter simple sequence repeat polymerase chain reaction (issr-pcr) for fast and accurate species identification. genome size estimation by flow cytometry was proposed as the first-choice method for quick accession screening. out of the 12 tested accessions, it was possible to identify six genotypes based on genome size estimation, whereas all species and varieties were identified using issr markers. flow cytometric analyses revealed that malva species possessed very small (1.45–2.77 pg/2c), small (2.81–3.80 pg/2c), and intermediate (11.06 pg/2c) genomes, but the majority of accessions possessed very small genomes. the relationships between the investigated accessions showed the presence of two clusters representing malvoid and lavateroid group of species. their results showed that flow cytometry and issr molecular markers can be effectively used in the identification and genetic characterization of malva species. until now, molecular studies using issr markers conducted in the malva genus have only included a few species (celka, et al., 2012). all primers used in issrpcrs for the malva genus revealed 100% polymorphism between all accessions. therefore, it was possible to identify all tested species. moreover, for malva verticillata taxon, it was possible to distinguish all studied varieties. the usefulness of most of the used issr primers was also confirmed in ocimum l., origanum l. and mentha l. identification (lo bianco, et al., 2017). the systematics of the malva genus and closely related genera is complicated. moreover, the relationships obtained from molecular studies do not confirm traditional classification (escobar garcía, et al., 2009). so far, only molecular analysis relying on rdna its sequences and issr markers have shed light on taxonomical relationships between malva species (escobar garcía, et al., 2009). phylogenetic analyses of rdna its sequences indicated the presence of two well-supported clusters within the mallow species (malvoid and lavateroid clades), which is consistent with the presented data. molecular markers (r apd) and morphometr y analysis were useful to study genetic diversity and population structure in malva species identification. all the species had distinct genetic differentiation. present results highlighted isolation and limited gene flow are the main deterministic factors that shape the malva population. we also reported high genetic diversity, which clearly shows the malva species can adapt to changing environments since high genetic diversity is linked to species adaptability. acknowledgements this work was supported by scientific research fund of education department of liaoning province (project no.: l202006), project name: study on pine needle extract as attractive agent for the transmission vector of pine wood nematode; science and technology project of shenfu reform and innovation demonstration zone (project no.: 2020jh14), project name: research and development of key technologies and processing products of traditional chinese medicine resources mining in liaoning mountainous areas. references akçin, ö.e.; özbucak, t.b. morphological, anatomical and ecological studies on medicinal and edible plant malva neglectawallr. 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(piperaceae). caryologia 73(4): 27-38. doi: 10.13128/caryologia-1133 received: november 12, 2020 accepted: november 15, 2020 published: may 19, 2021 copyright: © 2020 e. palchetti, m. gori, s. biricolti, a. masoni, l. bini, c. tani, s. falsini, e. corti, a. papini. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. author contributions: conceptualization, palchetti, biricolti and gori; methodology, gori, papini; software, papini; validation, palchetti, gori, papini, biricolti, calamai; formal analysis, papini, gori, biricolti, bini; investigation, gori, bini, falsini, corti, calamai; resources, palchetti; data curation, gori; writing— original draft preparation, all authors; project administration, palchetti; funding acquisition, palchetti. all authors have read and agreed to the published version of the manuscript. funding: this research was funded by the company tozzigreen, la tour 26 etage rue ravoninahitriniarivo – ankorondrano, antananarivo 101 madagascar. possible hybrid speciation for two malagasy species of piper l. (piperaceae) enrico palchetti1, massimo gori1,4, stefano biricolti1,*, alberto masoni1, lorenzo bini4, corrado tani2, sara falsini2, emilio corti2, alessio papini2,3 1 department of agriculture, food, environment and forestry (dagri) university of florence, piazzale delle cascine, 18, 50144 florence (fi), italy; e-mail: enrico.palchetti@ unifi.it 2 department of biology, university of florence, via micheli, 3, firenze, italy 3 cset tropical herbarium university of florence, italy; e-mail: alpapini@unifi.it 4 interdepartmental service centre for agricultural, chemical and industrial biotechnologies (cibiaci), university of florence, via romana, 21, 50125 florence, italy: e-mail: massimo.gori@unifi.it *corresponding author. e-mail: stefano.biricolti@unifi.it abstract. two new species of genus piper l. from madagascar: piper malgassicum papini, palchetti, m. gori & rota nodari and piper tsarasotrae papini, palchetti, m. gori & rota nodari, were analyzed to investigate their phylogenetic position and evolutionary history. both plastidial and nuclear markers were used for sequencing. the plastidial markers (ndhf and trnl intron) showed a close relationship between the two species with respect to the other species of piper. both species appeared phylogenetically related to the african p. guineense and the malagasyan/mascarenhas endemic p. borbonense. the nuclear marker (g3pdh) amplification produced two separate sets of sequences: “long” sequences and “short” sequences, characterized by some long deletions. analyzing together the nuclear sequences, we observed that the “long” sequence of p. tsarasotrae had a stricter relationship to the african accessions of p. guineense, while the accession of p. malgassicum was more strictly related to p. borbonense. on the contrary both “short” sequences of p. malgassicum and p. tsaratsotrae resulted phylogenetically related to asian accessions and more distantly related to the formerly cited species. this unexpected result was tentatively explained with a more ancient hybridization event between an ancestor of p. malgassicum and p. tsarasotrae (and possibly p. borbonense) and an asian species of piper. the asian contribution would have produced the ancestors carrying the “short” sequences. a more recent hybridization event would have led to the separation of p. malgassicum from p. tsarasotrae with an african pollen-derived genome contribution from p. guineense or, more probably, an ancestor thereof, to an ancestor of p. tsarasotrae. the chromosome numbers of p. tsarasotrae (2n = about 38) and p. malgassicum (2n = about 46), were more similar to the asian species than to the american species. unfortunately, no chromosome number of the african species p. guineense is currently available, to compare the chromosomal numbers. keywords: piper malgassicum, piper tsarasotrae, piperaceae, chromosomes, hybridization, dna sequences, g3pdh, trnl, ndhf, malagasy biodiversity. 28 enrico palchetti et al. 1. introduction genus piper l. (piperaceae) is one of the largest genera of angiosperms, with more than 2000 species (quijano-abril et al. 2006) and were considered belonging to a basal group of angiosperms, the so called “paleoherbs” (loconte et al. 1991). piper is a pantropical genus developing highly variable growth forms (isnard et al. 2012), with the highest biodiversity in the american continent with a number of species ranging from 500 (burger 1972; tebbs 1993), to 1100 (jaramillo and manos 2001), later increased to more than 1800 (ulloa ulloa et al. 2017), many of them with a small distribution area (quijano-abril et al. 2006). the separation of species is often tricky, due to the small size of the floral parts and hence the number of synonyms may be high (suwanphakdee et al. 2016), while other species tend to get naturalized (smith et al. 2008). while only two species are known as native of the african continent, p. guineense thonn. and p. capense l. f., more species are known of madagascar, even if some of them are known only for a single or few herbarium samples. the currently recognized species in madagascar are p. heimii c. dc, p. pachyphyllum baker and possibly p. borbonense (miq.) c. dc., described for the bourbon island, nowadays la reunion (weil et al. 2017), belonging to the mascarenhas islands. however, its presence in madagascar was affirmed by de candolle (1869; 1923). the fact that p. borbonense is cultivated makes more complex to understand its real distribution area (palchetti et al. 2018). piper malgassicum papini, palchetti, m. gori, rota nodari and piper tsarasotrae papini, palchetti, m. gori, rota nodari, were recently described as new malagasy species (palchetti et al. 2018) and are of economic interest, since their dried fruits are often mixed with p. borbonense to produce the typical malagasy spice called in local language “voatsiperifery” pepper. the aim of the investigation was to understand how the malagasyan species might have been originated and their relationships with the african and the asian species. this knowledge will help to understand how the malagasyan species used as spices may be related to p. nigrum with possible future biotechnological implications. the chosen method for anwering the research goal was the analysis of dna sequences both of nuclear and plastidial origin and the chromosome numbers of p. malgassicum and p. tsarasotrae. 2. materials and methods a first round of sample collection within the internal area of madagascar was conducted in 2016 and the samples have been submitted to analyses. the results have been reported in palchetti et al. (2018) but, in order to get a deeper knowledge about the genetic asset of the two species and to confirm the obtained results e second round of sample collection has been carried on in 2019. 4 new plants were collected in two different areas of the ambositra region in madagascar. the first 2 plants, belonging to the p. malgassicum type, were collected in the tropical rainy forest of vohiday and the second 2 plants, belonging to the p. tsarasotrae type, in the semi-dry area of the tsaratsotra village. these plants were compared with the samples of p. tsarasotrae and p. malgassicum which have been used for a previous investigation that included the description of the species (palchetti et al. 2018). samples were conserved either in ethanol 96% either as herbarium sample by the et (tropical herbarium of florence, cset, https://www.bio.unifi. it). some seeds were also germinated in florence for karyotyping. the dna used for this work was extracted from tissue conserved in ethanol 96% (murray et al. 1996; bressan et al. 2014). dna was extracted from 40 mg of the ethanol preserved leaves after drying under vacuum. the starting material was inserted in 2 ml tube, together with tungsten carbide beads, frozen in liquid nitrogen and finely ground in a tissue homogenizer (tissue lyser ®, qiagen). dna was extracted using invisorb spin plant mini kit (stratec molecular®) according to the manifacture’s guidelines. amplification of the trnl (uaa) intron (trnl) and the low copy nuclear gene glyceraldehyde 3-phosphate dehydrogenase (g3pdh) followed respectively the protocols by taberlet et al. (1991) and strand et al. (1997). two new primer pairs were designed using the chloroplast genome sequence of p. kadsura (genbank®: kt223569.1) as template to cover the entire nadh dehydrogenase f (ndhf) plastid gene: ndhf-f3_forward 5’-aggttcttatcgagccgctt-3’ and ndhff3_reverse 5’-gtaagaagaaatgcgccccc-3’ and ndhf-f10_forward 5’-cttcgccgtatgtgggcttt-3’ and ndhf-f10_reverse 5’-tcgaccaaaagcaagcaagag-3’. the amplicons have been directly and bi-directionally sequenced by using the corresponding primers for each amplified sequence. since direct sequencing of g3pdh showed fragments of extra peaked sequencing data, we proceeded with cloning with instaclone pcr cloning kit (thermo scientific®) of the g3pdh amplification products. several colonies for each cloned sample were amplified using t7 and sp6 primers whose sites are located at the boundaries of the cloning region. pcr products were purified using the qiaquick pcr purification kit (quiagen) and sent to the university of 29possible hybrid speciation for two malagasy species of piper l. (piperaceae) florence internal sequencing service cibiaci (www. cibiaci.unifi.it). manual correction and assembly of the sequences was performed using the software multalin (corpet 1988) and mega7 (kumar et al. 2016). unexpectedly, two dna sequences were obtained, after removing the cloning vector fragments, showing a different size: 965bp and 1058bp which were named “short” and “long” sequences respectively (figure 1). at a first sight only the long sequences of g3pdh have been considered as the right ones because, as figure 1. alignment of the long and short g3pdh sequences isolated from p .malgassicum and p. tsarasotrae using bioedit software (hall 1999). shaded fragments represent the primers used for amplification. 30 enrico palchetti et al. observed by smith et al. (2008), no paralogs have been detected in a great deal of other piper species and therefore other results have been discarded as pcr artifacts. in the present work a second thorough revision of the sequencing output has been carried on and showed the presence of overlapping peaks in all the samples and an additional round of analysis of the colonies confirmed the presence of the short sequences. to rule out any doubt, two additional specimen for each species has been collected and submitted to amplification and cloning in order to confirm the presence of the short sequence. as all the samples showed the same pattern, we decide to use also this “short” sequence to study the phylogeny of these two piper species, by comparing with all the accession present online. the sequences used during our investigation are available in genbank®: piper tsarasotrae g3pdh long sequence (mh234634), g3pdh short sequence (mt793801), trnl (mh234638), ndhf (mh234636) and piper malgassicum: g3pdh long sequence (mh234633), g3pdh short sequence (mt793800), trnl (mh234637), ndhf (mh234635). 2.1. phylogenetic analysis the dna sequences were aligned with clustalx 2.0 and checked by eye for manual adjustment. the plastidial and the nuclear sequences were aligned separately to produce matrices that were later combined with the software combinex2_0.py (python version 2.6.4; biopython 1.57), by a. papini, released under gpl license and available at w w w.unifi.it/caryologia/papiniprograms. html as implemented in bandara et al. (2013) and in simeone et al. (2016). the phylogenetic analysis was executed on both cpdna (ndhf and trnl) and nuclear sequences (g3pdh). maximum parsimony analysis was performed with paup* 4.0b1 (swofford 1998, 2001). the genbank sequences of p. humistratum görts & k. u. were used as outgroups both in the nuclear and the plastid genes matrix, following the previous phylogenetic analysis by smith et al. (2008). this sequences used as outgroup resulted belonging to the sister clade with respect to the clade containing the african species and the other related clades in smith et al. (2008). references of the other species used in the analysis are summarized (with genbank® codes) in table 1 in smith et al. (2008). all characters had equal weight and unordered state transitions. gaps were coded with the “simple indel coding” model (simmons and ochoterena 2000), with the software gapcoder (young and healy 2003) and added to the final matrix after the dna sequences as in papini et al. (2004). the evolutionary model implemented in mrbayes for treating gaps was the same as that proposed by lewis et al. (2001) for treating morphological data, the mk model, justified as simple absence/presence of the character without a priori assignment of different weights. we used mrmodeltest 2.0 (nylander 2004) to choose the best evolutionary model of dna sequences on the basis of the akaike information criterion (akaike 1974). the best model was used as settings with mrbayes 3.2.7 (ronquist et al. 2012) for bayesian inference. a maximum likelihood (ml) phylogenetic analysis was carried out with raxml (stamatakis 2014) and the resulting trees were edited with figtree (rambaut and drummond 2010). we mapped the support on the tree branches with the results of the bayesian phylogenetic analysis after removing the first trees with low likelihood values as “burn-in”, as in papini et al. (papini et al. 2007; papini et al. 2011). the remaining trees were used to produce a 50% majority-rule consensus tree in which the percentage indicated on branches was used as a measure of the bayesian posterior probability. 2.2. karyological analysis chromosomes images were obtained from somatic mitoses recorded from root tips of only one plant living in a pot. the procedure was the same as in mosti et al. (2011) and mousavi et al. (2013), with a pretreatment in 8-hydroxyquinoline and fixation in carnoy. then the material was hydrolyzed in hcl and then stained with lacto-propionic-orceine. we observed metaphase plates of meristematic cells, with the technique of fresh squashes of root tips. chromosome counts were made during direct observations with the microscope, and later recounted on enlarged digital images. images were recorded with a microscope leica dm rb fluo. 3. results amplification of two plastid fragments named ndhf and trnl intron was carried on and the amplicons correctly sequenced producing reads of 1860 bp and 920 bp, respectively. cloning of the amplicon of the nuclear gene g3pdh of p. malgassicum and p. tsarasotrae allowed to isolate two haplotypes, which were named “long” (1060bp for p. malgassicum and 1045bp for p. tsarasotrae) and “short” 965 bp (for both species) after their size. we used a total of 71 sequences, considering separately the short and long sequences of p. malgassicum and p. tsarasotrae for g3pdh and the plastid sequences 31possible hybrid speciation for two malagasy species of piper l. (piperaceae) matrix. the total alignment of the g3pdh region was 1127 nucleotides long including gaps. the final parts of the sequences were very variable and hence the alignment was ambiguous. for this reason, we excluded the characters from position 957 to 1127. the rest of the alignment was used for indels (gap) coding (with the 0.02 26hostmannianum_frenchguiana 70pingbienense_china 71nudifolium_costarica 37arboreum_honduras 54capense_kenya 66betle_cultivated 34umbellatum_tanzania 1humistratum_frenchguiana 67betle_tanzania 27auritum_costarica 3austrocaledonicum_newcaledonia 57thomsonii_china 23concepcionis_costarica 40avellanum_frenchguiana 36arboreum_cultivated_ghana 12malgassicum short 68wallichii_china 39obliquum_nicaragua 18aduncum_dominicanrepublic 22guazacapanense_mexico 41costatum_cultivated 25colonense_nicaragua 42augustum_frenchguiana 13tsarasotrae short 16aduncum_nicaragua 17hispidum_mexico 64_vietnam 24pseudofuligineum_honduras 51santum_mexico2 60chaudocanum_china 59caninum_cultivated_australia 52sanctum_nicaragua 50sanctum_mexico3 8guineense_cameroon2 32umbellatum_mexico 7guineense_cameroon1 46puberulum_cultivated 19_tanzania 15rothianum_australia 21yucatanense_mexico 45guahamense_cultivated 62submultinerve_china 44aequale_honduras 63nigrum_cultivated 38imperiale_costarica 11tsarasotrae long 58subpenninerve_malaysia 56semiimmersum_china 65sarmentosum_cultivated 43urophyllum_costarica 61flaviflorum_china 48methysticum_hawaii2 31umbellatum_honduras 6guineense_uganda1 47methysticum_hawaii1 30peltatum_frenchguiana 35umbellatum_cameroon 9guineense_kenya 28auritum_mexico 4borbonense_cultivated_reunion 29peltatum_costarica 69hancei_china 55porphyrophyllum_malaysia 49sanctum_mexico1 10guineense_uganda2 53capense_cameroon 33umbellatum_kenya 20amalago_honduras 14muricatum_malaysia 100/100 89/100 99/100 85/100 100/100 100/10098/100 88/100 100/100 45/100 2malgassicum long figure 2. maximum likelihood tree produced by raxml with nuclear sequences. the supports above or below the branches are, respectively, the bootstrap resampling support with maximum likelihood criterion produced by raxml, and the bayesian support calculated including the information derived from indels. in case the bayesian support is lower than 50, it is not indicated on the figure. 32 enrico palchetti et al. 0.005 27aduncum_dominicanrepublic 1humistratum_frenchguiana 40imperiale_costarica 21guineense_kenya 71 pingbienense_china 8submultinerve_china 52costatum_cultivated 49umbellatum_mexico 14betle_tanzania 65capense_cameroon 20guineense_uganda2 7 flaviflorum_china 61sanctum_mexico1 13betle_cultivated 44peltatum_costarica 69subpenninerve_malaysia 55urophyllum_costarica 16borbonense_cultivated_reunion 3new_nigrum 57guahamense_cultivated 62sanctum_nicaragua 48umbellatum_tanzania 2new_unguiculatum 9wallichii_china 60methysticum_hawaii2 38arboreum_cultivated_ghana 46umbellatum_cameroon 41obliquum_nicaragua 15sarmentosum_cultivated 19tsarasotrae 36yucatanense_mexico 28concepcionis_costarica 6hancei_china 68 thomsonii_china 42auritum_costarica 32colonense_nicaragua 10nigrum_cultivated 22guineense_uganda1 25muricatum_malaysia 53augustum_frenchguiana 50peltatum_frenchguiana 64santum_mexico2 67semiimmersum_china 11piper_vietnam 66capense_kenya 29aduncum_nicaragua 63sanctum_mexico3 56aequale_honduras 24guineense_cameroon2 43auritum_mexico 17malgassicum 23guineense_cameroon1 33pseudofuligineum_honduras 35amalago_honduras 18caninum_cultivated_australia 58puberulum_cultivated 39arboreum_honduras 47umbellatum_kenya 45umbellatum_honduras 30hispidum_mexico 54nudifolium_costarica 51avellanum_frenchguiana 34hostmannianum_frenchguiana 26rothianum_australia 31piper_tanzania 37guazacapanense_mexico 59methysticum_hawaii1 5chaudocanum_china 70porphyrophyllum_malaysia 12austrocaledonicum_newcaledonia 64/96 98/100 77/ 86/100 90/100 80/98 96/85 /100 figure 3. maximum likelihood tree produced by raxml with chloroplast sequences. the support indexes indicated on the tree are the same as in figure 2 (maximum likelihood bootstrap and bayesian support). 33possible hybrid speciation for two malagasy species of piper l. (piperaceae) software gapcoder), resulting in further 99 characters that were inserted after the nucleotide sequences. the plastid genes ndhf and trnl were inserted one after the other in the sequence, producing an aligned matrix of 2016 characters. the coding of indels resulted in further 115 characters. raxml applied on the nuclear g3pdh matrix (indels coding excluded) produced a maximum likelihood tree with bootstrap support obtained with 1000 replicates (figure 2). the support on branches corresponds to maximum likelihood bootstrap support (left) and bayesian support with gaps (on the right). the same method was using for the plastid matrix (figure 3). comparing the two maximum likelihood trees, the one based on nuclear dna data (g3pdh sequences) and that obtained with plastid markers, we could observe that in the first case p. malgassicum, clustered together and as sister group of p. borbonense (figure 2), another species from an island, la reunion, which lies relatively close to madagascar. this relationship is corroborated by 100% maximum likelihood bootstrap (mls) and bayesian (bs) support. the other malagasy species, p. tsarasotrae, typical of arid forest, was more strictly related to the entries of the african species p. guineense, with 100% mls and 100% bs. all these species formed a well characterized clade with 89% mls and 100% bs and their closest species appeared to be asian species p. caninum, (figure 2). the bs without considering gaps coding gave the same support in this clade. the “short” sequences of g3pdh of both p. tsarasotrae and p. malgassicum clustered together within a group of asian species, mainly originating from malaysia and australia with 98% mls and 100% bs (figure 2). the (phylogenetic) story told by the data obtained from chloroplast genome sequences was quite different: the malagasyan species p. tsarasotrae and p. malgassicum clustered together with the phytogeopraphically close p. borbonense with 90% mls and 100% bs, while the 5 accessions of the african p. guineense were in a more external condition with respect to the former group and separated in two groups, one from cameroon (nw africa) and one from uganda/kenya (central-east africa). all these species together formed a monophyletic group with 64% mls and 96% bs (95% bayesian support in the analysis without gaps). also in this case p. caninum, together with p. rothianum, was the outgroup to the african + malagasy species (figure 3) with 80% mls and 98% bs (99% without gaps). adding indels data to the matrix did not appear to increase the support value of nodes in the plastidial genes tree. the counted chromosome numbers varied from 2n=46±2 in p. malgassicum (figure 4a) to 2n=36± 2 in p. tsarasotrae (figure 4b). the uncertainty in the counts, that should be taken only as preliminary result, derived from the small size of the chromosomes (many of them less than 1 μm of length), the low amount of metaphases in the root tips of the plants cultivated in florence and the apparently small size of the mitotic spindle, leading a b figure 4. chromosomes. a) p. malgassicum number of chromosomes: 2n = about 46. bar = 5 μm; b) p. tsarasotrae: 2n = about 38. bar = 5 μm. 34 enrico palchetti et al. to partial overlapping of many of the small chromosomes. th e two currently known areal of the two species (two new localities discovered here) is shown in figure 5. 4. discussion th e fact that the phylogenetic history based on the chloroplast markers told a diff erent tale with respect to the tree produced with nuclear markers may be explained with a possible ancient hybridization/introgression event with pollen coming from an ancestor of the african p. guineense and reaching the ancestor of p. tsarasotrae, that would hence share some part of the nuclear genome with the african species. th e only species of piperaceae analyzed under the point of view of the type of plastid inheritance was a species of peperomia, which resulted to have only maternal plastidial inheritance (corriveau and coleman 1988). th e presence of the “short” g3pdh nuclear sequences may be related to a still more ancient hybridization event involving the ancestor of the malagasy species and some ancestor of asian origin. also in this case probably, with asian pollen entering in contact with the ancestoŕ s stigma of the malagasyan species. as a matter of fact the closest relatives to the african species sensu lato (including the malagasy and the reunion species) are asian, with the closest species (among those here sampled) apparently from malaysia (figure 3 and figure 4). apparently interspecifi c hybrids can be obtained in genus piper also experimentally (vanaja et al. 2008), while the hybrid origin of several andean species was already proposed by quijano-abril et al. (2006). th e presence of paralogs of g3pdh in angiosperms may represent a problem in several phylogenetic analysis (hurteau and spivack 2002); liu et al. 2009; sun et al 2012). however, here most of the indels were found in the introns of the gene and hence we are not able to assess the functionality of the short sequences. the preliminary results about the chromosome numbers scored about 2n=46+-2 in p. malgassicum and 2n=36+-2 in p. tsarasotrae. th e uncertainty in the counts was due to the small dimensions of the chromosomes that were observed in most of the species of the genus, together with stickiness (samuel 1987; samuel and morawetz 1989), the low amount of metaphases in the root tips of the plants cultivated in vitro and the apparently small size of the mitotic fuse, leading to partial overlapping of many of the small chromosomes. th e mitotic spindle can reach dimensions up to 60 μm (wühr et al. 2008; petry 2016), while in p. malgassicum and p. tsarasotrae it was about 15-20 μm (see figure 4). th e chromosome numbers in genus piper are very variable, ranging from 2n=26 to 2n=104, with some species apparently able to possess several possible chromosome numbers (samuel 1987). most new world species show a karyotype of 2n=26 and x=13 (samuel and morawetz 1989), with some exceptions having 2n=28 chromosomes (maugini 1953). in asia tetraploids 2n=52 would prevail (samuel 1987). no data was available for african and malagasy species up to the here presented results. however, the clear diff erence in karyotype between p. tsarasotrae and p. malgassicum, two species otherwise strictly phylogenetically related, may confi rm a possible hybridization/introgression event with a species with a diff erent chromosome number with respect to the ancestor of the malagasyan species. as a matter of fact, also nair et al. (1993) explained the observation of a triploid plant of p. nigrum (2n=78) as the result of a natural crossing between 2n=52 and 2n=104 plants. figure 5. geographical localization of the sampling area for p. tsarasotrae (yellow) and p. malgassicum (white). area of the sampling campaign of 2018 (a and b) and 2019 (a and b) for p. tsaratsotrae (a/a) and p. malgassicum (b/b) respectively. 35possible hybrid speciation for two malagasy species of piper l. (piperaceae) the progeny showed a range of variation from 2n=52 to 2n=104 and production of aneuploid viable pollen (nair et al. 1993). hybridization may influence diversity, including gene flow from one taxon to another (introgression) and the formation of new, stable hybrid taxa and, possibly, speciation (mallet 2007; vallejo‐marín and hiscock 2016). as preliminary guess, the two different chromosome numbers of the malagasyan species may have arisen as a consequence of hybridization of a 2n=52 species with a 2n=26 (p. tsarasotrae) and with another 2n=52 species (p. malgassicum), respectively, with following aneuploid reductions. the two species have close areals (they are almost sympatric), even if they tend to occupy different habitats, more arid p. tsarasotrae (not lianous habit) and more humid (lianous habit) p. malgassicum. such proximity of two closely related species may be considered another possible indication of a relatively fast speciation event. a discordance between plastid and nuclear inheritance inferred through dna sequencing has been often related to reticulate evolution and species of hybrid origin (garcía et al. 2014; stefanović et al. 2007; aubriot et al. 2018), even if the karyological data may be a decisive evidence, it has been rarely used in relationship to the dna sequence evidence as, for instance in selvi et al. (2002). here the hypothesis of an hybrid origin for the two investigated species may explain the presence of a double g3pdh sequence in both of them. the relationship between the malagasyan species and p. guineense with the asian species as members of piper s. s. was already proposed by jaramillo and callejas (2004) and jaramillo et al. (2008) as a result of a dispersal event, and our results do not disagree with this position. apparently, in africa and madagascar the conditions leading to the wide diversification observed in south-american piper (martines et al. 2015) are lacking or less capable of influencing the speciation process. 5. conclusions the surprising discrepancy between the nuclear and the plastid phylogeny could be explained with an ancestral introgression event due probably to pollen contribution from an ancestor of the african mainland p. guineense towards the ancestor of p. tsarasotrae. the presence of possible paralogs of the nuclear gene g3pdh, clustering together with more distantly related asian species lead to the hypothesis that a second more ancient hybridation/introgression event would have occurred between south asian species and the ancestor of the malagasyan species. the chromosome numbers observed in the malagasyan species would confirm different evolutionary history. further studies about the karyotypes of the malagasy species, the african p. guineense and p. borbonense will be necessary together with the investigation of the possible presence of short paralog sequences of g3pdh in p. borbonense. acknowledgments the authors would like to acknowledge dr. nicola gandolfi from the ngo tsiryparma, ambositra, madagascar, for the support given in the germplasm collection in field. references akaike h (1974) a new look at the statistical model identification. ieee trans autom control 19: 716– 723. https://doi.org/10.1109/tac.1974.1100705 aubriot x, knapp s, syfert mm, poczai p and buerki s (2018) shedding new light on the origin and spread of the brinjal eggplant (solanum melongena l.) and its wild relatives. am j bot 105: 1175–1187. https:// doi.org/10.1002/ajb2.1133 bandara n, papini a, mosti s, brown t, smith l (2013) a phylogeny of onobrychis and its relationships with allied genera of hedysareae. turk journ of bot. 37(6): 981-992. https://doi.org/10.3906/bot-1210-32 bressan e a, rossi ml, lee tsg, figueira a (2014) extraction of high-quality dna from ethanol-preserved tropical plant tissues.” bmc research notes 7: 268. https://doi.org/10.1186/1756-0500-7-268 burger w (1972) evolutionary trends in the central american species of piper (piperaceae). brittonia 24: 356-362. https://doi.org/10.2307/2805498 corpet f (1988) multiple sequence alignment with hierarchical clustering. nucleic acids res 16:10881– 10890. https://doi.org/10.1093/nar/16.22.10881 corriveau jl, coleman aw (1988) rapid screening method to detect potential biparental inheritance of plastid dna and results for over 200 angiosperm species. am j bot 75: 1443–1458. https://doi. org/10.2307/2444695 de candolle c (1869) piperaceae. in: de candolle c, editor. prodromus systematis naturalis regni vegetabilis, vol. 16. part 1. paris, france: masson, pp. 235-471 (in latin). de candolle c (1923) piperacearum clavis analytica. candollea 1: 65-415 (in latin). 36 enrico palchetti et al. garcía ma, costea m, kuzmina m, stefanović s (2014) phylogeny, character evolution, and biogeography of cuscuta (dodders; 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pre-anthesis and post-anthesis. programmed cell death hallmarks were examined in parallel to these stages. at pre-anthesis, the stigmatic papillae were ovoid and their dense cytoplasm were rich in insoluble polysaccharide and protein. at post-anthesis, vacuolization and enlargement were quite evident in papillae. besides, the protein content decreased, but reactive oxygen species increased in comparison to the pre-anthesis stage. although no significant change in superoxide dismutase activity was detected, catalase activity decreased and hydrogen peroxide content increased at post-anthesis. dapi stained nuclei appeared rounded and smooth appearance at preanthesis, however, some invaginations and fragmentation in nuclei were observed at post-anthesis. although, tunel staining was negative at pre-anthesis, while tunel positive reaction was significant in the nuclei of papillae at post-anthesis. in comparison to the pre-anthesis, the number of fragmented nuclei monitored by dapi and tunel staining increased at post-anthesis. keywords: programmed cell death, papillae, reactive oxygen species, sexual plant reproduction, tunel. 1. introduction brassica oleracea is a member of brassicaceae family consisting of 4060 species (bayer et al. 2019). it is an important agronomic plant due to its consumption as a vegetable (neik et al. 2017). flowers of b. oleracea have 4 sepals, 4 petals, 2 short and 4 long anthers, and one pistil (arın 2005). stigma is the pollen receptive surface of the pistil (edlun et al. 2004). there are two types of stigmas in angiosperms; wet and dry type. wet type stigmas produce stigmatic secretions while the dry typed stigmas are devoid of stigmatic secretion. in dry typed stigmas, a protein-based pellicle layer covers the papillae cuticle. despite this distinction between wet and dry stigmas, stigmatic papillae are characterized by the expression of various biomolecules such as the various organic matters such as insoluble polysaccharide and protein, enzymes, and 118 aslıhan çetinbaş-genç, cansu bayam, filiz vardar reactive oxygen species (ros) in both types. (mcinnis et al. 2006). for instance, stigmatic papillae contain proteins, lipids, carbohydrates that are necessary for pollen germination (edlund et al. 2004). also, stigmatic enzymes are necessary for stigma receptivity and function (souza et al. 2016). besides, ros regulates the stigma receptivity and plays as a signal molecule in the pollen germination process (zafra et al. 2010). during development, the expression of these biomolecules shows various changes due to several processes such as organ aging, pollination, cell death and etc. programmed cell death (pcd) is a genetically regulated complex process for plant lifespan. it has been proved that pcd is a necessary process both in developmental and defense processes for plants (serrano et al. 2010). so, it is investigated in two types; environmentally induced (epcd) and developmentally regulated (dpcd) (van hautegem et al. 2015). while both types are significant processes, dpcd particularly has an important function during plant life. dpcd occurs in various cells, tissues, or organs for various purposes. however, reproductive development is a rich arena as a showcase for dpcd in plants.  because dpcd can take place in sex determination, anther tapetum, megaspore, synergid, and antipodal cells, nucellus, endosperm, stylar transmitting tissue, stigmatic papillae or etc. (brighigna et al. 2006; vardar and ünal 2012; papini et al. 2011). dpcd is accompanied by various developmental stages at stigma during female reproductive organ development. for instance, stigma no longer required for a flower after pollination and it is eliminated by pcd (rogers 2006). the stigmatic branches of actinidia chinensis are degenerated by dpcd after pollination (ferradas et al. 2014). also, stigma goes pcd when incompatible pollen lands on the stigma. thus, pcd is involved in the pollen selection process of stigma (wu and cheung 2000). for instance, the stigmatic papillae undergo dpcd after incompatible pollination in olea europaea (serrano et al. 2010). characteristic hallmarks of dpcd in plants can be observed by various morphological, biological, and molecular methods. the fundamental descriptive hallmarks are cell shrinkage, cytoplasmic and nuclear breakdown, and dna fragmentation (serrano et al. 2010). also, ros accumulation is among the hallmarks of pcd causing harmful chain effects in the cell. since the accumulation of ros causes oxidative stress, they are balanced by scavenging mechanisms including antioxidants such as superoxide dismutase (sod) and catalase (cat) (apel and hirt 2004; pandhair and sekhon 2006). sod accelerates the conversion of superoxide, which is one of the reactive and toxic ros, to hydrogen peroxide (h2o2). cat catalyzes the deterioration of h2o2 thereby overcoming oxidative stress. so, changes in sod and cat enzyme activity and h2o2 content can give hint about the level of oxidative stress (wang et al. 2010). the aim of the present study is to investigate the morphological, biochemical, and molecular hallmarks of dpcd in stigmatic papillae of brassica oleracea l. the obtained results may provide new insights into the role of dpcd in stigma development and help to improve the knowledge on dpcd hallmarks in reproductive organs. to this end, we assayed different dpcd markers in stigmatic papillae excised from flowers at pre-anthesis and post-anthesis. 2. material and methods 2.1 determination of flower development stage flowers of b. oleracea l. were collected from the vicinity of akçakoca/düzce (turkey) in 2019. the stigma development was divided into 2 main stages (preanthesis and post-anthesis) correlated with some morphological markers of the flower such as the position of calyx and corolla, anther dehiscence, and the absence or presence of pollen on it. the flower buds with calyx covering half of the bud and collected 2-3 days before anthesis were accepted at pre-anthesis. at this stage, there were no pollen grains on the stigma, because the anthers were still indehiscent. the flowers with senescent petals and collected 2-3 days after anthesis were accepted at post-anthesis. at this stage, a lot of pollen grains were visible on the stigma due to anther dehiscence. 2.2 morphological and biochemical changes after fixation in acetic acid:alcohol solution (1:3, v/v), pistils were dehydrated and embedded in paraffin blocks. to investigate the morphological and biochemical changes, sections (8-10 μm) were stained with periodic acid-schiff (pas) (feder and o’brien 1968) for insoluble polysaccharides and, stained with coomassie brilliant blue (cbb) (fisher 1968) for proteins. images were captured using olympus bx-51 microscope and kamer am software. the optical density (od) of insoluble polysaccharide and protein contents of papillae were computed using image j software (rodrigo et al. 1997). 20 papillae were used for each group. 2.3 ros accumulation and antioxidant enzyme activity ros accumulation of papillae was determined according to previous studies (fabian et al. 2019). fresh 119morphological, biochemical and molecular hallmarks of programmed cell death in stigmatic papillae of brassica oleracea l. tissues were labeled by 20 μm 2’,7’-dichlorodihydrofluorescein diacetate (h2dcfda) and images were captured using olympus bx-51 f luorescence microscope and kamer am software. fluorescence intensities of 20 papillae were measured using the image j software. superoxide dismutase (sod) and catalase (cat) activities were detected according to li et al. (2000) and prochazkova et al. (2001), respectively. after homogenization of 0.03 g tissue in 1500 μl 50 mm pbs (ph 7.8) and centrifugation at 12,000×g for 15 min, supernatants were used as sod and cat enzyme source. to measure the spectrophotometric sod activity, 300 μl supernatant (same volume of 50 mm pbs for control) was added to 2400 μl measurement buffer containing 1500 μl of 50 mm pbs (ph 7.8), 300 μl of 130mm l-methionine, 300μl of 750μm nitro blue tetrazolium, 300 μl of 100 μm edta-na2. after incubation under light (50 μmol m-2 s-1) for 3 minutes, sod activity was measured at 560 nm. a non-incubated mixture was used as the blank. to measure the spectrophotometric cat activity, 200 μl supernatant was added to 2400 μl measurement buffer containing 1500 μl of 0.2 m pbs (ph 7.0) with 1% (w/v) pvp and 1000 μl of h2o2. cat activity was measured by the decrease in absorbance for 2 min at 240 nm. h2o2 content was measured according to junglee et al. (2014). after homogenization of 0.03 g tissue in 2000 μl buffer containing 0.1% trichloroacetic acid, 1 m ki, 10 mm phosphate saline buffer, and centrifugation at 12,000×g for 15 min, the supernatant was incubated in dark for 20 minutes. afterward, h2o2 content was measured at 390 nm, spectrophotometrically. 2.4 analysis of dna fragmentation to determine the dna fragmentation, 4’,6-diamidine-2’-phenylindole dihydrochloride (dapi) (schweizer 1976) and terminal deoxynucleotidyl transferase  dutp nick end labeling  (tunel)  (o’brien et al. 1997) test were performed. after fixation in pbs containing 4% paraformaldehyde, pistils were dehydrated and embedded in paraffin blocks. sections (8-10 μm) were stained with dapi and, tunel assay was conducted using the apoptag®  plus fluorescein  in situ  apoptosis detection kit (chemicon, temecula, ca, usa). to avoid false-positive tunel results, tunel results were evaluated considering the control slides included in the kit supplied by the company were used. images were captured using olympus bx-51 f luorescence microscope and kameram software. to evaluate the significant differences in nuclei undergoing pcd, percentages of dapi stained and tunel positive nuclei were presented counting approximately 300 nuclei for each treatment. 2.5 statistical analysis statistical analyses were performed by ibm spss 16.0 software and data were subjected to one-way analysis of variance (anova) with a threshold p value of 0.05. 3. results 3.1 morphological and biochemical changes the morphological and biochemical features of papillae were investigated to analyze their main differences at pre-anthesis and post-anthesis. papillae were ovoid and tightly packed cells at pre-anthesis. they had a thin wall, small vacuole (arrows, fig. 1c, e), and dense cytoplasm (dots, fig. 1c, e). papillae cells lost their tight alignment with the increase of their diameters during the development. in comparison with the pre-anthesis, the lengths of papillae were significantly increased by 84.12% at post-anthesis (fig. 1a). also, the widths of papillae were significantly increased by 42.31%, in comparison with the pre-anthesis (fig. 1b). at post-anthesis, it was remarkable that the vacuole was quite enlarged and covered a large part of the cell (arrows, fig. 1d, f ). moreover, organic matter contents of papillae such as insoluble polysaccharide and protein were changed at post-anthesis (fig. 1c-f ). cytoplasmic content was rich in insoluble polysaccharide and protein contents at preanthesis stage (fig. 1c, e). according to the od results of pas stained papillae, no significant change in insoluble polysaccharide content of papillae was detected between pre-anthesis and post-anthesis (fig. 1g). however, according to the od results of cbb stained papillae, protein contents of papillae were significantly decreased by 33.68% at post-anthesis, when compared with the pre-anthesis (fig. 1h). 3.2 ros accumulation and antioxidant enzyme activity to determine the ros accumulation difference of papillae at two development stages, stigmatic tissues were stained by h2dcfda. while ros accumulation was poor at pre-anthesis, an increase in ros accumulation was quite remarkable at post-anthesis (arrows, fig. 2a, b). to present the subtler differences, fluorescence intensities of h2dcfda labeled papillae were measured. when compared with the pre-anthesis, the fluorescence intensity of h2dcfda was significantly increased by % 31.69 at post-anthesis (fig. 2c). to reveal the effects of ros accumulation on the antioxidant system, changes 120 aslıhan çetinbaş-genç, cansu bayam, filiz vardar in sod-cat activity and h2o2 content were investigated. when compared to pre-anthesis, no significant change in sod activity was detected at post-anthesis (fig. 2d). however, h2o2 content was significantly increased by %23.21 (fig. 2e) and cat activity was significantly decreased by %37.5, at post-anthesis (fig. 2f). 3.3 analysis of nuclear dna fragmentation to determinate the nuclear morphology and dna fragmentation, dapi staining and tunel tests were performed. dapi stained nuclei were showed rounded and smooth appearance at pre-anthesis. the spherical nuclei of papillae emitted bright blue fluorescence and the chromatin was dispersed regularly (arrows, fig. 3a). however, nuclei lost their rounded appearance and some invaginations and fragmentation were observed at postanthesis (arrows, fig. 3b). in comparison with the preanthesis, the number of fragmented nuclei monitored by dapi staining was significantly increased about 11-fold at post-anthesis (fig. 3e). tunel assay results were in parallel with the dapi results. although tunel staining was negative at pre-anthesis, the tunel positive reaction was significant in the nuclei of papillae at postanthesis (arrows, fig. 3c, d). the number of fragmented nuclei monitored by tunel staining was significantly increased about 6-fold at post-anthesis (fig. 3f). 4. discussion dpcd is a major process during reproductive development in plants and occurs at various developmental stages (wang et al. 2020). atrophy of tapetum, nonfigure 2. ros accumulation and antioxidant enzyme activity of papillae at pre-anthesis and post-anthesis. a ros accumulation at pre-anthesis. b ros accumulation at post-anthesis. c fluorescence intensity of h2dcfda at pre-anthesis and post-anthesis. d change in sod activity. e change in h2o2 content. f change in cat activity. white arrows indicate the low ros accumulation and red arrows indicate the high ros accumulation. bar: 20 μm. figure 1. morphological and biochemical changes of papillae at pre-anthesis and post-anthesis. a length of papillae. b width of papillae. c pas stained papillae at pre-anthesis. d pas stained papillae at post-anthesis. e cbb stained papillae at pre-anthesis. f cbb stained papillae at post-anthesis. g od of pas stained papillae. h od of cbb stained papillae. black arrows indicate the vacuoles, red arrows indicate the cell wall and points indicate the cytoplasms. bar: 20 μm. 121morphological, biochemical and molecular hallmarks of programmed cell death in stigmatic papillae of brassica oleracea l. functional megaspores, nucellus, synergids, antipodal, and suspensor cells are some examples of dpcd in the development of reproductive organs (kurusu and kuchitsu et al. 2017; buono et al. 2019). also, dpcd may take place at stigmatic branches or stigmatic papillae during female reproductive organ development. especially papilla cell death is a good model for studying on pcd process (ye et al. 2020). serrano et al. (2010) have been reported that stigmatic papillae degenerated by dpcd after the pollination process in olea europaea. ferradas et al. (2014) have been described stigmatic branches of actinidia chinensis degenerated after pollination. besides, huang et al. (2020) have been reported the pcd in stigmatic papillae of raphanus sativus and brassica napa after pollination. according to our results, stigmatic papillae degenerated by dpcd after the anthesis stage. balk and leaver (2001) have been indicated the alterations in tapetal cell morphology of helianthus annuus, during dpcd. also, qiu et al. (2008) have been reported structural disintegration in non-functional megaspores of lactuca sativa during dpcd. serrano et al. (2010) have been reported the plasma membrane damage and alterations of cell morphology in stigmatic papillae during pcd. also, ferradas et al. (2014) have been indicated the progressive vacuolization and organelle disintegration in stigmatic branches during pcd. similarly, we detected some alterations in the shapes of papillae cells at post-anthesis. these alterations were probably related to both the increase of their diameters and dpcd process. during dpcd processes, researchers have been reported the vacuolization in the inner integument of brassica napus (wan et al. 2002), in tapetal cells of oryza sativa (ku et al. 2003) and in tapetum and filament of lathyrus undulatus (vardar and ünal 2012). besides, vacuolization was reported during dpcd processes ofstyle tissue of ficus carica (aytürk and ünal 2018), stigma of arabidopsis thaliana (gao et al. 2018), and anther and ovule of opuntia robusta (hernandezcruz et al. 2019). parallel to these literatures, it was remarkable that the vacuoles were large and covered the large part of the cell at the post-anthesis stage which we detected the dpcd. researchers have been reported the decrease in the protein content of cytoplasm in petals of nicotiana tabacum (serafini-fracassini et al. 2002), in tepals of iris and alstroemeria (wagstaff et al. 2005) and lilium candidum (mochizuki-kawai et al. 2015) during dpcd. similar to these findings, we detected the decrease in the protein content of papillae at the post-anthesis stage which we detected the dpcd. ros is the major regulator of plant growth and development due to its interaction capability with all cellular substances such as protein, lipid, signaling molecules, hormones and etc. (waszczak et al. 2018; sankaranarayanan et al. 2020). ros acts as cellular signaling molecules in lower doses. however excessive ros production leads to pcd (oracz and karpinsky 2016). yadegari and drews (2004) have been specified that ros plays vital role in the control and implementation of dpcd of aleurone and endosperm cells. also, hayashi et al. (2001) have indicated that ros accumulation in the central cell of the embryo sac acted as a signal molecule in dpcd of antipodal cells. besides, tripathi and tuteja (2007) have been reported that ros is accompanied to dpcd process in sepals, petals, and ovules. duan et al. (2014) have been specified that ros production caused cell wall alteration for the reception of pollen tubes in synergid cells of arabidopsis thaliana by causing dpcd in them. also, van durme and nowack (2016) have been implicated that ros signal regulates the dpcd of tapetal cells at the right time. ros also has a role in signaling networks promoting pollen germination and pollen tube growth on stigma. thus, the concentration of ros on the stigma affects the stigma receptivity and germifigure 3. analysis of nuclear dna fragmentation. a dapi stained spherical nuclei at pre-anthesis. b dapi stained degenerated nuclei at post-anthesis. c tunel negative reaction at pre-anthesis. d tunel positive reaction at post-anthesis. e dapi stained fragmented nuclei rate. f tunel positive nuclei rate. white arrows indicate the nuclear morphology and red arrows indicate the tunel positive nuclei. bar: 20 μm. 122 aslıhan çetinbaş-genç, cansu bayam, filiz vardar nation capability of pollen (zafra et al. 2010). breygina et al. (2020) and zhang et al. (2020) have been reported that proper doses of ros in stigma exudate are important for the communication between the pollen/pollen tube and female tissues at various stages. however, excessive ros accumulation may induce the dpcd of papillae. researchers have been reported ros-mediated dpcd occurred in papillae during incompatible pollination in the olea europaea (serrano et al. 2015). according to our results, the intense ros signal was quite remarkable in papillae cells at the post-anthesis stage that we detected the dpcd. one of the most commonly occurring and most stable ros is h2o2. it generated by the reduction of superoxide anions via sod. also, cat breakdown h2o2 to h2o and o2 (yanık et al. 2018). enzymes such as sod and cat that regulate the h2o2 content show differential expression during dpcd (singh et al. 2016). according to our results, no significant change in sod activity was detected at post-anthesis when compared to pre-anthesis. however, h2o2 content was significantly increased at post-anthesis. also, cat activity was significantly decreased at post-anthesis. researchers have been reported that the high h2o2 content of stigmatic papillae may be related to the stigmatic receptivity or dpcd process (serrano et al. 2012; xie et al. 2014). therefore, high h2o2 content in the post-anthesis indicates that the stigma is receptive at this stage. however, since dpcd occurs in papillae at post-anthesis, high h2o2 content is more likely to be related to pcd. besides, researchers have been indicated that high h2o2 content is involved dpcd process of petal and tapetal cells (tripathi and tuteja 2007; xie et al. 2014). also, researchers have been reported that ros increased due to the increased sod and decreased cat activities in sepals of daylily that undergoing dpcd (panavas and rubinstein 1998). dapi staining is one of the most commonly used methods for check the nuclei morphology. researchers have been reported the various alterations by dapi staining in chromatin, dna, and nucleus during dpcd; such as nuclei shrinkage and chromatin condensation in tapetal cells of lobivia rauschii and tillandsia albida (papini et al. 1999), chromosomal degradation in suspensor and endosperm of vicia faba (wredle et al. 2001) and, nuclear deformation and volume changes in synergid and antipodal nuclei of t. aestivum (an and you 2004). also, researchers have been reported the nucleus and dna degradation in suspensor and endosperm of phaseolus coccineus (lombardi et al. 2007), nucleus degeneration and chromatin fragmentation in synergids of malus domestica (tagliasacchi et al. 2007) and, chromatin condensation in non-functional megaspores of lactuca sativa (qiu et al. 2008) during dpcd. besides, nucleus and chromatin deformations in anther wall cells of lathyrus undulatus (vardar and ünal 2012) and, chromatin condensation in stamen primordia of cucumis sativus (pawełkowicz et al. 2019) were indicated as doped hallmarks by various researchers. also, shi et al. (2020) have been reported chromatin fragmentation in suspensor cells undergoing pcd of nicotiana tabacum. in parallel to these literatures, we detected the various invaginations and fragmentation in papillae nuclei at the post-anthesis stage which we detected the dpcd. similarly, ferradas et al. (2014) have been reported that dpcd occurs in stigmatic branches and papillae of actinidia chinensis after pollination, and chromatin condensation and nucleus degradation are quite remarkable during this dpcd process. besides, gao et al. (2018) have been shown the nucleus degradation in stigmatic papillae of arabidopsis thaliana during dpcd. also, tunel method is one of the most common and definitive methods used to determine pcd. it allows the determine pcd by marking the free 3 ’oh ends of dna formed by endonucleases in cells. researchers have been detected the tunel positive reaction in cells undergoing pcd such as in filament of hordeum vulgare (wang et al. 1999), in tapetal cells of zea mays (gonzález-sanchez et al. 2004) and, in suspensor and endosperm of phaseolus coccineus (lombardi et al. 2007). also, tunel positive nuclei were detected in anther wall cells and filament of lathyrus undulatus (vardar and ünal 2012), in style of ficus carica (aytürk and ünal 2018), in anther of opuntia robusta’s female flower (hernándezcruz et al. 2019) and in stamen primordia of cucumis sativus’s female flower (pawelkowicz et al. 2019). shi et al. (2020) have been reported tunel positive reaction in suspensor undergoing pcd of nicotiana tabacum. jimenez-duran et al. (2021) have been detected dna fragmentation by tunel assay during the dpcd process of marathrum schiedeanum’s central cell. at the post-anthesis stage, we also detected the tunel positive nuclei in papillae. similarly, ferradas et al. (2014) have been detected tunel positive nuclei in actinidia chinensis’s stigmatic branches and papillae undergoing dpcd. also, gao et al. 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4) to detect isolation by distance among the geranium species. we used traditional morphological and molecular methods to assess genetic diversity and genetic structure in the geranium species. a total of 129 amplified polymorphic bands were generated across 13 geranium species. the size of the amplified fragments ranged from 150 to 3000 bp. g. stepporum showed the highest values for the effective number of alleles (ne = 1.30) and shannon information index (i =0.35). significant anova results (p <0.01) showed differences in quantitative morphological characters in plant species. g. sylvaticum showed high genetic diversity. mantel test showed a significant correlation (r = 0.17, p=0.0002) between genetic distance and geographical distance, so isolation by distance (ibd) occurred among the geranium species. according to the scot markers analysis, g. kotschyi and g. dissectum had the lowest similarity, and the species of g. sylvaticum and g. pratense had the highest similarity. the present study revealed that a combination of morphological and scot methods could distinguish the species of geranium keywords: morphology, species identification, scot (start codon targeted). introduction genetic diversity helps to understand species characteristics and adaptation strategy in an ever-changing environment and aids in understanding the evolutionary relationship among species (erbano et al. 2015). several programs have been launched to conserve plant diversity while utilizing and 142 bo shi, majid khayatnezhad, abdul shakoor preserving plant genetic materials(gomez et al. 2005). given the importance of genetic diversity in conservation strategies and programs, it is necessary to study genetic diversity in plant species, particularly threatened and rare species (cires et al. 2013). population size is a pivotal factor to fathom genetic diversity because it disentangles the variation in a gene (ellegren and galtier 2016; turchetto et al. 2016). genetic variation and diversity are essential parameters for species survival; usually, individuals cannot exchange genetic materials due to geographical and genetic barriers. therefore, this could generate a scattered population. since these individuals have limited gene flow, there is a greater chance of a decline in population size (frankham 2005). around 325 species of geranium l. occur in the world (aedo et al. 1998). geranium species have medicinal and horticulture uses; henceforth, some systematic studies were conducted to better utilize geranium species in plant systematics and plant industry (aedo 1996). recent classification system divides geranium into three subgenera (yeo 2008). among them, subgenera geranium has 300 species (aedo and estrella 2006). g. sect. dissecta occurs in the eurasian, mediterranean, and himalaya regions. the majority of tuberosa (boiss.) members are found in western europe, central asia, and northwest africa. vegetative characters aid to classify tuberosa into subsections tuberosa (boiss.) yeo and mediterranea r. knuth (yeo 2008). previous studies identified the center of diversity of the g. subsect. tuberosa in iran and turkey (aedo and estrella 2006; aedo et al. 2007; esfandani-bozchaloyi et al. 2018a, 2018b, 2018c, 2018d). the geranium genus has twenty-two to twentyfive species in iran (schonbeck-temesy 1970; onsori et al. 2010). leaves and fruit morphology are valid characters to identify the geranium species (salimi moghadam et al. 2015). nonetheless, advancement in molecular science has revolutionized plant systematics and taxonomy to provide authentic results. start codon targeted (scot) polymorphism is one of the latest addition in molecular science. scot is a simple dna marker system. it works on the short conserved region in plant genes surrounding the atg (collard and mackill 2009) translation start codon (collard and mackill 2009). start codon targeted (scot) is affordable and produces reliable results and robust genetic profile of plant species (collard and mackill 2009, wu et al. 2013, luo et al. 2011). it is essential to mention that iran is the center of the diversity of geranium species. however, no study has been conducted to study genetic diversity via the scot molecular system. our study is the first attempt to utilize scot markers to check the genetic diversity in iran. we used 115 plant samples. our objectives were 1) to check genetic diversity among geranium species 2) genetic structure of the geranium 3) do the geranium species exchange genes? 4) to detect isolation by distance among the geranium species materials and methods plant materials we collected thirteen geranium species from different parts of iran (table 1, figure 1). morphological and molecular methods were used to study geranium species. one hundred fifteen plant samples (5-10 per plant species) were examined for morphometric analyses. we collected the following species for our study purpose. g. dissectum l. (sec. dissecta); g. persicum schönb.tem., g. tuberosum l., g. kotschyi boiss., g. stepporum p.h.davis (sec. tuberosa subsect. tuberosa (boiss.) yeo); g. platypetalum fisch. & c. a. mey., g. gracile ledeb. ex nordm., g. ibericum cav. (sec. tuberosa subsect. mediterranea r. knuth). g. columbinum l., g. rotundifolium l., g. collinum stephan ex willd, g. sylvaticum l., g. pratense (sec. geranium). different occurrence records were checked and correct identification of species was carried out by khayatnezhad in iran. (davis 1967, schonbeck-temesy 1970; zohary 1972, aedo et al. 1998b, janighorban 2009). we mentioned the sampling sites details in table 1. plant specimen vouchers were deposited in the herbarium of azad islamic university (haiu). morphometry we studied 21 qualitative and 19 quantitative plant morphology characters. data were transformed (mean= 0, variance = 1), before ordination (podani 2000). euclidean distance was implemented to cluster and ordinate plant species dna extraction and scot assay we isolated dna from fresh leaves. leaves were dried. the extraction of dna was carried out in accordance with the previous procedure. (esfandani-bozchaloyi et al. 2019). an agarose gel was used to validate the purity of the dna. 25 scot primers were used (collard & mackill (2009). among them, we selected ten primers that had simple, expanded, and rich polymorphism 143the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bands (table 2). overall, the polymerase chain reaction contained 25μl volume. this 25 volume included ten milliliters of tris-hcl buffer, 500 milliliters of kcl, 1.5 milliliters of mgcl2, 0.2 milliliters of each dntp, 0.2 milliliters of a single primer, 20 ng genomic dna, and three units of taq dna polymerase. (bioron, germany). we observed the following cycles and conditions for the amplification. at 94°c, a five-minute initial denaturation step was performed, followed by forty cycles of one minute at 94°c. then 1-minute cycle was at 52-57°c followed by two minutes at 72°c. in the end, the final extension step was performed for seven to ten minutes at 72°c. we confirmed the amplification steps while observing amplified products on a gel. a 100 base pair molecular ladder/standard was used to validate the scale of each band. (fermentas, germany). data analyses we used the ward methods and the unweighted pair group approach with arithmetic mean (upgma). multidimensional scaling and principal coordinate analysis were also used (podani 2000). analysis of variance table 1. geranium species and populations, their localities and voucher numbers. sp. no. of collected accessions locality latitude longitude altitude (m) 1. g. geranium dissectum 10 esfahan, ghameshlou, sanjab 37°07’48” 49°54’04” 165 2. geranium collinum 5 lorestan, oshtorankuh, above tihun village 37°07’08” 49°54’11” 159 5 east azerbaijan, ahar, kaleybar 38°52’93” 47°25’92” 1133 5 east azerbaijan, kaleybar, shojabad 38°52’93” 47°25’92” 1139 3. geranium rotundifolium 5 tehran, tuchal 35°50’36” 51°24’28” 2383 4. geranium columbinum 5 ardabil, khalkhal 35°42’29” 52°20’51” 2421 5. geranium sylvaticum 9 east azerbaijan , ahar, kaleybar 38°52’39” 47°25’92” 1133 6. geranium pratense 10 east azerbaijan, kaleybar, shojabad 38°52’39” 47°25’92” 1137 7. geranium platypetalum 8 hamedan, nahavand 38°52’39” 47°23’92” 1144 8. geranium gracile 9 mazandaran, tonekabon-jannat rudbar 36°48’47” 50°53’68” 1600 9. geranium ibericum 7 mazandaran, noshahr, kheyrud kenar forest 36°38’05” 51°29’05” 1250 10. geranium kotschyi 10 alborz, karajqazvin 35°49’23” 51°00’04” 1365 11. geranium tuberosum 8 kermanshah, islamabad 38˚52’39” 47°25’92” 1133 12. geranium stepporum 9 esfahan, fereydunshahr 35˚50’03” 51°24’28” 2383 13. geranium persicum 10 tehran, firuz kuh 35°43’15” 52°04’12” 1975 table 2. scot primers used for this study and the extent of polymorphism. note: tnb the number of total bands, npb: the number of polymorphic bands, ppb (%): the percentage of polymorphic bands, pi: polymorphism index, emr, effective multiplex ratio; mi, marker index; pic, polymorphism information content for each of cbdp primers primer name primer sequence (5’-3’) tnb npb ppb pic pi emr mi scot-1 caacaatggctaccacca 16 16 100.00% 0.37 3.88 8.56 1.65 scot-3 caacaatggctaccaccg 20 20 100.00% 0.55 6.23 8.23 2.47 scot-6 caacaatggctaccacgc 15 14 93.74% 0.47 5.66 7.56 3.67 scot-11 aagcaatggctaccacca 13 12 92.31% 0.34 3.21 5.60 5.55 scot-14 acgacatggcgaccacgc 10 10 100.00% 0.36 4.86 9.55 3.45 scot-15 acgacatggcgaccgcga 9 8 84.99% 0.43 4.91 7.43 4.85 scot-16 ccatggctaccaccggcc 13 13 100.00% 0.44 4.34 11.55 3.44 scot-17 catggctaccaccggccc 16 16 100.00% 0.37 3.88 8.56 1.65 scot-18 accatggctaccaccgcg 20 20 100.00% 0.55 6.23 8.23 2.47 scot-19 gcaacaatggctaccacc 15 15 100.00% 0.39 3.25 10.11 1.87 mean 13.4 12.9 97.78% 0.46 4.9 8.4 3.6 total 134 129 144 bo shi, majid khayatnezhad, abdul shakoor (anova) was used to determine the morphological differences between species and populations. pca analysis (podani 2000) was done to find the variation in plant population morphological traits. the past program, version 2.17, was used to perform multivariate and all required calculations (hammer et al. 2001). we encoded scot bands as present and absent. the appearance and absence of bands were indicated by the numbers 1 and 0. we calculated all necessary parameters to study genetic diversity. in addition to genetic diversity parameters, we also assessed the marker index (mi) of primers because mi detects polymorphic loci (ismail et al. 2019). marker index was calculated according to the previous protocol (heikrujam et al. 2015). the effective multiplex ratio (emr) and the number of polymorphic bands (npb) were calculated. gene diversity-associated characteristics of plant samples were calculated. nei’s gene diversity (h), shannon information index (i), number of effective alleles (ne), and percentage of polymorphism (p% = number of polymorphic loci/number of total loci) were measured (shen et al. 2017). unbiased expected heterozygosity (uhe), and heterozygosity were assessed with the aid of genalex 6.4 software (peakall and smouse 2006). neighbor-joining (nj) and networking were studied to fathom genetic distance plant populations (freeland et al. 2011). the mantel test was carried out to find the correlation between genetic and geographical distances (podani 2000). our goal was to know the genetic structure and diversity. therefore, we also investigated the genetic difference between populations by analyzing molecular variance (amova) in genalex 6.4 (peakall and smouse 2006). furthermore, gene flow (nm) was estimated through genetic statistics (gst) in pop gene ver. 1.32 (yeh et al. 1999). we also did structure analysis to detect an optimum number of groups. for this purpose, the evanno test was conducted (evanno et al. 2005). it is a common approach to measure genetic divergence or genetic distances through pair-wise fst and related statistics. the mantel test detects spatial processes that shape population structure. we used past software ver. 2.17 to calculate the mantel test ( (hammer et al. 2012 ). for the mantel test, scot data was used to measure nei genetic distance, whereas geographical data was used to calculate the geographic distances in past software. it is calculated based on the sum of the paired differences among both longitudes and latitudes coordinates of the studied populations. the mantel test, as originally formulated in 1967, is given by the following formula. where gij and dij are, respectively, the genetic and geographic distances between populations i and j, considering n populations. because zm is given by the sum of products of distances its value depends on how many populations are studied, as well as the magnitude of their distances results species identification and inter-relationship morphometry significant anova results (p <0.01) showed differences in quantitative morphological characters in plant species. different clustering and ordination methods showed similar patterns. therefore, upgma clustering and pca plot of morphological characters are presented here (fig. 2, 3). in general, plant samples of each species belong to a distinct section, were grouped, and formed a separate cluster. this finding indicates that the morphological characteristics examined may distinguish the geranium species into two main clusters or classes. we did not observe any intermediate types in the specimens. in general, the upgma tree produced two large groups (fig. 2). the morphological characters pca plot (fig. 3) clearly divided the species into distinct groups with no figure 1. map of iran shows the collection sites and provinces where geranium species were obtained for this study. 145the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers intermixing. this is consistent with the upgma tree that was previously described. species identification and genetic diversity ten scot primers were screened to study genetic relationships among geranium species; all the primers produced reproducible polymorphic bands in all 13 geranium species. an image of the scot amplification generated by scot-17 &14 primers is shown in figure 4. a total of 129 amplified polymorphic bands were generated across 13 geranium species. the size of the amplified fragments ranged from 150 to 3000 bp. g. stepporum showed the highest values for the effective number of alleles (ne = 1.30) and shannon information index (i =0.35) (table 3). we reported genetic difference among the geranium species as indicated by amova (p = 0.01) test results. 65% of the total variation was among species, and 35% was within species. pair-wise, fst values showed a significant difference among all studied species (table 4). moreover, genetic differentiation of these species was demonstrated by significant nei’s gst (0.44, p = 0.01) and d_est values (0.155, p = 0.01). high genetic diversity was observed within species (fig. 5) g. sylvaticum (sp5) showed high genetic diversity, as supported by diversity profiles (table 3). the pca plot successfully separated the species into groups. it shows the application of scot molecular markers to differentiate geranium species. pca results strongly support the amova and genetic diversity results. nm results showed 0.21 value. it indicates limited gene flow among geranium species. mantel test with 5000 permutations showed a significant correlation (r = 0.17, p=0.0002) between genetic distance and geographical distance, so isolation by distance (ibd) occurred among the geranium species. nei’s genetic identity and the genetic distance results showed genetic distances among the species (table is not included). g. sylvaticum and g. pratense (sect. geranium). were genetically identical (0.93). the lowest degree of genetic similarity occurred between g. kotschyi and g. dissectum (0.47). the species genetic structure to determine the optimum number of genetic groups, we used structure analysis followed by the evanno test. in the geranium population, we used the admixture model to show interspecific gene flow and ancestrally shared alleles. structure analysis followed by the evanno test produced δk =6. the structure plot (fig. 6) revealed figure 2. upgma clustering of morphological characters revealing species delimitation in subg. geranium. figure 3. pca plots of morphological characters revealing species delimitation in subg. geranium. figure 4. electrophoresis gel of studied ecotypes from dna fragments produced by scot-11 & scot-17. 146 bo shi, majid khayatnezhad, abdul shakoor more information about the genetic structure of the geranium species and shared ancestral alleles and gene flow between geranium species. this plot revealed that genetic affinity between g. sylvaticum and g. pratense (similarly colored) and g. ibericum and g. gracile (similarly colored) are due to shared common alleles. this is in agreement with the neighbor joining dendrogram presented before. the other species are distinct in their allele composition and differed genetically from each other. the low nm value (0.21) suggests limited gene flow between the geranium species and supports genetic stratification as indicated by k-means and structure analyses. the population assignment test also coincided with nm result. we could not detect substantial gene flow among the geranium species. however, we obtained scot and morphological trees (consensus tree) (figure not included). structure plot results showed the high degree of genetic stratification in the geranium species. discussion species identification and taxonomic consideration in phylogenetic systematics, ecology, biogeography, and biodiversity, plant species identification is a central table 3. genetic diversity parameters in the studied geranium species. (n = number of samples, na = number of different alleles, ne = number of effective alleles, i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations). pop n na ne i he uhe %p sp1 6.000 0.244 1.032 0.26 0.23 0.18 55.53% sp2 4.000 0.314 1.044 0.16 0.18 0.23 43.38% sp3 8.000 0.201 1.00 0.33 0.17 0.12 42.23% sp4 5.000 0.341 1.058 0.24 0.27 0.20 53.75% sp5 3.000 0.567 1.062 0.24 0.22 0.113 44.73% sp6 5.000 0.336 1.034 0.23 0.25 0.19 51.83% sp7 4.000 0.344 1.042 0.20 0.23 0.20 57.53% sp8 5.000 0.369 1.011 0.10 0.11 0.12 30.15% sp9 8.000 0.499 1.067 0.14 0.12 0.14 49.26% sp10 9.000 0.261 1.014 0.142 0.33 0.23 43.15% sp11 6.000 0.555 1.021 0.32 0.25 0.28 43.53% sp12 10.000 0.431 1.088 0.35 0.32 0.13 67.53% sp13 3.000 0.255 1.021 0.15 0.18 0.12 42.15% figure 5. pca plot of geranium species based on scot data. figure 6. structure plot of geranium species based on scot-11. 147the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers theme. several evolutionary processes operate to form new species. usually, gene flow occurs between phylogenetically closely related species (schluter 2001, duminil and di michele 2009, ji et al. 2020, sun et al. 2021, niu et al. 2021, zou et al. 2019). genetic diversity and species differentiation is the outcome of isolation by distance, local adaptation, and gene flow (freeland et al. 2011, frichot et al. 2013) the geranium is a relatively complex taxonomic group, and several morphological characters make it difficult to identify and classify geranium species (wondimu et al. 2017). given the complexity, it is necessary to explore other methods that could complement the traditional taxonomical approach (erbano et al. 2015). we examined genetic diversity in geranium by morphological and molecular methods. we mainly used scot markers to investigate genetic diversity and genetic affinity in geranium. our clustering and ordination techniques showed similar patterns. morphometry results clearly showed the utilization or significance of morphological characters in geranium species. pca results also confirmed the application of morphological characters to separate geranium species. the present study also highlighted that morphological characters such as length, bract length, and stipule length could delimit the geranium group. the geranium species highlighted morphological differences. we argue that such a dissimilarity was due to differences in quantitative and qualitative traits. present findings on morphological differences agree with the previous studies (jeiter et al. 2015; salimi moghadam et al. 2015; aedo and pando 2017). polymorphic information content (pic) values are helpful to detect genetic diversity. the current study recorded average pic values of 0.46. this value is sufficient to study genetic diversity in the population (kempf et al. 2016). the previous scientific data (kurata et al. 2019) supports our current high diversity results. interestingly, structure results showed the presence of shared alleles in geranium species. this existence of shared alleles is related to self-pollination in geranium (williams et al. 2000). some geranium members are also pollinated by bees, flies, and honey bees (lefebvre et al. 2019). present findings revealed limited gene flow, and it is quite logical to report low gene flow. similar low gene flow values were recorded while using rapd markers (fischer et al. 2000). other probable reasons for limited gene flow are geographical isolation (fischer et al. 2000) among the geranium species and population. low or limited gene flow results were according to the mantel test results. the mantel test indicated a positive correlation between genetic and geographical distances. therefore, it is concluded that isolation by distance and limited gene determines the geranium population genetic structure. scot data revealed a minimal amount of gene flow among the studied species. it was also supported by structure analysis as geranium species mostly had distinct genetic structures. reticulation analysis also showed some degree of gene f low in geranium species. we did not observe any intermediate forms in our extensive plant collection, but morphological variability within each species did occur to some extent. current findings showed a significant correlation between genetic and geographical distances. our findings revealed that isolation by distance (ibd) existed between geranium species (mantet test results). the magnitude of variability among na, ne, h, and i indices demonstrated a high level of genetic diversity among geranium species. dendrogram and principal component analysis results showed a clear difference among geranium species. this shows the high utilization of the scot technique to identify geranium species. our results have implications for conservation and breeding programs. conclusions the present study investigated the molecular variation of 13 species. molecular and morphometric analysis confirmed morphological and genetical difference between geranium species. this was first attempt to assess genetic diversity through scot molecular markers and morphometric analysis in iran. the current study reported two significant clusters. these two major groups were separated on the basis of genetic and morphological characters. the genetic similarities between 13 species was estimated from 0.47 to 0.93. scot molecular markers analysis, showed that g. kotschyi and g. dissectum had the lowest similarity. current study also reported correlation between genetic and geographical distances. this clearly indicated isolation mechanism involved in the ecology of geranium species. present results showed the potential of start codon targeted to assess genetic diversity and genetic affinity among geranium species. current findings have implications in biodiversity and conservation programs. besides this, 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mrna. rna (cambridge), 25(2): 205-218. caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 3 2021 firenze university press chromomycin a3 banding and chromosomal mapping of 45s and 5s ribosomal rna genes in bottle gourd ahmet l. tek*, hümeyra yıldız, kamran khan, bilge ş. yıldırım development of a protocol for genetic transformation of malus spp federico martinelli1,*, anna perrone2, abhaya m. dandekar3 cytogenetic and cytological analysis of colombian cape gooseberry genetic material for breeding purposes viviana franco-florez, sara alejandra liberato guío, erika sánchez-betancourt, francy liliana garcía-arias, víctor manuel núñez zarantes* palynological analysis of genus geranium (geraniaceae) and its systematic implications using scanning electron microscopy jun wang1,2,*, qiang ye1, chu wang2, tong zhang2, xusheng shi2, majid khayatnezhad3, abdul shakoor4,5 influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats yuri a. kirillov1, maria a. kozlova1, lyudmila a. makartseva1, igor a. chernov2, evgeniya v. shtemplevskaya1, david a. areshidze1,* the morphological, karyological and phylogenetic analyses of three artemisia l. (asteraceae) species that around the van lake in turkey pelin yilmaz sancar1,*, semsettin civelek1, murat kursat2 molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant jinxin cheng1,*, dingyu hu1, yaran liu2, zetian zhang1, majid khayatnezhad3 genetic variations and interspesific relationships in salvia (lamiaceae) using scot molecular markers songpo liu1, yuxuan wang1, yuwei song1,*, majid khayatnezhad2, amir abbas minaeifar3 development of female gametophyte in gladiolus italicus miller (iridaceae) ciler kartal1, nuran ekici2,*, almina kargacıoğlu1, hazal nurcan ağırman1 colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae) harshita dwivedi*, girjesh kumar statistical evaluation of chromosomes of some lathyrus l. taxa from turkey hasan genç1, bekir yildirim2,*, mikail açar3, tolga çetin4 use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater treatment and water of the kamniška bistrica river (slovenia) peter firbas1, tomaž amon2 the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bo shi1,*, majid khayatnezhad2, abdul shakoor3,4 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(1): 3-13, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1353 caryologia international journal of cytology, cytosystematics and cytogenetics citation: jelili a. badmus, samuel a. oyemomi, john o. fatoki, taofeek a. yekeen, olaniyi t. adedosu, peter i. adegbola, musibau a. azeez, elijah a. adebayo, agbaje lateef (2022) antihaemolytic and cytogenotoxic potential of aqueous leaf extract of annona muricata (l.) and its bio-fabricated silver nanoparticles. caryologia 75(1): 3-13. doi: 10.36253/caryologia-1353 received: june 29, 2021 accepted: march 31, 2022 published: july 6, 2022 copyright: © 2022 jelili a. badmus, samuel a. oyemomi, john o. fatoki, taofeek a. yekeen, olaniyi t. adedosu, peter i. adegbola, musibau a. azeez, elijah a. adebayo, agbaje lateef. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid jab: 0000-0003-4785-2609 jof: 0000-0003-3246-9172 tay: 0000-0002-2476-2283 pia: 0000-0002-4008-1728 maa: 0000-0002-0059-0309 eaa: 0000-0002-6574-7928 al: 0000-0001-5302-9892 anti-haemolytic and cytogenotoxic potential of aqueous leaf extract of annona muricata (l.) and its bio-fabricated silver nanoparticles jelili a. badmus1,3,*, samuel a. oyemomi1, john o. fatoki1, taofeek a. yekeen2,3, olaniyi t. adedosu1, peter i. adegbola1, musibau a. azeez2,3, elijah a. adebayo2,3, agbaje lateef2,3 1 department of biochemistry, ladoke akintola university of technology, ogbomoso, oyo state, nigeria 2 department of pure and applied biology, ladoke akintola university of technology, ogbomoso, oyo state, nigeria 3 nanotechnology research group (nano+), ladoke akintola university of technology, ogbomoso, oyo state, nigeria *corresponding author. e-mail: jabadmus@lautech.edu.ng abstract. nanotechnology is widely gaining worldwide application in biology and medicine because of its proven efficacy. annona muricata contains bioactive phytochemicals with an inherent ability to bio-fabricate metal ions nanoparticles (nps). annona muricata aqueous leaf extract and its green bio-fabricated silver nanoparticles were evaluated on red blood cells (rbc) for anti-haemolytic activity and cytogenotoxicity on allium cepa cells. the effects of a. muricata extract (am-e) and its biofabricated silver nanoparticles (am-agnps) were observed at 0.7, 7.0 and 70.0 µg/ml on h2o2-induced haemolysis in rbc and cyclophosphamide-induced cytogenotoxicity on a. cepa cells. results showed significant and concentration dependent anti-haemolytic activity of am-e relative to am-agnps. significant (p<0.05) reduction of mitotic index was observed in the groups treated with am-agnps compared with am-e, which indicates cytotoxic effect of the nanoparticles. the am-e protected a. cepa meristem root cells from cyclophosphamide-induced mitotic repression better than am-agnps. different degree of chromosomal abnormalities such as chromosome-bridge, sticky chromosome, and c-mitosis were observed in all the treatment groups with chromosome-bridge and sticky chromosome being prominent. this study revealed stronger anti-haemolytic efficacy of am-e at higher concentrations compared with am-agnps. chromosomal abnormalities observed in this study suggest greater chromosomal instability as influenced by the nanoparticles compared with the extract on onion cells. the protective effect of the extract against cyclophosphamide-induced chromosomal aberrations may be an indication of its potential as an anti-genotoxic agent. keywords: green synthesis, anti-haemolytic, annona muricata, allium cepa, silver nanoparticles, cytogenotoxicity. 4 jelili a. badmus et al. 1. introduction nanotechnology has captured a great scientific interest worldwide due to its wider objectives cum applications in biology and medicine (shaniba et al. 2017). its fundamental building block resides in the synthesis of nanoparticles (nps) which are products of creation, production, characterization, and manipulation of materials at nano-scale. it enables the amendment of materials at the atomic level with a view to obtain unique properties, which can be annexed for desired applications (gleiter, 2000). the distinct optical, electrical, catalytic properties of metal nanoparticles such as ag, zn, pt, au and pd, and their roles in biological and pharmaceutical applications are being studied intensively due to their unique amenability (jacob et al. 2012; firdhouse and lalitha, 2015; shaniba et al. 2017). silver nanoparticles (agnps) have found extensive use in pharmaceutical and cosmetic industries among other metal nanoparticles owing to their broad utility (sathishkumar et al. 2012; patil et al. 2017; annu et al. 2018; patra et al. 2018). biological synthesis of agnps from natural products viz. bacterial, fungi, yeast and plant extract, and their applications in biology and medicine have tagged them eco-friendly (lokina et al. 2014; shaniba et al. 2017; adebayo et al. 2019a,b). annona is a genus of flowering plants of annonaceae family known for its exotic fruits. four species of the genus such as a. muricata, a. squamosa, a. senegalensis and a. cherimola have been reported to have compelling pharmacological activities (santos-sánchez et al. 2018). the pharmacological activities of the genus have been related to considerable quantity of bioactive principles such as phenolic compounds (flavonoids and phenolic acids) (perrone et al. 2022). a. muricata is the one of the most studied species of the genus annona (santossánchez et al. 2018). a. muricata also known as soursoup is a typical tropical evergreen tree with heart shaped edible fruits and it is ubiquitous in most tropical countries (gavamukulya et al. 2017). pharmacological and traditional uses of the leaf, bark, root, stem, fruit, and seed extracts include hypoglycemic, anti-cough and analgesic (hardoko et al. 2015; coria-tellez et al. 2018). it has also been found useful as antispasmodic, sedative (mishra et al. 2013; moghadamtousi et al. 2015), anti-malarial (somsak et al. 2016), antioxidant (balderrama-carmona et al. 2020), anti-inflammatory (abdul wahab et al. 2018) and anticancer (yang et al. 2015; najmuddin et al. 2016; coriatellez et al. 2018). it contains phytochemicals such as flavonoids, cardiac glycosides, saponins, alkaloids, tannins, phytosterol, and terpenoids giving it the ability to reduce metal ions (vijayameena et al. 2013). previous study from our laboratory have indicated that the physicochemical property of silver nanoparticles synthesized using a. muricata aqueous leaf extract is within a normal range (badmus et al. 2020). the nanoparticles displayed robust biomedical applications such as antidiabetic, antioxidant, antimicrobial and anti-proliferative potential. generally, agnps have wide applications in household materials, food, pharmaceutical and cosmetic industries. the increase and unregulated disposal of the nanoparticles will elevate environmental availability and bioaccumulation (mcgillicuddy et al. 2017). there is a dearth of scientific evaluation of toxicological capability and implication of some identified nanoparticles with strong biomedical presentations. therefore, this research was designed to study the anti-haemolytic, and cytogenotoxic potential of silver nanoparticles synthesized using an aqueous leaf extract of a. muricata on red blood cell and a. cepa cell chromosomes respectively. 2. materials and methods 2.1 collection of plant materials the leaves of annona muricata were collected from ologundudu, ondo state, nigeria and identified by a taxonomist at the department of pure and applied biology, ladoke akintola university of technology, ogbomoso, oyo state, nigeria. a sample of the plant was deposited in herbarium unit of the department with voucher number lho 250. 2.2 extract preparation aqueous extraction of a. muricata leaf was carried out using the method as earlier reported by yekeen et al. (2017a) with slight modification. the leaves were pulverized and 6 g of it was soaked in 100 ml distilled water. the soaked sample was heated with continuous stirring for 30 min at 40 °c. the mixture was filtered with whatman no. 1 filter paper and stored in a refrigerator at 4 °c until use. 2.3 green synthesis of silver nanoparticles (agnps) a. muricata aqueous extract (1 ml) was added to 40 ml of 1 mm agno3 in glass container while 40 ml of am-e and 1 mm agno3 solutions were separately kept in containers as controls. the controls and the reacting mixture of the extract and agno3 were placed in sunlight for a complete synthesis of nanoparticles (yekeen et al. 2017a). a complete change of colour of reacting mixture, an indicator of synthesized nanoparticles was 5anti-haemolytic and cytogenotoxic potential of aqueous leaf extract of annona muricata observed after 30 min. 2.4 determination of anti-haemolytic activity anti-haemolytic activities of am-e and am-agnps were carried out using the method of joujeh et al. (2017). the blood sample collected from a male wistar rat through heart puncture was spun at 5000 rpm for 5 min. the plasma was discarded and the precipitate was washed 3 times with phosphate buffer saline (ph 7.4). five percent of erythrocyte was prepared in phosphate buffer saline. samples (biosynthesized nanoparticles and the extract) (500 µl) at different concentrations (700, 350 and 175 µg/ml) were added to 1 ml of 5% erythrocyte and incubated for 20 min at room temperature (25 °c). next, 500 µl of h2o2 was added and spun at 5000 rpm for 5 min. the absorbance of free haemoglobin content in the supernatant was read at 540 nm while the percentage inhibitions of h2o2-induced haemolysis of the nanoparticles and the extract were calculated using the equation 1. % 𝑖𝑖𝑖𝑖ℎ𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑖𝑖𝑜𝑜 ℎ𝑎𝑎𝑎𝑎𝑎𝑎𝑖𝑖𝑎𝑎𝑎𝑎𝑎𝑎𝑖𝑖𝑎𝑎 = 𝐴𝐴𝑖𝑖𝑎𝑎(𝐶𝐶𝑖𝑖𝑖𝑖𝑖𝑖𝐶𝐶𝑖𝑖𝑎𝑎–𝑆𝑆𝑎𝑎𝑎𝑎𝑆𝑆𝑎𝑎𝑎𝑎) 𝐴𝐴𝑖𝑖𝑎𝑎(𝐶𝐶𝑖𝑖𝑖𝑖𝑖𝑖𝐶𝐶𝑖𝑖𝑎𝑎) × 100 (1) 2.5 allium cepa cytogenotoxicity assay onion bulbs (240) of approximately the same size were bought at a local market, owode-egba, ogun state, nigeria. the method earlier reported by badmus et al. (2013) and yekeen et al. (2017a, b) was adopted in this study and the experimental set up as described in table 1. the onions were sundried for two weeks to minimize moisture and aid root growth. the outer scales of the onion bulbs were carefully peeled without affecting the primordial root ring. fifteen onions were used for each group as indicated in table 1. the base of each onion bulb was suspended in each container (100 ml beaker) separately containing the control and test solutions at different concentrations. all samples were placed in a dark cupboard at 25 ± 2 °c to reduce the fluctuation of dividing cells. the controls and test solutions were changed at 24 h intervals. five onions per group were respectively harvested at 48 h and 72 h of growth, and their roots were fixed in ethanol: acetic acid (3:1, v/v) for microscopic evaluation. 2.5.1 microscopic evaluation the fixed roots were hydrolyzed in 1 n hcl at 65 °c for 3 min. the tip of two roots was squashed on each of the six slides per group and chopped carefully to ease the scoring process. aceto-orcein was used to stain the prepared slides for 15 min. five slides were analyzed per group, in which 1000 cells were scored per slide at x1000 magnification for normal and abnormal chromosome behaviour during cell division using various template as earlier reported (badmus et al. 2013; yekeen and adeboye 2013; yekeen et al. 2017a,b). 2.5.2 macroscopic evaluation after 72 h, the length of the roots of five onions with best growth selected from each of the concentrations was measured with ruler in cm. 2.6 statistical analysis all the data obtained in this study were expressed as mean ± sd. comparison between treatments was done by analysis of variance (anova) on statistical package for social sciences (spss) 21.0. software duncan’s multiple range post hoc test was performed to measure variation between the mean with significant difference considered at p<0.05. 3. results 3.1 biosynthesis of silver nanoparticles the colour change from colourless to brown of the reaction mixture after 30 min exposure to sunlight (uv table 1. experimental design of cytogenotoxic evaluation. groups treatments 1 distilled water only (negative control) 2 100 µg/ml cyclophosphamide (positive) 3 0.17 µg/ml agno3 4 0.7 µg/ml am-agnps 5 7.0 µg/ml am-agnps 6 70.0 µg/ml am-agnps 7 100 µg/ml cyclophosphamide + 0.7 µg/ml a.m-agnps 8 100 µg/ml cyclophosphamide + 7.0 µg/ml a.m-agnps 9 100 µg/ml cyclophosphamide + 70.0 µg/ml a.m-agnps 10 0.7 µg/ml am-e 11 7.0 µg/ml am-e 12 70.0 µg/ml am-e 13 100 µg/ml cyclophosphamide. + 0.7 µg/ml am-e 14 100 µg/ml cyclophosphamide. + 7.0 µg/ml am-e 15 100 µg/ml cyclophosphamide. + 70.0 µg/ml am-e 16 100 µg/ml of cyclophosphamide. + 1 mm agno3 6 jelili a. badmus et al. rays) is an indication of bio-reduction of silver ion to agnps 3.2 anti-haemolytic activity of aqueous leaf extract of a. muricata and its fabricated silver nanoparticles the results in table 2 show that the bio-fabricated nanoparticle and extract exhibited anti-haemolytic activity in an inverse concentration dependent manner. the activities of the extract and silver nanoparticles were stronger at lower concentration with the extract showing significantly (p<0.05) higher activity compared with their biosynthesized silver nanoparticles. 3.3 effects of aqueous leaf extract of a. muricata, its fabricated silver nanoparticles and cyclophosphamide on root length of a. cepa the root lengths assessed after 72 h of exposure revealed that the average root length of the treated groups am-agnps (groups 4, 5, 6), cyclo + am-agnps (7, 8 and 9) decreased in a concentration dependent manner and significantly (p<0.05) lower than that of the control group. non-significant (p>0.05) increase in the mean root length was observed in the groups treated with 0.7 and 7.0 µg/ml am-e only (group 10, 11), while at 70 µg/ml am-e only (group 12) significant (p<0.05) increase was observed compared to the control group and cyclophosphamide treated group (group 2) (table 3). whereas non-significant (p>0.05) difference in average root length was observed in the cyclo + am-e (13, 14 and 15) treated groups relative to the control (group 1). the mean root length of am-agnps and cyclo + am-agnps at 0.7 and 7.0 µg/ml decreased significantly (p<0.05) relative to the group treated with cyclophosphamide alone. no significant difference was observed between the root lengths of the cyclophosphamide (group 2) treated group and the control. no growth of roots was observed in the onions treated with agno3 alone and cyclophosphamide + agno3. 3.4 cytogenotoxic effects of aqueous leaf extract of a. muricata, its fabricated silver nanoparticles and cyclophosphamide on a. cepa cells the cytogenotoxic effects of am-agnp and a. muricata extract on a. cepa cells are, respectively revealed in tables 4 and 5 after 48 and 72 h exposure. at 48 h, a concentration dependent reduction in the total number of dividing cells was observed in each of the treated groups compared with the negative control group. the mitotic index (mi) value of the treatment groups was lower than that of the control group whereas, a complete cell growth arrest was observed in groups treated with agno3 solution alone and the highest concentration of am-agnps only and in combination with cyclophosphamide. furthermore, the lesser mi value was observed for both am-e and am-agnps singly and when combined with cyclophosphamide compared to group treated with cyclophosphamide only. mitotic index values lesser than the half of the negative control were recorded table 2. anti-haemolytic activity of am-agnp and am-e. concentration (µg/ ml) am-e (%) am-agnps (%) 175 65.44 ± 0.6 49.22 ± 0.9 350 61.64 ± 0.6 45.92 ± 0.7 700 49.64 ± 0.8 34.77 ± 1.7 data were mean ± sd of triplicate experiments conducted at different time. am-e (aqueous leaf extract of a. muricata); am-agnps (a. muricata-fabricated silver nanoparticles). table 3. effects of aqueous leaf extract of a. muricata and its fabricated silver nanoparticles on a. cepa root growth. groups root length (cm) mean ±sd 1 (distilled water only (negative control)) 1.60±0.80 a 2 (100 µg/ml cyclophosphamide (positive)) 1.42±0.50 a 3 (0.17 µg/ml agno3) 0 4 (0.7 µg/ml am-agnps) 1.57±0.71 a 5 (7.0 µg/ml am-agnps) 1.26±0.64 b 6 (70.0 µg/ml am-agnps) 0.16±0.07 b 7 (100 µg/ml cyclophosphamide + 0.7 µg/ml a.magnps ) 1.58±0.64 a 8 (100 µg/ml cyclophosphamide + 7.0 µg/ml a.magnps) 0.67±0.32 b 9 (100 µg/ml cyclophosphamide + 70.0 µg/ml a.magnps) 0.27±0.13 b 10 (0.7 µg/ml am-e) 1.63±0.71 a 11 (7.0 µg/ml am-e) 1.79±0.98 a 12 (70.0 µg/ml am-e) 2.48±1.14 b 13 (100 µg/ml cyclophosphamide. + 0.7 µg/ml am-e) 1.55±0.68 a 14 (100 µg/ml cyclophosphamide. + 7.0 µg/ml am-e) 1.75±0.74 a 15 (100 µg/ml cyclophosphamide. + 70.0 µg/ml am-e) 1.61±0.73 a 16 (100 µg/ml of cyclophosphamide. + 0.71 µg/ml agno3) 0 data are presented as mean ± sd of triplicate experiment. mean ±sd with different superscript are significantly different at p<0.05. agno3: silver nitrate, a.m-e: annona muricata extract, amagnps: annona muricatasilver nanoparticles, positive control: cyclophosphamide, negative control: distilled water, sd: standard deviation. 7anti-haemolytic and cytogenotoxic potential of aqueous leaf extract of annona muricata for both the 70.0 µg/ml am-e treated group and in combination with cyclophosphamide. higher mitotic inhibition values were observed in am-e, cyclophosphamide + am-e and cyclophosphamide + agno3 treated groups relative to the other groups. the mitotic inhibition values were lower in the groups treated with 0.7 µg/ml of agnps and agnps + cyclophosphamide relative to the group treated with cyclophosphamide only. the highest percent proportion of prophase was observed in the negative control group whereas the least was observed in the group treated with 70.0 µg/ml am-e alone and when combined with cyclophosphamide. the 7.0 µg/ml am-e + cyclophosphamide treated group had higher percentage proportion of prophase compared to the negative control. a reduction was observed in the percentage of metaphase in all the treated groups relative to the negative control group. anaphase stage was higher in the group treated with 0.7 µg/ml agnps relative to the other groups and the control. telophase in the 0.7 µg/ ml agnps + cyclophosphamide and am-e only treatment groups was higher than the control and the other groups, whereas it was higher in the control group than the other treated groups. at 72 h, reduction in the total number of dividing cells of the treatment groups relative to negative control was observed. cumulative numbers of dividing cells were lower in the 0.7 µg/ml am-e treated group than half of the positive and negative control. the mi values at 72 h and 48 h were found to be significantly reduced in the treated groups relative to the negative control group. the reduction in mitotic index values was concentration dependent except in the 0.7 µg/ml am-e and table 4. cytogenotoxic effect of a. muricata extract-mediated silver nanoparticles on allium cepa roots meristerimatic cells at 48 h compared with control (positive and negative) conc (µg ml-1) no of dividing cells mitotic index (%) mitotic inhibition (%) prophase metaphase anaphase telophase cm sb cb vc f no. of a/d % aberrant per cell scored control 412 8.24 227 106 35 44 cyclo 100 288 5.76 30.10 136 50 35 36 1 16 14 0.11 0.62 agno3 0.17 am-agnps 0.7 303 6.06 26.46 134 47 37 31 38 16 0.18 1.08 7 285 5.70 30.83 127 39 33 38 30 18 0.17 0.96 70 cyclo + am-agnps 100 + 0.7 315 6.30 23.54 125 74 32 47 27 10 0.12 0.74 100 + 7 283 5.66 31.31 147 63 12 16 36 9 0.16 0.90 100 + 70 am-e 0.7 267 5.34 35.19 144 38 33 46 1 5 0.02 0.12 7 249 4.98 39.56 132 46 27 36 1 7 0.03 0.16 70 224 4.48 45.63 105 43 35 37 2 2 0.02 0.08 cyclo + am-e 100 + 0.7 271 5.42 34.22 135 56 27 30 16 7 0.08 0.46 100 + 7 246 4.92 40.29 121 57 21 35 1 11 0.05 0.24 100 + 70 199 3.98 51.70 105 36 15 37 6 0.03 0.12 cyclo + agno3 100 + 170 cyclo: cyclophosphamide, conc: concentration, cm: c-mitosis, sc: sticky chromosome, cb: chromosome bridge, vc: vagrant chromosome, f: fragmentation, no. of a/d: number of aberration per dividing cell, positive control: cyclophosphamide, negative control: distilled water. 8 jelili a. badmus et al. table 5. cytogenotoxic eff ect of a. muricata extract-mediated silver nanoparticles on allium cepa root meristerimatic cells at 72 h compared with control (positive and negative) concentration (µg ml-1) no of dividing cells mitotic index (%) mitotic inhibition (%) prophase metaphase anaphase telophase cm sb cb vc f no. of a/d % aberrant per cell scored control 244 4.88 144 46 17 37 cyclo 100 207 4.14 15.16 112 35 19 32 5 4 0.04 0.18 agno3 0.17 am-agnps 0.7 216 4.32 11.48 121 43 11 29 1 11 0.06 0.24 7 142 2.84 41.80 75 29 3 31 4 0.03 0.08 70 cyclo + am-agnps 100 + 0.7 198 3.96 18.85 100 39 9 34 1 4 11 0.08 0.32 100 + 7 167 3.34 31.56 78 37 9 16 19 8 0.16 0.54 100 + 70 am-e 0.7 83 1.66 65.98 45 22 9 6 1 0.01 0.02 7 200 4.00 18.03 104 36 17 38 1 4 0.03 0.10 70 175 3.50 28.28 95 20 9 43 2 6 0.05 0.16 cyclo + am-e 100 + 0.7 213 4.26 12.70 121 29 24 37 2 0.01 0.04 100 + 7 229 4.58 6.15 157 18 20 33 1 0.00 0.02 100 + 70 cyclo + agno3 100 + 170 cyclo: cyclophosphamide, conc: concentration, cm: c-mitosis, sc: sticky chromosome, cb: chromosome bridge, vc: vagrant chromosome, f: fragmentation, no. of a/d: number of aberration per dividing cell, positive control: cyclophosphamide, negative control: distilled water. figure 1. representative photomicrographs of normal stages of mitotic cell divisions in treated allium cepa root cells and observed chromosomal aberrations. 9anti-haemolytic and cytogenotoxic potential of aqueous leaf extract of annona muricata am-e+ cyclophospahamide treatment groups where lower values were observed. complete cell growth arrest was demonstrated in 70.0 µg/ml am-agnps alone, 70.0 µg/ml am-agnps + cyclo and agno3 solution treated groups at 48 h. chromosomal aberrations, including chromosomebridge, sticky chromosome, and c-mitosis were observed in different degrees in all the treated groups with chromosome-bridge and sticky chromosome being prominent in most of the treated groups (figure 1). 4. discussion 4.1 biosynthesis and characterization of silver nanoparticles a change in colour of silver nitrate solution in the presence of the aqueous leaf extract of annona muricata from colourless to brown that confirms the synthesis of am-agnps nanoparticles has been previously reported (badmus et al. 2020). physicochemical characterization results of am-agnps similar to the previous study (badmus et al. 2020) indicated that the nanoparticles absorbed maximally at 420 nm and ftir showed that the synthesized silver nanoparticles was possible because of amide and hydroxyl groups of the aqueous leaf extract. zeta potential of the nanoparticles was -27.2 mv, dls indicated 86.8 nm size with polydispersity index of 0.329 and xrd/saed presented crystalline nature of the nanoparticles with face centre cubic (fcc) phase (santhosh et al. 2015; gavamukulya et al. 2020; badmus et al. 2020) 4.2 anti-haemolytic effects of aqueous leaf extract of a. muricata and its fabricated silver nanoparticles the erythrocyte model for assessing the anti-haemolytic activity of test compounds can reveal the toxicity of an agent and can serve as an indicator of membrane toxicity (zohra and fawzia, 2014). blood cells are easily isolated and the test method using the blood cell can mimic other cell membrane (farag and alagawany, 2018). the haemolytic ability of the test compound is proportional to the concentration, chemical constituent and potency of the compound (zohra and fawzia, 2014). in this study, the am-e and am-agnps demonstrated a concentration dependent anti-haemolytic activity. the extract showed a better protection against h2o2induced haemolysis of the red cell membrane compared to am-agnps. this implies that am-agnps is best used at lower concentration because a high concentration as used in this study is toxic to rbc membrane (raja et al. 2016; hamouda et al. 2019). anti-haemolytic activity of plant extract has been shown to be related to the constituent antioxidant agents such as polyphenolic compound (ramchoun et al. 2015; karim et. 2020). the bioactive compounds of the plant are responsible for the synthesis of the nanoparticles and also confer the biomedical property such as anti-haemolytic on the synthesized nanoparticles (kuppusamy et al. 2016; badmus et al. 2020). the toxic influence of rbc membrane at high concentration by am-agnps could be attributed to the ag component of the nanoparticles as earlier reported by choi et al. (2011) and hamouda et al. (2019) to cause the death of red blood cells, even at low concentration. clinical outcome of haemolysis can cause anaemia and contribute to blood coagulation abnormalities. however, nanoparticles including sliver have been shown to protect blood against coagulation (lateef et al. 2018; elegbede and lateef, 2019; azeez et al. 2020). these activities are indicative of biomedical applications of nanoparticles in blood disorder. 4.3 effects of aqueous leaf extract of a. muricata and its fabricated silver nanoparticles and cyclophosphamide on root length macroscopic evaluation helps to determine the root sprouting or root growth inhibition effect exerted by the test solution on the onion roots while microscopic evaluation helps to study the harmful qualitative and quantitative effect (cytotoxic effect on onion meristem cells) (yekeen et al. 2017a). observations of a. cepa root growth inhibition after 48 and 72 h were used as indicator of the cytotoxic nature of am-agnps and am-e. the extract did not show inhibition of root length, but did protect the roots from cyclophosphamide-induced root length reduction. the protective effect of am-e on the root growth inhibition imposed by cyclophosphamide and increased mean root length when am-e was used alone may be credited to the ability of the extract to induce root sprouting (yekeen et al. 2017a). contrarily, the am-agnps reduced the root length in a concentration dependent manner attesting to its mitodepressive capability. the reduction of root length was augmented in the presence of both am-agnps and cyclophosphamide at both 48 and 72 h. root growth inhibition observed in this experiment at both 48 and 72 h exposure to cyclophosphamide together with am-agnps and am-agnps alone is an indication of the genotoxicity nature which can be linked to the presence of heavy metals constituent (yekeen et al. 2017a). stunted growth, hardness, and colouration of the roots observed at 72 h 10 jelili a. badmus et al. exposure in addition to the aforementioned observations confirm the mitodepressive effect on the onion roots meristem cells. 4.4 cytogenotoxic effect of aqueous leaf extract of a. muricata and its fabricated silver nanoparticles and cyclophosphamide the study of chromosome behavior during cell division in order to establish health safety status of a given compound has been the focus of scientists employing a. cepa test. a. cepa assay is widely used to study normal and the abnormal chromosome response when the onions base is suspended in a test solution. the assay reveals the effect of a test substance at a minute level of interaction with genetic material, which makes it a robust tool for effective assessment of genotoxic compounds (bonciu et al. 2018). it is reproducible, sensitive, fast, cheap, and effective in monitoring genetic materials response on exposure to environmental pollution and mutagenic compounds (badmus et al. 2013; bhat et al. 2017). prophase stage of cell division dominated the other cell division stages in all the treated and control groups. an increase in prophase number compared to other stages of cell division has been related to delay in the breaking down of its nuclear membrane (pankaj et al. 2014). cell division at the root tip of the onion meristematic region was assessed using mitotic index (badmus et al. 2013). the reduction in the mitotic index values of the treated groups compared with the negative control revealed the cytotoxicity potential of cyclophosphamide, am-agnps and am-e at both 48 and 72 h. the reduction of mi by any agent compared with the untreated control is known to relate to cytotoxicity of the tested compound (asita and matebest, 2010; yekeen et al. 2017a). the depression of mi could be linked to the inhibition of dna synthesis due to the blockage of g2 phase of the cell cycle, which prevents the cell from entering m-phase during the cell cycle (badmus et al. 2013; obute et al. 2016; yekeen et al. 2017a). inhibition of mitotic activities is employed for tracing cytotoxic substance (singh and roy, 2016). as earlier reported, mi reduction might be as a result of the adverse effects of the extract and am-agnps on the microtubule (yekeen et al. 2017a). this was corroborated by the total cell arrest obtained when a. cepa was treated with agno3 and the highest concentration of agnps with or without cyclophosphamide. however, the ability of agnps to induce cell arrest could be an indication of its capability as an agent of antiproliferation against uncontrolled cell division in cancer cell (chukwujekwu and van staden, 2014). in addition, reduction of mitotic activity in this study could be because of impaired synthesis of nucleoprotein coupled with low level of atp to power spindle elongation, movement of chromosome and microtubule dynamics (yekeen et al. 2017a). the structural changes of chromosome due to an exchange or a break of chromosomal materials are termed chromosome aberration (preston, 2014). chromosome aberration (ca) could be as a result of improper or unrepair oxidation of dna deoxyribose sugar and a nitrogenous base leading to the breaking of the double strand (badmus et al. 2013). ca observed in cells could be either lethal or viable and can induce somatic or inherited genetic effects (chang-hui, 2019). various chromosomal abnormalities such as chromosomebridge, sticky chromosome, and c-mitosis were observed in different degrees in all the treatment groups with chromosome-bridge and sticky chromosome being prominent in most of the treatment groups (figure 1). kuchy et al. (2016) reported that bridge formation could be linked to chromosome breaks, stickiness, or a reunion of already broken ends of chromosomes. olorunfemi et al. (2012) reported that sticky chromosome effect is irreversible and ultimately result in cell death. therefore, total growth inhibition observed with agno3 and the highest concentration of am-agnps with or without cyclophosphamide treatment may be due to sticky chromosome formation. the total root growth inhibition as observed in agno3 treated group shows that the presence of ag in am-agnps is responsible for root inhibition and chromosomal aberration observed in amagnps treated groups. 5. conclusion the green fabricated nps using plants have been shown by several studies to have robust biomedical applications. their actions have been linked to increase surface area due to the reduced size. this study established the anti-haemolytic activity of am-e and amagnps. am-e demonstrated a better anti-haemolytic activity relative to am-agnps at tested concentrations suggesting the toxic potential of biosynthesized agnps to rbc at high concentration. the cytotoxicity of amagnps was revealed through reduction of mi value and increased root growth inhibition of the treatments, suggesting the possibility of employing the biogenic particles as anti-proliferative agent in cancer study. induction of ca observed at both 48 and 72 h in this study shows the genotoxic potential of both am-e and am-agnps. while considering the possible influence of am-agnps in disease therapy, its cytogenotoxic potential should 11anti-haemolytic and cytogenotoxic potential of aqueous leaf extract of annona muricata be robustly evaluated before its exposure to human. in addition, there should be restrain in disposing any synthesized nanoparticles into the environment because their toxicity could be far reaching at high concentration. geolocation information the research was carried out in ogbomoso (210214), oyo state, nigeria. references abdul wahab sm, jantan i, haque ma, arshad l 2018. exploring the leaves of annona muricata l. as a source of potential anti-inflammatory and anticancer agents. front 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fawzia a, 2014. hemolytic activity of different herbal extracts used in algeria. int. j. life sci. pharma res. 508, 495-500. caryologia. international journal of cytology, cytosystematics and cytogenetics 74(2): 79-88, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1051 caryologia international journal of cytology, cytosystematics and cytogenetics citation: maria v. smirnova, anna v. korovkina (2021) toxic and genotoxic effects of aqueous extracts of polygonum weyrichii fr. schmidt on the allium test taken as an example. caryologia 74(2): 79-88. doi: 10.36253/caryologia-1051 received: august 13, 2020 accepted: july 14, 2021 published: october 08, 2021 copyright: © 2021 maria v. smirnova, anna v. korovkina. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. toxic and genotoxic effects of aqueous extracts of polygonum weyrichii fr. schmidt on the allium test taken as an example maria v. smirnova*, anna v. korovkina federal research centre «kola science centre of the russian academy of sciences», laboratory of medical and biological technologies 184209, murmansk region, apatity, 14fersman str., russia *corresponding author. e-mail: zbe3do4et@mail.ru abstract. the plant poligonum weyrichii fr. schmidt is a promising plant in murmansk region because it is a valuable source of flavonoid compounds. the aim of the study is to investigate, using a sensitive and the well-established allium test, toxic and genotoxic effects of aqueous extracts of inflorescences and leaves of the middle tier, which differ in concentration (20, 50, 80 and 100%). according to the observations, aqueous extracts of inflorescences and leaves of p. weyrichii of 50%, 80% and 100% concentration have a mitodepressive effect on the cells of the root meristem of allium cepa l., and inhibit the root growth, causing chromosomal abnormalities. the further investigations are necessary on selection of aqueous extracts concentrations of p. weyrichii. keywords: allium test, polygonum weyrichii, oxidative stress, aqueous extract, flavonoids. introduction the plants containing flavonoids are the promising sources for fitopreparations because of a wide spectrum of their medicinal effect. these valuable capillary strengthening, cholagogue, antiphlogistic, immunomodulatory, diuretic, antimicrobial, anticarcinogenic medications, like many others, are used in medicine. of special interest is the antioxidant effect of flavonoids, their ability to prevent free radicals from causing much severe pathologies (siasos et al. 2013). the oxidative stress caused by unfavourable exogenic factors, by activation of the endogenic active forms of oxygen, and by weakening of antioxidant protection of an organism, is currently considered to be an important pathogenetic link in appearance and development of many diseases (vazhappilly et al. 2019). it can be explained by the key role of redox reactions in the cells of the organism in normal and general pathological processes. oxidation induced by free radicals, which develops most readily in the membrane lipid phase enriched with unsaturated bonds, is the biochemical basis of the universal mechanism of the cell damage caused by different 80 maria v. smirnova, anna v. korovkina damaging factors. genetic and environmental risk factors cause imbalance in the oxidants-antioxidants system, free radicals accumulation, oxidative stress development, and, as a consequence, imbalance in organ and tissues functioning. flavonoids are a large group of polyphenols, which is synthesized by plants. a group of substances participates in many key processes of plants growth and development; however, flavonoids play the most significant role in the mechanism of non-specific plant adaptation to unfavorable environmental factors (brunetti et al. 2013). it is possible due to their antioxidant activity. as membrane mechanisms of damage and adaptation of plants and animals are uniform, plant ergogenic aids are successfully applied in medical practice. however, flavonoid compounds can act as prooxidants. this activity is expressed in much greater oxidation, in the formation of other radicals under redox transformations, which, in the long run, can cause a mutagenic effect. this action depends mainly on solubility, the oxidizingto-reducing agent ratio in the environment, the presence of metals with variable valence, ph, and many other factors (decker 2009). flavonoid synthesis in different organs is a common adaptive plant reaction to the effect of damaging factors of the environment. the climatic conditions of murmansk region are characterized by a short summer period, a short vegetation period, high humidity and extreme lighting conditions; reverse flipping between polar day and polar night (marshall et al. 2016). as the region is located in high latitudes, the geomagnetic field is unstable and cosmic radiation activity is high. taking into account the data on more intensive synthesis of flavonoids by plants experiencing the effect of the extreme factors of the different nature (yang et al. 2018), we suppose that the arctic regions may be an important source of valuable medicinal raw materials. the increasing incidence of diseases and a complicated course of different pathologies, which are mainly caused by oxidative stress among the residents of murmansk region in recent years (statistical yearbook…, 2019) indicate the urgent need in new sources of plant flavonoids capable of increasing the nonspecific protection of the human organism from physical, chemical and biological effects. p. weyrichii was introduced in the kola peninsula in 1920. this plant was often attributed to fam. aconogonon (meinh.) rchb. as a. weyrichii (f. schmidt) h. hara (tsvelev 1987, 2012; hassannejad and ghafarbi 2017), and recently, based on the molecular-phylogenetic data, it was included into fam. koenigia l. (k. weyrichii (f.schmidt) t. m. schust. et reveal) (schuster et al. 2015). as this plant is more widely known as p. weyrichii in the resource management and phytochemistry literature, we also use p. weyrichii in this study. the representatives of this species have successfully naturalized in the local conditions and are widely distributed over the industrial area of the region. p. weyrichii is a perennial herbaceous plant, frostand coldresistant, being distinguished for rapid growth and high productivity of the green mass. the leaves and inflorescences of p. weyrichii contain 3, 4% to 5, 6% of flavonoids by weight of dried tissue (korovkina and zhirov 2019), so this plant can be used as a possible source of flavonoid compounds. the utilization of any plant for medical purposes can lead to negative consequences, so it is necessary to study the ways of producing the flavonoids compounds, their concentrations and toxicity in order to have the reliable data on its safe and efficient application in medicine. the aim of this study is, using the allium test, to estimate genotoxicity and toxicity of the aqueous extracts of the inflorescences and leaves of p. weyrichii, of different concentrations. materials and methods plant material the plant material was the leaves of the middle tier and the inflorescences of naturalized p. weyrichii growing in the protected area of the n.a. avrorin polaralpine botanical garden-institute of the kola science centre, the russian academy of sciences (pabgi, ksc ras) located near the town of kirovsk, murmansk region (67°36’, 33°40’), russia. the kirovsk site (pabgi) is located at about 340m above sea level, near the khibiny mountains. the plant material was collected in the flowering season, on 20. 07. 2019, p. weyrichii was identified by experienced biologists working at pabsi, ksc ras. the extraction technique the inflorescences and the leaves of the middle tier of the p. weyrichii were the plant material to be used in the experiment. according to the standards of drying and storage of medicinal plants (waterhouse, 2001), the plant material was dried, ground into powder, 1mm mesh sieved, additionally dried for 3 h at 60°c to stabilize its mass. to produce the plant aqueous extracts, 81toxic and genotoxic effects of aqueous extracts of polygonum weyrichii fr. schmidt on the allium test the ground plant raw materials composed of leaves of the middle tier and inflorescences, was placed into perforated infantry glasses and then into infudirkas heated before in a boiling water bath for 15 minutes, filled with distilled water of room temperature, which was used, taking into account the corresponding water saturation coefficient given in ofs “determination of water absorption coefficient and consumption coefficient of medicinal plant raw materials”, and was drawn on a boiling water bath for 15 minutes. then the material was cooled for 45 minutes at room temperature, strained, and distilled water was added to reach the required volume. the aqueous extracts were used to make the allium test. to make the allium test, it was necessary for the aqueous extracts of leaves of the middle tier and inflorescences to be watered with distilled water to reach the concentration of 20, 50, 80 and 100 μg ml. hydrogen peroxide 1% (akwu et al., 2019) was used as the positive control, distilled water was used as the negative control. to produce alcoholic extracts, the material was drawn in 70% ethanol for 24 h at room temperature; the plant material-to-solvent ratio was 1:10. the alcoholic extracts were used to quantify flavonoids. quantifying the flavonoids the method based on the complexation reaction of f lavonoids with aluminum chloride, was applied to determine the total flavonoid content (belikov and shraiber, 1970). the 0.05 ml extract was mixed with 0.1 ml of 2% solution of alcl3 in 96% ethanol, and the volume was adjusted to 2.5 ml with 70% ethanol. absorbance at 415 nm of the analyzed solutions was measured using a kfk-3-01 “zomz” spectrophotometer. the calibration curve was obtained using the solutions of routine in 70% ethanol-water mixture (100 – 1000 μg ml). the total flavonoid complex (tfc) is calculated by the formula w (flavonoids) = (k*a415*v1*v2)/(m*v3*106)mq/g, where k – calibration coefficient, a415 – absorbance at 415 nm µg; v1 – extract volume, ml; v2 – dilution volume, ml; v3 – analyzed sample volume, ml; м – mass of dried plant material, g. the allium test onion (a. cepa l., 2n=16, fam. amaryllidaceae), class kupido, was purchased in the shop. before the experiment, the bulbs were preserved in a dark cool place for 14 days and then were selected to be of similar diameter, were examined and pilled from old scales. the experiment was made in accordance with fiskesjo (fiskesjo, 1997), with the bulbs being preliminary sprouted in distilled water for 24 h. then 40 bulbs were selected, 5 bulbs per each concentration and per each control, with roots of 2-3 mm long, which were placed into test tubes filled with aqueous extracts of inflorescences and leaves of p. weyrichii of 20, 50, 80 and 100% concentrations. the aqueous extracts and controls were changed for new ones once a day, with the roots being cut and placed into ethanol solution (96%) and glacial acetic acid (3:1) for 24 h. the roots were placed into sealed test tubes, in 80% alcohol. in total, it took 96 h to make the experiment in darkness at room temperature, in an encrypted form. the cytotoxic and genotoxic effects of the plant extracts were analyzed at a microscopic level using only the meristematic part of the a. cepa roots. to prepare the medications, the roots were subject to hydrolysis and simultaneous colouring in ceramic crucibles, in the aceto-orcein solution heated to a boil over the flame of a spirit lamp. once cooled, the crucibles were left in the dye (medvedeva et al., 2014) for 24-72 h at temperature of 4°с. three roots were used per each concentration and per each control, to prepare 3 squashed medications. calculation was made of 1000 cells, with phases and chromosome aberrations marked, with 40x and 100x amplifications (with immersion) using the “mikromed-1 microscope, var.1-20”. simultaneously with cells calculation, the photographs were taken with the help of the toupcam 2.0 camera. in total, over 52000 cells were calculated. to assess the root growth, new bulbs were placed in aqueous extracts of inflorescences and leaves of the similar concentrations and controls for 48 h, and the length of all the roots was measured. in total, 217 roots were measured (fiskesjo, 1985; wierzbicka and antosiewicz, 1988; o’hare et al., 1995). the mitotic index (mi) was calculated by using the following equations (bakare et al. 2000): mi= the total number of dividing cells/the total cell number) × 100. statistical analysis statistical analyses were done using the program statistica 8.0 and microsoft excel. the differences in the mitotic rate between the experimental and control groups (the negative control) were tested applying the mann-whitney non-parametric test. the significance 82 maria v. smirnova, anna v. korovkina level was taken as p≤0.05. results and discussion the study presents the primary estimation which has been for the first time made over the genotoxicity of aqueous extracts of the p. weyrichii inflorescences and leaves because this plant contains a great amount of biologically active compounds, which means that it can be used in the development of medications and biological supplements. the flavonoid content in the inflorescences samples was equal to 5.9% of all the dried tissue (5.9mg ml), and in the leaves samples – 4.4% (4.4 mg ml). a 24-h experiment has revealed the root growth inhibition, root dying the colour of the solution, and root roughening in 20%, 50% and 100 % concentrations of aqueous extracts of inflorescences and 50% and 80% concentrations of aqueous extracts of leaves. the next day, the root growth was observed in 20% concentrations of the inflorescences aqueous extracts and in 50% and 80% concentrations of aqueous extracts of leaves, as well as sediment and slime on the bulb bottoms in 80% and 100% concentrations of aqueous extracts of inflorescences. in 96 h, all the roots died in 100% concentrations of aqueous extracts both of inflorescences and leaves (fig. 2 a, b) except 20% concentration o aqueous extracts of inflorescences and 20% and 50% concentrations of aqueous extracts of leaves. to estimate the toxicity, we measured the length of the roots because this is the indicator of the toxicity of the substances tested. this indicator is easily correlated with the microscopic data, and takes no time to be recorded (fiskesjö 1985; sobrero and ronco 2004; konuk et al. 2007). the concentration-dependent inhibition of t he root growth in the aqueous extract of inflorescences was observed after exposure in these extracts for 48 h (fig. 1). in addition, the roots bending was observed in 20% and 50% concentrations of the inflorescences extracts after a 24 h exposure and in 50% and 80% concentrations of the leaves extracts after a 48-h exposure (fig. 2 c, b). according to levan (1949), the phenomenon like this is due to the toxic effect of the substances. of all the mitotic disorders, the most recurrent ones were bridges in telophase and anaphase, chromatin budding in mns, chromosomes lagging in metaphase, chromosome fragments and their sticking. also observed were cells-ghosts with damaged chromatin or enucleated cells, cells with apoptotic bodies, giant cells and cells with damaged membranes. a factual, concentration-dependent, reduction in the mi compared to negative control was observed in the solutions of all the aqueous extracts of leaves and inflorescences (p<0, 05) (table 1). there was also the significant reduction in the mi in the aqueous extract of inflorescences compared to the aqueous extract of leaves. the aqueous extracts of plants are composed of complex mixtures of chemical substances, which effect the processes taking place in the living organisms in synergy, can be antagonists or act additively, which determines the different changes in genetic material. to assess these changes is possible by a number of tests on different objects, such as rodents (carver et al. 1983), drosophila, cells hela (lu et al 2009), erythrocytes (bhagyanathan and thoppil 2015; aktar et al. 2019), leukocytes (palmieri et al. 2016), different plants, etc. in this study we used the allium test because it is an express-test, and is effective and sensitive when used in biological monitoring. it allows us to assess the environmental pollution, toxicity of different compounds, particles, physical factors and plant extracts (levan 1938; frescura et al. 2012; frescura et al. 2013; kuhn et al 2015; bolsunovsky et al. 2019; bernardes et al. 2019). the allium test allows us to assess the cytoand genotoxicity of the factors of different nature, not spending a lot of physical and economical resources (teixeira et al. 2003), to avoid solving the problems connected with ethical use of plant objects; it provides with large amounts of data and the results are correlated with those obtained on cell lines (tedesco and laughinghouse 2012). it should be also noted that, in comparison with other plant objects figure 1. dependence of the root length (mm) of allium cepa l. on the concentration of aqueous extracts of p. weyrichii (exposure 48 h). abbreviation: nc, negative control (distilled water); pc, positive control (h2o2 1%). abbreviations: nc, the negative control (distilled water); pc, the positive control (h2o2 1%). 83toxic and genotoxic effects of aqueous extracts of polygonum weyrichii fr. schmidt on the allium test used in genotoxicity tests (smirnova et al. 2012; bonea and bonciu 2017; daphedar and taranath 2018), the chromosomes and cells of a. cepa l. are rather great in size, which makes it easy, using primitive equipment, to count phases and mitotic disorders and assess even some changes in cells (e.g., membrane breakdown). in testing, a number of parameters are taken into account, which allows us to get a distinct cytoand genotoxicity pattern. these parameters are as follows: the mitotic index (mi) and a number of changes in genetic material which are classified and in scientific literature into 2 categories clastogenic (chromatid fragments, mns, ring chromosomes, bridges); aneugenic (chromosomes ‘sticking’, c-mitosis, nucleus buds, (sharma et al. 1990, kurás 2004) giant cells appearance)). these changes are related to disruption of the dna molecule or chromosomes breakdown, to mitotic spindle breakdown and finalizing the cytokinesis. there is a separate category of turbagenic changes, which include laggard chromosomes, vagrants chromosomes (bonciu et al. 2018). the mi is an indicator of cell division, and the index reduction is related to mitodepressive effect of the substances tested (akinboro and bakare 2007; sharma and vig 2012), which was observed in the study when the concentrations of aqueous extracts of leaves and inflorescences increased. it is due to the intervention into the mitotic cycle and indicates the possible cytostatic and cytotoxic effect. the decrease in the mitotic cell activity can be related to inhibition of the dna synthesis in the s-phase (el-ghamery et al. 2000) or to blocking in the g2-phase of the cell cycle, which results in finalizing the entry of a cell into mitosis (bruneri 1971; christopher and kapoor 1988; sudhakar et al. 2001). the similar effect of plant extracts has been already described as a concentration-dependent decrease in the mi in both the studies of the different, potentially medicinal and useful in industry plants, and those which are well known in medicine and production (oyedare et al. 2009; i̇lbaş et al. 2012; ping et al. 2012; oyeyemi and bakare 2013; pesnya et al. 2017; de abreu et al. 2019; madike et al. 2019). the flavonoid plant compounds, namely glycosides and alkaloids, as well as polyphenolic compounds possess high antioxidant activity and, in high concentrations, can induce cytotoxic effects in the test eukaryotic systems, showing the so-called prooxidant effect (ono and nakane 1990; wong and mclean 1999; lu et al. 2009; samuel et al. 2010). the decrease in the mi, the absence of roots after 96-h bulb incubation in the inflofigure 2. death of roots in 100% aqueous extracts of inflorescences after 96 h (a, b) and bending of roots after exposure to aqueous extract of inflorescences and leaves 20, 50 and 80% after 48 h (c, d). table 1. cytological effects in the meristematic cells of roots allium cepa. sample code/ indicator mi(%) b c-m bi ds sc mns cg cl сdm nc 72.3±6.2 5 2 pc 10.8±3.1 2 1 56 12 10 2 182 inflorescences 20% 6.0±2.1a 1 151 3 1 88 50% 4.4±2.7a 14 1 40 3 8 3 4 80% 3.5±0.9a 8 56 96 100% 2.0±0.4a 6 60 253 leaves 20% 22.1±6.6a 10 1 162 1 8 1 50% 5.3±1.7a 23 20 42 80% 5.0±1.0a 46 3 100% 3.3±0.9a 23 25 28 abbreviations: nc, negative control (distilled water); pc, positive control (1% hydrogen peroxide (h2o2); mi – mitotic index, b – bridges, c-m – cmitosis, bi – buddings at interphase, ds – destroyed spindle, sc – sticky chromosomes, mns – micronuclei, cg – cells – ghosts, cl – ‘laggard’ cromosomes; сdm –cells with damaged membranes. a statistically different, when compared with untreated control 84 maria v. smirnova, anna v. korovkina rescences and leaves concentrations higher than 50% indicate that the roots are not growing due to the inhibiting effect of the aqueous extracts of high concentrations. the oxidation and antioxidant stress results in protein, lipids and genetic material damage (melo et al. 2016; zhou et al. 2016; singh and patra 2018; zhou et al. 2018). roots roughening and dying the color of the solution (dark brown hue) in aqueous extracts of leaves of 80% and 100% concentrations (the roots were longterm hydrolyzed) can be related to a high content of tanning agents, including also tannins contained in a great amount in p. weyrichii. in studying the plant aqueous extracts genotoxicity, the most well-known and often observed are bridges in the anaphase and telophase, lagging chromosomes, chromatin budding or breakdown in the inter-phase, mns and c-mitosis, as a variant of breakdown or spindle inhibiting in the metaphase and disorder in microtubes functioning alongside with sticking and lagging chromosomes and fragments (fiskesjo 1988; pesnya et al. 2011; prajitha and thoppil 2016; costalonga et al. 2017: madić et al. 2017; bibi et al. 2019). in this study we have observed, practically, all types of effects. the breakdowns of chromosomes in meristematic cells in the aqueous extracts of p. weyrichii are seen in fig. 4, disorders are seen in fig. 5. the mitotic phases normal in the control (distilled water), are presented in fig.3. the bridges in the anaphase (fig. 4 (4, 11) can appear in the translocation process and in uneven exchange due to the presence of dicentric chromosomes, as well as due to disintegration between chromosomes and chromatids, followed by their joining (el-ghamery et al., 2000), which results in chromosome mutations at the structural level (devi and thoppil, 2016). the sticking of chromosomes occurs as a result of chromatin defect and is considered to be an irreversible process resulting in the cell death (fig. 4 (13, 14) (pawlowski et al. 2012). the apoptotic cells and cells with the damaged membranes (fig. 5) are observed in 50% and 100% concentrations of aqueous extracts of inflorescences after a figure 3. the mitotic phases are normal. 1) anaphase; 2) telophase; 3) interphase; 4) prophase; 5) metaphase. figure 4. chromosome and mitotic abnormalities in the roots of allium cepa. 1) chromatin budding in the interphase; 2) micronucleus in the interphase; 3) chromosome lagging in the telophase; 4) a bridge in the anaphase; 5) bridges in the telophase; 6) fragments and a lagging chromosome in the metaphase; 7) chromosome lagging and their possible sticking in the telophase; 8) a lagging chromosome in the metaphase; 9) spindle destruction in the metaphase (c-mitosis); 10) chromatin fragments in the interphase; 11) chromatin destruction in the interphase, fragments and bridges in the anaphase; 12) spindle destruction in the metaphase, chromosome lagging; 13) bridges, chromosome lagging and sticking in the anaphase; 14) chromosome sticking in the metaphase and lagging. 15) a bridge in the telophase and chromatin bulgling in the nucleus which is formed; 16) giant cells; 17) nucleus-free cells (possibly due to chromatin destruction; 18) a cell-ghost and a cell with destroyed chromatin (a). 85toxic and genotoxic effects of aqueous extracts of polygonum weyrichii fr. schmidt on the allium test 24and 18-h exposure, respectively. it can be supposed that apoptosis, as the mechanism of the programmed cell death has proven to be a reaction to a stress impact, and the damage of the cell membranes was induced by membrane ferments or by decrease in the cellulose content (sultan and celik 2007; 2010). the deviations in mitosis include also elongated cells, giant cells and cells-ghosts that appear due to cytoskeleton damage in the interphase, as well as deformed nuclei and bulged chromatin, which appear in spindle and cytokines inhibiting (mushtaq et al. 2019). for instance, giant cells and cells-ghosts (fig. 4 (10) and fig. 4 (18) were observed in positive control of 1% hydrogen peroxide after a 48-72 h exposure and in 100% concentration of the aqueous extract of leaves after a 24-h exposure, and nucleus-free cells were observed in 50% concentration of aqueous extract of leaves after a 24-h exposure. the nuclear buds (fig. 4 (1) appear due to displacement or bulging of the genetic material from aneuploid cells (fernandes at al. 2007). the flavonoid compounds in p. weyrichii, such as avicularin, hespedin, quercetin, hypricide, quercetin3-rhamnosyl, caempferol, mirecitin, epigallocatechin can explain the effects observed in the study (adegoke et al., 1968). it is also known that some plant components, for instance, flavonoids and tanning agents, can modulate the activity of many genotoxicants (jafferey and rathore 2007). in (korovkina et al. 2020), the extract of p. weyrichii inflorescences is characterized by high antioxidant activity and possesses a great amount of phenol and other compounds if compared to the leaves extract, which confirms the results obtained by this study. conclusion the study of aqueous extracts is carried out for the first time with inflorescences and leaves of p. weyrichii, and we believe that it contributes to understanding of the effect of natural extracts of potential medicinal plants on living organisms, as well as to the assessment of their toxicity and genotoxicity. the results of the study show that aqueous extracts of inflorescences and leaves of p. weyrichii of 50%, 80% and 100% concentrations have a mitodepressive 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apoptosis via the anti-oxidative stress pathway. nan fang yi ke da xuexuebao. 38:949-955. caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 1 2021 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 74(3): 9-19, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1248 caryologia international journal of cytology, cytosystematics and cytogenetics citation: federico martinelli, anna perrone, abhaya m. dandekar (2021) development of a protocol for genetic transformation of malus spp. caryologia 74(3): 9-19. doi: 10.36253/caryologia-1248 received: march 11, 2021 accepted: august 10, 2021 published: december 21, 2021 copyright: © 2021 federico martinelli, anna perrone, abhaya m. dandekar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. development of a protocol for genetic transformation of malus spp federico martinelli1,*, anna perrone2, abhaya m. dandekar3 1department of biology, university of florence, sesto fiorentino, florence, 50019, italy 2department of biological, chemical and pharmaceutical sciences and technologies (stebicef), university of palermo, viale delle scienze, palermo, 90128, italy 3department of plant sciences, university of california, one shields avenue, mail stop 4, davis, ca 5616, usa *corresponding author. e-mail: federico.martinelli@unifi.it abstract. a protocol to produce transgenic shoots of malus x domestica cv greensleaves was optimized using two gene constructs previously used to create parthenocarpic tomato, ino-iaam and defh9-iaam. the aim was to obtain sufficient nº of transgenic shoots for in vitro multiplication, transfer to soil, grafting and testing for parthenocarpy in the next years. we investigated the effects of two modifications of a previous published protocol: 1) co-transformation with an agrobacterium containing “vip” genes in the gene construct and 2) two different hormones or hormone combinations. more shoot regeneration was obtained with a combination of three hormones (ba:naa:tdz) during co-cultivation instead of iba and no co-transformation was performed using the vip gene. for the defh9-iaam transgene, 21.04% regeneration was achieved for this treatment instead of 8.95% achieved with “iba treatment” and 4.42% with the agrobacterium co-transformation treatment. more shoot regeneration occurred with the combination of three hormones (ba:naa:tdz) instead of with only iba and no co-transformation was performed using vip gene. experiments using ino-iaam confirmed the results shown for the defh9-iaam transgene. the regenerated shoots were multiplied in selective media containing kanamycin and roots were obtained. keywords: apple, greensleaves, genetic transformation, malus, organogenesis, tdz introduction traditional genetic improvement in woody fruit species used selection and breeding, resulting in relatively few genotypes and a restricted germplasm base. this genetic uniformity has increased vulnerability of woody crops to insect pests and pathogens and caused excessive use of chemicals (norelli et al. 1994) genetic transformation provides an alternate approach through introduction of genes encoding desirable traits (jia et al. 2019), bypassing the long periods required for genetic crosses and selection. once a useful transgenic plant is isolated (assuming the transgene expression is stable), vegetative propagation allows rapid production of the desired trans10 federico martinelli, anna perrone, abhaya m. dandekar genic line. genetic improvement of an elite cultivar can occur because there is no sexual reproduction. since production of most fruit tree species is based on a few cultivars, the impact of genetically transforming them is important.the characterization of induced overll metabolism changes using omic tools has been previously done (tosetti et al. 2010; rizzini et al. 2010). the most widely produced commercial transgenic tree crop is papaya (carica papaya l.) resistant to prsv (papaya ringspot virus), while transgenic apple is not yet on the market. this is partially due to the absence of efficient regeneration protocols for important commercial cultivars of malus x domestica. protocols developed for one cultivar are often not suitable for other cultivars of the same species. in some cases, genetic transformation has been obtained only from seedling material (mante et al. 1991). the time required for transformation and evaluation of phenotype is generally much longer for tree crops (three to 20 years) than for herbaceous species. space requirements can be large and evaluation of transgenic tree crops, expensive and time-consuming. however, conventional breeding for new cultivars has the same requirements. among molecular genetic approaches, genetic transformation is probably the most important tool to increase the speed of cultivar creation, because it avoids some disadvantages of conventional breeding, like loss of desirable characteristic in the offspring. in addition, the small number of cultivars produced for each woody species increases the impact of genetic improvement of one of them. for example, over 50% of world and united states apple production is based on red delicious, golden delicious, granny smith, gala and fuji. an improvement of one of these cultivars can have a significant impact on total production. methods for plant transformation fall into three main groups:1) biological vectors (virusor agrobacterium-mediated transformation; 2) direct dna transfer (chemical-, electricalor microlaser-induced permeability of protoplasts or cells; and 3) non-biological vector systems (microprojectiles, microinjection or liposome fusion). the availability of an efficient protocol for regeneration is an important step for recovery of transgenic plants. there are efficient regeneration systems for many herbaceous species (tomato, arabidopsis, tobacco). however, systems for many woody fruit crops are either not available or suitable only for juvenile material of zygotic origin, which makes them useless for transforming elite cultivars. dandekar (1992) considered two important conditions for regenerating transgenic plants:1) the regenerating cells must be accessible to agrobacterium and 2) the regenerated plants must originate from single cells. direct adventitious regeneration is preferred to intermediate proliferation of callus because callus can be a source of somaclonal variation, requiring extensive field tests to ensure that regenerated plants are true to type. also, a pluricellular origin for regenerated plants can produce chimeric plants with variable expression. genetic transformation of single cells or protoplasts can overcome this situation (oliveira et al. 1994; hidaka and omura, 1993). previous work on genetic transformation of apple has focused on genes to improve two kinds of traits: 1) disease resistance against viruses, bacteria, insects and fungi and 2) modification of agronomic phenotypic features, such as columnar growth, rooting ability, freezing tolerance or toxin resistance. plant resistance to a pathogen is often caused by a hypersensitive response, involving elicitor recognition that activates a cascade of host genes and eventually leads to a generalized response known as systemic acquired resistance (sar). previous studies attempted to confer disease resistance by introducing specific resistance genes rather than by activating plural defence mechanisms (schuerman and dandekar, 1993). most research has focused on virus-induced disease. some used genes encoding viral coat proteins to increase tolerance to specific viruses such as prsv (papaya ringspot virus) in papaya (fitch et al. 1993) and ctv (citrus tristeza virus) in citrus (ghorbel et al. 2001). in apricot, the regenerated plants were of zygotic origin and resistance has not yet be recovered from transformed commercial cultivars. resistance to insects, bacteria and fungi has been developed in actinidia deliciosa against botrytis cinerea (nakamura et al. 1999) and in walnut against cydia pomonella (dandekar et al. 1998). a japanese persimmon cultivar was transformed with the cryia (c) from bacillus thuringiensis and biossays with two different lepidopteran pests showed significative resistance to these pathogens. pear, like apple, is severally affected by fire blight (erwinia amylovora) and pear cultivars with increased resistance were recovered that expressed d5c1 (puterka et al. 2002). the rol a, b or c genes were used to improve rooting in kiwifruit (rugini et al. 1991) and in apple rootstocks such as m26 (welander et al. 1998). in citrus, the juvenile phase was shortened and precocious flowering was promoted using floral genes such as leafy (lfy) and apetala1 (ap1) from arabidopsis (pena et al. 2001). progeny of the transgenic lfy and ap1 trees had a generation time of one year from seed to seed, but only the ap1 trees had fully normal development. in peach, greater branching and shorter internodes were obtained using strains of agrobacterium with a silenced auxin 11improved apple transformation protocol synthesis gene and intact ipt gene for cytokinin synthesis (smigocki and hammerschlag, 1991). among tree fruits, apple is used frequently for transgenic research because optimized transformation protocols exist for the elite cultivars greesleaves (james et al. 1993) and delicious (sriskandarjah et al. 1994). recently, transgenic apple trees with reduced scab susceptibility were obtained by introducing a gene for puroindoline-b from wheat, effective against new races of scab that are resistant to the vf gene (faize et al. 2004). other researchers transformed apple using genes from the biocontrol fungus trichoderma atroviride encoding the antifungal proteins endochitinase or exochitinase (n-acetyl-beta-d-hexosaminidase) driven by a modified camv35s promoter (bolar et al. 2001). exochitinase was less effective than endochitinase and the enzymes acted synergistically to reduce disease. the level of expression of endochitinase correlated negatively with apple tree growth, while exochitinase had no consistent effect on growth. transgenic lines, especially one expressing low levels of endochitinase activity and moderate levels of exochitinase activity, were selected for high resistance in growth chamber trials and negligible reduction in vigor (bolar et al. 2000, 2001). other researchers used t4 lysozyme, attacin or cecropin mb39 genes to enhance resistance of transgenic “royal gala” apple trees against erwinia amylovora (liu et al. 2001). transgenic trees were evaluated for fire blight resistance, delayed fruit softening and scab resistance (bolar et al. 2000). apple fruit shelf life was improved by altering ethylene biosynthesis using sense or antisense cdna encoding acc-synthase and acc-oxidase (dandekar et al. 2004). ethylene biosynthesis was also down-regulated in gala apple using a sam-k gene encoding a s-adenosylmethionine hydrolase (samase). resistance to codling moth was obtained using a chemical version of the bacillus thuringiensis cryac gene (dandekar et al. date). another important objective of genetic improvement in apple is regulation of tree growth. apple growth has been modified using rola genes isolated from agrobacterium rhizogenes. apple rootstock m26 transformed with rola had reduced internode length, dry matter and leaf area. when the scion gravestein was grafted onto transformed m26, the scion showed reduced stem and internode length without altered leaf area and relative growth rate (zhu and welander, 1999). rolb promotes rooting through increased auxin sensitivity (delbarre et al. 1994). this gene has been successfully inserted into the apple rootstocks m26 (welander et al. 1998) and jork9 (sedira et al. 2001). self-incompatibilty restricts fertilization and fruit set in apple and makes pollinator plants necessary for orchard productivity. transgenic plants created with deleted pisitil s-rnase proteins, which are responsible for self-incompatibility, produced normal fruit and seeds after selfing (broothaerts et al. 2004). this work tested different hormone combinations and co-cultivation with different agrobacterium harboring vip genes to improve regeneration of transgenic apple shoots. we used two plasmid constructs containing ovule-specific promoters to induce expression of the iaam gene, which is involved in auxin biosynthesis. the resulting trees will be evaluated for the presence of seeds, since these gene constructs were used successfully to cause parthenocarpy in tomato cv micro-tom. materials and methods binary vectors, plant materials and treatments two binary vectors were used to transform apple cv greensleaves. the first, pdu04100, contained the iaam gene (involved in auxin biosynthesis) in a sense orientation, under the control of ovule-specific promoter “ino,” isolated from arabidopsis ovary integument (meister et al. 2004). the second, pdu04160, contained iaam in a sense orientation under the control of another ovulespecific promoter, def h9, isolated from anthirrium majus ovary (martinelli et al. 2019). apple cv greensleaves was cultured in vitro in shoot multiplication medium (a17) under controlled temperature (18 to 25°c) and 16-hour photoperiod (fluorescent light) with no bacterial or fungi contamination. the plants were subcultured and separated every ? months. the effects of several treatments on transformation efficiency were studied: ba:naa:tdz (5:1:1, (mg/l)) iba (3 mg/l) cotransformation with agrobacterium containing the vip1 gene construct as described (escobar and dandekar 2003, raman et al. 2019). the a17 shoot multiplication medium consisted of 30 g/l sorbitol, 431 g/l ms salts (macroand micronutrients), 100 mg/l myo-inositol, 1 ml/l 1000x ms vitamin stock, 1 ml/l of 1mg/ml iba, 1 ml/l of 1 mg/ml ba and 8 g bactoagar, ph 5.8. rooting of shoots the apple cv greensleaves shoots used for genetic transformation were rooted using a two-phase method: root induction and root emergence. shoots were trans12 federico martinelli, anna perrone, abhaya m. dandekar ferred from a17 medium to ri medium and placed under a 16-hour photoperiod for two to five days (fluorescent light). next the shoots were transferred to re medium without cutting off the base and placed under a 16-hour photoperiod (fluorescent light) for four to five weeks until roots emerged and leaves were fully expanded. root induction media (ri) was identical to a17 medium, except the ba was omitted. re medium omitted both ba and iba. agrobacterium preparation agrobacterium from frozen stock was inoculated into yep medium containing 50 mg/ml rifampicin, 50 mg/ml kanamycin sulfate and 20 mg/ml gentamicin sulfate and incubated overnight at 28ºc. the next day, five ml yep medium was inoculated with bacteria from the plate and incubated with shaking at room temperature for two to three hours. afterward, 10 μl tetracycline were added to five ml yep medium, swirled, combined with agro-yep suspension and incubated overnight at room temperature with shaking. the od at a420 was determined using 100 μl bacterial suspension from the overnight growth and 900 μl yep. the bacterial cells were centrifuged at 5000 g for 15 min at room temperature, resuspended in im medium to od420 = 0.5 and incubated at room temperature with shaking for five hrs. agrobacterium growth medium (yep) consisted of 5 g/l bacto yeast extract, 10 g/l bacto peptone and 10 g/l nacl, ph 7.2. virulence induction medium (im) consisted of 431 g/l ms salts, 1 ml/l 1000 x ms vitamins, 2% sucrose, 100 mg/l myo-inositol, 1 mm proline and 100 μm acetosyringone, ph 5.2. genetic transformation protocol leaf discs were cut from leaves of shoots grown in re media for four to five weeks and placed immediately in petri dishes containing co-cultivation medium solution with no hormone. the leaf discs were incubated with agrobacterium suspension for 10 to 20 minutes, blotted onto sterile whatman filter paper to remove excess bacteria, then transferred to co-cultivation medium supplemented with 200 μm acetosyringone and 1 mm proline (24 discs per plate). plates were incubated in the dark at 21ºc for three days and transferred to regeneration medium. plates were checked weekly for regenerants and the explants were transferred to fresh medium monthly. as soon as they appeared, regenerated shoots were transferred to a17 medium supplemented with 200 μg/ml cefotaxime and 100 μg/ml kanamycin and incubated under 16 hours photoperiod at room temperature. the regenerated shoots were divided grown separately in single tubes (20 ml) in fresh selective a17 until sufficient material was produced for biochemical and molecular analyses. the first co-cultivation medium (cc) was composed of 30 g/l sorbitol, 431 g/l ms salts (macro and micro-elements), 100 mg/l myo-inositol, 1 ml 1000 x ms vitamin, 3 ml/l 1 mg/ml iba, and 3 g/l gelrite, ph 5.8. the second co-cultivation medium (cc) was the same, except that the hormones were 5 ml/l 1mg/ ml ba, 1 ml/l 1 mg/ml naa and 1 mg/ml tdz. in regeneration medium (rg) the hormone were 5 ml/l 1 mg/ml ba, 1 ml/l 1 mg/ml naa, 1 ml/l 1 mg/ml tdz, 200 μg/ml cefotaxime and 100 μg/ml kanamycin. histochemical mug assay fifty to 100 mg tissue was ground in 100 μl extraction buffer in a microcentrifuge tube using a plastic pellet pestle and centrifuged five to 10 min at 14000 rpm at 4 ºc at room temperature. fifty μl supernatant was transferred to microcentrifuge tubes containing 450 μl of extraction buffer. two hundred μl 4 mm mug were added, mixed and immediately added to 800 μl .02 m na2co3 (time 0). time 0 and remaining samfigure 1. objective and scheme of the two ovary-specific gene constructs used for genetic transformation of ‘micro-tom’ tomato. role of gene iaam in auxin biosynthesis. also the mechanism to induce parthenocarpy is described briefly. 13improved apple transformation protocol ples were incubated at 37ºc for 30 min. afterward, 200 μl of the remaining supernatant was added to 800 μl .02 m na2co3 and mixed (time 30). samples were analyzed under ultraviolet light and the fluorescence of times 30 and 0 were compared to a control with a fluorometer. dilutions were made to read fluorescence using .02 m na2co3. the extraction buffer consisted of 50 mm napo4, ph 7; 10 mm edta, ph 8; 01% triton x-100; .01% sodium luryl sarcosine; 7μl/10 ml 2-β-mercaptoethanol; .02 m na2co3 and 4 mm mug (4-methylumbellifery glucoronide). rooting and soil transfer of transgenic shoots the same procedure described previously to generate shoots used for genetic transformation was also used to root transgenic shoots, although ri and re media were supplemented with 200 μg/ml cefotaxime and 100 μg/ ml kanamycin. transgenic shoots produced expanded roots and were acclimated. statistical analysis for each treatment, 20 petri dishes containing 12 explants were used. three parameters were calculated for each petri dish: 1) the percentage of regeneration (explants forming on at least one shoot/total explants used), 2) the nº of regenerated shoots/total explants used, 3) the nº of groups of shoots/ total explants used. means were calculated for each treatment and spss statistical software was used to analyse the data with anova univariate and duncan t-test (p=005). results different hormone combinations were used to improve the genetic transformation protocol, using two constructs containing the iaam gene driven by def h9 or ino, two ovule-specific promoters previously used to transform tomato. different in vitro plant culture factors were studied for each construct. two different hormone combinations were used during co-cultivation with the def h9-iaam construct. the first was the same combination of hormones used for regeneration (ba:naa:tdz at 5, 1 and 1 mg/l, respectively). the second was 3 mg/l iba to induce callus formation before regeneration. the effect of co-transformation with two agrobacterium strains was tested: 1) with a construct containing a “vip1” gene, and 2) with agrobacterium containing the ino-iaam construct. the vip1 gene increases the number of transformed cells and also their regeneration capacity. co-cultivation was also studied in two experiments using the construct ino-iaam. for all transformation experiments, the regeneration percentage and nº single shoots regenerated were measured to determine transformation efficiency. the number of shoot groups were also counted, although it was unclear whether such groups derived from one or several transformation events. generally, each group formed two to six shoots, of which only one was maintained in culture for confirmation of transformation. iba in co-cultivation or co-transformation produced fewer regenerants, a lower percentage of regeneration, and fewer shoots (single or groups) than ba-naatdz treatment (table 1, figures 2 and 3). leaf discs transformed with ino-iaam showed similar results: more table 1. transformation of “greensleaves” apple using construct defh9-iaam. the construct, date and number assigned and a description of the experiments are included. percentage of regeneration and number of single or grouped shoots regenerated were determined. the letters on the side of the numbers in the same column indicate significative differences calculated using the duncan test (p=005). treatment % regeneration nº of shoots nº of group of shoots control 21.04 b 1.52 b 0.72 b iba 8.95 a 0.78 a 0.04 a vip 4.42 a 0.47 a 0 a figure 2. a) genetic transformation of ‘greensleaves’ apple with the defh9-iaam gene construct. percentage of regeneration (explants forming at least one shoot/total explants) for each treatment. b). genetic transformation of ‘greensleaves’ apple with the defh9-iaam gene construct. number of shoots/total explants and number of group of shoots/total explants are indicated for each treatment. 14 federico martinelli, anna perrone, abhaya m. dandekar regeneration was obtained when co-transformation was not used (figures 3, 4). all shoots transformed with one of the two ovule-specific constructs were transferred into a selective propagation medium containing 100 mg/l kanamicin and 200 mg/l cefotaxime to select for transgenic shoots and avoid “escapes”. each single shoot was separated and grown separately, except in groups of indistinct shoots, where only one was chosen and propagated. the shoots with healthy growth were analyzed with a mug assay to confirm the presence of the marker gene “gus” in the constructs. transgenic shoots were more fluorescent than control shoots (difference between time 30 and time 0; table 3; fig. 5). discussion transformation of woody fruit species expressing marker genes has occurred in apple (james et al. 1993), citrus (vardi et al. 1990) and vitis (scorza et al. 1995). perennial transgenic plants that express genes of agronomic interest have been obtained in actinidia (rugini et al.. 1991) and apple (norelli et al. 1994). usually, agrobacterium-based methods were used because figure 3. regeneration of shoots after genetic transformation of leaf discs. on the right (a) treatment with the combination ba:naa:tdz (5:1:1) during co-cultivation; on the left (b) treatment with agrobacterium “vip” in co-transformation. table 2. transformations of “greensleaves” apple using construct ino-iaam. the construct, date, assigned number and description of the experiments are indicated. percentage of regeneration and number of single or grouped shoots regenerated were measured. the letters on the side of the numbers in the same column for the same date experiment indicate significative differences calculated using the duncan test (p=005). experiment n. treatment % regeneration nº of shoots nº of groups of shoots 1 control 32.73 b 1.44 b 1.33 b 2 vip 17.67 a 0.84 a 0.33 a 3 control 26.13 b 0.90 b 0.93 b 4 vip 7.73 a 0.40 a 0.25 a figure 4. genetic transformation of ‘greensleaves’ apple leaf discs using the construct ino-iaam. the explants were cultivated in ms medium containing the combination ba:naa:tdz (ratio 5:1:1) either during co-cultivation or regeneration. figure 5. a) transformation of ‘greensleaves’ apple using a leaf disc infected by two agrobacterium strains simultaneously: one containing the construct ino-iaam and the second with a “gene vip” b) transformation of ‘greensleaves’ apple using a leaf disc infected by two agrobacterium strains simultaneously: one containing the construct ino-iaam and the second with a “gene vip”. 15improved apple transformation protocol table 3. measurements of fluorescence of five single shoots regenerated from each transformation treatment and ten control greensleaves cultured in vitro. the construct, treatment, nº assigned to the shoot, presence of fluorescence at uv (“+” means fluorescence, “-”not fluorescence) and concentration at the beginning (time 0) and end of the mug assay (time 30) were indicated. construct treatment nºpetri (nºplant) uv fluorescence total concentration total concentration defh9-iaam 1 2 (4) + 23700 159000 1 1 (3) + 28200 463000 1 6 (2) + 31300 260000 1 5 (4) + 253000 241000 1 7 (3) + 276000 245000 2 2 (3) + 75500 102000 2 4(10) + 68600 542000 2 5(4) + 27100 451000 2 8(2) + 46700 532000 2 3 (1) + 77600 746000 3 4 (8) + 67200 442000 3 3(6) + 67500 578000 3 2(5) + 43500 876000 3 3(5) + 87100 783000 3 8(4) + 85000 903000 ino-iaam 1 3(5) + 11000 613000 1 2(3) + 41700 403000 1 5(10) + 76500 338000 1 7(3) + 76500 338000 1 13(4) + 13300 141000 2 2(5) + 13300 141000 2 2(7) + 31300 1650000 2 11(5) + 12800 418000 2 2(4) + 58800 157000 2 4(6) + 55300 223000 3 9(4) + 12300 183000 3 11(7) + 82800 197000 3 3(4) + 30300 841000 3 15(6) + 31400 229000 3 2 (3) + 56300 437000 4 7 (11) + 68300 649000 4 12 (2) + 63900 726000 4 13 (4) + 92600 968000 4 4 (5) + 74300 319000 4 15 (3) + 28500 274000 control 1 312 347 2 367 386 3 396 455 4 474 606 5 452 537 6 573 612 7 627 429 8 391 621 9 482 430 10 619 329 16 federico martinelli, anna perrone, abhaya m. dandekar of their greater transformation efficiency and more stable integration of the transgene into the host plant genome. agrobacterium strain lba4404 has been used widely and the kanamycin-sensitive strain eha105 was used to transform walnut (mcgranahan et al. 1990) and apple (dandekar et al. 2004). the virulence of agrobacterium strains against different crops can vary. different alleles of vir g genes can increase virulence (ghorbel et al. 2001). the expression of vir genes is also stimulated by different environmental factors, like ph, temperature and osmotic conditions. the length of in vitro cocultivation of explants with bacteria influences transformation efficiency, which generally increases with time. however, co-cultivation of more than three to four days can make it difficult to control agrobacterium growth (petri et al. 2004). the efficiency of transformation can be increased if the medium contains phenolic compounds like acetosyringone or osmoprotectants such as betaine phosphate and proline. these metabolites stimulate induction of the virulence genes (james et al. 1993). two gene constructs, ino-iaam and def h9-iaam, previously used to transform micro-tom tomato, were used to test different hormone combinations to improve a genetic transformation protocol for ‘greensleaves’ apple. a secondary objective was to create transgenic plants that might be tested in the future for parthenocarpy, since this feature might counter the auto-incompatibility of many apple cultivars. in addition, malus spp. are sensitive to adverse environmental conditions for pollination and/or fertilization. a parthenocarpic apple orchard would have several benefits. no pollination or fertilization would be needed for fruit set, making fruit set resistant to inclement weather, which would allow consistent production of high-quality fruit. there are currently transformation protocols for many apple cultivars, such as greensleaves (james et al. 1993), delicious (sriskanadarjah et al. 1994), royal gala (yao et al. 1995) and marshal mcintosh (bolar et al. 1999). however, these protocols would benefit from more efficient regeneration of transgenic shoots. while a protocol for transformation of apple cv greensleaves has been developed (james et al. 1993), the transformation rate is only one to three % of the total explants. a recent and reliable procedure for grape transformation has been developed using meristematic bulk (mb tissue) made using mechanical and chemical treatments. mb tissue has a high regenerative competence and can be transformed efficiently by agrobacterium (xie et al.. 2016). this protocol should be tried in apple. our protocol used mature leaf discs. the developmental stage of the explant is an important factor influencing genetic transformation. juvenile material regenerated better than old material in citrus (12 to 80% vs 6%; cervera et al. 1998). in apple, genetic transformation rates are < 3% (dandekar et al. 2004); in pear cultivars, < 1 to 43% depending on genotype (zhu and welander, 2000); while in prunus, protocols that regenerate transformed buds from 30% of explants were obtained almost thirty years ago (mante et al. 1991). our protocol tested two hormones, ba (benzyl adenine) and naa (naphthalene acetic acid) for their ability to stimulate regeneration of transgenic shoots. our work was based on preliminary evidence that ‘greenleaves’ leaf explants regenerated three to four times more shoots per explant with diphenyl urea thidiazuron (tdz) combined with other medium changes, such as concentration of silver nitrate. the concentration of tdz used is critical because high concentrations may cause “condensed” axillary shoots that do not elongate or proliferate in culture. in these experiments, a combination of 1 mg/ml tdz, 5 mg/ml ba and 1 mg/ml naa were used to regenerate transgenic shoots. cotransformation with an agrobacterium strain containing “vip” genes did not increased the percentage of transgenic shoots regenerated. using iba instead of the combination ba:tdz:naa during co-cultivation increased the amount of callus without increasing regeneration of transgenic shoots. other factors that can affect regeneration were evaluated. these included the biological source of the explants (leaf age, maturity and position on the stem, explant orientation) or environmental conditions (nitrogen concentration, growth regulators, incubation time and temperature; oliveira et al. 1996). here, we used mature leaf discs. young leaves are very useful as an explant source and morphogenesis occurred mainly at the cut edges of midribs, or in association with vascular tissues. regeneration ability may be affected by stress induced by genetic transformation itself (oliveira et al. 1996). a factor that greatly affects the regeneration capability is the amount, type and timing of the antibiotics used to kill agrobacterium (sain et al. 1994). together with the gene of interest, other genes are transferred to allow selection of transformed cells. among these, antibiotic resistance genes are common, such as the neomycin phosphotransferase gene (nptii) that confers resistance to aminoglycoside antibiotics (miki and mchugh, 2004). carbenicillin and kanamycin are used widely as selection antibiotics and can yield quite different results in different species. for example, in citrus, pear, walnut or olive, 100 mg/l kanamycin is used for selection, but in prunus, the concentrations are usually five to 10 mg/l. in apple, alternate periods of selection and non-selection, or selection applied only on the regener17improved apple transformation protocol ated shoots, were used (james et al. 1993). selection of transformed shoots is also complicated by the presence of escapes (non-transformed shoots) due to inactivation of antibiotics by transformed cells or by the persistance of agrobacterium in the explants. because of public concern with introducing antibiotic resistance genes into food, methods have been developed to eliminate them from the selection process (zuo et al. 2002). for instance, a reporter gene such as gus (β-glucoronidase gene) can be used to evaluate transformation efficiency by visual selection. to avoid bacterial contamination, gus genes that cannot be spliced out by the host cells were used. in prunus, this method is still complicated by intrinsic gus-like activity of the plants. the number of transformants obtained is usually underestimated by at least 25% when based on the expression of screenable marker genes (oliveira et al. 1996). kanamycin resistance is still a common strategy for selecting transgenic shoots, but the strong selection required to avoid escapes or chimeras reduces the number of cells that both received the dna and regenerated buds. an innovative approach has improved transformation efficiencies tenfold over kanamycin selection in recalcitrant species. this method is based on giving transformants a metabolic advantage, rather than on killing non-transformed cells (joersbo, 2001). it is hypothesized that necrosis produced by antibiotics in non-transformed tissues could inhibit regeneration from transformed adjacent tissues (joersbo, 2001). using regeneration-promoting genes, combined with hormone-free regeneration medium, could also substitute for traditional antibiotic marker genes. with no growth regulators, only transformed cells can regenerate, allowing simple screening for putative transformants without using a marker gene. much work is devoted to identifying regeneratingpromoting genes, presumably related to cytokinin synthesis, that enable the embryogenic or organogenic transition (zuo et al. 2002). the ipt gene, from agrobacterium, must be used under the control of a inducible promoter, because constitutive over-expression of this gene can cause phenotypic growth disorder (kunkel et al. 1999). conclusions explants transformed with either ino-iaam or def h9-iaam transgenes regenerated more shoots on combination of three hormones (ba:naa:tdz) than on iba and co-transformation had no effect. in experiments using def h9-iaam, the percentage of regeneration for the hormone combination was significatively greater than for the other two treatments (21.04% vs 8.95 and 4.42%, respectively). the number of transgenic shoots was also greater with the hormone combination (1.52% vs 0.78 and 0.47%, respectively). experiments using ino-iaam confirmed these results. co-transformation with agrobacterium containing vip genes was deleterious to production of regenerants, possibly due to a lower concentration of agrobacterium containing the ino-iaam or def h9-iaam transgene during infection. most shoots regenerated in selection medium containing 100 mg/l kanamycin at were transgenics with significantly greater fluorescence in the mug assay than untransformed, regenerated greensleaves. this suggests that this concentration of kanamycin provided a good balance between selection of transgenic shoots and allowing reasonable regeneration efficiency. author contributions mf and amd designed and conceived the research work. mf performed the experimental work and statistical analysis. mf mainly wrote the article. ap and amd reviewed and discussed results. all authors contributed significantly on the writing of the manuscript. references bolar j.p., norelli j.l., harman g.e., brown s.k., aldwinckle, h.s. 2001. synergistic 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implications using scanning electron microscopy jun wang1,2,*, qiang ye1, chu wang2, tong zhang2, xusheng shi2, majid khayatnezhad3, abdul shakoor4,5 influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats yuri a. kirillov1, maria a. kozlova1, lyudmila a. makartseva1, igor a. chernov2, evgeniya v. shtemplevskaya1, david a. areshidze1,* the morphological, karyological and phylogenetic analyses of three artemisia l. (asteraceae) species that around the van lake in turkey pelin yilmaz sancar1,*, semsettin civelek1, murat kursat2 molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant jinxin cheng1,*, dingyu hu1, yaran liu2, zetian zhang1, majid khayatnezhad3 genetic variations and interspesific relationships in salvia (lamiaceae) using scot molecular markers songpo liu1, yuxuan wang1, yuwei song1,*, majid khayatnezhad2, amir abbas minaeifar3 development of female gametophyte in gladiolus italicus miller (iridaceae) ciler kartal1, nuran ekici2,*, almina kargacıoğlu1, hazal nurcan ağırman1 colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae) harshita dwivedi*, girjesh kumar statistical evaluation of chromosomes of some lathyrus l. taxa from turkey hasan genç1, bekir yildirim2,*, mikail açar3, tolga çetin4 use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater treatment and water of the kamniška bistrica river (slovenia) peter firbas1, tomaž amon2 the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bo shi1,*, majid khayatnezhad2, abdul shakoor3,4 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(3): 65-75, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1330 caryologia international journal of cytology, cytosystematics and cytogenetics citation: jinxin cheng, dingyu hu, yaran liu, zetian zhang, majid khayatnezhad (2021) molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant. caryologia 74(3): 65-75. doi: 10.36253/caryologia-1330 received: june 07, 2021 accepted: july 16, 2021 published: december 21, 2021 copyright: © 2021 jinxin cheng, dingyu hu, yaran liu, zetian zhang, majid khayatnezhad. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant jinxin cheng1,*, dingyu hu1, yaran liu2, zetian zhang1, majid khayatnezhad3 1china people’s police university, langfang, hebei,065000, china 2beijing forestry university, beijing 100083, china 3department of environmental sciences and engineering, ardabil branch, islamic azad university, ardabil, iran *corresponding author. e-mail: jinxin_cheng@163.com abstract. alcea l. is one of the largest genera of malvaceae family with nearly 70 species worldwide mainly distributed in sw asia. according to the latest revision of the family, it is represented by 34 species in the flora of iran, among them, 15 species are endemic. it is tough to accurate germplasm/ plant recognition by using morphological characteristics because of its propagation, growing and using. we conducted a molecular data analysis on these plant species due to their importance. we examined 156 plants from 14 species in 16 regions that were selected randomly for this investigation. it has been 119 polymorphic bands (94.33%) were resulted from 128 bands of 10 primers in amplification of genomic dna. issr primers have a great capacity to detect polymorphic loci among alcea species, as evidenced by the high average pic and mi values found. the genetic similarity of 14 species was calculated and ranged between 0.635 to 0.990. inter-simple sequence repeats (issr) markers research revealed that alcea tarica pakravan & ghahreman and alcea kopetdaghensis lljin had the least similarity, while alcea semnanica pakravan and alcea mazandaranica pakravan & ghahreman had the most. the current study attempts to answer three questions: 1) can issr markers identify alcea species? 2) what is the genetic structure of these taxa in iran? and 3) what is the inter-relationship between these taxa? the current study discovered that issr markers can be used to identify species. keywords: population structure, gene flow, network, genetic admixture. introduction it is vital to determine the precise boundaries of a species in order to gain a better understanding of any scientific investigations. as a result, in the context of biology, species delimitation is a topic that receives a lot of attention (collard and mackill 2009; wu et al. 2013). however, establishing the criterion that could be used to resolve species borders is a contentious issue (esfandani-bozchaloyi et al. 2018a, 2018b, 2018c, 2018d). (pandey et al. 2008). 66 jinxin cheng et al. furthermore, the analysis of wild population genetic structure and the study of intra-specific levels of genetic diversity are critical for the creation of successful conservation measures. the malvaceae family includes the perennial herb alcea l., which has its primary centers of diversity in the western mediterranean basin and the middle east (zohary 1963a, b, hutchinson 1973, riedl 1976, heywood et al. 1978). in europe, there are only a few species of alcea (escobar et al. 2009). the flora of iran has 34 species, 15 of which are endemic, according to the most recent revision of the family (pakravan 2008) alcea species are usually tall-growing hemicryptophytes that grow annually, biennially, or perennially. the stem is erect, rarely branching at the base, and occasionally acaulescent. the leaves might be simple, lobed, palmatipartite, or palmatisect in shape. the sepals are five in number and are connate at the base. petals are pentamerous and come in a variety of colors. mericarps come in a variety of shapes and sizes, each with a sterile upper chamber and a single seeded bottom chamber. (ghahreman et al. 2001, pakravan & ghahreman 2006, pakravan 2006, 2008). the mucilage that containing the plants of the malvaceae family are sources of carbohydrates, which are used in medicine (azizov et al. 2007). the species of this family, especially alcea rosea has been used as diuretic, demulcents, emollient, aperients, and in the treatment of burning sensation, skin disease, and constipation (shaheen et al. 2010). delimitation of alcea and althaea ganera has been a challenging task in taxonomic history of malvaceae. alcea has been traditionally included in althaea based on epicalyx characteristics (bentham & hooker 1862, baker 1890, candolle 1837, edlin 1935, willdenow 1800). however, characteristics of staminal column and fruit features led to consider alcea and althaea as two separate taxa (alefeled 1862; boissier 1867; iljin 1949). molecular-phylogenetic data also support the monophyly and distinctness (as suggested by morphological data) of alcea but they are of limited use in determining relationships between species and species delimitations (escobar garsia et al. 2012). the taxonomic complexity of alcea is remarkable (zohary 1963a,b, riedl 1976, townsend 1980). alcea has so far proposed two infrageneric classifications, each of which is divided into a few informal groups. despite the fact that it has a significant number of species, no formal subgeneric categorization has been established. due to uniformity and pronounced plasticity in morphological characters of this genus (especially in flower and fruit characters), some traits such as leaf sequence, mericarp shape, relative length of calyx versus epicalyx, and indumentum morphology are more applicable in taxonomy of alcea (escobar garcia et al. 2012). for researching genetic diversity, molecular markers are a useful tool. random amplified polymorphic dna (rapd) and inter simple sequence mutation (issm) are two sophisticated genetic markers. for diversification assessments, issr markers have been routinely used (pharmawati et al. 2004). the rapd approach is rapid, simple, and does not require any prior sequence awareness. then uses a single primer of any nucleotide sequence, the approach detects nucleotide sequence polymorphism (moreno et al. 1998). a single 16-18 bp. long primer consists of a repeating sequence attached at the 3’ or 5’ end of 2-4 arbitrary nucleotides is used to amplify dna for issr markers. the method is faster, easier, and less affordable than rapd, and it is more repeatable (esfandani-bozchaloyi et al. 2017a, 2017b, 2017c, 2017d; collard and mackill 2009; wu et al. 2013). the current study used new gene-targeted molecular markers, namely issr markers, to assess the genetic diversity and connections among different alcea species. we conducted a genetic research of 156 collected specimens of 14 alcea species because this is the first study on the usage of issr markers in the alcea genus. we try to answer the following questions: 1) is there infra and interspecific genetic diversity among studied species? 2) is genetic distance among these species correlated with their geographical distance? 3) what is the genetic structure of populations and taxa? 4) is there any gene exchange between alcea species in iran? materials and methods plant materials a total of 156 individuals were sampled representing 16 geographical populations belonging to 14 alcea species in east azerbaijan, lorestan, kermanshah, mazandaran, esfahan, tehran, khorasan, semnan, fars, golestan provinces of iran during july-agust 2016-2019 (table 1). we utilized 156 botanical accessions (three to twelve samples of each group) from 16 different populations with various eco-geographic attributes for issr analysis which were extracted and stored in -20 until further use. more information about geographical distribution of accessions are in table 1 and fig. 1. during several field excursions to the all part of iran as well as survey to the several herbaria {herbarium of iranian research institute of plant protection (iran), herbarium of tehran university (tuh), herbarium of shahid beheshti university (sbuh), and some herbaria of natural resources research centers in most provinces of iran such as: east and west azerbaijan], some new 67molecular identification and genetic relationships among alcea (malvaceae) species by issr markers information were obtained. the specimens were identified using the identification keys and descriptions of the alcea species in the relevant floras [taxonomical studies in alcea of south-western asia (zohary 1963a, b), flora orientalis (boissier 1967), flora palestina (zohary 1972), flora iranica (riedl 1976), flora of iraq (townsend et al. 1980), and the taxonomic revision of alcea and althaea in turkey (uzunhisarcikli & vural 2012). dna extraction and issr assay in every one of the tested populations, fresh leaves were also used in random from one to twelve plants. silica gel powder was used to dry them. to extract genomic dna, the ctab activated charcoal procedure was applied (esfandani-bozchaloyi et al. 2019). a 0.8 percent agarose gel was used to test the purity of the isolated dna. 22 primers from the ubc (university of british columbia) series were evaluated for dna amplification for the issr study. based on band reproducibility, ten primers were chosen for issr study of genetic diversity (table 2). pcr reactions were carried in a 25μl volume containing 10 mm tris-hcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). the following program was used to perform the amplifications and reactions in a techne thermocycler (germany): 94°c for 5 minutes, then 40 cycles of 1 minute at 94°c, 1 minute at 52-57°c, and 2 minutes at 72°c. a final extension step of 7-10 minutes at 72°c finished the reaction. running the amplification results table 1. voucher details of alcea species in this study from iran. no sp. locality latitude longitude altitude (m) sp1 alcea aucheri (boiss.) alef. esfahan:ghameshlou, sanjab kermanshah, islamabad 38°52’37” 47°23’92” 1144 sp2 alcea angulata freyn & sint. tehran, damavand 32°50’03” 51°24’28” 1990 sp3 alcea rhyticarpa (trautv.) iljin khorasan, mashhad 29°20’07” 51°52’08” 1610 sp4 alcea sulphurea (boiss.& hohen.) alef. tehran, tochal 38°52’37” 47°23’92” 1144 sp5 alcea striata (dc.) alef. kermanshah, islamabad esfahan, semirom 33°57’12” 47°57’32” 2500 sp6 alcea loftusii (baker) zohary lorestan, oshtorankuh, above tihun village 34°52’37” 48°23’92” 2200 sp7 alcea gorganica (rech. f., aellen & esfand.) zohary golestan, gorgan 38°52’37” 47°23’92” 1144 sp8 alcea popovii iljin tehran, chalous 35°50’03” 51°24’28” 1700 sp9 alcea mazandaranica pakravan & ghahreman mazandaran province, kelardasht, rodbarak 36°14’14” 51°18’07” 1807 sp10 alcea tarica pakravan & ghahreman tehran, damavand 32°36’93” 51°27’90” 2500 sp11 alcea ghahremanii pakravan & assadi east azerbaijan, arasbaran 37°07’02” 49°44’32” 48 sp12 alcea kopetdaghensis lljin khorasan, koppeh dagh 28°57’22” 51°28’31” 430 sp13 alcea iranshahrii pakravan, ghahreman & assadi fars, estahban 30°07’24” 53°59’06” 2178 sp14 alcea semnanica pakravan semnan, damghan 28°57’22” 51°28’31” 288 figure 1. map of iran shows the collection sites and provinces where 14 alcea species were obtained for this study; sp1= a. aucheri; sp2= a. angulata; sp3= a. rhyticarpa; sp4= a. sulphurea; sp5= a. striata; sp 6= a. loftusii ; sp7= a. gorganica; sp8= a. popovii; sp9= a. mazandaranica; sp10: a. tarica; sp11: a. ghahremanii; sp12= a. kopetdaghensis; sp13= a. iranshahrii; sp14= a. semnanica. 68 jinxin cheng et al. over a 1 percent agarose gel and staining with ethidium bromide revealed the amplification products. a 100-bp molecular size ladder was used to assess the fragment size (fermentas, germany). data analyses molecular analyses the collected issr bands were coded as binary characters (presence = 1, absence = 0) and utilized to analyze genetic diversity. the upgma (unweighted paired group using average) ordination methods were utilized to sort the plant specimens into groups (podani 2000). to quantify the capability of each primer to distinguish polymorphic loci amongst these genotypes, two measures, polymorphism information content (pic) and marker index (mi), were utilized to assess its discriminatory ability (powell et al. 1996). mi is calculated for each primer as mi = pic × emr, where emr is the product of the number of polymorphic loci per primer (n) and the fraction of polymorphic fragments (β) (heikrujam et al. 2015). for each primer, the effective multiplex ratio (emr) and the number of polymorphic bands (npb) were computed. parameter like nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism (p% = number of polymorphic loci/number of total loci) were determined (weising et al, 2005, freeland et al. 2011). shannon’s index was calculated by the formula: h’ = -σpiln pi. rp is defined per primer as: rp = ∑ ib, were “ib” is the band informativeness, that takes the values of 1-(2x [0.5-p]), being “p” the proportion of each genotype containing the band. genalex 6.4 software is used to analyze the percentage of polymorphic loci, the mean loci by accession and population, uhe, h’, and pca (peakall & smouse 2006). neighbor joining (nj) clustering and neighbor-net networking were based on nei’s genetic distance between populations (freeland et al. 2011, huson & bryant 2006). the mantel test was used to see if there was a link between the analyzed populations’ geographical and genetic distances (podani 2000). the comparison of genetic divergence or genetic distances, estimated by pairwise fst and related statistics, with geographical distances by mantel test is one of the most popular approaches to evaluate spatial processes driving population structure. the mantel test, as originally formulated in 1967, where gij and dij are, respectively, the genetic and geo-graphic distances between populations i and j, considering populations. because zmis given by the sum of products distances its value depends on how many populations are studied, as well as the magnitude of their distances. the zm-value can be compared with a null distribution, and mantel originally proposed to test it by the standard normal deviate (snd), given by snd =zm/var(zm)1/2 (mantel 1967). these analyses were done by past ver. 2.17 (hammer et al. 2012), darwin ver. 5 (2012) software. to show genetic differences between the populations, the amova (analysis of molecular variance) test (with 1000 permutations) was utilized, which was implemented in genalex 6.4 table 2. issr primers used for this study and the extent of polymorphism. primer name primer sequence (5’-3’) tnb npb ppb pic pi emr mi issr-1 dbdacacacacacacaca 15 13 93.84% 0.66 4.66 11.33 4.67 issr-2 ggatggatggatggat 12 11 94.91% 0.48 5.21 12.50 5.65 issr-3 gacagacagacagaca 16 14 95.74% 0.67 5.66 9.57 5.37 issr-4 agagagagagagagagyt 13 12 92.31% 0.54 8.21 10.23 4.55 issr-5 acacacacacacacacc 17 17 100.00% 0.47 7.32 11.55 4.18 issr-6 gagagagagagagagarc 11 10 96.89% 0.43 6.56 9.34 7.17 issr-7 ctctctctctctctctg 13 12 95.81% 0.34 4.21 6.78 5.59 issr-8 cacacacacacacacag 12 12 100.00% 0.47 3.37 9.55 3.45 issr-9 gtgtgtgtgtgtgtgtyg 11 9 93.89% 0.53 6.56 8.34 6.11 issr-10 cacacacacacacacarg 11 11 100.00% 0.59 4.22 10.11 4.33 mean 12.8 11.9 94.33% 0.55 5.32 10.66 5.7 total 128 119 note: tnb the number of total bands, npb: the number of polymorphic bands, ppb (%): the percentage of polymorphic bands, pi: polymorphism index, emr, effective multiplex ratio; mi, marker index; pic, polymorphism information content for each of caat boxderived polymorphism (cbdp) primers. 69molecular identification and genetic relationships among alcea (malvaceae) species by issr markers (peakall & smouse 2006). this approach considers the equal amount of gene flow among all populations. the genetic structure of populations was studied by bayesian based model structure analysis (pritchard et al. 2000), and maximum likelihood-based method of k-means clustering of genodive ver. 2 (2013). data were evaluated as dominating markers for structure analysis (falush et al. 2007). under the correlated allele frequency model, we used the admixture ancestry model. after a 105 burn-in period, a markov chain monte carlo simulation was ran 20 times for each value of k. using the delta k value, the evanno test was run on the structure result to determine the right number of k. (evanno et al. 2005) results species identification and genetic diversity to examine genetic links among alcea species, ten issr primers were tested; all of the primers yielded replicable polymorphic bands in all 14 alcea species. figure 2 depicts the issr amplification induced by the issr-5 primer. across 14 alcea species, a total of 119 amplified polymorphic bands were produced. the amplified fragments were between 100 and 3000 bp in length. with an average of 11.9 polymorphic bands per primer, issr-5 had the most and lowest number of polymorphic bands, with 17 and 9 respectively. the average pic of the 10 issr primers was 0.55, ranging from 0.34 (issr-7) to 0.67 (issr-3). the mi of the primers ranged from 3.45 (issr-8) to 7.17 (issr-6) on average, with an average of 5.7. issr primers had an emr ranging from 6.78 (issr7) to 12.50 (issr-2), with an average of 10.66 per primer (table 2). the primers with the highest emr values were thought to be more useful in separating the genotypes. the genetic parameters for all 14 alcea species amplified with issr primers were calculated (table 3). unbiased expected heterozygosity (h) ranged from 0.15 (alcea popovii) to 0.39 (alcea aucheri), with a mean of 0.28. shannon’s information index (i) showed a similar pattern, with the greatest value of 0.39 in alcea aucheri and the lowest value of 0.10 in (alcea popovii) , with a mean of 0.27. the number of alleles (na) observed in alcea rhyticarpa ranged from 0.201 to 0.645 in alcea kopetdaghensis. the effective number of alleles (ne) in figure 2. electrophoresis gel of alcea species from dna fragments produced by issr-5 and issr-3; sp1,14= a. aucheri; sp2,15= a. angulata; sp3,16= a. rhyticarpa; sp4,17= a. sulphurea; sp5,18= a. striata; sp 6,19= a. loftusii; sp7,20= a. gorganica; sp8,21= a. popovii; sp9,22= a. mazandaranica; sp10,23: a. tarica; sp11,24: a. ghahremanii; sp12,25= a. kopetdaghensis; sp13,26= a. iranshahrii; sp14,27= a. semnanica. table 3. genetic diversity parameters in the studied alcea species. sp n na ne i he uhe %p sp1 alcea aucheri 5.000 0.462 1.095 0.398 0.48 0.39 76.55% sp2 alcea angulata 8.000 0.399 1.167 0.322 0.398 0.344 65.77% sp3 alcea rhyticarpa 8.000 0.201 0.095 0.23 0.27 0.22 42.23% sp4 alcea sulphurea 5.000 0.341 1.058 0.24 0.27 0.20 53.75% sp5 alcea striata 5.000 0.455 1.077 0.277 0.24 0.22 55.05% sp6 alcea loftusii 8.000 0.499 1.067 0.24 0.23 0.24 49.26% sp7 alcea gorganica 6.000 0.555 1.020 0.22 0.25 0.28 43.53% sp8 alcea popovii 10.000 0.431 1.088 0.20 0.22 0.25 41.53% sp9 alcea mazandaranica 3.000 0.255 1.021 0.25 0.28 0.22 47.15% sp10 alcea tarica 9.000 0.261 1.024 0.292 0.23 0.23 43.15% sp11 alcea ghahremanii 12.000 0.287 1.253 0.266 0.254 0.28 51.99% sp12 alcea kopetdaghensis 3.000 0.645 1.062 0.24 0.224 0.213 44.73% sp13 alcea iranshahrii 8.000 0.499 1.067 0.24 0.281 0.24 49.26% sp14 alcea semnanica 12.000 0.287 1.233 0.271 0.284 0.292 51.91% abbreviations: n = number of samples, na= number of different alleles; ne = number of effective alleles, i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations. 70 jinxin cheng et al. these species ranged from 0.095 (alcea rhyticarpa) to 1.253 (alcea ghahremanii). the amova test revealed a substantial genetic difference (p = 0.001) between the species investigated. it was discovered that 67 percent of overall variance was found across species, whereas 33 percent was found within species (table 4). significant nei’s gst (0.245, p = 0.001) and d est (0.765, p = 0.001) values further indicated the genetic difference of these species. in comparison to within-species genetic diversity, these findings demonstrated a larger distribution of genetic variety within alcea species. species identification and inter-relationship because the results of other clustering and ordination approaches were similar, pca plot and upgma clustering are provided here (figure 3-4). plant samples from different species were put together and formed various groups in general. this study demonstrates that the examined species were divided into several groups based on molecular characteristics. we didn’t find any intermediate forms in the specimens we looked at. the dendrogram based on issr data was constructed by upgma analysis, grouping all of the alcea species into two major clusters (fig. 4). the first major cluster divided into two minor clusters of which the first minor cluster again divided into two sub-minor clusters. the first subminor cluster consisted of a. aucheri; a. rhyticarpa and a. striata. the second sub-minor cluster was represented by a. angulata; a. sulphurea. the second major cluster divided in to two minor clusters of which the first minor cluster consisted of a. loftusii, a. gorganica and a. popovii. the second sub-minor cluster was represented by a. kopetdaghensis, a. mazandaranica, a. tarica, a. ghahremanii, a. iranshahrii; and a. semnanica. this is consistent with the amova and genetic diversity metrics previously reported. genetically, the species are distinct from one another. issr molecular markers can be employed to taxonomist alcea species, according to these findings. popgene software’s nm analysis similarly yielded a mean nm=0.476, which is considered a very low amount of gene flow that among samples analyzed. isolation by distance (ibd) occurred among the alcea species tested, as the mantel test with 5000 permutations revealed a strong correlation (r = 0.76, p=0.0002) between genetic distance and geographical distance. the genetic identity of nei, as well as the genetic distance between the species tested, were discovered (table 5). the results revealed that alcea semnanica and alcea mazandaranica had the most table 4. analysis of molecular variance (amova) of the studied species. source df ss ms est. var. % φpt among pops 29 1601.364 45.799 15.194 67% within pops 122 454.443 1.905 2.884 33% 67% total 151 2033.807 17.020 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance; φpt: proportion of the total genetic variance among individuals within an accession, (p < 0.001). figure 3. pca plots of based on issr data revealing species delimitation in alcea species; sp1= a. aucheri; sp2= a. angulata; sp3= a. rhyticarpa; sp4= a. sulphurea; sp5= a. striata; sp 6= a. loftusii ; sp7= a. gorganica; sp8= a. popovii; sp9= a. mazandaranica; sp10: a. tarica; sp11: a. ghahremanii; sp12= a. kopetdaghensis; sp13= a. iranshahrii; sp14= a. semnanica. figure 4. dendrogram generated using the unweighted pair group method with arithmetic average (upgma) analysis showing relationships among different alcea species using issr data. 71molecular identification and genetic relationships among alcea (malvaceae) species by issr markers degree of genetic resemblance (0.99). between alcea tarica and alcea kopetdaghensis, there was the least genetic affinity (0.63). the low nm value (0.47) implies little gene flow or ancestrally shared alleles between the species investigated, as well as considerable genetic divergence between and within alcea species. to determine the ideal number of genetic groups, we used structure analysis followed by the evanno test. in the species analyzed, we employed the admixture model to show interspecific gene flow and/or ancestrally shared alleles. structure analysis followed by evanno test produced δk = 10 (fig. 5). the structure plot (fig. 6) produced more detailed information about the genetic structure of the species studied as well as shared ancestral alleles and/ or gene flow among alcea species. this plot revealed that genetic affinity between alcea aucheri and a. sulphurea (similarly colored, no. 1, 4), as well as a. gorganica; a. popovii and a. semnanica; (no. 7,8,14) table 5. the matrix of nei genetic similarity (gs) estimates using issr molecular markers among 14 alcea species. sp1= a. aucheri; sp2= a. angulata; sp3= a. rhyticarpa; sp4= a. sulphurea; sp5= a. striata; sp 6= a. loftusii; sp7= a. gorganica; sp8= a. popovii; sp9= a. mazandaranica; sp10: a. tarica; sp11: a. ghahremanii; sp12= a. kopetdaghensis; sp13= a. iranshahrii; sp14= a. semnanica sp1 1.000 sp1 sp2 0.887 1.000 sp2 sp3 0.891 0.744 1.000 sp3 sp4 0.738 0.787 0.842 1.000 sp4 sp5 0.705 0.742 0.745 0.775 1.000 sp5 sp6 0.778 0.891 0.744 0.936 0.838 1.000 sp6 sp7 0.599 0.702 0.808 0.875 0.836 0.862 1.000 sp7 sp8 0.754 0.785 0.676 0.829 0.733 0.800 0.709 1.000 sp8 sp9 0.757 0.741 0.758 0.816 0.740 0.785 0.676 0.725 1.000 sp9 sp10 0.737 0.890 0.722 0.719 0.853 0.741 0.758 0.834 0.746 1.000 sp10 sp11 0.807 0.799 0.755 0.812 0.774 0.990 0.722 0.768 0.800 0.721 1.000 sp11 sp12 0.782 0.744 0.636 0.834 0.750 0.799 0.755 0.720 0.785 0.635 0.839 1.000 sp12 sp13 0.702 0.757 0.703 0.778 0.691 0.744 0.636 0.829 0.741 0.750 0.799 0.642 1.000 sp13 sp14 0.751 0.774 0.732 0.790 0.750 0.797 0.812 0.774 0.990 0.675 0.727 0.728 0.684 1.000 sp14 figure 6. structure plot of issr data in alcea species. sp1= a. aucheri; sp2= a. angulata; sp3= a. rhyticarpa; sp4= a. sulphurea; sp5= a. striata; sp 6= a. loftusii; sp7= a. gorganica; sp8= a. popovii; sp9= a. mazandaranica; sp10: a. tarica; sp11: a. ghahremanii; sp12= a. kopetdaghensis; sp13= a. iranshahrii; sp14= a. semnanica. figure 5. evanno test produced δk = 10 of issr data in alcea species. 72 jinxin cheng et al. due to shared common alleles. this is in agreement with upgma dendrogram presented before. the other species are distinct in their allele composition. the neighbornet diagram (fig. 7) also revealed almost complete separation of the studied species within the network, supporting the amova results. populations 1, 2 and 12,13 are distinct and stand separately from the other populations at a great distance. populations 6 and 7 and populations 10 and 11 show a closer genetic affinity and are placed close to each other. discussion in the biology of long-term evolution of a group of animals or species, genetic diversity plays a crucial role. the foundation for a taxon’s presence, development, and evolution. to recognize the taxonomy, origin, and evolution of a taxon, it is necessary to investigate its genetic diversity. in addition, such study could provide a theoretical foundation for the conservation, expansion, exploitation, and breeding of germplasm resources (lubbers et al. 1991). the current study provided fascinating information about genetic variability, genetic stratification, and morphological difference in iran’s north and west. the degree of genetic variability within a species is significantly connected with its reproduction method; the higher the degree of open pollination/cross breeding, the greater the genetic variability in the taxon under study (meusel et al. 1965). a primer’s pic and mi features aid in establishing its efficacy in genetic diversity analysis. the ability of a marker technique to resolve genetic variability, according to sivaprakash et al. (2004), may be more directly connected to the degree of polymorphism. pic values ranging from zero to 0.25 indicate relatively low genetic variation among genotypes, 0.25 to 0.50 indicate a mid-level of genetic diversity, and ≥ 0.50 indicate a high level of genetic diversity (tams et al. 2005). the pic values of the issr primers in this study ranged from 0.34 to 0.66, with a mean value of 0.55, indicating that issr primers have a good level of competence in detecting genetic diversity among alcea species. in the alcea taxon, all ten primer pairs demonstrated good polymorphism. for the species under investigation, a total of 128 alleles were discovered. the total number of polymorphic bands per primer varied from 9 to 17, and the average allele number in loci was 11.9. occurrence of high polymorphism could be explained for species in different climatic zones with varying selection pressure during the course of evolution (mishra et al. 2011). in most studies, population size is limited to several vegetative accession (meusel et al. 1965; uotila 1996). this population could be showed genetic drift, whose effect are observed in the high level of fis and low level of genetic diversity. the isolation of the population and absence the gene flow led to fragmentation of the alcea populations. between genetic diversity parameters and population size were showing positive correlations that confirmed various studies (leimu et al. 2006). there are two reasons for the positive correlation between genetic diversity and population size (leimu et al. 2006). 1a positive connection may confirmed the existence of an extinction vortex, in which declining population reduces genetic variety, resulting in inbreeding depression. plant fitness separates populations depending on habitat quality changes, which is the second cause. low levels of genetic variation, according to booy et al. (2000), can impair plant fitness and limit a population’s capabilities to react to environmental changes by selection and adaptation. genetic diversity (33%) was obtained within populations, whereas 67% of genetic variation obtained between the evaluated populations. the breeding system in plant species is one of the primary elements controlling the distribution of genetic variation (duminil 2007). couvet (booy et al. 2000) shown that one migrant each generation is insufficient to ensure long-term persistence of tiny populations, and that the number of migrants is determined by family background characteristics and population genetics (vergeer et al. 2003). for the lack of distinctions across isolated groups, there are two explanations. the initial theory proposed that genetic variety figure 7. neighbor-net of issr data in alcea species. sp1= a. aucheri; sp2= a. angulata; sp3= a. rhyticarpa; sp4= a. sulphurea; sp5= a. striata; sp 6= a. loftusii; sp7= a. gorganica; sp8= a. popovii; sp9= a. mazandaranica; sp10: a. tarica; sp11: a. ghahremanii; sp12= a. kopetdaghensis; sp13= a. iranshahrii; sp14= a. semnanica 73molecular identification and genetic relationships among alcea (malvaceae) species by issr markers in and between populations demonstrates gene flow patterns, resulting in group splitting (dostálek et al. 2010). geographically close communities are far more successfully associated via gene flow than populations segregated by considerable distance, according to the next objective. merely a few research have investigated into alcea’s genetic diversity thus far. kazemi et al. (2011) found a 93 percent polymorphism ratio with strong genetic resemblance (0.31 to 0.75) within a. rosea species in iran using rapd identifiers analysis. utilizing rapd markers, oztürk et al. (2009) evaluated the genetic profiles of 18 alcea species and found wide difference (0.13 to 0.69) throughout them. according to badrkhani et al. (2014), the sequence-related amplified polymorphism (srap) identifier was used to evaluate the genetic diversity and genetic similarity links among 14 alcea species were collected from the northwest of iran. seventeen srap primer pairings produced 104 segments, with an average of 5.7 polymorphic fragments per primer. the percentage of polymorphism spanned from 50% (me2-em6) to 100% (me2-em6), with an average polymorphism information content value of 0.3. the genetic similarity between a. sophiae and a. flavovirens was the lowest (0.17), while the highest was identified between a. digitata and a. longipedicellata (0.68). using upgma, two primary clusters were discovered, neither of which corresponded to the species’ geographical origin. according to their findings, srap markers may be suitable for analyzing genetic diversity in alcea. so far, only morphological data has been used to define iranian alcea species. however, due to the very small number of characteristics, the genus has a challenging taxonomy. according to pakravan’s (2008) study on alcea, only the leaf sequence and carpel structure are valuable traits. escobar garcia et al. (2012) with using three molecular markers (nrdna its and the plastid spacers psbatrnh and trnl-trnf), showed that a phylogeny of alcea and test previous infrageneric taxonomic hypotheses as well as its monophyly with respect to althaea, a genus with which it has often been merged. they also go into morphological variation and the use of morphological features as phylogenetic association indicators. while molecular findings indisputably corroborate the circumscription of alcea deduced from morphology, they are of limited usefulness in clarifying interspecific relationships, implying that alcea’s great species diversity is attributable to swift and early radiation. their research establishes the first alcea phylogeny and intends to pave the way for future research into the processes that underpin species radiation in the irano-turanian region. in conclusion, the results of this study showed that to evaluate the genetic diversity of the alcea genus in the irano-turanian region, a main center of species diversity for many medium-sized to large genera that remains greatly understudied. issr-derived primers were more successful than those produced from all other molecular markers. in addition, alcea species were clearly distinguished from one another in the dendrogram and pca, demonstrating that the issr approach is more effective in identifying alcea species. acknowledgment key r&d projects in hebei province(19275416d). key r&d projects in china people’s police university (zdx202101). references azizov u.m., mirakilova d.b., umarova n.t., salikhov s.a., rakhimov d.a. and mezhlumyan l.g.2007. chemical composition of dry extracts from alcea rosea. chemistry of natural compounds 43:508-511. alefeld, f.g.c. 1862. ueber die malveen. oesterreichische botanische zeitschrift 12:246-261. boissier p.e. 1867. flora orientalis, vol. 1. basel, geneva, leiden. baker eg. 1890. synopsis of genera and species of malveae. journal of botany 28:140-371. bentham g. & hooker jd. 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genetic transformation of malus spp federico martinelli1,*, anna perrone2, abhaya m. dandekar3 cytogenetic and cytological analysis of colombian cape gooseberry genetic material for breeding purposes viviana franco-florez, sara alejandra liberato guío, erika sánchez-betancourt, francy liliana garcía-arias, víctor manuel núñez zarantes* palynological analysis of genus geranium (geraniaceae) and its systematic implications using scanning electron microscopy jun wang1,2,*, qiang ye1, chu wang2, tong zhang2, xusheng shi2, majid khayatnezhad3, abdul shakoor4,5 influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats yuri a. kirillov1, maria a. kozlova1, lyudmila a. makartseva1, igor a. chernov2, evgeniya v. shtemplevskaya1, david a. areshidze1,* the morphological, karyological and phylogenetic analyses of three artemisia l. (asteraceae) species that around the van lake in turkey pelin yilmaz sancar1,*, semsettin civelek1, murat kursat2 molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant jinxin cheng1,*, dingyu hu1, yaran liu2, zetian zhang1, majid khayatnezhad3 genetic variations and interspesific relationships in salvia (lamiaceae) using scot molecular markers songpo liu1, yuxuan wang1, yuwei song1,*, majid khayatnezhad2, amir abbas minaeifar3 development of female gametophyte in gladiolus italicus miller (iridaceae) ciler kartal1, nuran ekici2,*, almina kargacıoğlu1, hazal nurcan ağırman1 colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae) harshita dwivedi*, girjesh kumar statistical evaluation of chromosomes of some lathyrus l. taxa from turkey hasan genç1, bekir yildirim2,*, mikail açar3, tolga çetin4 use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater treatment and water of the kamniška bistrica river (slovenia) peter firbas1, tomaž amon2 the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bo shi1,*, majid khayatnezhad2, abdul shakoor3,4 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(3): 53-63, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1139 caryologia international journal of cytology, cytosystematics and cytogenetics citation: pelin yilmaz sancar, semsettin civelek, murat kursat (2021) the morphological, karyological and phylogenetic analyses of three artemisia l. (asteraceae) species that around the van lake in turkey. caryologia 74(3): 53-63. doi: 10.36253/caryologia-1139 received: november 23, 2020 accepted: may 26, 2021 published: december 21, 2021 copyright: © 2021 pelin yilmaz sancar, semsettin civelek, murat kursat. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid pys: 0000-0002-6134-622x sc: 0000-0003-1398-585x mk: 0000-0002-0861-4213 the morphological, karyological and phylogenetic analyses of three artemisia l. (asteraceae) species that around the van lake in turkey pelin yilmaz sancar1,*, semsettin civelek1, murat kursat2 1fırat university, faculty of sciences, department of biology, 23119 elazıg, turkey 2bitlis eren university, faculty of arts and sciences, department of biology, 13000 bitlis, turkey *corresponding author. e-mail: peyilmaz@firat.edu.tr abstract. artemisia is one of the biggest genera in the family asteraceae, with around 500-600 taxa at specific and sub-specific levels and organised in five subgenera. due to the high number of taxa, a lot taxonomists are trying to solve the problem of its classification and phylogeny but its natural classification still has not been achieved. the aim of this study is to try to solve the problematic systematic relationship between three different artemisia species growing in close proximity to each other in the light of morphological, karyological and molecular data. the roots, stems, leaves, flowers structures of the plant samples collected from different populations belong to these species were investigated within the framework of morphological studies. additionally, the chromosome counts and karyotype analysises of these species were made and idiograms were drawn in the karyological studies. in the context of phylogenetic studies, its (its15.8s-its2) and trnt trnl3’ regions of 22 individuals belonging to 3 taxa were studied. according to results of phylogenetic anlysis, it has been found that there is completed speciation genetic isolation mechanism between the species artemisia spicigera, artemisia taurica and artemisia fragrans that inhibit gene flow. also artemisia fragrans and artemisia spicigera species are very similar to each other in terms of morphological characteristics. however, since populations of the species artemisia fragrans are autopolyploid, the dimensional values of their morphological squares are larger than those of the species artemisia spicigera. this study is important as it is the first molecular based study relating with some species of artemisia growing naturally in turkey. keywords: artemisia, karyology, morphology, phylogeny, cpdna trnt-trnl3’, r-dna its. introduction artemisia is one of the biggest genera in the family asteraceae, with around 500-600 taxa at specific and sub-specific levels and organised in five subgenera (vallès et al., 2011). the majority of the members of this genus have a high economic value (chehregani et al., 2010; hayat et al., 2010). due 54 pelin yilmaz sancar, semsettin civelek, murat kursat to the high number of taxa, a lot taxonomists are trying to solve the problem of its classification and phylogeny but its natural classification still has not been achieved (mcarthur et al., 1981; torrel et al., 1999; torrell and vallès 2001; vallès et al., 2003; kurşat, 2010; kurşat et al., 2011). the genus is currently divided into five main groups [artemisia, absinthium (mill.) less., dracunculus (besser) rydb., seriphidium besser and tridentatae (rydb.) mcarthur] but subgeneric classification is subject to rearrangements in the light of recent molecular studies (torrell et al., 1999; vallès et al., 2003). although a lot of investigation have been made of the genus artemisia, enhancing the available morphologic and karyological data (kawatani and ohno 1964; vallès 1987a; torrell et al., 1999; torrell and vallès 2001; torrell et al., 2001; vallès and mcarthur 2001; vallès et al., 2001; kreitschitz and vallès 2003; inceer and hayirlioglu-ayaz 2007; pellicer et al., 2007; chehregani and hajisadeghian 2009; nazirzadeh et al., 2009; chehregani et al., 2010), still the chromosome numbers of some species of the genus remain unknown or doubtful. the genus has two basic chromosome numbers, the largely predominating x=9 and the less extended x=8. x = 9 is not only the most common basic number in the genus artemisia, but in the tribe anthemideae and the family asteraceae as well (mcarthur and sanderson 1999; oliva and vallès 1994; schweizer and ehrendorfer 1983; solbrig 1977; vallès and siljak-yakovlev 1997). a high percentage of artemisia species are polyploid. this phenomenon is present in all of the major groups into which the genus is divided. both basic chromosome numbers (x=8 and x=9) show polyploidy, with levels up to 12x for x=9 and 6x for x=8 (vallès and mcarthur 2001). the gene regions that have been used for phylogeographic and phylogenetic inferences in plants come from the single copy portions (lsc and ssc) of the chloroplast genome, and internal transcribed spacer (its) regions of nuclear ribosomal dna (rdna). several molecular methods have been used to determine the genetic diversity and relationships among different artemisia species, including karyotyping (mcarthur and pope 1979), cpdna restriction site variation analysis (kornkven et al., 1999), polymerase chain reaction restriction fragment length polymorphism (pcr–rflp) analysis of several genes (lee et al., 2009; mahmood et al., 2011), microsatellite (ssr) polymorphism analysis (tripathi et al., 2009; shafie et al., 2011) and random amplified polymorphic dna (rapd) analysis (mcarthur et al., 1998a; mcarthur et al., 1998b; sangwan et al., 1999). nevertheless, very few of artemisia species have been verified with molecular phylogenetic studies based on nucleotide sequence data in turkey, so far (koloren et al., 2016). so, the aim of this study is to try to solve the problematic systematic relationship between three different artemisia species growing in close proximity to each other in the light of morphological, karyological and molecular data (using of rdna its and trnt-trnl3’ regions sequence data). additionally, the first molecular data for artemisia spicigera, artemisia taurica, and artemisia fragrans from turkey has been submitted to the genbank databases. material and methods morphological evaluation plant specimens were collected from around the van lake during the vegetative, flower and seed season in 2010, collected by m. kurşat, ş. civelek and p.y. sancar. morphological examinations consist of instant observations on the samples in the field and macroscopic and microscopic examinations on the samples that have been converted into herbarium material in the laboratory. in order to determine the minimum and maximum values of the examined characters, 10 samples were taken from each locality and measurements were made. measurements of small structures were made with a ruler under a stereo microscope. measurements of macroscopic structures were made using the ruler again with the naked eye. the herbarium materials of the collected samples are stored in firat university herbarium (fuh). the list of the examined specimens, localities, collected date and voucher numbers were given in table 1. karyology meristematic cells of root tips are used in the caryological studies. the seeds (about 15-20 seeds for each type) were germinated on moist filter paper in petri dishes between 20-25°c. the actively growing root tips, 1  cm in length, were excised from the germinating seeds and pretreated with aqueous colchicine (0.05%) for 3-3.5  h at room temperature. then, the root tips were fixed with farmer (1:3 glacial acetic acid–absolute ethanol) for at least 24 h at 4°c, hydrolysed in 0.1 n hcl at room temperature for 1 min, and subsequently rinsed in tap water for 3–5 min. then they were stained in feulgen for 1 h and mounted in 45% acetic acid (gedik et al., 2014). digital photographs from at least five well-spread metaphase plates from each species were taken using an olympus bx51 microscope (olympus optical co. ltd, tokyo, japan), and were recorded with an olympus camedia c-4000 digital camera (olympus optical 55the morphological, karyological and phylogenetic analyses of three artemisia species co. ltd) (figure 1). the short arm, long arm and total lengths of each chromosome were measured and the relative lengths (rl), arm ratios (ar), and centromeric indices (ci) were determined from images of selected cells. levan et al. (1964) was used for the classification of chromosomes. the number of somatic chromosomes, ploidy level, karyotype formula, morphometric parameters, a1 and a2 values (the intrachromosomal asymmetry index a1 and the interchromosomal asymmetry index a2) were determined for each taxa (romero zarco 1986) (table 2). idiograms of haploid chromosomes were drawn (figure 2). the examined taxa and characteristics of somatic chromosomes are given in the results section. genomic dna isolation, pcr, and sequencing genomic dna isolation was performed manually as described ctab method by doyle and doyle (1987). in pcr studies conducted by using trnatrnd primers and its4-its5 primers, trnt trnl3’ and its (its1table 1. information of artemisia populations location in field. taxa locality altitude lat lon date voucher number a . s pi ci ge ra ant valley slope 5.5 km after passing aktuzla 1555m 39°.21’-42°.15’ 10.10.2010 yilmaz sancar, kurşat and civelek 5007,5008,5022 hınıs varto highway, 24 km before varto roadside, slopes 1780m 39°.13’-41°.42’ 10.10.2010 yilmaz sancar, kurşat and civelek 5009,5024 van hakkâri road, after 1 km of gürpınar road separation, çavuştepe locality roadside 1799m 38°.20’-43°.25’ 25.11.2010 yilmaz sancar, kurşat and civelek 5013 a . f ra gr an s kuzgun koran pass hills 2142m 38°.23’-42°.47’ 09.10.2010 yilmaz sancar, kurşat and civelek 5001,5002,5010,5011 between edremit and gürpınar, 15 km before gürpınar 1714m 38°.19’-43°.14’ 09.10.2010 yilmaz sancar, kurşat and civelek 5003,5012 muradiye şelale location 1788m 39°.03’-43°.25’ 10.10.2010 yilmaz sancar, kurşat and civelek 5005,5016 between malazgirt aktuzla, around nurettin village roadside slopes 1728m 38°.50’-43°.25’ 26.11.2010 yilmaz sancar, kurşat and civelek 5017 after passing aktuzla 5.5 km, ant valley slopes (roadside slopes) 1555m 39°.21’-42°.15’ 26.11.2010 yilmaz sancar, kurşat and civelek 5006,5019,5020,5021,5023 a . t au ri ca van-hakkari highway, after 1 km of gürpınar crossroads, çavuştepe area, roadsides 1799m 40°.25’-43°.20’ 25.11.2010 yilmaz sancar, kurşat and civelek 5004,5027,5028, figure 1. somatic metaphase in a. spicigera (2n=18), and haploid idiogram (scale bars: 1 μm). 56 pelin yilmaz sancar, semsettin civelek, murat kursat 5.8s-its2) region for 22 samples has been multiplied (taberlet et al., 1991). the sequence of primers that were used to amplified both trnt trnl3’ region and its (its1-5.8s-its2) region were given in table 3 (taberlet et al., 1991). the following protocol on a bıorad thermal cycler : 2 min 95 °c initial denaturation, 35 cycles of 1 min 95 °c denaturation, 40 s 60 °c (for trn region) and 55 °c (for its region) annealing and 1 min 72 °c extension, followed by a 5 min final extension at 72 °c. pcr products were monitored in agarose gel with a 1 % ratio. two-way reading was applied to the amplification products. pcr purification process was realized before sequence analysis. the purification and sequencing process was realized by the macrogen company. table 2. somatic chromosome numbers (2n), ploidy level, karyotype formula, ranges of chromosome length, total karyotype length (tkl), and asymmetry indexes (a1, a2) of the studied taxa. taxon 2n ploidy level karyotype formula chromosome length range (μm) tkl (μm) a1 a2 a. spicigera 1 18 2x 1m+5m+3sm 4,94-5,56 46,72 0,27 0,04 a. spicigera 2 18 2 x 2m+5m+2sm 4,26-4,81 40,79 0,23 0,02 a. spicigera 3 18 2 x 1m+6m+2sm 4,64-5,14 43,02 0,24 0,04 a. spicigera 4 18 2 x 1m+6m+2sm 4,69-4,97 43,40 0,25 0,02 a. spicigera 5 18 2 x 2m+6m+1sm 4,55-5,50 42,28 0.26 0,03 a. spicigera 6 18 2 x 3m+4m+2sm 4,42-5,25 41,40 0,22 0,03 a. spicigera 7 18 2 x 1m+4m+4sm 4,34-5,33 44,50 0,28 0,04 a. taurica 1 36 4 x 3m+13m+2sm 3,48-4,07 67,31 0,22 0,05 a. taurica 2 36 4 x 4m+12m+2sm 4,57-5,51 92,47 0,25 0,05 a. taurica 3 36 4 x 4m+11m+3sm 4,44-5,50 89,70 0,23 0,06 a. fragrans 1 36 4 x 3m+10m+5sm 4,10-5,35 85,51 0,35 0,08 a. fragrans 2 36 4 x 3m+11m+4sm 3,13-3,53 59,52 0,24 0,01 a. fragrans 3 36 4 x 2m+14m+2sm 3,40-3,94 66,85 0,25 0,05 a. fragrans 4 36 4 x 2m+10m+6sm 4,81-6,29 100,08 0,26 0,07 a. fragrans 5 36 4 x 1m+14m+3sm 3,97-4,65 77,57 0,27 0,05 a. fragrans 6 34 4 x 3m+12m+2sm 3,98-4,89 75,78 0,20 0,07 a. fragrans 7 36 4 x 3m+13m+2sm 3,67-4,14 70,40 0,24 0,04 a. fragrans 8 36 4 x 1m+13m+4sm 3,43-4,75 70,15 0,29 0,08 a. fragrans 9 36 4 x 3m+12m+3sm 3,98-4,99 82,53 0,23 0,07 a. fragrans 10 36 4 x 2m+11m+5sm 3,83-4,79 76,09 0,28 0,07 a. fragrans 11 36 4 x 2m+10m+6sm 3,65-4,51 71,96 0,27 0,06 a. fragrans 12 36 4 x 3m+12m+3sm 3,40-4,49 70,52 0,27 0,08 figure 2. somatic metaphase in a. taurica (2n=36), and haploid idiogram (scale bars: 1 μm). 57the morphological, karyological and phylogenetic analyses of three artemisia species the obtained data was uploaded to ncbi and genbank accession numbers were taken. the genbank accession numbers were given in table 4. phylogenetic analysis phylogenetic analysis was conducted using the program molecular evolutionary genetics analysis software (mega x) (kumar et al., 2019). in order to evaluate the data of chromatograms (sequencing), finch tv 1.4 version is used. dna sequence alignments of 22 individuals, variable sites, number of parsimony informative sites, genetic distance, nucleotide diversity, and divergence within species were computed by mega x version. dna sequence alignment of all the individuals is made subject to statistical analysis within the scope of this program. ultimately, phylogenetic trees were constructed by maximum parsimony method with 100 bootstrap replicates (nei and kumar, 2000; kumar et al., 2019). results morphological results it was observed that a. taurica and a. fragrans species had a larger size compared to a. spicigera species in accordance with the environmental conditions it grows and the number of chromosomes and ploidy levels. detailed morphological measurements of the studied species are as in table 5. karyological results a. spicigera k.koch this taxa general spread is the eastern anatolia region in turkey. samples were collected from three populations and 6 individuals of three different localities (table 1). the samples were labelled “p.y. 5007-50085009-5013-5022-5024”. the number of chromosomes in all the samples examined was 2n=2x=18 and it consists of 4m, 10m and 4sm chromosomes. the metaphase chromosome length is 1.46–3.06 μm and longest to shortest chromosome ratio is 2.0:1. chromosome arm ratios are 1.28–2.27 μm, the centromeric index is 30.55– 43.83 μm, and relative lengths are 4.20–8.78 μm (table 2, figure 1,). secondary structures and satellite chromosomes (sat-chromosome) were not observed in this specimens. a. taurica willd. the species a. taurica shows the wide distribution in the steppes of central, eastern and southeastern anatolia in turkey. samples were collected from 6 individuals of three different localities (table 1). the samples were labelled “p.y. 5004-5027-5028”. the number of chromosomes in all the samples examined was 2n=4x=36 and it consists of 2m, 14m and 2sm chromosomes. the metaphase chromosome length is 3.40–3.94 μm. chromosome arm ratios are 1.27–2.57 μm, the centromeric index is 33.09–49.20 μm, and relative lengths are 5.08–5.89 μm (table 2, figure 2). secondary structures and satellite chromosomes (sat-chromosome) were not observed in this specimens. table 3. the base sequences of the primers used (taberlet et al., 1991). primers base sequences (5’ – 3’) its 5 (f): 5’ gaa agt aaa agt cgt aac aag g 3’ its 4 (r): 5’ tcc tcc gct tat tga tat gc 3’ trn a (f): 5’ cat tac aaa tgc gat gct ct 3’ trn d (r): 5’ ggg gat aga gga ctt gaa c 3’ table 4. genbank accession numbers for the rdna its (its1-5.8sits2) and trnt-trnl3’ regions of the studied samples specimens genbank accesion numbers its region trnt-trnl3’ region artemisia fragrans 1 mt159779 mt648006 artemisia fragrans 2 mt159780 mt648007 artemisia fragrans 3 mt159781 mt648008 artemisia fragrans 4 mt159782 mt648009 artemisia fragrans 5 mt159783 mt648010 artemisia fragrans 6 mt159784 mt648011 artemisia fragrans 7 mt159785 mt648012 artemisia fragrans 8 mt159786 mt648013 artemisia fragrans 9 mt159787 mt648014 artemisia fragrans 10 mt159788 mt648015 artemisia fragrans 11 mt159789 mt648016 artemisia fragrans 12 mt159790 mt648017 artemisia spicigera 1 mt159791 mt648018 artemisia spicigera 2 mt159792 mt648019 artemisia spicigera 3 mt159793 mt648020 artemisia spicigera 4 mt159794 mt648021 artemisia spicigera 5 mt159795 mt648022 artemisia spicigera 6 mt159796 mt648023 artemisia spicigera 7 mt159797 mt648024 artemisia taurica 1 mt159798 mt648025 artemisia taurica 2 mt159799 mt648026 artemisia taurica 3 mt159800 mt648027 58 pelin yilmaz sancar, semsettin civelek, murat kursat a. fragrans willd. this taxa only spread is the eastern anatolia region in turkey and this species a new record for turkey (kursat et al., 2014). samples were collected from five populations and 14 individuals of four different localities (table 1). the samples were labelled “p.y. 5001-5002-5003-50055006-5010-5011-5012-5016-5017-5019-5020-5021-5023”. the number of chromosomes in all the samples examined was 2n=4x=36 and it consists of 4m, 10m and 4sm chromosomes. the metaphase chromosome length is 1.46–3.06 μm and longest to shortest chromosome ratio is 2.0:1. chromosome arm ratios are 1.28–2.27 μm, the centromeric index is 30.55–43.83 μm, and relative lengths are 4.20–8.78 μm (table 2, figure 3). secondary structures and satellite chromosomes (sat-chromosome) were not observed in this specimens. phylogenetic results in this part of the study, a phylogenetic tree displaying the phylogenetic position of three artemisia species table 5. comparison in terms of key features that distinguish of the species of a. spicigera, a. taurica and a. fragrans characters a. spicigera a. fragrans a. taurica stem length (cm) 20–50 20-75 20–45(–60) dimensions lower leaves (cm) 0.5–2.5 × 0.3–1.5 2.5-4 × 1–2 1–2.5 × 0.5–1.2 dimensions of cauline leaves (cm) 0.5–1.5 × 0.3–1.5 1-2.5 × 0.5–1.5 0.5–2.5 × 0.3–1 dimensions of floral leaves (cm) 0.1–1 × 0.1–0.2 0.1–1.2 × 0.1–0.8 0.1–1 × 0.1–0.4 orientation of synflorescence branches usually horizontal usually ascendant usually horizontal capitula length (mm) 1–3 mm long 1–5 mm long (1–) 3–5 mm long, outer phyllaries dimensions (mm) 0.2–0.4 × 0.2–0.4 0.5–0.8 × 0.3–0.5 0.6–0.9 × 0.5–0.8 middle phyllaries dimensions (mm) 1.2–2.2 × 0.5–1 1–1.2 × 0.8–1.5 1–2.2 × 1.3–1.7 inner phyllaries dimensions (mm) 3–3.4 × 0.5–1 3.3–3.8 × 1–1.2 4–4.2 × 1.2–1.5 corolla colour yellow or red yellow or red yellow or pinkish red or purplish red corolla dimensions (mm) 2.5–3.2 × 0.5–0.8 1.7–3.5 × 0.2–0.6 2.8–3.3 × 0.5–1 pistil length (mm) 1.8–3 2.1–3.2 3.1–3.9 ovarium dimensions (mm) 0.4–0.6 × 0.2–0.4 0.5–0.8 × 0.3–0.6 0.7–1 × 0.2–0.7 style length (mm) 1.2–1.6 1.5–1.9 1.5–2.2 forks length of bifid stigma (mm) 0.2–0.5 0.3–0.7 0.4–0.7 stamens length (mm) 2.2–3.2 3–3.5 3–4.2 filaments length (mm) 0.8–1.3 1.3–1.6 1–1.5 anhters dimensions (mm) 1.4–1.7 × 0.1–0.3 2–2.5 × 0.2–0.5 2–2.7 × 0.1–0.3 number of flowers in capitula 3-5 5-8(-10) achenes (cypselas) dimensions (mm) 1.2–2.2 × 0.5–1.2 2–2.5 × 0.8–1.4 1.8–2.7 × 0.8–1.4 somatic chromosome number 2n=2x=18 2n=4x=36 2n=4x=36 , 2n=6x=54 figure 3. somatic metaphase in a. fragrans (2n=36), and haploid idiogram (scale bars: 1 μm). 59the morphological, karyological and phylogenetic analyses of three artemisia species with respect to each other was constructed (figure 4). populations of these species were collected from 9 different regions cultivated in van lake around and 22 individuals were included in the analyzes. i̇n addition to, the reference base sequences of two individuals belong to species a. sieberi (kj004347.1) and a. maritima (nc045093.1) were also included in our analysis to demonstrate the accuracy of the study (shahzadi et.al., 2020.) haplocarpa scaposa (eu846325.1 and dq444824) was used as an outgroup (mckenzie et.al. 2006; mckenzie and barker 2008). sequence data of plants used as outgroup and sister group were taken from ncbi. within the scope of the studies, dna isolation of 22 individuals from leaf tissue was made by ctab method, then rdna its (its1-5.8s-its2) region and non-coding trnt-trnl3’ region of cpdna was amplified in pcr using specific primers (table 2). the base sequences of the obtained regions were analyzed and their genetic characteristics were compared and information was obtained about the proximity and distance of taxa to each other. for a more accurate visualization of the results of the alignment, about 50-100 base from the head and the end were not evaluated by us. as a result of the research done from ncbi for artemisia genus, the base length of the its (its1-5.8s-its2) region was found to be 700-750 bp, trnt-trnl3’ in total and the base length was 900-1000 bp in total, and in our study, it was found to be of similar length in accordance with the literature. the analyses were performed with the x version of the mega program and the method that would give the best result for us was selected from the “find best dna models” step of the program. as a result, it was decided that maximum parsimony method would give the most accurate result of the tree drawn with tamura 3-parameter step. in addition, two different dna regions were evaluated at the same time and a complex tree has been obtained to achieve a more accurate result (figure 4). as a result of the calculations made with the maximum parsimony (mp) method, in both the separate and co-evaluations of the sequences of the its and trn regions of the examined individuals, in total ~1745 base pairs were taken into consideration and the number of variable regions (v) 204, the number of conserved regions (c) was 1507 parsimony number (pi) 34, and gc ratio was 41.6%. these calculated values are given in table 6. discussion artemisia is one of the most complex genera and it is represented by the large number of species, diverse morphological types, ploidy and complicated genetic relationships (winward and mcarthur 1995). because of this, the clarification of the genus’s taxonomy using classical botanical tools and morphological characteristics has many difficulties (torrel et al., 1999). therefore, table 6. pcr amplified region length and summary statistics from the rdna its (its1-5.8s-its2) and the cpdna (trnt-trnl3’) dataset of genus artemisia. molecular diversity parameters its (its1-5.8sits2) region trnt-trnl3’ region co-evaluated of its (its1-5.8sits2) and trnttrnl3’ regions total sample count 22 22 22 total characters ~725 ~1020 ~1745 gc ratio (%) 52.7 34.3 41.6 protected regions (c) 577 930 1507 regions with variation (v) 142 62 204 parsimony informative regions (pi) 14 20 34 figure 4. maximum parsimony tree obtained from the co-evaluation of sequences of the its (its1-5.8s-its2) and trnt-trnl3’ regions of individuals. 60 pelin yilmaz sancar, semsettin civelek, murat kursat usage of molecular markers and caryological data is a valuable and promising addition to the traditional morphology-based classification (turuspekov et al., 2018). in this study a phylogenetic systematic study is conducted by using the morphological, karyological and phylogenetic data of three species in artemisia that grow around the van lake in turkey. 22 individuals of taken from 8 different populations belong to taxa of the a. spicigera, a. taurica and a. fragrans were examined morphological measurements, karyotype analysis and analysing the base slice of the regions being obtained, it was tried to get information about the closeness and distance of taxa with each other. this research is important as it is the first molecular based study relating with some artemisia species growing naturally that around the van lake in turkey. additionally, the first molecular data about these species from turkey has been submitted to the genbank international databases. a lot of research has been carried out to better understand the morphological, karyological, anatomical, and phylogenetic analysis of the genus artemisia and its relationships to the other (four) subgenera, absinthium, dracunculus (besser) rydb, seriphidium besser ex less. and tridentatae (rydb), in different parts of the world. polyploidy is currently considered a prominent force in plant evolution and represents the most common mode of sympatric speciation in plants (wendel and doyle 2005). polyploids, moreover, may have superior levels of adaptability and higher probabilities of survival than their diploid relatives (thompson and lumaret 1992; soltis and soltis 2000). most of the artemisia that colonize extreme and arid habitats are polyploids. this fact supports the hypothesis that polyploids have more tolerance of extreme environmental conditions (pellicer et al., 2007). chromosome data currently available show polyploidy to be the most significant evolutionary trend in chromosome number within asteraceae (chehregani et al., 2010). accordig to chehregani et al. (2010), the highest variation in chromosome number was observed in a. spicigera. in this species; different chromosome numbers (2n=2x=18, 2n=3x=27, 2n=4x=36, 2n=5x=45, 2n=6x=54 and 2n= 8x= 72) were identified in different populations that collected from different parts of iran. however, in our study, the number of chromosomes in all studied populations was found to be 2n=2x=18. but tabur et al. (2014), the number of chromosomes in all a. spicigera populations they work from turkey have recorded as 2n=2x=18. accordingly, the ploidy level of a. spicigera kind in turkey, we can say that 2x. the phylogenetic relationship among the different artemisia species collected from different regions of pakistan based on the chloroplast gene rps11 was investigated by mahmood et al. (2011). the molecular phylogenetic analyses of the hawaiian artemisia and its worldwide divergence based on nuclear and chloroplast dna markers were reported by hobbs and baldwin. (2013). as discussed by haghighi et al. (2014), the phylogenetic relationships among artemisia species based on nuclear its and chloroplast psba-trnh dna markers using three sections of artemisia, dracunculus and serphidium propose that the its and cpdna psba-trnh markers are practicable in the systematic revision of troubled taxa at the intra-genus level in plants. furthermore, pellicer et al. (2014) performed phylogenetic analysis of the annual artemisia within its major lineages and suggested that annual artemisia have been specially misidentified at a subgeneric level and verified that they are phylogenetically restricted to basal grades. however, to date, very few artemisia species have been verified with molecular phylogenetic studies based on the nucleotide sequence data in turkey (koloren et al., 2016). civelek et al. (2010) have carried out a revisionary study of the genus artemisia in turkey. according to results of the revisionary study based on the morphological features, it was observed that growing around the lake van that in the populations thought to belong to the a. spicigera species, there are some groups showing significant morphological differences from this species. these groups were found to be similar to a. spicigera and a. taurica in terms of morphological, but it has been accepted that they were closer to a. spicigera. in these populations, a new variety (a. spicigera var. vanensis) belonging to the species a. spicigera was made, but the variety was not certain as it was not published (nomen nudum). the researchers stated that they are not sure about the accuracy of this systematic arrangement and stated that these populations should be studied in detail. to solve this systematic problems, in flora of turkey specified to be very close to each other a. spicigera and a. taurica species of, planned to investigate detailed morphological and cytogenetic aspects and the research was conducted. while these studies continue, after literature search and cytogenetic observations in these populations, have been identified as belonging to the species a. fragrans case a new record for the flora of turkey and published (kursat et al., 2014). however, it has been stated that a molecular study is needed to confirm these results. for this purpose, it was decided to phylogenetically evaluate the populations of a. spicigera, a. taurica and a. fragrans species around the van lake with various molecular markers. gramer in molecular studies, its (its1-5.8s-its2) in rdna and trn regions in cpdna were amplified with specific primers and ana61the morphological, karyological and phylogenetic analyses of three artemisia species lyzed with mega program. in the phylogenetic family tree created after the analysis, it was observed that the examined individuals of the species in question were completely separated from each other, and the individuals of each species were grouped among themselves. according to these results, it has been determined that there is no gene flow between the populations of these species and they are completely independent from each other. according to the morphological and caryological data, it has been molecularly proven that the populations considered as a. spicigera var. vanensis (nomen nudum) are correct to be published as a. fragrans species. conclusions according to this study results, it has been found that there is complete speciation genetic isolation mechanism between the species a. spicigera, a. taurica and a. fragrans that inhibit gene flow. also a.fragrans and a. spicigera species are very similar to each other in terms of morphological characteristics. however, since populations of the species a. fragrans are autopolyploid, the dimensional values of their morphological squares are larger than those of the species a. spicigera. this study is so important as it is the first molecular based study relating with some species of artemisia growing naturally in turkey. acknowledgements this work was supported by the firat university scientific research projects coordination unit [grant number: ff.2090]. references chehregani a, atri m, yousefi s, jalali f. 2010. polyploidy variation in some species of the genus  artemisia  l (asteraceae) in iran. caryologia. 63(2):168–175. chehregani a, hajisadeghian s. 2009. new chromosome counts in some species of asteraceae from iran. nordic j. of bot. 27(3):247–250. civelek s, yilmaz o, bagci e, kirbag s, gur n, turkoglu i, tabur s, kursat m. 2010. the researches of taxonomical, chemical (flavonoids and essential oils), karyological, palynological and antimicrobial activities on taxa of the genus artemisia l. 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(arecaceae) is one of the best known and typical species of the mediterranean basin. this is due to its wide distribution and to its numerous uses since the dawn of civilization. c. humilis has been studied in relation to morphological and genetic diversity (giovino et al. 2014, giovino et al. 2015a, guzmán et al. 2017), seed lipids composition (giovino et al. 2015b), ethnobotany (okkacha et al. 2013). this species naturally occurs in western and central mediterranean. it is widespread in the iberian peninsula, morocco, algeria, south france, sardinia, tunisia, sicily and peninsular italy (euro+med 2006, castroviejo and galan 2007, pignatti 2017). the eastern distribution limit of this species lies in calabria, italy. linnaeus (1753) reported c. humilis as common in spain. 86 antonio giovino, annalisa marchese, gianniantonio domina the original material used for lectotypification of this name is presumptively collected in europe (moore and dransfield 1979). to date, the dwarf palm is considered a species threatened by human activities and environmental and climatic changes and for this reason deserves a special attention in conservation management strategies (blach‐ overgaard et al. 2010; giovino et al. 2016). chamaerops humilis is extremely variable in height, leaf colour, presence of thorns, size and shape of fruits (beccari 1921, zagolin 1921, maire 1957). this large morphological variation is at the basis of the description of numerous varieties. despite the kew checklist of palms (govaerts and dransfield 2005) reports more than 20 intra-specific epithets among varieties and sub-varieties, giovino et al. (2014) reports that many morphological traits appear to be related to environmental conditions. this scenario is complicated by the fact that a large part of known varieties has been described on cultivated plants of unknown origin. whilst a great number of names are used in the horticultural field, only two varieties are widely accepted in floras: c. humilis l. var. humilis and c. humilis var. argentea andré (= c. humilis var. cerifera becc.). they are mainly distinguished by leaves color: c. humilis l. var. humilis, has green leaves and c. humilis var. cerifera becc. has grey and waxy leaves (maire 1957). the aim of this contribution is to investigate the taxonomic value of c. humilis var. argentea andré and to clarify if the morphological variability observed in high altitude populations studied in high atlas and anti atlas in morocco is merely due to environmental factors or if it is of genotypic nature and transmitted to progenies. materials and methods a preliminary assessment of the morphological variability of c. humilis in all the countries where it naturally occurs was performed. we were not able to find any wild individual in the maltese archipelago, where this species was recorded by haslam et al. (1977). after this preliminary evaluation, seven representative populations were chosen for this study. in order to exclude variability due to the environment, plants grown from seeds collected in nature were studied ex-situ under the same environmental conditions in a collection field at the crea-dc research center bagheria (n sicily). these seeds were collected from three populations in morocco (terketen, benimellal, and touama) (figure 1), one population in spain (valencia), one in algeria (sidi belattar near mostaganem), one in tunisia (cap serrat), and one in sardinia, italy (porto tangone) (table 1). seed collections were done on at least six individuals occurring in the central part of wild populations. in december 2013, seeds were treated with either sulphuric acid, water or mechanical scarification as described in giovino et al. (2015b). after, seeds were germinated on humid sand for about 100 days (mgt) in a cold greenhouse with temperatures between 12 °c and 16 °c at relative humidity of 90%. after germination plants were transplanted on pots of 1.6 ℓ containing a mixture of sand (70%), red soil (25%) and commercial garden soil (5%). pots were maintained on open air from april to november and irrigated with 1 dripper 2 ℓ/h providing 2 min irrigation per day for three days a week. after two years plant were transplanted in 7 ℓ pots containing the same substratum. figure 1. elevation of the collecting localities in morocco. mar: terketen; mar1: beni mellal; mar2: touama. 87morphological and genetic variation of chamaerops humilis (arecaceae) in relation to the altitude for morphological analysis, the selection of characters was done on the basis of rhouma (1994, 2005), hammadi et al. (2009), rizk and el sharabasy (2007). measures were done in spring 2018 with a digital caliper. for each character three measures were performed and their arithmetic average registered. per each population 31 individuals were measured. on the whole 12 quantitative characters were measured; nine continuous: height of the stem (cm), height of the plant (cm), crown diameter (cm), stem diameter (mm), petiole length (cm), petiole width (cm), leaf length (cm), leaf width (cm), leaf thickness (mm) and three discrete ones: no. of leaf segments, no. thorns on the petiole, and wax coverage density. this latter was estimated on percentage of coverage on the upper surface of the leaf. the measures used for statistics are presented in the supplemental data (file 1). according to the methodology adopted in giovino et al. (2015c), domina et al. (2017a, 2017b), and domina (2018) these characters were subjected to a principal component analysis, with the individuals a priori assigned to the eight populations (figure 2). each character was also subjected to univariate analysis (analysis of variance or kruskal– wallis test with corrections for multiple comparisons, pearson correlation coefficients, tukey’s hsd, honestly significant difference, test and bonferroni, respectively), table 1. sampled populations, environmental characteristics and seeds collection data. code locality latitude longitude altitude m a.s.l. habitat bioclimate date collector mar terketen, morocco 31°27’44.47”n 7°24’23.39”w 1420 mediterranean steppe humid 21.10.2012 a. giovino mar1 beni mellal, morocco 32°25’54.78”n 6°30’41.76”w 430 mediterranean maquis semiarid 21.10.2012 a. giovino mar2 touama, morocco 31°31’46.14”n 7°28’59.82”w 990 mediterranean steppe humid 21.10.2012 a. giovino spa valencia, spain 40°16’15.12”n 0°17’12.04”e 130 mediterranean maquis semiarid 15.10.2012 p. ferrer galego alg sidi belattar, algeria 36°01’43.07”n 0°09’03.03”e 200 mediterranean maquis semiarid 5.9.2013 a. mostari tun cap serrat, tunisia 37°11’ 55.8”n 09°15’45.9”e 20 mediterranean maquis humid 5.9.2013 r. el mokni sar porto tangone, italy 40°28’19.23”n 8°22’52.98”e 50 mediterranean maquis semiarid 25.06.2013 a. giovino figure 2. principal components analysis based on the 12 considered morphological characters, with 7 a priori defined groups based on the geographical distribution of the sampled populations. pc1 eigenvalue 645.724, % variance 76.48, pc2 eigenvalue 132.387, % variance 15.68. mar: terketen, morocco; mar1: beni mellal, morocco; mar2: touama, morocco; spa: valencia, spain; alg: sidi belattar, algeria; tun: cap serrat, tunisia; sar: porto tangone, sardinia, italy. 88 antonio giovino, annalisa marchese, gianniantonio domina using past version 4.03 (hammer et al. 2001; hammer 2020). pearson correlation coefficients (r) among the 12 characters measured are presented in the supplemental data (file 2). a discriminant analysis for the seven a priori recognized groups was performed. the scatter plot of specimens along the first two canonical axes is shown (figure 3). the range of each continuous numerical character is represented using box-and-whisker plots (figure 4). for molecular analysis a total of 35 genotypes were used for the characterization: 5 from terketen (morocco); 5 from beni mellal (morocco); 4 from touama (morocco); 6 from valencia (spain); 6 from mostaganem (algeria); 3 from cap serrat (tunisia), and 6 from porto tangone (sardinia, italy). genomic dna was extracted from 40 mg of dried leaf sample using doyle & doyle (1987) protocol. a set of 10 microsatellite markers was employed, including 6 microsatellites showing trinucleotide repeats (arranz et al. 2013) and 4 showing dinucleotide repeats recently isolated in fan palm by giovino et al. (submitted 2020) following pcr conditions and thermal cycles reported in giovino et al. (2014) and giovino et al. (submitted 2020) (table 2). pcr products were analyzed using an abi 3130 genetic analyzer (applied biosystems) and allele sizes were established using genemapper, version 4.0 software (applied biosystems). basic genetic parameters including the number of alleles per locus (na), observed (ho) and expected heterozygosity (he), the total number of null alleles (fnull), the polymorphic information content (pic) value and the deviation from the hardy-weinberg equilibrium (hwe), inferred by sequential bonferroni correction, in the 36 selected genotypes, were calculated using cervus 3.0 software (marshall et al. 1998; kalinowski et al. 2007). a neighbor-net graph was constructed based on calculations of reynold’s genetic distances with splitstree (huson and bryant 2006) in order to study the genetic diversity and relationships between palm genotypes (figure 5). results morphological characterisation single morphological characters (figure 4) show continuous variation and do not distinct population groupings. the population from cap serrat shows the largest intra-population variation. only the leaf length distinguishes, in part, some populations from the others. univariate analysis shows that this character discriminates moroccan populations from the others. figure 3. discriminant analysis based on the 12 considered morphological characters, with 7 a priori defined groups based on the geographical distribution of the sampled populations. axis 1: eigenvalue 22.424, % variance 74.11; axis 2: eigenvalue 5.635, % variance 18.62. mar: terketen, morocco; mar1: beni mellal, morocco; mar2: touama, morocco; spa: valencia, spain; alg: sidi belattar, algeria; tun: cap serrat, tunisia; sar: porto tangone, sardinia, italy. 89morphological and genetic variation of chamaerops humilis (arecaceae) in relation to the altitude the principal components analysis (figure 2) and the discriminant analysis (figure 3) show three well distinct groups across the populations studied. the cases correctly classified by discriminant analysis according to the groups assigned a priori were 91.4%. molecular characterisation a total of 71 ssr alleles were identified across the 10 loci (table 2) in 35 dwarf palm genotypes and all individuals were differentiated. locus37 showed the highest figure 4. box-plots of the 9 continuous morphological characters considered (a: height of the stem (cm); b: height of the plant (cm); c: crown diameter (cm); d: stem diameter (mm); e: petiole length (cm); f: petiole width (cm); g: leaf length (cm); h: leaf width (cm); i: leaf thickness (mm). for each sample, the 25–75% quartiles are drawn using a box. the median is shown with a horizontal line inside the box. the whiskers are drawn from the top of the box up to the largest data point less than 1.5 times the box height from the box, and similarly below the box. values outside the inner fences are shown as circles, values further than 3 times the box height from the box are shown as stars. mar (fuchsia): terketen, morocco; mar1 (yellow): beni mellal, morocco; mar2 (grey): touama, morocco; alg (light blue): sidi belattar, algeria; spa (orange): valencia, spain; tun (blue): cap serrat, tunisia; sar (red): porto tangone, sardinia, italy. 90 antonio giovino, annalisa marchese, gianniantonio domina number of observed alleles per locus (16) while locus16 and locus23 the lowest (3); the average number of alleles per locus was 7.1. seven ssr markers were found highly informative (pic > 0.5) and three reasonably informative (0.25 < pic < 0.5); pic average was 0.62. for eight ssr loci the expected heterozygosity (he) was higher than the observed heterozygosity, except for locus35 and locus44, which deviated from hardy weinberg equilibrium. mean he resulted 0.67 and the mean ho was 0.54. interestingly, a rare allele of 97 bp at the locus37 was found restricted to two genotypes from terketen (mar1 and mar8). neighbor-net method cluster analysis (figure 5) showed that the moroccan genotypes separated from all genotypes of the other geographical areas. overall genotypes from beni mellal presented an intermediate genetic proximity with other populations. two genotypes mar6 and mar7 from terketen showed closer relationship with touama genotypes. algerian, sardinian, spanish, and tunisian genotypes shared closer relationships and genotypes from sardinia grouped together. discussion the large intrapopulational morphological variation observed testifies a large genetic variability among the studied populations. this variation was proven by the molecular analysis. a rich assortment of ssr alleles was found indicating a greater genetic diversity than that previously identified on 705 sicilian dwarf palm genotypes using 28 ssr markers (giovino et al. 2014). this information is useful for acquiring new knowledge on the species and for planning a more extensive work on the whole area of distribution of this species in order to acquire a more detailed knowledge to preserve chamaerops humilis genetic diversity in the future. as concern the presence of a rare allele restricted to some genotypes of the terketen population, it is possible to speculate that this allele may reflect an adaptation to particular environment conditions or stresses. it has been shown in many species that ssr diversity is adaptive, influenced by natural and anthropic selection and correlated with ecological-edaphic and genetic factors (marchese et al. 2010, marchese et al. 2017). natural selection plays an essential role in controlling the length of a repeat (li et al. 2000). king and soller (1999) proposed that changes in length of ssrs functionally integrated into the genome can influence plant adaptive fitness. however, further molecular studies are needed, including a greater number of genotypes representative of all dwarf palm populations, to confirm the uniqueness of this allele. in the neighbor-net cluster analysis the moroccan genotypes grouped together and appeared separated from algerian, sardinian, spanish, and tunisian genotypes which seemed to share closer relationships, among this latter group the sardinian genotypes grouped jointly. in general, the neighbor-net cluster analysis was in agreement with the discriminant analysis. according to the observed variability, it seems opportune to distinguish the populations of high altitude of atlas and anti-atlas as a distinct subspecies. a careful bibliographic research has allowed us to ascertain that the name chamaerops humilis var. figure. 5. split tree of 35 chamaerops humilis genotypes of five putative populations based on nei and li’s genetic distance. mar: terketen, morocco; mar1: beni mellal, morocco; mar2: touama, morocco; spa: valencia, spain; alg: sidi belattar, algeria; tun: cap serrat, tunisia; sar: porto tangone, sardinia, italy. table 2. ssr locus name, number of alleles (no), observed (ho) and expected heterozygosities(he), polymorphic information content (pic) of 10 microsatellite loci in a sample of 35 accessions of chamaerops humilis. locus name allele no h exp ho pic locus19 6 0,64 0,36 0,55 locus25 12 0,84 0,44 0,8 locus27 9 0,85 0,47 0,82 locus15 5 0,63 0,58 0,57 locus16 3 0,5 0,36 0,43 locus23 3 0,26 0,14 0,25 locus35 6 0,65 0,83 0,58 locus37 16 0,9 0,72 0,88 locus44 4 0,58 0,97 0,49 locus48 7 0,83 0,55 0,79 mean 7,1 0,668 0,542 0,62 91morphological and genetic variation of chamaerops humilis (arecaceae) in relation to the altitude argentea andré rev. hort. 57: 231 (1885) quoted by the large part of repertoires and floras (e.g. maire 1957, fennane 2014) has never been published either in that place or, as far as we have been able to verify, in other sources. so, the first validly published name that can refer to this entity is c. humilis var. cerifera becc. described on material cultivated in naples of dubious origin. the study of original material housed in fi, where the herbarium by beccari is housed (cuccuini and nepi 2006) was not sufficient to dispel the doubts because the single specimen found (beccari n. 384) consists only of badly preserved fruits. in any case the only known populations in nature with grey leaves, due to the high concentration of waxes on their surface, are found exclusively in morocco at high altitudes, thus the taxon described by beccari, by reasonable assumption, refers to these populations. the following new combination is proposed: chamaerops humilis subsp. cerifera (becc.) giovino & domina subsp. nov. (≡c. humilis var. cerifera becc. in webbia 5(1): 65 1921). type: lectotype (here designated): beccari n. 384, chamaerops humilis l. v. cerifera becc.; italia, napoli, n. 1919, ruffo, ex ruffo principe di s. antimo (fi). this subspecies differs from c. humilis subsp. humilis by its leaves glaucous-silvery, dull, covered with persistent scaly hairs. the individuals in form of compact tufts with short stems and smaller leaves blades commonly found in high altitude populations in high atlas and anti atlas are due to the effect of environmental and anthropozoogenic degradation. conclusion the obtained results agree with those by garcíacastaño et al. (2014): c. humilis has a large morphological and genetic variation throughout its distribution range. the southern populations from morocco are isolated from the resting ones and in particular high-altitude populations are well distinct from the morphological and genetic points of view due to a speciation underway moved by ecological and spatial separation to which they are subjected. thus, it is here proposed to discriminate these populations within a separated subspecies. these results encourage about the possibility of cultivating chamaerops humilis subsp. cerifera also in environments with lower temperatures than the mediterranean coasts where c. humilis subsp. humilis has been confined so far. such cultivations would have primary interest as ornamental but the possibility of extraction of medicinal principles is not excluded. acknowledgements we thank mohamed rejdali and pasquale marino for their support during field work in morocco, abbassia mostari, pedro pablo ferrer-galego and ridha el mokni for providing us the study material and seeds from algeria, spain, and tunisia respectively. a special thanks to enrico banfi for nomenclatural advice. reference andré é-f. 1885. les palmiers cultivés. rev hort. 57:230–232. arranz se, avarre jc, balasundaram c, bouza c, calcaterra nb, cezilly f et al. 2013. permanent genetic resources added to molecular ecology resources database 1 december 2012–31 january 2013. molec ecol res. 13:546–549. beccari o. 1921. recensione delle palme del vecchio mondo. webbia 5(1):5–70. blach‐overgaard a, svenning jc, dransfield j, greve m, balslev h. 2010. determinants of palm species distributions across africa: the relative roles of climate, non‐climatic environmental factors, and spatial constraints. ecography 33(2):380–391. chong j, xia j. 2018. metaboanalystr: an r package for flexible and reproducible analysis of metabolomics data. bioinformatics 27:4313–4314. cuccuini p, nepi c. 2006. the palms of odoardo beccari. quad bot amb appl. 17/1:3–249. domina g, greuter w, raimondo fm. 2017a. a taxonomic reassessment of the centaurea busambarensis complex (compositae, cardueae), with description of a new species from the egadi islands (w sicily). israel j pl sci 64(1-2):48–56. domina g, scibetta s, scafidi f, giovino a. 2017b. contribution to the identification of dianthus rupicola (caryophyllaceae) subspecies using morphological and molecular approaches. phytotaxa 291:17–32. domina g. 2018. host-driven morphological variability in orobanche crenata (orobanchaceae). turk j bot. 42:502–509. euro+med 2006. euro+medplantbase the information resource for euro-mediterranean plant diversity. published on the internet http://ww2.bgbm.org/europlusmed/ [last accessed 21/11/2018]. fennane m, ibn tattou m, el oualidi j 2014. flor pratique du maroc, 3. inst. sci. rabat, rabat. 92 antonio giovino, annalisa marchese, gianniantonio domina garcía-castaño jl, terrab a, ángeles ortiz m, stuessy tf, talavera s. 2014. patterns of phylogeography and vicariance of chamaerops humilis l. 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(arecaceae) in the area of tlemcen (western algeria). res j pharm biol chem sci 4(2):910–918 pignatti s. 2017. chamaerops l. pp. 381-382 in: flora d’italia, 1, 2° ed. edagricole, milano. zagolin a. 1921. ricerche sul polimorfismo del frutto della chamaerops humilis l. n giorn bot ital., n.s., 28:36–66. 93morphological and genetic variation of chamaerops humilis (arecaceae) in relation to the altitude supplemental data file 1. mean of the measures on morphological characters used for statistical analysis. code population h ei gh t o f t he st em ( cm ) h ei gh t o f t he pl an t ( cm ) c ro w n di am et er ( cm ) st em d ia m et er (m m ) pe tio le w id th (c m ) pe tio le w id th (c m ) le af le ng th (c m ) le af w id th (c m ) le af th ic kn es s (m m ) n o. o f l ea f se gm en ts n o. th or ns o n th e pe tio le w ax c ov er ag e de ns ity % mar_1 terketen, morocco 7,5 17 27 34,5 3,9 3,2 8,7 11,8 1 9 0 95 mar_2 terketen, morocco 8 20 22 29,9 3,1 3,5 9,7 13,7 0,8 8 5 95 mar_3 terketen, morocco 6 20 28 28,6 0,9 4,1 11,5 14 0,8 9 8 95 mar_4 terketen, morocco 4,5 15,5 19 34,5 0,8 3,3 9,5 11 0,7 10 1 95 mar_5 terketen, morocco 9 19 24 47,5 3,3 2,7 8,8 10,6 0,5 9 2 95 mar_6 terketen, morocco 6 20 21 29,4 4,2 4,2 12,7 15,5 1,1 9 0 95 mar_7 terketen, morocco 6 26,5 31 23,8 3,7 6 13,6 17,7 0,7 11 4 95 mar_8 terketen, morocco 8 20 23 31,5 3 4,7 10,3 13 0,9 11 0 95 mar_9 terketen, morocco 11,5 29 36 34,5 3,4 6 11,3 15 0,8 11 6 95 mar_10 terketen, morocco 9,5 22 24 20,5 3,2 3,7 9,4 10,5 0,5 8 9 95 mar_11 terketen, morocco 7 18,5 26 34,9 3,3 5 9 11,5 1,2 9 10 95 mar_12 terketen, morocco 7,5 23 30,5 34,9 3,9 4,5 10,2 13,2 0,9 11 7 95 mar_13 terketen, morocco 8 21,5 28 27,3 2,7 3,2 9 10,5 0,4 9 4 95 mar_14 terketen, morocco 6 16 20 31,7 3,2 2,7 5,2 10,7 0,7 9 4 95 mar_15 terketen, morocco 8 20 21 29,5 2,9 2,8 11,4 13,7 0,8 10 5 95 mar_16 terketen, morocco 7,5 23 24 26,2 3,1 2,7 10 10,2 0,4 9 11 95 mar_17 terketen, morocco 8 22,5 26,5 34,8 2,5 4,4 10,7 10,1 0,5 9 1 95 mar_18 terketen, morocco 9 21 29,2 34,2 3,1 4,3 9,7 8,5 0,7 8 3 95 mar_19 terketen, morocco 4 12 16 33,5 2,8 2 6,5 9,5 0,5 7 15 95 mar_20 terketen, morocco 8 21 32 34,4 3,9 4,5 9,8 12,2 1 10 5 95 mar_21 terketen, morocco 6,5 27,5 32 33,4 3 9,5 14,3 19,2 0,5 9 9 95 mar_22 terketen, morocco 8 21,5 30 38,5 3,1 4,2 9,5 10,2 0,4 9 13 95 mar_23 terketen, morocco 10 25 31 28,2 3 5,5 12,3 14,5 0,5 11 12 95 mar_24 terketen, morocco 6 2,6 30,5 35,4 3 4,5 10,5 13,2 0,5 10 12 95 mar_25 terketen, morocco 10 23,5 35,5 44,8 2,6 4,7 10 14,8 0,5 10 6 95 mar_26 terketen, morocco 4 21 23 27,8 3 3,8 9,7 13,2 0,6 9 1 95 mar_27 terketen, morocco 6 20 29,5 30,1 3,5 4,8 10,2 13,8 0,6 9 11 95 mar_28 terketen, morocco 7 21 29 24,1 3,1 3,7 9,5 14 0,6 9 7 95 mar_29 terketen, morocco 4 20 30 24,4 2,7 5 12,3 16,8 0,6 8 4 95 mar_30 terketen, morocco 9 22 28,5 34,2 3,4 5,3 12 15,2 0,8 11 7 95 mar_31 terketen, morocco 6 24 34,5 29,8 3,3 6,3 12,3 14,2 0,8 8 11 95 mar1_1 beni mellal, morocco 4 20 30 14,3 3,8 2 16,8 11 0,9 4 5 55 mar1_2 beni mellal, morocco 4 17 31 26,5 4,6 4 15 14 0,8 10 6 55 mar1_3 beni mellal, morocco 3,5 17,5 29 4,3 3,7 2,3 16,7 12,7 0,7 5 3 55 mar1_4 beni mellal, morocco 4 15 20 16,4 4 2,2 12 12,3 1 7 5 65 mar1_5 beni mellal, morocco 3 15,5 17 16 3,3 2,7 10,3 9 0,6 6 5 65 mar1_6 beni mellal, morocco 4 14 17 20 3,5 2 10,2 9,3 1,3 7 3 65 mar1_7 beni mellal, morocco 4 14 17 20 2,5 1,5 9 6 0,6 6 0 65 mar1_8 beni mellal, morocco 3,5 21,5 41 20 3,4 5,3 16,3 12 0,7 6 6 55 mar1_9 beni mellal, morocco 4 14,5 23 24 1,2 3,2 11,7 8 0,8 5 0 55 mar1_10 beni mellal, morocco 5 18,5 25 18,3 3,5 3,2 15,2 12,3 0,8 11 5 65 mar1_11 beni mellal, morocco 2 12,5 21 17 3,3 2,8 12,2 8 0,7 7 3 75 mar1_12 beni mellal, morocco 3 18 18 23 3,9 2,2 13,7 9 0,9 6 3 65 mar1_13 beni mellal, morocco 3 19 25 18 3,8 3 14,5 9 0,7 6 6 75 mar1_14 beni mellal, morocco 4 16,5 24 19 3,5 2,9 12,6 9 0,9 6 3 55 mar1_15 beni mellal, morocco 4 17,5 23 17,5 3,3 2,6 11 9,3 0,9 5 3 55 94 antonio giovino, annalisa marchese, gianniantonio domina code population h ei gh t o f t he st em ( cm ) h ei gh t o f t he pl an t ( cm ) c ro w n di am et er ( cm ) st em d ia m et er (m m ) pe tio le w id th (c m ) pe tio le w id th (c m ) le af le ng th (c m ) le af w id th (c m ) le af th ic kn es s (m m ) n o. o f l ea f se gm en ts n o. th or ns o n th e pe tio le w ax c ov er ag e de ns ity % mar1_16 beni mellal, morocco 3 18 22 23 3,6 2,9 13 9,2 0,7 7 5 65 mar1_17 beni mellal, morocco 3 16,5 20 17 3,8 3,2 12,8 9,2 0,7 6 3 65 mar1_18 beni mellal, morocco 3,5 16 24 23 3,7 2,9 12,5 8,7 0,9 6 3 76 mar1_19 beni mellal, morocco 3 17 23 23 2,9 3,2 16 12,5 0,7 6 5 65 mar1_20 beni mellal, morocco 4 15 26 21 3,6 4 16 13,5 0,9 6 3 55 mar1_21 beni mellal, morocco 4 15,5 25 18 3,5 3,6 17 13,2 0,6 7 3 65 mar1_22 beni mellal, morocco 3,5 16,5 19 23 3,2 2,8 12,7 8,3 0,7 6 5 76 mar1_23 beni mellal, morocco 3 18 25 18,5 3 3,1 15,1 12 0,9 6 3 65 mar1_24 beni mellal, morocco 5 19 24 19 2,9 2,7 12 9,8 0,9 6 5 65 mar1_25 beni mellal, morocco 4 18 19 19 3,3 3,5 16,6 13,2 0,6 7 0 55 mar1_26 beni mellal, morocco 3,5 17,5 23 18 3,5 3,4 17,2 13 0,7 6 5 65 mar1_27 beni mellal, morocco 4 18 24 18,5 3,6 3,3 12 8,2 0,9 5 3 65 mar1_28 beni mellal, morocco 3,5 18,5 22 21 3 4 15,5 13,8 0,7 6 5 75 mar1_29 beni mellal, morocco 4 17 20 22 2,9 2,8 14 12,6 0,9 6 3 55 mar1_30 beni mellal, morocco 3 18 24 23 3,4 3 13,7 8,9 0,8 7 3 65 mar1_31 beni mellal, morocco 4 17 24 26 3,6 2,7 12 10,3 1,1 7 3 55 mar2_1 touama, morocco 3 17,5 25 20 3,4 3,5 14,3 10,7 0,4 7 0 55 mar2_2 touama, morocco 3 13 15 19 3,1 2,2 10,5 8 0,4 7 0 65 mar2_3 touama, morocco 3 16 16 22 2,8 2,3 12,7 10,7 0,7 6 0 75 mar2_4 touama, morocco 3 15 18 16 3,6 2,7 11,7 12,7 0,7 9 0 75 mar2_5 touama, morocco 4 14 20 12 3,6 2,7 10,3 10 0,9 6 0 75 mar2_6 touama, morocco 3 17 23 12 2,6 5,3 13,7 10 0,7 7 0 65 mar2_7 touama, morocco 5 14 10 25 2,3 3 8,7 6 1,4 5 0 55 mar2_8 touama, morocco 4 16 22 20 2,6 3 12,6 10,8 0,6 6 0 55 mar2_9 touama, morocco 4 17 23 22 2,7 3,1 13,5 10 0,7 5 0 65 mar2_10 touama, morocco 3 17 18 18 2,6 2,8 10,5 9,6 0,9 7 0 55 mar2_11 touama, morocco 3 18 22 20 3 2,9 11,9 12,5 0,6 6 0 65 mar2_12 touama, morocco 5 16 23 22 2,6 4 12,7 10,7 0,7 5 0 75 mar2_13 touama, morocco 4 16 18 20 2,8 2,8 12 10 1 7 0 55 mar2_14 touama, morocco 3 17 20 18 2,8 2,7 10,7 9,8 0,6 5 0 65 mar2_15 touama, morocco 3 17 23 22 3 3 13,5 9,5 0,7 6 0 55 mar2_16 touama, morocco 5 18 20 18 4 2,8 11,9 9 0,9 7 0 75 mar2_17 touama, morocco 5 17 18 20 2,8 3 12,7 10,5 1 6 0 65 mar2_18 touama, morocco 4 16 22 25 2,8 3 10,8 9,8 0,6 5 0 75 mar2_19 touama, morocco 4 17,5 23 23 3,1 2,7 12,2 9,6 0,7 6 0 55 mar2_20 touama, morocco 3 18 22 22 3 2,6 13,1 8,7 1 7 0 65 mar2_21 touama, morocco 3 17 20 19 2,8 2,9 12,7 10,7 0,9 6 0 75 mar2_22 touama, morocco 3 18 20 21 4 3,1 12,2 10,5 0,6 6 0 55 mar2_23 touama, morocco 4 17,5 22 20 2,6 2,5 13,4 9 0,7 7 0 65 mar2_24 touama, morocco 5 16 23 20 3 2,6 9,6 7 0,9 6 0 75 mar2_25 touama, morocco 4 16 22 18 2,8 2,7 12,4 10,7 0,6 6 0 55 mar2_26 touama, morocco 3 14 24 20 3,2 2,8 11,3 10,3 0,6 5 0 75 mar2_27 touama, morocco 3 15 21 22 3,2 4,2 13,7 10 0,7 6 0 55 mar2_28 touama, morocco 5 15,5 20 19 2,8 3,2 15 11,4 0,7 7 0 75 mar2_29 touama, morocco 4 17,5 20 21 2,9 3,6 15,3 10,5 0,6 6 0 55 mar2_30 touama, morocco 4 17 22 22 3 3,6 12,8 10,5 0,9 7 0 55 mar2_31 touama, morocco 3 17 18 19 2,6 3 13 11,7 0,7 7 0 65 95morphological and genetic variation of chamaerops humilis (arecaceae) in relation to the altitude code population h ei gh t o f t he st em ( cm ) h ei gh t o f t he pl an t ( cm ) c ro w n di am et er ( cm ) st em d ia m et er (m m ) pe tio le w id th (c m ) pe tio le w id th (c m ) le af le ng th (c m ) le af w id th (c m ) le af th ic kn es s (m m ) n o. o f l ea f se gm en ts n o. th or ns o n th e pe tio le w ax c ov er ag e de ns ity % alg1 sidi belattar, algeria 7,5 29 31 19,8 3,6 7,3 21,3 18,3 0,7 8 8 25 alg2 sidi belattar, algeria 7 25,5 41 25,1 3,8 6,2 21,7 19,7 0,6 10 8 25 alg3 sidi belattar, algeria 7 23 27 19,8 3,9 5 15 16 0,9 7 8 25 alg4 sidi belattar, algeria 6,5 22,5 24,5 21 3,7 6 18,3 14,2 0,8 5 6 25 alg5 sidi belattar, algeria 6 26 32,5 24 3,3 7,2 21,5 15,2 0,6 6 6 25 alg6 sidi belattar, algeria 2,5 25,5 35 14,3 3,8 6,7 24,2 8,3 0,4 4 7 25 alg7 sidi belattar, algeria 3,5 21 29 12 3,9 5,2 20,7 12,2 0,8 6 7 25 alg8 sidi belattar, algeria 4 22 37 19,2 4,2 6,2 20,7 11,3 0,8 7 7 25 alg9 sidi belattar, algeria 4 18 31 12,3 3,8 2,2 16 7,3 0,9 4 2 30 alg10 sidi belattar, algeria 7 28 41 24,6 3,2 6 22 10,3 0,8 6 7 35 alg11 sidi belattar, algeria 5 18 27 13,8 3,4 4 14,5 8,7 0,5 7 6 35 alg12 sidi belattar, algeria 5 20 26 29,1 3,1 10 3,8 6,8 0,5 10 4 35 alg13 sidi belattar, algeria 7 18 28 21,7 3,7 5,2 19,2 5,3 0,8 6 5 45 alg14 sidi belattar, algeria 8 29,5 34 35,4 3,6 7,7 19,3 13,7 0,7 12 13 45 alg15 sidi belattar, algeria 3 22 38 19,5 3,9 4,8 8,7 8,3 0,9 7 8 45 alg16 sidi belattar, algeria 6 30 33 14,8 3,1 5,8 20,3 7,3 0,7 4 5 45 alg17 sidi belattar, algeria 5 23 30 23 3,2 4,7 15 10,7 0,6 7 1 45 alg18 sidi belattar, algeria 5 21 29 34,3 3,1 5,5 15,8 11,7 1,3 7 5 45 alg19 sidi belattar, algeria 7 16 26,5 26 4 3,8 12,7 7 0,6 9 4 45 alg20 sidi belattar, algeria 5,5 27,5 30 14,5 3,5 5,2 21 7,2 0,6 7 6 45 alg21 sidi belattar, algeria 4 20 30 25 3,9 4,5 16 9,8 0,8 7 2 45 alg22 sidi belattar, algeria 4,5 20 28 24,8 3,6 6 17,2 14,8 1 8 4 45 alg23 sidi belattar, algeria 5,5 28,5 33,5 17,3 3,5 6,2 22 12,3 1 5 6 45 alg24 sidi belattar, algeria 4 22 30 12,4 4 6,5 20 6 0,8 7 9 45 alg25 sidi belattar, algeria 5 28 28 15,7 3,7 6,2 18,2 9,7 0,9 6 5 45 alg26 sidi belattar, algeria 7 23 39 25,8 4,3 5,3 21,8 14,3 0,7 8 8 35 alg27 sidi belattar, algeria 4 21 24 31 4,4 3,3 14,5 8 1,2 6 4 35 alg28 sidi belattar, algeria 4 26 31 16,2 5,2 2,7 17 16 0,7 9 3 35 alg29 sidi belattar, algeria 6 20 25 20,5 4,1 3,7 11,8 11,7 1,1 10 3 35 alg30 sidi belattar, algeria 5 25 29 15,8 3,8 4,5 19,7 8 0,8 8 3 35 alg31 sidi belattar, algeria 6 21 36 20,3 3,6 5,7 17,2 12,8 0,5 6 7 35 spa1 valencia, spain 6,5 24 27,5 23,6 4,1 5,2 16 16,2 1,6 9 7 35 spa2 valencia, spain 4 28 29,5 15,3 3,7 7,2 23 24,5 0,9 6 11 35 spa3 valencia, spain 3,5 30 34 13,1 3,5 5,7 29 19,3 1,2 6 7 35 spa4 valencia, spain 3 21 24 16,9 4,3 3,3 16 18 2,8 6 5 35 spa5 valencia, spain 6 24 32 18,8 4,4 4,3 19,5 15 1,8 4 7 35 spa6 valencia, spain 5 20,5 24 18,4 3,1 6,7 19,2 18,2 2 7 8 35 spa7 valencia, spain 2,5 20,5 24 13,5 4,1 3,5 14,7 12,8 0,9 5 5 35 spa8 valencia, spain 4 27 27 14,2 4,4 4,8 23,7 9,7 1,9 4 4 30 spa9 valencia, spain 6 29,5 25 29,3 3,8 4,7 19,2 15,8 1,3 5 7 25 spa10 valencia, spain 3,5 27 30 19,3 3,4 7 19,7 16 2 6 9 25 spa11 valencia, spain 4,5 26 29,3 18,5 3,8 7,8 22,2 15 1,5 7 11 25 spa12 valencia, spain 4 24,5 30 23 3,8 7,5 20,7 16 1,3 7 10 25 sp13 valencia, spain 5,5 28,5 24 21,7 4,7 5,7 5,8 14 1,6 7 11 25 spa14 valencia, spain 4 35 39 33,8 6,2 7,8 26 24 2,3 9 11 25 spa15 valencia, spain 4 23 26 21,7 2,9 4,3 17 10 1,4 7 7 35 spa16 valencia, spain 4 27,5 33 20,1 4,3 6,5 19 15,8 1,7 7 10 35 96 antonio giovino, annalisa marchese, gianniantonio domina code population h ei gh t o f t he st em ( cm ) h ei gh t o f t he pl an t ( cm ) c ro w n di am et er ( cm ) st em d ia m et er (m m ) pe tio le w id th (c m ) pe tio le w id th (c m ) le af le ng th (c m ) le af w id th (c m ) le af th ic kn es s (m m ) n o. o f l ea f se gm en ts n o. th or ns o n th e pe tio le w ax c ov er ag e de ns ity % spa17 valencia, spain 4,5 27 20,5 22,2 4,2 4,7 17,5 14,7 0,9 8 6 35 spa18 valencia, spain 4 21 28,5 22,3 4,9 4,5 17,7 20,7 1,6 8 11 35 spa19 valencia, spain 3,5 21,5 27 22,5 4,3 4,7 15,5 14,3 1,6 7 7 35 spa20 valencia, spain 2,5 20,5 24 13,2 4,1 2,2 16 11,7 1,4 6 3 35 spa21 valencia, spain 3,5 20 24,5 15,4 5,4 2,3 15,7 12 1,1 6 3 35 spa22 valencia, spain 3,5 18 23 14,5 5,1 2,5 13,5 13 1,3 7 2 35 spa23 valencia, spain 5 22,5 26 17,3 3,9 2,8 19,2 14,5 1,3 6 8 35 spa24 valencia, spain 5 28 28 19,1 4,2 7 22,3 14,3 1,4 7 11 35 spa25 valencia, spain 3 30 29,5 18 5,4 4,5 23,2 14,2 1,7 6 3 35 spa26 valencia, spain 4 33 36 12,6 4,3 6,7 27,7 22,5 2,5 6 10 35 spa27 valencia, spain 4 25,5 26 17 4,5 5,5 20 11,8 1,5 6 3 35 spa28 valencia, spain 3 28 23 11 3,7 5,7 19,5 13,2 0,9 7 8 35 spa29 valencia, spain 3,5 26,5 21 12,8 4,1 6 16,3 11,8 0,9 7 6 35 spa30 valencia, spain 4 27 25,5 13,4 3,8 5,8 13,3 12,2 0,7 6 10 35 spa31 valencia, spain 3 27,5 22 11,5 3,4 5,5 12,5 12,2 1 5 7 35 tun1 cap serrat, tunisia 3 31 33,5 20,3 4,2 8,3 27,3 23 0,8 7 8 25 tun2 cap serrat, tunisia 4 41 46 33,5 4,3 11,7 30 20 0,6 7 11 25 tun3 cap serrat, tunisia 2 24.5 22 17,7 3,6 2,3 16,3 11,8 1,2 4 0 25 tun4 cap serrat, tunisia 2 16 26 18,1 3,7 3 13 11,3 1,3 7 0 25 tun5 cap serrat, tunisia 2 18 28 20,6 4,7 3,3 16,3 16,3 0,8 10 5 25 tun6 cap serrat, tunisia 6 36,5 43 20,2 4,3 5,8 22,2 22 0,9 9 11 25 tun7 cap serrat, tunisia 5 28,5 32 22,8 3,9 6,7 20,3 10 0,6 8 7 25 tun8 cap serrat, tunisia 7 37,5 38 27,5 5 11,3 25,8 25,7 0,7 10 10 25 tun9 cap serrat, tunisia 4,5 27,5 34 18 5,1 8,8 24,8 15,7 1 5 5 25 tun10 cap serrat, tunisia 6,5 35 38 36 5,9 10,5 24,2 17,7 1 11 11 25 tun11 cap serrat, tunisia 5 30,5 40 22 4,3 8,2 26,8 19 0,5 9 7 25 tun12 cap serrat, tunisia 5 22 35 16,4 3,8 3,2 17,8 13,7 0,7 5 1 25 tun13 cap serrat, tunisia 3 23 37 16,7 3,7 5,2 21,3 8 0,7 3 0 25 tun14 cap serrat, tunisia 6 32 41,5 28,8 4,9 8,3 24,5 18 0,8 9 8 25 tun15 cap serrat, tunisia 6,5 33 34 20 4,2 6,5 24,2 12 0,7 4 5 25 tun16 cap serrat, tunisia 6 26,5 26 20,1 3,9 4,2 18,2 11,7 0,6 5 3 25 tun17 cap serrat, tunisia 6,5 47 56 32,3 4,8 12,7 31 23,7 1,7 12 10 25 tun18 cap serrat, tunisia 10 43 48 42 5,3 11,3 33,3 21,3 1 8 9 25 tun19 cap serrat, tunisia 7 38 41 28,3 4,7 7,7 22,7 15 0,7 6 10 25 tun20 cap serrat, tunisia 9 46,5 50 47 5,4 15,8 31,7 23 0,9 10 11 25 tun21 cap serrat, tunisia 7 44 45 38,3 4,6 12 25,8 22,3 1 10 12 25 tun22 cap serrat, tunisia 11 54,5 60 42,5 5,6 18 38,3 20,7 0,7 12 11 25 tun23 cap serrat, tunisia 7,5 38 43 35 4,8 8,7 16,3 25 1,1 10 7 25 tun24 cap serrat, tunisia 8 49,5 45 38 6 12,7 38,3 14 1,1 8 7 25 tun25 cap serrat, tunisia 6 41 49 28,1 5,2 12,8 31,8 13 1,5 5 9 25 tun26 cap serrat, tunisia 7 30 33 30 4,2 6,7 19,3 15,7 1,1 6 7 25 tun27 cap serrat, tunisia 7,5 38 44 44,2 5,1 12 27,3 10 1,1 9 7 25 tun28 cap serrat, tunisia 5,5 35 37 23 4,4 7,8 26,2 15 0,7 4 8 25 tun29 cap serrat, tunisia 7 38,5 50 34,4 6 14,8 32,2 20 1,4 12 12 25 tun30 cap serrat, tunisia 5 31 40 19 4,4 5,7 23 12 1,3 6 9 25 tun31 cap serrat, tunisia 3 27 34 16 3,4 5,5 24 11 1 3 3 25 sar1 porto tangone, sardinia 10 29,5 30 16 4,8 4,3 14 16,5 3,7 8 6 25 97morphological and genetic variation of chamaerops humilis (arecaceae) in relation to the altitude code population h ei gh t o f t he st em ( cm ) h ei gh t o f t he pl an t ( cm ) c ro w n di am et er ( cm ) st em d ia m et er (m m ) pe tio le w id th (c m ) pe tio le w id th (c m ) le af le ng th (c m ) le af w id th (c m ) le af th ic kn es s (m m ) n o. o f l ea f se gm en ts n o. th or ns o n th e pe tio le w ax c ov er ag e de ns ity % sar2 porto tangone, sardinia 3 19 22 11,5 3,1 2,7 12,5 9,5 2 4 1 25 sar3 porto tangone, sardinia 4,5 20,5 24 16,8 4,5 2,8 14,8 15,2 5 9 7 25 sar4 porto tangone, sardinia 4 22 12,5 12,5 4 1,3 17,7 9 4,3 5 0 35 sar5 porto tangone, sardinia 2,5 18,5 14 9,8 3,9 1 10,8 8,3 3,4 4 0 35 sar6 porto tangone, sardinia 5,5 21 21,5 12,7 3,6 2,3 12,8 10,2 5,7 5 5 35 sar7 porto tangone, sardinia 3,5 21 27,5 12,9 3,4 2,7 13,7 15,7 4,7 8 3 35 sar8 porto tangone, sardinia 3,5 20,5 26,5 14,8 3,9 2,7 15,5 15,8 9,3 5 0 30 sar9 porto tangone, sardinia 5 26 23 15,9 3 4 16 13,7 3,2 3 5 25 sar10 porto tangone, sardinia 4 26 27,5 23,8 3,9 4,5 16,7 17,3 4,3 11 5 25 sar11 porto tangone, sardinia 3 22,5 29,5 10,6 3 3,3 16,3 13,3 3,5 5 3 30 sar12 porto tangone, sardinia 4,5 28 26 18,5 4,8 5,3 19,8 17 3,7 8 7 35 sar13 porto tangone, sardinia 5 25 26,5 17 3,8 3,8 20 12,5 4,7 8 5 30 sar14 porto tangone, sardinia 4 32 27,5 9,9 4,5 4,5 22 13,7 4,5 5 3 35 sar15 porto tangone, sardinia 4,5 30 27,5 16,9 4,7 4,2 21,3 13 5,6 3 5 25 sar16 porto tangone, sardinia 6 27 23 22 3,8 3,6 18,9 14,3 4,7 5 3 25 sar17 porto tangone, sardinia 5 25 27,5 21,8 3,9 4,1 19,1 15 4,3 4 0 30 sar18 porto tangone, sardinia 4,5 19 26,5 10,7 4,5 4,5 22 13,6 4,7 8 5 30 sar19 porto tangone, sardinia 4 32 27,5 16,7 4,7 4,1 20,6 14,7 5,5 5 3 35 sar20 porto tangone, sardinia 6 22 27,5 10,4 3,8 3,8 17,3 11,6 4,7 5 3 40 sar21 porto tangone, sardinia 5 27 23 18,4 4 4,2 14,1 8,9 4 4 0 30 sar22 porto tangone, sardinia 4,5 25 27,5 16,9 4,5 4,5 16,3 15,4 5,6 5 3 25 sar23 porto tangone, sardinia 4 19 26,5 23 4,3 3,8 22,2 13,4 4,5 4 5 30 sar24 porto tangone, sardinia 6 31 19 17,5 3,8 3,6 19,5 16,2 4,7 5 5 35 sar25 porto tangone, sardinia 4 22 27,5 16,9 4,2 4,5 22,6 15,3 4,3 3 3 25 sar26 porto tangone, sardinia 4,5 24 26,5 20,3 4,5 4,2 19,4 16,3 4 4 0 30 sar27 porto tangone, sardinia 5 28 27,5 18,6 3,9 3,8 19,2 16,2 4,7 8 5 35 sar28 porto tangone, sardinia 3,5 31 23 17,5 3,8 3,6 22 14 3,8 5 0 30 sar29 porto tangone, sardinia 6 24 26,5 18,3 4,5 4,5 21,6 15,8 4,1 4 3 25 sar30 porto tangone, sardinia 5,5 28 23 17,4 4,1 4,1 23 15,8 4,6 5 5 30 sar31 porto tangone, sardinia 7 22 23 15,8 3,9 3,7 9,8 13,3 3,4 8 4 35 98 antonio giovino, annalisa marchese, gianniantonio domina supplemental data file 2. pearson correlation coefficients (r) among the 12 characters measured. all correlations were significant with p > 0.0001 (student’s t test) with the lone exception of leaf thikness. h stem h plant crown diameter stem diameter petiole width petiole length leaf length leaf width leaf thikness no. leaf segments no. thorns hair density h stem 3,42e-08 1,82e-08 3,39e-23 0.048467 1,14e-07 0.079418 1,98e-01 0.24308 7,31e-13 3,29e-06 0.0058902 h plant 0.4303 1,55e-41 7,09e-02 4,91e-22 9,76e-52 2,97e-45 1,70e-21 0.0277 0.0065374 1,04e-14 3,97e-13 crown diameter 0.45465 0.77937 1,41e-06 7,02e-14 6,31e-52 3,46e-31 2,11e-15 0.18042 1,34e-02 2,91e-18 1,30e-04 stem diameter 0.64769 0.29956 0.4178 0.3676 7,78e-10 0.44561 0.0001304 4,70e-02 3,28e-20 2,50e-03 9,37e-03 petiole width 0.13412 0.63638 0.54079 0.061459 3,93e-12 9,67e-23 9,64e-12 6,58e-01 0.14084 5,07e-06 9,39e-19 petiole length 0.4396 0.82722 0.82799 0.47882 0.51553 4,07e-33 8,69e-17 0.082782 4,18e-04 2,50e-19 7,00e-05 leaf length 0.11934 0.79836 0.71441 0.052045 0.64331 0.72809 5,46e-16 0.013342 0.39458 3,73e-06 1,16e-26 leaf width 0.28525 0.63096 0.56117 0.25682 0.49385 0.57859 0.55578 0.0043571 6,97e-04 1,80e-12 5,84e-03 leaf thikness -0.079577 0.14947 -0.091265 -0.30507 0.26751 -0.11803 0.16776 0.19284 5,25e-01 0.14078 2,41e-05 no. leaf segments 0.52635 0.1841 0.32128 0.61759 0.1003 0.36163 -0.058079 0.35603 -0.27094 2,85e-05 1,18e-02 no. thorns 0.41007 0.53844 0.59605 0.34151 0.38371 0.60805 0.40892 0.52061 -0.10031 0.38944 0.0071865 hair density 0.18638 -0.53018 -0.37405 0.32569 -0.60164 -0.38043 -0.67882 -0.33146 -0.39111 0.32287 -0.182 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(2): 21-35, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1057 caryologia international journal of cytology, cytosystematics and cytogenetics citation: shaimaa s. sobieh, dina m. fahmy (2021) assessment of antimitotic and programmed cell death stimulation potentials of galium sinaicum (delile ex decne) boiss. at the cellular level. caryologia 74(2): 21-35. doi: 10.36253/ caryologia-1057 received: august 21, 2020 accepted: july 20, 2021 published: october 08, 2021 copyright: © 2021 shaimaa s. sobieh, dina m. fahmy. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid sss: 0000-0002-1286-7352 assessment of antimitotic and programmed cell death stimulation potentials of galium sinaicum (delile ex decne) boiss. at the cellular level shaimaa s. sobieh1,*, dina m. fahmy2 1 botany department, faculty of women for art, science and education, ain shams university, cairo, egypt 2 natural product unit, medicinal and aromatic plant department, desert research center, cairo, egypt * corresponding author. e-mail: shimaa.sobieh@women.asu.edu.eg, abstract. galium sinaicum is a wild medicinal plant in saint catherine, egypt. to distinguish apoptotic effect of  g. sinaicum  ethanol extract (gsee). the role of gsee in inducing programmed cell death (pcd) of allium cepa root meristematic cell(acr) was examined. cells was subjected to gsee in definite concentrations (0.1,0.3, 0.5%) and duration (6, 12h), then pcd induction was assessed. application of gsee arrested the mitotic division of acr with metaphase accumulation. electron microscopy analysis demonstrated ultrastructural alterations of organelles verifying pcd hallmarks. protein electrophoresis analysis of acr revealed a change in protein profile of allium cepa root, also quantitative analysis showed significant increase in nuclease activity enzymes that stimulated dna laddering fragmentation. additionally, cell proliferation of mcf7 and bhk21 was arrested by gsee. apoptotic effect of g. sinaicum may be attributed to the presence of potent phenolic compounds such as querectin and rutin as established by hplc phenolic fingerprint analysis. keywords: galium sinaicum, programmed cell death (pdc) hallmarks, allium cepa, mcf-7, bhk21, hplc. introduction when conventional medicine reaches an edge of success, patients search for alternatives. traditional phyto-medicine gains more and more consideration, especially with respect to alternative treatment of cancer. an intensive survey of plants especially which have an appropriate history of cancer treatment in folklore began in the later 1950s mainly since the national cancer institute (nci) adopted random selection screening program, as novel compounds may be found anywhere from plant kingdom. about 60% of currently used anticancer agents are derived from natural sources (newman et al. 2002; cragg et al. 2005). therefore, many researchers investigate extensively the mechanism of interaction between phytochemicals and cancer cells (kaufman et al. 1999; amirghofran et al. 2006a). 22 shaimaa s. sobieh, dina m. fahmy phytochemicals may modulate cell signalling pathways thereby inhibiting cancer development or progression and induce apoptosis in malignant cells (stevenson and hurst 2007). apoptosis is a programmed cell death and a highly organized physiological mechanism to destroy injured or abnormal cells. apoptotic cells manifest morphological features and characteristic molecular expression. provoking apoptosis in cancer cell is recognized as an efficient strategy for cancer therapy. apoptosis also seems to be a dependable marker for the evaluation of potential agents for cancer inhibition (taraphdar et al. 2001). in past decades, research on traditionally tumour inhibitory medicinal plants have yielded an impressive array of novel healing plants via authorizing their apoptosis mechanism (taraphdar et al. 2001).epidemiological studies suggest that consumption of diets containing fruits and vegetables, which are major sources of phytochemicals and micronutrients, may reduce the risk of developing cancer (reddy et al. 1997). developed studies with such plants with respect to their abilities to induce apoptosis and figure out their mechanism of action may provide a good aim of work and valuable information for their possible application in cancer therapy or prevention. thus, considering the importance of screening apoptotic inducers from plants, galium sinaicum was elected to be screened for its apoptotic enhancement activity. galium sinaicum (delile ex decne) boiss. is a wild herbal plant habituated in saint catherine protectorate, south saini, egypt. g. sinaicum belongs to rubiaceae characterized by presence of anthraquinones and lignens in root, and flavonoids in aerial part (el-gamal et al. 1999) like other galium sp.for instance g. verum and g. mollugo and g. spurium , g. aparine g. odoratum (yang et al. 2011, vlase et al. 2014, bradic et al. 2018) in folk medicine galium species are used to coagulate milk, also as diuretics, choleretics, against diarrhea and in the treatment of some stomach complaints, gout, epilepsy and as anticancer remedy in europe, africa and australia (güvenalp et al. 2006a; güvenalp et al. 2006b; gîrd et al. 2015). additionally, in pakistan, g. aparine is used as herbal cure for cancer disease (tariq et al. (2017). no data have been previously stated on g. sinaicum regarding its phenolic content and its potential as antiproliferative and apoptotic inducing agent. thus, phytochemical components and potential antiproliferative activity on breast cancer (mcf-7) and normal kidney cell of baby hamster (bhk21) cell line as well as meristematic cell of root of allium cepa l. of g. sinaicum were investigated. materials and methods herbal material: aerial part of galium sinaicum (delile ex decne) boiss. was collected in spring from wadi gemal saint katherine protectorate, south saini egypt. it was identified and authenticated by saint katherine protectorate members. arial parts of galium sinaicum were collected, packed in paper bags and coded in the field. the aerial parts of the plant materials were airdried at room temperature and grinded to coarse powder. tested models cell lines: breast adenocarcinoma cell line (mcf7 (atcc®  htb22™)) and normal baby hamster kidney fibroblasts cell line (bhk 21[c-13] (atcc® ccl10™)) were used in this study. cell lines were obtained from the american type culture collection (atcc, minnesota, u.s.a.). the cell lines were maintained at the national cancer institute, cairo, egypt, by serial subculturing. onions bulbs: root tips of allium cepa l. (2n = 16, variety giza 20) (acr) were obtained from desert research center, cairo, egypt. preparation of extract a weight of 200gm of powdered plant was extracted by maceration technique in ethanol: water (8:2) (200ml×4). extract was filtered, concentrated under reduced pressure using rotary evaporator (buchi, r200, switzerland) at 40oc. the dry extract (gsee) stored at 4oc for further investigation (harborne 1973). measurement of programmed cell death (pcd) as stated before, pcd involves cell with changes only after the point of no return. there are several techniques to obtain information about cell death and induction of pcd such as measurement of mitotic indices, morphological changes, and dna profile etc. evaluation of antiproliferation activity sulphorhodamine-b (srb) assay was performed to assess anti-proliferative potency of gsee as reported by vichai and kirtikara (2006). mcf-7 and bhk21 cells were planted in 96-well microtiter plates at initial concentration of 3x103 cell/well in a 150µl rpmi 1640 and left for 24h to attach to the plates. gsee were prepared 23assessment of antimitotic and programmed cell death stimulation potentials of galium sinaicum at the cellular level from the stock solutions by serial dilution (0, 12.5, 25, 50 & 100 µg/ml) in dmso to give a volume of 100μl in each well. the assay for both cell lines was completed in triplicates and the culture plates were kept at 37°c with 5% co2 for 48h. after 48h of incubation, the cells were fixed with 50μl 10% cold trichloroacetic acid for 1h at 40c. the plates were washed with distilled water using automatic washer (tecan, germany) and stained with 50μl 0.4% srb dissolved in 1% acetic acid for 30 minutes at room temperature. subsequently, the plates were washed with 1% acetic acid and air-dried. the dye was solubilized with 100μl/well of 10m tris base (ph 10.5). absorbance value was measured spectrophotometrically at 570nm with a microplate reader (sunrise tecan reader, germany). cell survival was measured as the percentage absorbance compared to the control (non-treated cell). the ic50 values were calculated by graphpad prism 5 software (graphpad software inc., san diego, ca). mitotic analysis using a. cepa root five a. cepa bulbs were used for each concentration. a. cepa bulbs were germinated in distilled water at room temperature. when the roots reached 3-4cm, they were treated with 0.1, 0.3 and 0. 5% of gsee for 6 and 12h, then the roots were fixed in carnoy’s fixative (ethyl alcohol: glacial acetic acid 3:1 (v/v)). for the untreated group, a. cepa bulbs were germinated in distilled water. the roots were taken out of carnoy’s fixative after 24h, hydrolyzed with 1n hcl at 60°c for 5 min, washed with distilled water and stained using schiff ’s stain for 1h in the dark. preparations were carried out using feulgen’s squash technique. slides were examined at 400x magnification. the number of cells in the mitotic division was scored and the mitotic index (mi) was calculated according to fiskesjö (1997). mitotic phase indices were also determined. the cytological abnormalities were scored in the mitotic cells. the most common abnormalities were photographed. statistical analysis was carried out using graphpad (version 4.0; san diego, usa, www. graphpad.com) with five replicates for each group. the significance was analysed using tukey’s and dunnett’s tests (significance was accepted at p ≤ 0.05 and p ≤ 0.01). data were expressed as mean± standard deviation (sd). electron microscopy investigation transmission electron microscopy is the method of choice when making detailed examination of the structural changes within cells. ultrastructural changes such as chromatin condensation and appearance of vacuoles containing remnants of cell organelles. acr tips (not more than 1mm) of control and treated groups were fixed in 2% glutaraldehyde in 0.05 mol/l sodium cacodylate buffer (ph 6.9) for 2h at 4°c. the tissue was rinsed in sodium cacodylate buffer and postfixed in 4% osmium tetraoxide for 1 hour at 4°c. the tissue was rinsed in sodium cacodylate buffer and dehydrated in graded ethanol water series. following passage through a graded propylene oxide/ethanol series, the root tips were gradually infiltrated with resin by placing them for 24h in each of series of resin/propylene oxide mixtures, followed by three changes in 100% epon 812. then materials were embedded in freshly prepared resin mixture and polymerized in oven at 60°c for 48 h (glińska and gabara 2000). sections (1μm) were cut with reichert ultra-microtome, mounted on copper grids and stained with 0.5% uranyl acetate for 30 min and lead citrate for 30min as described by reynolds (1963). observations were carried out using jeol tem 1010 transmission electron microscope at 80 kv, electron microscope unit, center for mycology and the regional biotechnology, al azhar university, cairo, egypt. measurement of the endonuclease activity nuclease activity was determined by the release of acid soluble nucleotides from single-stranded (ss) or double strand (ds) calf thymus dna, following the method of blank and mckeon (1989). a weight of 3 g of acr samples was ground in liquid nitrogen with a mortar and pestle. ice-cold buffer (0.05 m tris-hcl, ph 7.5/0.15m nacl/1 mm n-ethylmaleimide; 10 ml/g of leaf ) was added and the suspensions were homogenized in an ice bath for 1min. aliquots of homogenates were centrifuged for 10min at 6000 rpm in an eppendorf microcentrifuge at 6°c. the supernatant solutions were frozen and stored at -70°c. double-stranded calf thymus dna (sigma) was dissolved (1mg/ml) in sterile distilled water, boiled for 10min and placed on ice immediately. assay mixtures containing dna calf thymus, reaction buffer (0.2m nacl, 0.002m zncl2, 0.06m ch3coona, ph (4.6)) and 0.1ml cytosolic homogenate were incubated for 10 in at 37°c. the reactions were stopped by the addition of 2ml of 15% perchloric acid on ice for 10min, then centrifuged for 15min at 2000rpm. the absorbency was recorded at 260nm. native calf thymus dna solution without sample homogenate was used as blank units /ml = ((as – ab) ×dilution × 1242) / 10 one unit is the amount of enzyme liberating 1µg (0.033 a260) of acid-soluble nucleotides from heat-dena24 shaimaa s. sobieh, dina m. fahmy tured dna per minute at 37°c and at ph 4.6. where 1242 is a factor derived by dividing the reaction volume, by the a260 of 1μg, which is 0.033, and dividing by 0.1ml (volume of enzyme sample used). detection of dna fragmentation to examine dna fragmentation as a marker of programmed cell death, dna was extracted by ctab reagent from acr cells treated with gsee and untreated cells by the method of doyle and doyle (1987). acr cells (0.5 g) were frozen in liquid nitrogen and ground into a fine powder. each sample was incubated for 60min at 65°c in 9ml pre-warmed ctab extraction buffer, then mixed with an equal volume of chloroform–isoamyl alcohol (24:1). after gently shaking for 5 min, the mixture was centrifuged for 15min at 10000 rpm. the chloroform–isoamyl alcohol extraction was repeated when necessary. dna was recovered by centrifugation for 10min at 10000 rpm, followed by washing with 70% ethanol, and dissolved in 1ml te buffer. to detect dna fragmentation, samples were run on a 1% (wt/vol) agarose gel and stained with 0.5μg of ethidium bromide per ml (tada et al. 2001). the bands were visualized under uv transilluminator then were photographed for further analysis using gene tools syngene ver. 4.00(a) gel documentation system software. protein electrophoresis of a. cepa root distribution of protein pattern was detected using continuous polyacrylamide gel electrophoresis (sdspage) using hoefer (se 245) dual vertical mini-gel. a weight of 1g of each treatment was extracted with 1 ml of extraction buffer contained 1.21 g tris hcl, 1ml 10% sds, 0.5ml β-mercaptoethanol and 5 g sucrose completed to 50 ml distilled water (ph 8.0). protein pellets were precipitated by cooling centrifuge at 4000 rpm for 15 min., pellets were dissolved with 0.5µl sample buffer contained 1.2ml tris hcl, 2ml 10% sds, 1 ml glycerol, 0.5ml 0.4% bromophenol, 0.5 ml β-mercaptoethanol, 4.8ml distilled water. the homogenate was boiled in water bath for 90 seconds, loaded on gel. bio rad low molecular weight protein marker (97 to 14.4 kda, catlog number 1610304) was loaded on gel as protein standard. the gel was forced to run at 70 volts, 40ma. finally, gel was stained with coomassie brilliant blue g-250 stain and destained according to laemmli (1970). gel was photographed and scanned for further documentation system using gel pro analyzer version 3.1 for windows95-nt (media cybernetics 1993-1997). high performance liquid chromatography (hplc)fingerprint the instrumentation used for hplc analysis consisted of agilent 1260 series. chromatographic column was used: eclipse plus c18 column (4.6 mm x 250 mm i.d., 5 μm). mobile phase flow rate was set by 1.0 ml min1; sample volume was 10 µl. the mobile phase consisted of water (a) and 0.02% tri-floro-acetic acid in acetonitrile (b). the mobile phase was programmed consecutively in a linear gradient as follows: 0 min (80% a); 0-5min (80% a); 5-8min (40% a); 8-12min (50% a); 12-14min (80% a) and 14-16min (80% a). monitoring the uv spectrum of the peaks was done by ultraviolet detector at 280nm. phenolic compounds were identified by comparing their relative retention times with those of the standards mixture chromatogram. the concentration of an individual compound was calculated based on peak area measurements, then converted to mg phenolic g-1 dry weight. all chemicals and solvents used were hplc spectral grade. results extraction yield about 200g of dry powder of aerial parts of g. sinaicum was macerated by 80% ethanol. the percentage yield of the crude extract used for the assays was 13g%. effect of gsee on the viability of mcf-7 and bhk21 cell viability percentage was assessed by srb assay after 48h. of incubation. gsee decreased cell viability in a concentration-dependent manner in both mcf-7 and bhk21 as compared to untreated controls (fig. 1). ic50 value of gsee was 35µg/ml and 39.5µg/ml against mcf7 and bhk21, respectively (table 1) (a) (b) figure 1. antiproliferative effect of gsee (0-100 µg/ml) using srb method on (a) and mcf-7 (b) bhk21 cell lines. 25assessment of antimitotic and programmed cell death stimulation potentials of galium sinaicum at the cellular level mitotic analysis using a. cepa root a remarkable decrease in the mitotic indices of acr cells was clearly found after the application of gsee. this remarkable decrease was dose and time dependent (table 2). treatment with gsee has stopped the progress through mitosis of many cells and they began to accumulate in mitosis. concentration of 0.5% of gsee exerted the most potent effect on the mitotic index; it decreased the mitotic index to the minimum proportion (0.92) after 6 h exposure, while the maximum proportion of mitotic index (2.08) was achieved after exposure to 0.1% of gsee for 6h. concentration 0.5% of gsee caused the complete arrest of the mitotic process after 12h of treatment. the statistical analysis of the data revealed that all doses of gsee induced a highly significant reduction in mitotic index of acr cells as compared with their control (table 2). the obvious accumulation in metaphase stage was noted after treatment with all concentrations of gsee, this accumulation was on the expense of prophase and anaphase stages (table 2). as well, treatment of acr cells with gsee resulted in a high increase in the total percentage of abnormalities exceeded its counterpart control, this increase was concentration and time dependent (table 2). the spindle apparatus was the target of the treatment as can be demonstrated by the induction of spindle disturbance as a common type of aberrations. spindle disturbance included several forms of spindle disturbance like disturbed metaphase, disturbed anaphase and diagonal configuration. stickiness was also detected during the study (fig. 2). electron microscopy induction of pcd in acr treated with gsee was followed up by transmission electron microscope. root tip cells of control possessed a large-rounded nucleus with intact nuclear membrane, dense cytoplasm and wellorganized organelles (fig. 3a). the electron micrographs after treatment with gsee showed changes in sub-cellular organelles and induction of some pcd hallmarks. fig. 3b revealed the early stage of pcd as confirmed by the formation of dilated endoplasmic reticulum, intensification in the vacuolar system and presence of organelles with intact membrane inside the vacuole. the next stage of pcd was illustrated by the most striking aspects of pcd where obvious disintegration of nuclear membrane and formation of clotting chromatin was observed (figs. 3c, d). the result of prolonged immersion of acr in the highest concentration (0.5%) of gsee showed the final stage of pcd in which the vacuolar system extremely expanded, the shrinkage nucleus and undifferentiated organelles with intact membranes were observed only around the periphery of the cell (fig 3e). table 1. ic50 of antiproliferative activity of gsee (n=3). antiproantiproliferative activity on mcf-7, ic50 (µg/ml of extract) 35 antiproantiproliferative activity on bhk21, ic50 (µg/ml of extract) 39.5 table 2. mitotic and phase indices and percentage of mitotic abnormalities of acr cells treated with gsee. treatment mitotic index ± sd mitotic abnormalities prophase metaphase ana-telophase control 6 hrs 3.26±0.70 5.32±0.15 32.45±0.31 34.30±0.19 33.25±0.15 12 hrs 3.83±0.23 5.89±0.39 32.10±0.57 35.66±0.66 32.24±0.70 0.1% 6 hrs 2.08*±0.19 15.33**±0.07 24.62*±0.82 48.31**±25 27.07*±0.38 12 hrs 1.93**±0.08 23.04**±0.96 20.32**±0.41 51.11**±0.10 28.57*±0.70 0.3% 6 hrs 1.56**±0.73 25.98**±0.67 27.25*±0.13 50.91**±0.09 21.84**±0.23 12 hrs 0.93**±0.24 29.12**±0.53 23.03*±0.68 59.43**±0.70 17.54**±0.70 0. 5 % 6 hrs 0.92**±0.36 34.57**±0.21 23.96*±0.74 59.98**±0.37 16.06**±0.49 12 hrs 0.00±0.00 00.00±0.00 00.00±0.00 00.00±0.00 00.00±0.00 *statistically significant at p≤0.05 **statistically significant at p≤0.01 26 shaimaa s. sobieh, dina m. fahmy dna fragmentation the induction of internucleosomal dna cleavage in acr cells was analyzed by agarose gel electrophoresis. gsee successfully cleaved the genomic dna of acr cells and gave a genome-specific fingerprint of dna fragments (fig. 4). agarose gel electrophoresis showed the presence of the ladder pattern of degraded dna. gsee produced multiple-bands profiles. therefore, fig. 4 verified that the induction of ladder pattern was dose dependent. endonuclease activity nuclease activity assay was carried out using nuclease extracts from acr cells treated with gsee for 6 and 12h. a dose and time dependent increase in nuclease activity was found. table 3 revealed that the nuclease (a) (b) (c ) (d) (e ) (f) (g) (h) (i) (j) (k) (l) figure 2. the most common types of mitotic abnormalities induced in acr after treatment with different doses of gsee. a: disturbed metaphase; b-c: c-metaphase; d: sticky diagonal metaphase; e: sticky metaphase; f: sticky metaphase &anaphase; g: disturbed anaphase; h-j: disturbed diagonal anaphase; k: sticky disturbed anaphase; l: severe sticky anaphase. 27assessment of antimitotic and programmed cell death stimulation potentials of galium sinaicum at the cellular level (a) (b) (c ) (d) (e ) figure 3. electron micrographs of control and treated acr with gsee. (a): electron micrograph of control a. cepa root tip showing large nucleus (n), nucleolus (nu), cell wall (cw), mitochondrion (m) and free ribosomes (r); (b): formation of dilated endoplasmic reticulum (arrow) and the presence of organelle inside the vacuole (arrowhead); (c): disintegration of nuclear envelope (line); (d): formation of clotting chromatin (cc); (e): final stage of cell death with the unusual shrinkage of the cell contents, expanding of vacuolar system (v) and the cytoplasm was observed only around the periphery of the cell. 28 shaimaa s. sobieh, dina m. fahmy activity increment was highly significant as compared with their corresponding control and their results were treatment dependent. the nuclease activity increased more than four folds of the control reaching 4.15 units g-1 fresh weight after 6h exposure to 0.1% of gsee while 12h exposure to 0.5% has increased the nuclease activity more than sixteen folds reaching 16.83 units g-1 fresh weights. protein electrophoresis of a. cepa root the alteration in the protein banding profile of acr treated with gsee was recorded in table 4 and fig. 5. the total number of bands ranged from 7 to 9 bands. figure 4. induction of dna fragmentation in acr cells after treatment with different doses of gsee. c: control; 1:acr treated with 0.1% gsee for 6h; 2: acr treated with 0.1% gsee for 12h; 3: acr treated with 0.3% gsee for 6h; 4: acr treated with 0.3% gsee for 12h; 5: acr treated with 0.5% gsee for 6h; 6: acr treated with 0.5% gsee for 12h. table 3. nuclease enzyme activity (units g-1 fresh weight) in acr cells during the induction of programmed cell death by gsee. time/ concentration control 0.1% 0.3% 0.5% 6h 1.72±0.18 4.15*±0.27 7.29**±1.16 10.23**±0.11 12h 1.75±0.09 5.13**±0.14 11.61**±0.31 16.83**±0.23 *statistically significant at p≤0.05 **statistically significant at p≤0.01 figure 5. protein banding pattern of acr cells after treatment with gsee. m: marker; 1: control; 2:acr treated with 0.1% gsee for 6h; 3: acr treated with 0.1% gsee for 12h; 4: acr treated with 0.3% gsee for 6h; 5: acr treated with 0.3% gsee for 12h; 6: acr treated with 0.5% gsee for 6h; 7: acr treated with 0.5% gsee for 12h. table 4. effect of gsee on protein banding pattern of acr cells. band no. mol.wt. kda marker control 0.1% 0.3% 0.5% 6h 12h 6h 12h 6h 12h 1 97.40 + + 2 88.23 + + + 3 78.64 + 4 66.20 + + + 5 62.80 + + + 6 57.61 + + + + 7 51.70 + + + + + + 8 47.45 + + + 9 45.00 + + + + 10 40.21 + + + + + + 11 35.51 + + + + + 12 31.00 + + + 13 29.00 + + + + + + 14 27.07 + + + 15 21.50 + + + + + + 16 19.72 + + + + + 17 18.23 + + + + 18 14.40 + sum 7 9 10 7 10 9 9 + present band absent band 29assessment of antimitotic and programmed cell death stimulation potentials of galium sinaicum at the cellular level the new bands induced in the treated samples were 11. most of these bands were with low molecular weight. treatment with gsee caused the completely disappearance of protein band with molecular weight of 78.64 kda, while some bands were disappeared in some treatments only (table 4). high performance liquid chromatography (hplc) fingerprint qualitative phytochemical analysis of gsee was performed by high performance liquid chromatography to determine the biologically active compounds. a total of 15 phytochemicals; ten phenolic acids: gallic acid, chlorogenic acid, catechin, caffeic acid, syringic acid, coumaric acid, vanillin, ferulic acid, propyl gallate, cinnamic acid, four flavonoids: rutin, naringenin, 4 .̀7-dihydroxyisoflavone, quercetin, and one alkaloid: caffeine was identified. table 5 shows the abundant identified compounds. discussion herbal drugs, including plants, herbal complexes, and herbal products were used thousand years before era of modern drugs. herbal plants are used all over the world in different methods both in allopathic and traditional systems (pal and shukla 2003; smith-hall et al. 2012). based on ethnopharmacological approaches, revival of drugs with herbal origin specially to treat cancer and immunologic and cns diseases is highly considerable. there are also several studies investigating the antiproliferative effects of galium species on various cancer types. amirghofran et al. (2006b) reported that g. mite extract exhibited cytotoxic effects against human leukemia cells. moreover, anti-cancer effect of g. verum aqueous extract was investigated on drug-sensitive and -resistant laryngeal carcinoma cell lines. g. verum was found to be cytotoxic against all tested laryngeal carcinoma cell lines (schmidt et al. 2014a). in additional study by schmidt et al. (2014b), sublethal doses of g. verum aqueous extract acted as strong inhibitor on the motility of human head and neck cancer cell lines also, the fractional extract of petroleum ether had promising cytotoxic effects on colon cancer ht29 (pashapour et al. 2020). furthermore, aslantürk et al. (2017) proved that g. aparine ethyl acetate and methanol extracts have cytotoxic and apoptotic inducing effect on mcf-7 and caco-2 cancer cells. in this study, effect of gsee on the viability of mcf7 and bhk21 was assessed by srb method. after 48h. incubation, gsee arrested cell proliferation of both mcf-7 at 35µg/ml and bhk21 at 39.5µg/ml in a concentration dependent pattern. the american national cancer institute (nci) guidelines set the limit of activity for crude extracts at 50% inhibition (ic50) of proliferation of less than 30µg/ml after exposure time of 72h. (abdelhameed et al. 2012), moreover, a crude extract with ic50 less than 20µg/ml is considered highly cytotoxic (mahavorasirikul et al. 2010). regarding this scale, gsee showed moderate antiproliferative effect against cancer and normal cell after 24h of exposure. accordingly, at this level of investigation, gsee is considered as having an impact on mcf-7 and bhk21 cell viability that can be explained by different mechanisms rather than causing cell toxicity. cancer developed when unusual cell proliferation is triggered. apoptosis is a well-established self-destruct system which is essential to normal tissue development and homeostasis (vaux and korsmeyer 1999). apoptosis and its related signalling pathways antagonize the progression of tumor growth (lowe and lin 2000). thus, induction of apoptosis is a highly desirable goal for launching cancer control strategy (reed and pellecchia 2005). the current work documented the program cell death process in acr evoked by 0.1%,0.3% and 0.5% of gsee. depression in mitotic division is the first sign to table 5. high performance liquid chromatography fingerprint of gsee. peak no. compound ret. time [min] conc. [mg/g] 1 gallic acid 3.135 7.92 2 chlorogenic acid 3.502 9.13 3 catechin 3.785 5.05 4 caffeine 4.046 0.38 5 caffeic acid 4.924 1.75 6 syringic acid 5.360 0.86 7 rutin 5.650 3.20 8 coumaric acid 7.667 0.30 9 vanillin 8.121 0.72 10 ferulic acid 8.774 0.17 11 naringenin 9.439 2.65 12 propyl gallate 10.414 0.64 13 4`.7-dihydroxyisoflavone 10.519 0.77 14 quercetin 10.704 3.41 15 cinnamic acid 11.291 1.79 the table contains the most abundant identified compounds in retention time order. 30 shaimaa s. sobieh, dina m. fahmy the induction of cell death. depression in mitotic index was previously reported by several medicinal plant extracts (shalini and velavan 2017; karaismailoğlu 2017). in present study mitotic indices analysis elucidates a consecutive decrease in mitotic index as reported by rubeena & thoppil (2018). this decrease may be due to dna damage and/or spindle damage. mccollum et al. (2005) interpreted the decrease in mi after treatment with arsenic trioxide as result of the g2 phase delay. zhou and elledge (2000) cleared that the cell-cycle control system can readily detect dna damage and arrest the cycle at dna damage checkpoint. this checkpoint prevents and /or delays entry into mitosis by inactivating the cdc25 protein phosphatase. inactivated cdc25 protein phosphatase blocks the dephosphorylation and activation of m phase cyclin dependent kinases (m-cdk) (hanamata et al. 2020).the pervious explanation indicates that gsee might delay the progression of cell cycle at dna damage checkpoint and inhibit most cells to enter mitosis (m phase). on the other hand, only a few numbers of cells entered mitosis and accumulated or arrested at metaphase stage, these cells might be unable to perform metaphase checkpoint (the mitotic checkpoint). that may cause stabilizing activity of mitotic cyclin dependent kinases (m-cdk) all time and preventing cells to exit from mitosis (tawab et al. 2014). drugs causing alterations in microtubules prevent alignment of the daughter chromosomes and consequently lead to stop of mitosis at metaphase and anaphase, which can be finally followed by apoptosis (safarzadeh et al. 2014; yanık et al. 2017; huang et al. 2019). metaphase accumulation and induction of spindle disturbance as a common feature of mitotic abnormalities verified the ability of gsee to abolish and block metaphase to anaphase transition. gsee might disrupt the equilibrium between polymerization and depolymerization of microtubules. this disruption might target tubulin subunit resulting in failure of cytokinesis (murata et al. 2013). besides, the disassembly of mitotic spindle induces a strong signal that greatly prolongs metaphase stage. therefore, any kinetochore that is not attached to spindle sends out a negative signal to the cell-cycle control system, blocking cdc20-anaphase, promoting complex (cdc20-apc) activation, and sisterchromatid separation. thus, sister-chromatid separation cannot occur until the last kinetochore is attached (shah and cleveland 2000). so, the data in the present study confirm that both entry into and exit from mitosis is blocked in treated cells suggesting that gsee may interfere with the balance between cyclin condensation required for entry into mitosis and exit from mitosis. pelayo et al. (2003) declared that plant cells unable to perform checkpoint adaptation may instead induce a program of cell death or may simply fail to proliferate, remaining inactive in mitosis depending on the stimulus and features of cell damage. ultrastructural analyses illustrate the changes in cytoplasm organelles in relation to nucleus and provide a confirmatory approach to study cell death processes. adamakis and eleftheriou (2019) proved the changes in cell ultrastructure of pisum sativum during the induction of pcd by tungsten. ultrastructural analysis of acr treated with 0.1%,0.3% and 0.5% gsee showed that the first detectable events occurred in the cytoplasm is the accumulation of endoplasmic reticulum and its lumen appears dilated as compared with their control. the same finding was also seen in the nucleus of tillandsia by brighigna et al. (2006) and proved by tawab et al. (2014) who elucidated that allim cepa cells treated with punica granatum lose contact with their neighbours because of formation of dilated endoplasmic reticulum. this aspect was previously explained by madeo et al. (1997) who affirmed that the formation of dilated endoplasmic reticulum could be ultimately leading to the programmed cell death in yeast cells. the nucleus displays an irregular shape (pycnosis), with disintegrated nuclear envelope and highly condensed chromatin “clotting chromatin”. this nuclear morphology has been described in other forms of plant programmed cell death, including aerenchyma formation in response to hypoxic stress (gunawardena et al. 2001) and as a response of a. cepa root cells to punica granatum polyphenol extract by tawab et al. (2014). furthermore, the formation of clotting chromatin is a microscopical marker for both apoptotic and nonapoptotic cell death as previously reported by schwartz (1992). the presence of disintegrated nuclear envelope is resembled to the late stage of animal programmed cell death as previously proved by gao et al. (2018). the concentration of 0.5% gsee were able to increase the size of vacuolar system. some vacuoles appeared to have cellular debris indicating the presence of autophagic and autolysis processes (fuzinatto et al. 2007; papini et al. 2014). domínguez et al. (2004) reported that the cytoplasm of wheat aleurone cells undergoing pcd showed rapid vacuolation; they thought that vacuolar collapse was indicative of a high hydrolytic activity. vacuolar collapse has been hypothesized to be a common feature to many forms of plant pcd by jones (2001) and tawab et al. (2014). all the previous controlled changes in nuclear structure, cytoplasm, as well as the presence of organelle in complete integrity case in the periphery of the cell are leading to assumption that gsee is considered as a programmed cell death stimulus which is concurring with shahid et al. (2017). 31assessment of antimitotic and programmed cell death stimulation potentials of galium sinaicum at the cellular level different concentrations (0.1%,0.3% and 0.5%) of gsee altered the electrophoretic pattern of acr protein. the present variations in protein patterns of the treated acr might be resulted from changes in gene expression, these changes in gene expression occurs at transcriptional or transnational level (hopkins 1999). variation in either structural or regulatory genes can induce changes in the protein profile. herein, most new protein bands were with low molecular weight. farr and cohen-fix (1999) suggested that low molecular weight proteins are believed to be proteolysis enzymes and some of which block the cell cycle progression. avila and devarenne (2013) explained the critical role of proteolysis enzymes activity during the induction of programmed cell death in tomato cell culture by chemical treatment. additionally, large increase in normal proteolytic activity during the senescence of different plants have been documented (renxian et al, 2013; karmous et al. 2014). moreover, the newly induced protein with molecular mass of 21 kda almost in all treated roots might be required for preventing cells to exit from mitosis because it blocks cell cycle progression at metaphase by determining the substrate specificity of cyclosome/anaphase promoting complex, this conclusion was confirmed by tawab et al. (2004). additionally, the presence of protein band with molecular weight of 57.8 might be cyclin dependent kinase inhibitors that have been formerly found in plants and called putative ckis as reported by jasinski et al. (2002). in addition, proteins with molecular mass of 18, 29, 31, 35 and 51kda could be considered as protein bands for several types of nucleases enzymes that are responsible for dna degradation as was reported by yupsanis et al. (2004). dna fragments is one of the hallmarks for apoptosis matilla 2020, subsequently, detection of dna fragmentation is currently considered as one of the most frequently used techniques in the study of programmed cell death (hanna et al. 2013; shi et al. 2020). different concentrations (0.1%,0.3% and 0.5%) of gsee dna was degraded into multimers of 180 bp. this active degradation of genomic dna during plant programmed cell death has been obtained by several stimuli (lombardi et al. 2007; tawab et al. 2014). ning et al. (2002) suggested that this pattern of dna fragmentation may be a universal marker of nuclear change during plant programmed cell death. this systematic dna fragmentation was associated with significant increase in nucleases activity (sakamoto and takami 2014; matilla 2020). langston et al. (2005) who stated that the nucleic acid catabolism must be catalysed by endonucleases enzymes which are having task of digesting both single-stranded dna (ssdna) and double stranded dna (dsdna). the increase in nuclease activity signifies the representing of some specific endonucleases with strong activity accumulated in the treated cells (ning et al. 2002). this explanation can be confirmed by the protein electrophoresis result in the present work as the novel proteins with molecular weights of 29 and 51kda have been induced in almost all treated acr suggesting that those new proteins may be zn2+-dependent nucleases which are responsible for dna fragmentation (sodmergen et al. 1991). other types of nucleases may be existed with molecular weights of 18 and 31kda in treated acr that might cause dna fragmentation and then cell death as stated by hosseini and mulligan (2002) who stated that those nucleases activities were increased in parallel with the increment of dna fragmentation and cell death. plant phenolics which is a well-known as a natural antioxidant induced dna fragmentation in different human cancer cell lines (hl-60, ml-1, u-937, thp-1) resulting into apoptosis as reported previously by taraphdar et al. (2001). moreover, inone et al. (1994) showed that tannic acid and caffeic acid induced dna fragmentation in hl-60 cells. liu et al. (2013) proved the ability of chlorogenic acid to cause g0/g1 arrest and form dna ladder pattern consequently induce apoptosis in apl hl60 cell line. the fragmentation of nuclear dna into specific segments by nuclease enzymes, proved by ladder formation, which confirms a point of no return to the cell to die. this result was considered the final hallmark of cell death (tawab et al. 2014; matilla 2020). flavonoids are naturally occurring molecules that are abundant in higher plants. there have been reports of flavonoids inducing apoptosis in cancer cells (wang et al. 1999; park et al. 2008). hplc fingerprint of gsee identified different phenolic and flavonoid compounds with evidenced apoptotic potential such as gallic acid, chlorogenic acid, catechin, caffeic acid, rutin, naringenin, 4 .̀7-dihydroxyisoflavone, quercetin (you and park 2010; chen et al. 2013; bao et al. 2016; zhang et al. 2016). in conclusion, the current study could ensure the ability of galium sinaicum to reprogram the cancer cell (mcf-7) and normal cells (bhk21 and acr) to enter the death program by arresting cell division, that was clarified by microscopical hallmarks of pcd and finally forming dna fragmentation with an increment in the endonuclease’s enzymes, all the previous features may be attributed to the presence of phenolic and flavonoid compounds. author contributions both authors suggested the point of the work and planned the experimental design to achieve this point. 32 shaimaa s. sobieh, dina m. fahmy both authors supplied the financial support for the work. the writing of the manuscript was done by both authors. acknowledgements both authors thank saint katherine protectorate members for helping them in collecting tested plant (galium sinaicum) from saint katherine, egypt and the identification of it. also, they thank prof. dr., muhammad raeef desert research center for supporting them allium cepa. references abdel-hameed es, salih a, bazaid sa, shohayeb mm, el-sayed mm, el-wakil ea. 2012. phytochemical studies and evaluation of antioxidant, anticancer and antimicrobial properties of conocarpus erectus l. growing in taif, saudi arabia. eur j med plants. 2: 93-112. adamakis is, eleftheriou ep. 2019. structural evidence of programmed cell death induction by tungsten in root tip cells of pisum sativum. plants. 8: 62; doi. org/10.3390/plants8030062. amirghofran z, bahmani m, azadmehr a, javidnia k. 2006a. induction of apoptosis in leukemia cell lines by linum persicum and euphorbia cheiradenia. j cancer res clin oncol. 132: 427-432. amirghofran z, bahmani m, azadmehr a, javidnia k. 2006b. anticancer effects of various iranian native medicinal plants on human tumor cell lines. neoplasma. 53: 428-433. aslantürk ös, çelik ta, karabey b, karabey f. 2017. active phytochemical detecting, antioxidant, cytotoxic, apoptotic activities of ethyl acetate and methanol extracts of galium aparine l. british j pharmaceutal res. 15(6): 1-16. avila j, devarenne tp. 2013. ubiquitination of the tomato cell suppressor adi3 by the ring e3 ubiquitin ligase adbil. biochem bioph reas comm. 430: 119124. bao l, liu f, guo hb, li y, tan bb, zhang wx, peng yh. 2016. naringenin inhibits proliferation, migration, and invasion as well as induces apoptosis of gastric cancer sgc7901 cell line by downregulation of akt pathway. tumor biol. 37: 11365-11374. blank a, mckeon ta. 1989. single-strand preferring nuclease activity in wheat leaves is increased in senescence and is negatively photoregulated. proc nat aca sci usa. 86: 3169-3173. bradic j, petkovic a, tomovic m. 2018. phytochemical and pharmacological properties of some species of the genus galium l. 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checkpoints in perspective. nat. 408: 433439. caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 1 2021 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 74(2): 45-56, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1236 caryologia international journal of cytology, cytosystematics and cytogenetics citation: ruonan zheng, shuhua zhao, majid khayatnezhad, sayed afzal shah (2021) comparative study and genetic diversity in salvia (lamiaceae) using rapd molecular markers. caryologia 74(2): 45-56. doi: 10.36253/caryologia-1236 received: march 05, 2021 accepted: april 28, 2021 published: october 08, 2021 copyright: © 2021 ruonan zheng, shuhua zhao, majid khayatnezhad, sayed afzal shah. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. comparative study and genetic diversity in salvia (lamiaceae) using rapd molecular markers ruonan zheng1,*, shuhua zhao2, majid khayatnezhad3, sayed afzal shah4 1 zhengzhou railway vocational and technical college,  zhengzhou,henan,  450052,  china 2 zhengzhou central hospital, zhengzhou, henan, 450007, china 3 department of environmental sciences and engineering, ardabil branch, islamic azad university, ardabil, iran 4 department of biological sciences, national university of medical sciences, the mall, abid majeed road, rawalpindi, punjab, 46000, pakistan *corresponding author. e-mail: zhengruonan2021@163.com; majidkhayatnezhad126@ gmail.com abstract. salvia has a high degree of environmental compatibility and is widespread around the world, especially in tropical and temperate regions. it is represented by 61 species including 19 endemic species. salvia species are mostly shrubs or subshrubs, occasionally herbs, typically perennial, sometimes biennial or annual, and often aromatic. the genus has high medicinal, commercial and horticultural value. it is the largest and one of the taxonomically complicated genus of lamiaceae. to determine the genetic diversity and understand the species, we produced both morphological and molecular data using 145 randomly collected plants representing 30 species from 18 provinces of iran. a total of 107 reproducible bands were generated by 10 of 25 random amplified polymorphic dna (rapd) primers, with an average of 10.7 bands/ primer and 44% polymorphism. largest number of effective alleles (ne), genetic diversity (h), and shannon index (i) were shown by salvia reuterana. our data depicted highest similarity between s. suffruticosa and s. hydrangea and lowest between s. aristata and s. oligphylla. s. limbata showed relatively low level of genetic variation. finally, the neighbor joining (nj) trees based on rapd markers data divided the populations into two different clusters, indicating their genetic difference which is discussed in details. keywords: gene flow, genetic variation, random amplified polymorphic dna (rapd), salvia. introduction one of the most important aspect of biological diversity for conservation strategies is the genetic diversity, particularly in rare, and narrow endemic species (mills and schwartz 2005; tomasello et al. 2015). most authors 46 ruonan zheng et al. agree that longstanding evolutionary potential of a species necessitates maintenance of genetic diversity (falk and holsinger 1991). similarly, most geneticists regard population size as a significant factor in preserving genetic diversity (ellegren and galtier 2016; turchetto et al. 2016). in fragmented populations, this is critical as they are more susceptible due to allelic resources’ loss and bigger population differentiation through genetic drift (reduces heterozygosity and subsequent allele fixation) and inbreeding depression (develop homozygosity within populations) (frankham 2005). understanding of genetic variability and inter-, and intra-population diversity of rare and endemic species is therefore necessary for their conservation and management (e.g. cires et al. 2012, 2013; jing, et al. 2021). salvia l. of family lamiaceae (mentheae-salviinae) is recognized as the largest genus, comprising about 1000 species distributed in central and south america (500 species), western asia (200 species), and eastern asia (100 species) (walker et al. 2004; will and claßenbockhoff 2017). iran is considered one of the major regions for diversity of salvia in southwest asia represented by 19 endemic species out of 61 (jamzad 2012). the name of the genus ‘salvia’ comes from the latin name ‘salvio’ which means to recover or save (wang et al. 2011). salvia is one of the groups of herbs most valued for its richness in essential oil and biologically active compounds (erbano et al. 2015). in industries such as pharmaceuticals, the use of the salvia has been widely increased since it has pharmacological potentials including anti-inflammatory, and antiplatelet properties (erbano et al. 2015). salvia species have been used against different ailments including diabetes, acquired immunodeficiency syndrome (aids), liver disease, and alzheimer’s disease (sepehry javan et al. 2012). members of the genus have economical value in the perfumery industry, cosmetics, spices, and flavoring agents (wang et al. 2011). plant genetic diversity is a crucial feature regarding their breeding and domestication. some researcher have thus attempted to evaluate the variability in various salvia species using issr and rapd techniques (song et al. 2010; wang et al. 2011; sepehry javan et al. 2012; zhang et al., 2013; peng et al. 2014; erbano et al. 2015). they have documented high polymorphism in markers data and reported the utility of these techniques for assessing the genetic diversity in salvia (song et al. 2010; javan et al. 2012). kharazian (2010) studied the taxonomy and morphology of 42 salvia spinosa l. accessions (lamiaceae) from iran. the hair frequency and indumentum of the base and surface of the stem, the shape of the leaf, the leaf margin, and the leaf apex were all linked to the morphological diversity of this species. cluster analysis revealed that there was variation among the accessions. hence, its morphological diversity  may be attributed to polymorphism, hybridization, and new varieties. (kharazian 2010, zou et al. 2019; niu et al. 2021; sun et al. 2021). issr and rapd molecular techniques were used to assess the genetic relationships among twenty-one ecotypes of eight salvia species in iran (yousefiazarkhanian et al. 2016). the findings of their marker parameter analysis revealed no significant differences between the two marker systems. issr and rapd markers were shown to have identical efficiency in identifying genetic polymorphisms and a strong ability to distinguish closely related salvia ecotypes. for genetic diversity and relationship study of nine salvia species in iran, etminan et al., (2018) used inter-simple sequence repeats (issr) and start codon targeted (scot) markers. twenty-one issr and twenty scot primers, amplified  350 and 329 loci, respectively,  all of which were polymorphic. the average polymorphism information quality (issr, 0.38; scot, 0.40), average band informativeness (issr, 16.67; scot, 16.45), and resolving power (issr, 9.75; scot, 12.52) found within salvia accessions demonstrated a high level of genetic diversity. their findings suggested that scot markers can be reliably used to assess genetic diversity and relationships among salvia species. issr is a simple and efficient marker system for identification of genetic diversity for plant germplasm collection (peng et al. 2014). issr molecular markers have been used to show polymorphism and distinguish germplasms of salvia miltiorrhiza bunge by incorporating phenotypic characters (zhang et al. 2013). molecular markers are commonly used in genetic analysis, fingerprinting, linkage mapping, germplasm characterization, and molecular breeding. rapd analysis using pcr along with short arbitrary sequence primers has been reported sensitive to detecting variation at level of individuals (williams et al. 1990). the benefits of this method are: a) a large number of samples are tested easily and efficiently using less quantity of material; b) the dna amplicons are independent of ontogenetic expression; c) several genomic regions may be sampled with likely infinite numbers of markers (soniya et al. 2001; esfandani-bozchaloyi et al. 2017 a, 2017b, 2017c, 2017d). this study has been carried out to evaluate the genetic diversity and relationships among the iranian salvia species based on rapd data. this is the first step towards using rapd markers on a broader sampling of iranian salvia and aims at answering the following questions: 1) is there infraand interspecific genetic diversity among salvia species? 2) is genetic distance 47comparative study and genetic diversity in salvia (lamiaceae) using rapd molecular markers correlated with distribution of these species? 3) what is the populations’ genetic structure? 4) is there any genetic exchange within salvia species? materials and methods plant sampling a total of 145 individuals were collected from ecogeographically different populations representing 30 salvia species in east azerbaijan, lorestan, kermanshah, guilan, mazandaran, golestan, yazd, esfahan, tehran, arak, hamadan, kurdistan, ilam, bandar abbas, ghazvin, khorasan and ardabil provinces of iran during july–august 2017–2019 (table 1; figure 1). all of these samples were used during rapd and morphometric analysis and stored for further use in -20°c. morphological studies three to twelve samples from each species were used for morphometric analysis. a total of 22 morphological (9 qualitative, 13 quantitative) characters were examined. the obtained data were standardized (mean= 0, variance= 1) and used to assess euclidean distance for clustering and ordination analyses (podani 2000). morphological characters studied were: basal leaf shape, basal leaf length, basal leaf width, stem leaf length, stem leaf width leaf surface, bract shape, bract length, bract color, pedicel length, calyx length. table 1. voucher details of salvia species in this study from iran by khayatnezhad. no sp. locality latitude longitude altitude (m) sp1 salvia aristata aucher ex benth. east azerbaijan, kaleybar, shojabad 38°52’37” 47°23’92” 1144 sp2 salvia eremophila boiss. esfahan, ghameshlou, sanjab 32°50’03” 51°24’28” 1990 sp3 salvia santolinifolia boiss. fars, jahrom 29°20’07” 51°52’08” 1610 sp4 salvia tebesana bunge khorasan, tabas 38°52’373 47°23’92” 2234 sp5 salvia bracteata banks & sol lorestan, oshtorankuh, above tihun village 33°57’12” 47°57’32” 2500 sp6 salvia suffruticosa montb. & aucher hamedan, nahavand 34°52’373 48°23’92” 2200 sp7 salvia dracocephaloides boiss. east azerbaijan, kaleybar, cheshme ali akbar 38°52’373 47°23’92” 1144 sp8 salvia hydrangea dc. ex benth. arak, komayjan, pass of chehregan village, the margin road 35°50’03” 51°24’28” 1700 sp9 salvia multicaulis vahl. mazandaran, haraz road, emam zad-e-hashem 36°14’14” 51°18’07” 1807 sp10 salvia syriaca l. esfahan, fereydunshahr 32°36’93” 51°27’90” 2500 sp11 salvia viridis l. guilan, sangar, road sid 37°07’02” 49°44’32” 48 sp12 salvia mirzayanii rech. f. & esfand. boushehr, dashtestan 28°57’22” 51°28’31” 430 sp13 salvia macrosiphon boiss. yazd, khatam 30°07’24” 53°59’06” 2178 sp14 s. sharifii rech. f. & esfand. bandar abbas, hormozgan 28°57’22” 51°28’31” 288 sp15 salvia reuterana boiss. hamedan, alvand 34°46’10” 48°30’00” 1870 sp16 salvia palaestina benth. kermanshah, islamabad 35°37’77” 46°20’25” 1888 sp17 salvia sclareopsis bornm. ex hedge ilam, ilam 33°47’60” 46°07’58” 1250 sp18 salvia spinose l. guilan, lahijan 37°07’02” 49°44’32” 48 sp19 salvia compressa vent. bandar abbas, hormozgan 28°57’22” 51°28’31” 288 sp20 salvia sclarea l. esfahan:, ghameshlou, sanjab 32°36’93” 51°27’90” 2500 sp21 salvia aethiopis l. azerbaijan, 78 km from mianeh to khalkhl. 37°38’53” 48°36’11” 1500 sp22 salvia microstegia boiss. & bal. tehran, darband 35°36’93” 51°27’90” 1700 sp23 salvia xanthocheila boiss. ex benth. ardabil, khalkhal 37°38’53” 48°36’11” 1958 sp24 salvia limbata c. a. mey. guilan,gole rodbar, road sid 37°09’45” 49°55’39” 15 sp25 salvia chloroleuca rech. f. & aell. golestan, ramian 37°09’45” 55°55’39” 1320 sp26 salvia virgate jacq. golestan, ramian 37°09’45” 55°55’39” 1320 sp27 salvia nemorosa l. mazandaran, chalos 36°14’14” 51°18’07” 1807 sp28 salvia urmiensis bunge kurdistan, sanandaj 35°20’53” 53°30’20” 2344 sp29 salvia oligphylla aucher ex benth. ghazvin to hamedan just after avaj 35°36’93” 51°27’90” 2100 sp30 salvia verticillata l. mazandaran jadeh chalous 36°14’14” 51°18’07” 1807 48 ruonan zheng et al. dna extraction and rapd assay in each of the populations studied, fresh leaves from one to twelve plants were used randomly. leaves were dried with silica gel prior to dna extraction (esfandanibozchaloyi et al. 2019). by running on 0.8 percent agarose gel, the quality of extracted dna was examined. a total of 25 operon technology decamer rapd primers (alameda, canada) belonging to opa, opb, opc, opd sets were used. among them, ten primers were selected with simple, enlarged and rich bands of polymorphism (table 2). pcr reactions were performed in a 25μl volume mixture containing the following component: tris-hcl buffer (10 mm) at ph 8; kcl (50 mm); mgcl2 (1.5 mm); dntps (0.2 mm); primer (0.2 μm); genomic dna (20 ng) and of taq dna polymerase (3 u). in techne thermocycler (germany), the amplification reactions were carried out with the following pcr settings: 5 min initial denaturation at 94 °c; 40 cycles of 1 min at 94 °c; 1 min at 52-57 °c and 2 min at 72 °c. the reaction was completed by 7-10 min extension at 72 °c. the pcr  amplified  products were detected by  running  on 1% agarose gel, preceded by staining with ethidium bromide. the size of fragments was measured using a ladder with a molecular size of 100 bp (fermentas, germany). data analyses morphological studies morphological characters (mean = 0, variance = 1) were first standardized and used to determine the euclidean distance between taxa pairs (podani 2000). the ordination methods of upgma (unweighted paired group using average) were used for clustering the samples (podani 2000). in order to demonstrate morphologifigure 1. map of iran showing sampling localities for salvia. sp1= salvia aristata; sp2= s. eremophila; sp3= s. santolinifolia; sp4= s. tebesana; sp5= s. bracteata ; sp 6= s. suffruticosa; sp7= s. dracocephaloides; sp8= s. hydrangea; sp9= s. multicaulis; sp10: s. syriaca; sp11: s. viridis; sp12= s. mirzayanii; sp13= s. macrosiphon; sp14= s. sharifii; sp15= s. reuterana; sp16= s. palaestina; sp17= s. sclareopsis; sp18= s. spinose; sp19= s. compressa; sp20= s. sclarea; sp21= s. aethiopis; sp22= s. microstegia; sp23= s. xanthocheila; sp24= s. limbata; sp25= s. chloroleuca; sp26= s. virgate; sp27= s. nemorosa; sp28= s. urmiensis; sp29= s. oligphylla; sp30= s. verticillata. table 2. rapd primers used for this study and the extent of polymorphism. primer name primer sequence (5’-3’) tnb npb ppb pic pi emr mi opa-05 5’-aggggtcttg-3’ 12 12 100.00% 0.46 3.86 10.55 4.45 opa-06 5’-ggtccctgac-3’ 9 8 84.99% 0.33 4.51 8.43 2.85 opb-01 5’-gtttcgctcc-3’ 9 9 100.00% 0.44 3.34 10.55 4.44 opb-02 5’-tgatccctgg-3’ 10 10 100.00% 0.47 3.18 9.56 2.65 opc-04 5’-ccgcatctac-3’ 11 11 100.00% 0.35 5.23 8.23 5.47 opd-02 5’-ggacccaacc-3’ 14 13 93.74% 0.47 4.66 8.56 3.67 opd-03 5’-gtcgccgtca-3’ 13 12 92.31% 0.44 4.21 6.60 3.55 opd-05 5’ -tgagcggaca-3’ 13 13 100.00% 0.47 4.32 9.55 2.45 opd-08 5’-gtgtgcccca-3’ 11 9 82.89% 0.33 6.56 9.34 2.11 opd-11 5’-agcgccattg-3’ 10 10 100.00% 0.49 4.25 11.11 3.87 mean 11.2 10.7 93.68% 0.44 4.6 9.3 3.5 total 112 107 note: tnb the number of total bands, npb: the number of polymorphic bands, ppb (%): the percentage of polymorphic bands, pi: polymorphism index, emr, effective multiplex ratio; mi, marker index; pic, polymorphism information content for each of cbdp primers. 49comparative study and genetic diversity in salvia (lamiaceae) using rapd molecular markers cal variation between populations, anova (analysis of variance) was performed, while pca (principal components analysis) bi-plot was employed to identify the most variable characters (podani 2000). past software version 2.17 (hammer et al. 2012) was used for multivariate statistical analyses of morphological data. molecular analyses the obtained rapd bands were coded as binary characters (absence = 0, presence = 1) and used for the study of genetic diversity (powell et al. 1996; heikrujam et al. 2015). for each primer, the number of polymorphic bands (npb) was determined followed by the effective multiplex ratio (emr). other parameters such as nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism (p% = number of polymorphic loci/number of total loci) were also determined (weising et al, 2005; freeland et al. 2011). the formula for calculation of shannon’s index was: h’ = -σpiln pi. rp is defined per primer as: rp = ∑ ib, where “ib” is the band informativeness, that takes the values of 1-(2x [0.5-p]), being “p” the proportion of each genotype containing the band. the percentage of polymorphic loci, uhe, h’ and pca were determined by genalex 6.4 software (peakall and smouse 2006). for generating neighbor joining (nj) clusters and neighbor-net networking, nei’s genetic distance between populations was employed (freeland et al. 2011; huson and bryant 2006). the mantel test determined the correlation between the geographical and genetic distances of the populations (podani 2000). these tests were performed in past ver. 2.17 (hammer et al. 2012) and darwin software ver. 5 (2012). as implemented in genalex 6.4 (peakall & smouse, 2006), the amova (analysis of molecular variance) test (with 1000 permutations) was used to evaluate population genetic differences. gene flow was estimated by calculating nm, an estimate of gene flow from gst in popgene ver. 1.32 (1997) as: nm = 0.5 (1 gst)/gst. this method considers the equal amount of gene flow among all populations (yeh et al. 1999). results species identity and relationships – morphometry anova test showed substantial differences (p <0.01) between the studied species in quantitative morphological characteristics. pca analysis was conducted to determine the most variable characters among the taxa analysed. it showed that over 77 % of the overall variance was composed of the first three variables. characters such as seed shape, calyx shape, calyx length, bract length and basal leaf shape have shown the highest association (>0.7) in the first pca axis with 55 per cent of total variance. characters affecting pca axis 2 and 3 respectively were seed colour, leaf surface, corolla length, filament length, nut width, basal leaf length. different ordination and clustering methods generated similar results. therefore, pcoa plot of morphological characters are presented here (fig. 2). samples of each species were separately grouped. this finding indicates that the studied species were divided into different classes by both quantitative and qualitative morphological features. among the studies sample we did not find any  intermediate forms. species identification and genetic diversity ten rapd primers were screened in order to study genetic relationships within salvia. all primers generated reproducible polymorphic bands in 30 salvia species. figure 3 shows an image of the amplification of the rapd created by the opd-03 primer. in total, 107 amplified polymorphic bands were produced across 30 species. the size range of the amplified fragments was 150 to 3000 bp. the highest and lowest numbers of polymorphic bands were 13 for opd-02, opd-05 and 8 for opa-06, with an average of 10.7 polymorphic bands per primer. the pic of the 10 rapd primers ranged from 0.32 (opd-08) to 0.48 (opd-011) with an average of 0.44 per primer. mi of the primers ranged from 2.11 (opd08) to 5.47 (opc-04) with an average of 3.5 per primer. emr of the rapd primers ranged from 6.60 (opd-03) to 11.11 (opd-011) with an average of 9.3 per primer (table 2). the primers with the high emr values were considered to be more informative in distinguishing the genotypes. genetic parameters were determined for all the 30 salvia species amplified with rapd primers (table 3). the range of unbiased expected heterozygosity (h) was 0.099 (salvia limbata) to 0.31 (s. reuterana) (mean: 0.18). a similar trend was depicted by shannon’s information index (i), with the highest value of 0.39 found in s. reuterana and the lowest value of 0.13 found in s. limbata (mean: 0.27). the observed number of alleles (na) varied between 0.201 in s. nemorosa and 1.28 in s. eremophila. the range of effective number of alleles (ne) was 1.00 (s. nemorosa) to 1.670 (s. santolinifolia). amova test revealed substantial genetic variation (p = 0.01). it showed that 81% of total variation was interspecific and 19% was intra-specific (table 4). in 50 ruonan zheng et al. addition, genetic differentiation of was demonstrated by significant nei’s gst (0.29, p = 0.001) and d_est values (0.137, p = 0.01). compared to intra-species, these results revealed a greater distribution of interspecific genetic diversity. two main clusters were produced in the nj tree (fig. 4). the first main cluster comprised two sub-clusters: s. xanthocheila and s. verticillata were separated from the rest of the species and join the others with a great distance and comprised the first sub-cluster. the second sub-cluster comprised of s. limbata, s. aethiopis, s. sclarea and s. virgata. the second main cluster also comprised two sub-clusters: three species including s. sclareopsis, s. macrosiphon and s. sharifii were placed close figure 2. pcoa plots of morphological characters revealing species delimitation in the salvia. sp1= salvia aristata; sp2= s. eremophila; sp3= s. santolinifolia; sp4= s. tebesana; sp5= s. bracteata ; sp 6= s. suffruticosa; sp7= s. dracocephaloides; sp8= s. hydrangea; sp9= s. multicaulis; sp10: s. syriaca; sp11: s. viridis; sp12= s. mirzayanii; sp13= s. macrosiphon; sp14= s. sharifii; sp15= s. reuterana; sp16= s. palaestina; sp17= s. sclareopsis; sp18= s. spinose; sp19= s. compressa; sp20= s. sclarea; sp21= s. aethiopis; sp22= s. microstegia; sp23= s. xanthocheila; sp24= s. limbata; sp25= s. chloroleuca; sp26= s. virgate; sp27= s. nemorosa; sp28= s. urmiensis; sp29= s. oligphylla; sp30= s. verticillata. figure 3. electrophoresis gel of studied ecotypes from dna fragments produced by opd-03. 1= salvia aristata; 2= s. eremophila; 3= s. santolinifolia; 4= s. tebesana; 5= s. bracteata ; 6= s. suffruticosa; 7= s. dracocephaloides; 8= s. hydrangea; 9= s. multicaulis; 10: s. syriaca; 11: s. viridis; 12= s. mirzayanii; 13= s. macrosiphon; 14= s. sharifii; 15= s. reuterana; 16= s. palaestina; 17= s. sclareopsis; 18= s. spinose; 19= s. compressa; 20= s. sclarea; 21= s. aethiopis; 22= s. microstegia; 23= s. xanthocheila; 24= s. limbata; 25= s. chloroleuca; 26= s. virgate; 27= s. nemorosa; 28= s. urmiensis; 29= s. oligphylla; 30= s. verticillata;l = ladder 100 bp, arrows are representative of polymorphic bands. 51comparative study and genetic diversity in salvia (lamiaceae) using rapd molecular markers to each other, while close genetic affinity between other species. relationships obtained from rapd data usually agree well with the relationship of species obtained from morphological data. this is in accordance with the parameters of amova and genetic diversity previously reported. salvia species are genetically well distinguished from each other. the species are well distinguished from each other genetically. these findings show that rapd molecular markers can be used in the taxonomy of salvia. the nm analysis by popgene software also produced mean nm= 0.288, that is deemed a low value of gene flow. a strong association (r = 0.16, p=0.0002) between geneticand geographical distance was demonstrated by mantel test with 5000 permutations. it indicates that  isolation by distance  (ibd) has occurred among these species. the results of nei’s genetic identity and the genetic distance (table 5) show the highest genetic similarity table 3. genetic diversity parameters in the studied salvia species. sp n na ne i he uhe %p s. aristata 8.000 0.333 1.016 0.19 0.12 0.22 48.23% s. eremophila 12.000 1.287 1.233 0.271 0.184 0.192 51.91% s. santolinifolia 5.000 0.358 1.670 0.18 0.20 0.29 43.50% s. tebesana 6.000 0.299 1.029 0.231 0.18 0.23 44.38% s. bracteata 5.000 0.462 1.095 0.288 0.25 0.22 62.05% s. suffruticosa 8.000 0.399 1.167 0.259 0.234 0.133 32.88% s. dracocephaloides 8.000 0.477 1.187 0.256 0.233 0.148 31.26% s. hydrangea 8.000 0.313 1.026 0.144 0.13 0.26 49.23% s. multicaulis 12.000 1.144 1.322 0.28 0.284 0.192 50.91% s. syriaca 5.000 0.358 1.117 0.28 0.15 0.12 44.30% s. viridis 6.000 0.458 1.039 0.28 0.18 0.23 49.38% s. mirzayanii 5.000 0.455 1.077 0.277 0.24 0.22 55.05% s. macrosiphon 8.000 0.499 1.067 0.14 0.13 0.14 49.26% s. sharifii 9.000 0.261 1.014 0.142 0.23 0.23 43.15% s. reuterana 6.000 0.555 1.021 0.39 0.35 0.31 68.53% s. palaestina 4.000 0.344 1.042 0.20 0.23 0.20 57.53% s. sclareopsis 5.000 0.369 1.011 0.15 0.18 0.12 42.15% s. spinose 9.000 0.261 1.014 0.142 0.33 0.23 43.15% s. compressa 6.000 0.555 1.021 0.29 0.25 0.28 43.53% s. sclarea 10.000 0.431 1.088 0.33 0.22 0.13 57.53% s. aethiopis 3.000 0.255 1.021 0.15 0.18 0.12 42.15% s. microstegia 3.000 0.288 1.024 0.23 0.15 0.17 44.30% s. xanthocheila 9.000 0.352 1.083 0.23 0.22 0.14 45.05% s. limbata 5.000 0.369 1.011 0.13 0.11 0.099 29.15% s. chloroleuca 6.000 0.244 1.032 0.26 0.23 0.18 55.53% s. virgata 4.000 0.314 1.044 0.16 0.18 0.23 43.38% s. nemorosa 8.000 0.201 1.00 0.33 0.17 0.12 42.23% s. urmiensis 5.000 0.341 1.058 0.24 0.27 0.20 53.75% s. oligphylla 3.000 0.567 1.062 0.24 0.224 0.113 44.73% s. verticillata 5.000 0.336 1.034 0.23 0.25 0.19 51.83% abbreviations: n: number of samples; na: number of different alleles; ne: number of effective alleles, i: shannon’s information index, he: gene diversity, uhe: unbiased gene diversity, p%: percentage of polymorphism, populations. table 4. analysis of molecular variance (amova) of the studied species. source df ss ms est. var. % φpt among pops 28 1601.364 79.789 12.154 81% 81% within pops 130 234.443 4.777 2.888 19% total 158 1955.777 14.060 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance; φpt: proportion of the total genetic variance among individuals within an accession, (p < 0.001). 52 ruonan zheng et al. ta bl e 5. th e m at ri x of n ei g en et ic s im ila ri ty ( g s) e st im at es u si ng r a pd m ol ec ul ar m ar ke rs a m on g 30 s al vi a sp ec ie s. s p1 = sa lv ia a ri st at a; s p2 = s. e re m op hi la ; s p3 = s. s an to lin ifo lia ; sp 4= s . t eb es an a; s p5 = s. b ra ct ea ta ; sp 6 = s. s uff ru tic os a; s p7 = s. d ra co ce ph al oi de s; sp 8= s . h yd ra ng ea ; s p9 = s. m ul tic au lis ; s p1 0: s . s yr ia ca ; s p1 1: s . v ir id is ; s p1 2= s . m ir za ya ni i; sp 13 = s. m ac ro si ph on ; s p1 4= s . s ha ri fii ; s p1 5= s . r eu te ra na ; s p1 6= s . p al ae st in a; s p1 7= s . s cl ar eo ps is ; s p1 8= s . s pi no se ; s p1 9= s . c om pr es sa ; s p2 0= s . s cl ar ea ; s p2 1= s . a et hi op is ; s p2 2= s . m ic ro st egi a; s p2 3= s . x an th oc he ila ; s p2 4= s . l im ba ta ; s p2 5= s . c hl or ol eu ca ; s p2 6= s . v ir ga te ; s p2 7= s . n em or os a; s p2 8= s . u rm ie ns is ; s p2 9= s . o lig ph yl la ; s p3 0= s . v er tic ill at a. sp 1 sp 2 sp 3 sp 4 sp 5 sp 6 sp 7 sp 8 sp 9 sp 10 sp 11 sp 12 sp 13 sp 14 sp 15 sp 16 sp 17 sp 18 sp 19 sp 20 sp 21 sp 22 sp 23 sp 24 sp 25 sp 26 sp 27 sp 28 sp 29 sp 30 sp 1 1. 00 0 sp 2 0. 70 8 1. 00 0 sp 3 0. 66 7 0. 71 2 1. 00 0 sp 4 0. 66 6 0. 73 7 0. 84 2 1. 00 0 sp 5 0. 64 9 0. 80 7 0. 78 6 0. 75 4 1. 00 0 sp 6 0. 61 7 0. 78 2 0. 84 6 0. 92 8 0. 79 3 1. 00 0 sp 7 0. 59 9 0. 70 2 0. 80 8 0. 87 5 0. 83 6 0. 86 2 1. 00 0 sp 8 0. 73 5 0. 70 6 0. 61 8 0. 70 8 0. 82 3 0. 93 6 0. 76 4 1. 00 0 sp 9 0. 75 0 0. 79 7 0. 81 6 0. 88 4 0. 78 5 0. 67 6 0. 69 9 0. 75 6 1. 00 0 sp 10 0. 77 9 0. 79 8 0. 75 2 0. 75 4 0. 74 1 0. 75 8 0. 74 6 0. 75 3 0. 79 5 1. 00 0 sp 11 0. 71 9 0. 92 0 0. 74 1 0. 75 8 0. 74 6 0. 75 3 0. 63 5 0. 81 6 0. 88 4 0. 72 1 1. 00 0 sp 12 0. 81 2 0. 77 4 0. 87 6 0. 72 2 0. 63 5 0. 81 6 0. 63 2 0. 75 2 0. 75 4 0. 63 5 0. 83 9 1. 00 0 sp 13 0. 83 4 0. 75 0 0. 79 9 0. 75 5 0. 63 2 0. 75 2 0. 66 7 0. 71 2 0. 77 9 0. 75 0 0. 79 9 0. 64 2 1. 00 0 sp 14 0. 77 8 0. 69 1 0. 74 4 0. 63 6 0. 66 7 0. 71 2 0. 66 6 0. 73 7 0. 67 5 0. 67 5 0. 72 7 0. 72 8 0. 68 4 1. 00 0 sp 15 0. 71 0 0. 68 8 0. 75 7 0. 70 3 0. 66 6 0. 73 7 0. 64 9 0. 80 7 0. 69 1 0. 68 1 0. 74 6 0. 79 6 0. 67 6 0. 72 2 1. 00 0 sp 16 0. 82 9 0. 73 3 0. 80 0 0. 68 1 0. 64 9 0. 80 7 0. 61 7 0. 78 2 0. 73 4 0. 73 3 0. 80 0 0. 70 9 0. 77 0 0. 75 4 0. 77 0 1. 00 0 sp 17 0. 81 6 0. 74 0 0. 78 5 0. 62 4 0. 61 7 0. 78 2 0. 59 9 0. 70 2 0. 74 4 0. 74 0 0. 78 5 0. 67 6 0. 69 9 0. 75 6 0. 73 5 0. 77 8 1. 00 0 sp 18 0. 73 0 0. 61 4 0. 84 3 0. 75 9 0. 59 9 0. 70 2 0. 73 5 0. 70 6 0. 71 9 0. 95 3 0. 74 1 0. 75 8 0. 74 6 0. 75 3 0. 79 5 0. 79 9 0. 75 6 1. 00 0 sp 19 0. 70 1 0. 80 0 0. 75 1 0. 77 4 0. 73 2 0. 79 0 0. 75 0 0. 79 7 0. 81 2 0. 77 4 0. 99 0 0. 72 2 0. 63 5 0. 81 6 0. 88 4 0. 81 2 0. 75 0 0. 79 9 1. 00 0 sp 20 0. 76 4 0. 72 3 0. 68 3 0. 65 9 0. 67 9 0. 75 4 0. 77 9 0. 79 8 0. 83 4 0. 75 0 0. 79 9 0. 75 5 0. 63 2 0. 75 2 0. 75 4 0. 70 3 0. 67 5 0. 72 7 0. 75 5 1. 00 0 sp 21 0. 78 5 0. 62 4 0. 61 7 0. 78 2 0. 73 4 0. 79 9 0. 84 3 0. 74 1 0. 69 0 0. 69 1 0. 74 4 0. 63 6 0. 66 7 0. 71 2 0. 77 9 0. 79 8 0. 68 1 0. 74 6 0. 68 4 0. 71 1 1. 00 0 sp 22 0. 84 3 0. 75 9 0. 59 9 0. 70 2 0. 74 4 0. 77 8 0. 77 4 0. 84 3 0. 77 8 0. 68 8 0. 75 7 0. 70 3 0. 66 6 0. 73 7 0. 67 5 0. 80 8 0. 73 3 0. 80 0 0. 84 8 0. 77 4 0. 71 2 1. 00 0 sp 23 0. 82 5 0. 72 2 0. 64 1 0. 81 4 0. 73 5 0. 70 6 0. 75 0 0. 79 9 0. 71 0 0. 73 3 0. 80 0 0. 68 1 0. 64 9 0. 80 7 0. 69 1 0. 66 5 0. 74 0 0. 78 5 0. 84 6 0. 75 7 0. 70 7 0. 87 4 1. 00 0 sp 24 0. 86 0 0. 75 9 0. 73 2 0. 79 0 0. 75 0 0. 73 5 0. 70 6 0. 71 9 0. 75 4 0. 74 1 0. 75 8 0. 74 6 0. 75 3 0. 79 5 0. 79 9 0. 79 9 0. 95 3 0. 74 1 0. 69 0 0. 65 7 0. 64 5 0. 72 6 0. 73 5 1. 00 0 sp 25 0. 72 6 0. 64 7 0. 67 9 0. 75 4 0. 77 9 0. 75 0 0. 79 7 0. 81 2 0. 77 4 0. 99 0 0. 72 2 0. 63 5 0. 81 6 0. 88 4 0. 81 2 0. 77 8 0. 77 4 0. 77 70 0. 77 8 0. 69 1 0. 74 4 0. 63 6 0. 66 7 0. 75 7 1. 00 0 sp 26 0. 85 8 0. 70 3 0. 69 5 0. 68 1 0. 68 9 0. 77 9 0. 79 8 0. 83 4 0. 75 0 0. 79 9 0. 75 5 0. 63 2 0. 75 2 0. 75 4 0. 70 3 0. 70 6 0. 75 0 0. 79 9 0. 71 0 0. 68 8 0. 75 7 0. 70 3 0. 66 6 0. 69 0 0. 79 7 1. 00 0 sp 27 0. 83 6 0. 68 1 0. 68 6 0. 75 6 0. 70 1 0. 95 3 0. 74 1 0. 69 0 0. 69 1 0. 74 4 0. 63 6 0. 66 7 0. 71 2 0. 77 9 0. 79 8 0. 79 7 0. 69 1 0. 74 4 0. 82 9 0. 73 3 0. 80 0 0. 68 1 0. 64 9 0. 67 3 0. 75 5 0. 76 8 1. 00 0 sp 28 0. 69 1 0. 74 4 0. 82 9 0. 74 0 0. 78 5 0. 62 4 0. 99 0 0. 77 8 0. 68 8 0. 75 7 0. 70 3 0. 66 6 0. 95 3 0. 74 1 0. 75 8 0. 74 6 0. 75 3 0. 79 5 0. 79 9 0. 79 9 0. 95 3 0. 74 1 0. 69 0 0. 65 7 0. 64 5 0. 77 9 0. 79 8 1. 00 0 sp 29 0. 53 8 0. 82 6 0. 78 6 0. 77 2 0. 68 6 0. 75 0 0. 79 9 0. 71 0 0. 73 3 0. 80 0 0. 68 1 0. 64 9 0. 80 7 0. 69 1 0. 66 5 0. 82 5 0. 73 3 0. 80 0 0. 73 0 0. 61 4 0. 84 3 0. 75 0 0. 79 9 0. 71 0 0. 68 8 0. 75 7 0. 70 3 0. 72 3 1. 00 0 sp 30 0. 72 1 0. 79 4 0. 75 4 0. 71 7 0. 79 5 0. 69 1 0. 74 4 0. 82 9 0. 74 0 0. 78 5 0. 62 4 0. 61 7 0. 78 2 0. 73 4 0. 79 9 0. 67 6 0. 74 0 0. 78 5 0. 77 0 0. 64 1 0. 82 5 0. 72 2 0. 81 6 0. 74 0 0. 78 5 0. 62 4 0. 61 7 0. 65 6 0. 76 5 1. 00 0 53comparative study and genetic diversity in salvia (lamiaceae) using rapd molecular markers (0.93) between s. suffruticosa and s. hydrangea. lowest of genetic similarity was shown between s. aristata and s. oligphylla (0.53). lower nm value (0.288) is an indicator of limited gene flow or ancestrally shared alleles between different species and indicating high genetic differentiation among and within salvia species. discussion genetic diversity is a fundamental element of biodiversity and its conservation is indispensable for longterm survival of any species in unstable environments (mills and schwartz 2005; tomasello et al. 2015). genetic diversity is non-randomly distributed among different populations and is influenced by numerous factors such as geography, dispersal mechanisms, breeding systems, life span etc. changes in environment often lead to variation in genetic diversity levels among populations, and under adverse circumstances, populations with little variability are generally deemed less adapted (falk and holsinger 1991; olivieri et al. 2016). most authors recognize that genetic diversity is fundamental to preserving the long-term evolutionary potential of a species (falk and holsinger 1991). experimental and field research has shown that habitat fragmentation and population decline have reduced the effective population size in the last decade. similarly, majority of geneticists regard population size as a significant factor in preserving genetic variation (turchetto et al. 2016). in fragmented populations, this is very important because it is more vulnerable because of allelic richness  loss and increased population differentiation via genetic drift and inbreeding depression (frankham 2005). information of interand intra-population genetic diversity is therefore important for their conservation and management (e.g. esfandanibozchaloyi et al. 2018a, 2018b, 2018c, 2018d). we used morphological and molecular r apd molecular data to test species relationships in salvia in the current analysis. morphological studies showed that both quantitative (the anova test result) and qualitative characters are well distinguished from each other (the pca plot result). furthermore, pca analysis suggests that morphological characters such as bract length, stipule length, bract shape, calyx shape, petal shape, stem-leaf length and width, petal length and width may be used in the delimitation of species groups. this morphological differentiation is attributed to quantitative and qualitative characters. genetic structure and gene flow a primer’s pic and mi characteristics assist in assessing its usefulness in the study of genetic diversity. sivaprakash et al. (2004) asserted that the ability to overcome genetic diversity by a marker technique could be figure 4. nj tree of rapd data revealing species delimitation in the salvia. 54 ruonan zheng et al. more explicitly linked to the degree of polymorphism. in general, the pic value 0 to 0.25 suggests a very low genetic diversity among genotypes, a mid-level of genetic diversity (tams et al. 2005). in this study, the rapd primers’ pic values ranged from 0.33 to 0.49, with a mean value of 0.44, indicating a moderate level ability of rapd primers in determining genetic diversity. somewhat similar but low pic values have been reported for issr and rapd in salvia species (yousefiazar-khanian et al. 2016); rapd and aflp in african plantain (ude et al. 2003), aflp in wheat (bohn et al. 1999) and scot markers (etminan et al. 2018; pour-aboughadareh et al. 2017, 2018). in heikrujam et al. (2015), cbdp markers were shown to be more efficient than scot regarding the average pic which was higher. in our analysis, the rapd markers reflect success in estimating genetic diversity of salvia species regarding average percentage polymorphism (93.68%), average pic value of rapd markers (0.44), average mi (3.5) and average emr of rapd markers (9.3), which were higher than other reported markers on salvia (wang et al. 2009; song et al. 2010; yousefiazar-khanian et al. 2016; etminan et al. 2018; souframanien and gopalakrishna 2004). gene flow is inversely correlated with the gene differentiation but is very significant for population evolution, and occurs through pollen grains and seeds between populations (song et al. 2010). the observed gene flow (nm) between salvia species was 0.288 in the current study, indicating low genetic differentiation. generally, the pollinators of old world salvia are insects (claßen-bockhoff et al. 2004). at the lower elevations, bees and at the higher altitudes insects such as flies are the major pollinators of bi-labiate flowers such as salvia (pellissier et al. 2010). according to moein et al., (2019) srap marker’s genetic structure revealed that despite the existence of limited gene flow, two separate ecotypes were produced which may be the result of reproductive isolation triggered by altitudinal gradient and dissimilar niches through parapatric speciation (que et al. 2014). in conclusion, the findings of this study showed that the primers derived from rapd were more efficient than the other molecular markers in assessing the genetic diversity of salvia in iran. in addition, the salvia species in the dendrogram and pcoa were clearly distinguished from each other, suggesting the greater efficiency of the rapd technique in the identification of the genus. acknowledgment the authors thank anonymous reviewers for valuable comments on an earlier draft. references bohn m, utz hf, melchinger ae.1999. genetic similarities among wheat cultivars determined on the basis of rflps, aflps and ssrs and their use for predicting progeny variance. crop sci. 39: 228–237. cires e, cuesta c, fernández prieto ja. 2012. conservation genetics of the endangered endemic ranunculus cabrerensis subsp. muniellensis (ranunculaceae) in the northwest of spain. bol. ci. nat. ridea 52: 117134. cires e, cuesta c, fernández prieto ja. 2013. genetic diversity and structure in fragmented populations of the endangered species ranunculus cabrerensis (ranunculaceae): implications for conservation. biologia 68(1): 30-40. chen sy, dai tx, chang yt, wang ss, ou sl, chuang wl, cheng cy, lin yh, lin ly, ku hm 2013. genetic diversity among ocimum species based on issr, rapd and srap markers. australian journal of crop science 7(10): 1463-1471. claßen-bockhoff r, speck t, tweraser e, wester p, thimm s, reith m. 2004. the staminal lever mechanism in salvia l. 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(2019). gene2vec: gene subsequence embedding for prediction of mammalian n 6 -methyladenosine sites from mrna. rna (cambridge), 25(2), 205-218. doi: 10.1261/ rna.069112.118 caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 1 2021 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 74(4): 39-50, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1114 caryologia international journal of cytology, cytosystematics and cytogenetics citation: neda atazadeh, masoud sheidai, farideh attar, fahimeh koohdar (2021) molecular phylogeny and morphometric analyses in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f. caryologia 74(4): 39-50. doi: 10.36253/caryologia-1114 received: october 10, 2020 accepted: november 27, 2021 published: march 08, 2022 copyright: © 2021 neda atazadeh, masoud sheidai, farideh attar, fahimeh koohdar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. molecular phylogeny and morphometric analyses in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f. neda atazadeh1,*, masoud sheidai1, farideh attar2, fahimeh koohdar1 1 department of plant sciences and biotechnology, faculty of life sciences and biotechnology, shahid beheshti university, tehran, iran 2 central herbarium of tehran university, school of biology, college of science, university of tehran, p. o. box: 14155, tehran, iran *corresponding author. e-mail: atazadeh_neda@yahoo.com abstract. taxonomy and molecular phylogeny of the genus cousinia are complicated and unresolved mainly because of disagreement between morphological and molecular phylogenetic studies. the genus cousinia has approximately 700 species, which makes it one of the most varied genera found in central and southwest asia. section cynaroideae, containing 89 species, is considered the largest section of the genus. identification and delineation as well as classifying the section and the species’ relationships within the genus cousinia generally remain debatable. therefore, the present study aims to: 1) identify and delineate the species within the two sections cynaroideae and platyacanthae; 2) study the species relationships based on both morphological and molecular features (internal transcribed spacer (its) marker); 3) study the sectional delimitation and its monophyly; and 4) estimate the divergence time of the studied sections. to this end, 50 cousinia species occurring in iran were investigated for the first time. a maximum parsimony tree of the morphological features separated the species of the two sections from each other. however, the its-based phylogenetic tree did not delimit the two studied sections. the relationships among the studied cousinia species in the genetic trees were generally not congruent with the obtained morphological tree. the divergence time of the studied species within the cynaroideae and platyacanthae sections determined using bayesian evolutionary analysis sampling trees (beast) was estimated to be around 3.5 million years ago (mya). keywords: cousinia, cynaroideae, its, phylogeny, platyacanthae. introduction the genus cousinia cass. of the tribe cardueae (family asteraceae) has approximately 700 species, which makes it one of the most diverse genera following senecio l. (c. 1500 species) and vernonia schreb. (c. 1000 species) in central and southwest (sw) asia (tscherneva 1962; rechinger 1972, 1979; frodin 2004; attar and ghahreman 2006; susanna and garcia-jacas 2007; attar 40 neda atazadeh et al. and djavadi 2010; mehregan and assadi 2016; minaeifar et al. 2016; rastegar et al. 2017, 2018). this genus has the greatest prevalence in the flora iranica area, with more than 400 different species distributed in sw asia, of which, 379 are considered endemic. these species are distributed in mountainous areas of iran, afghanistan, and turkmenistan (rechinger 1986; knapp 1987). the genus cousinia is not considered to be monophyletic, and comprises the arctium-cousinia complex as well as the genus arctium l. (susanna et al. 2003; lopez-vinyallonga et al. 2009). although the exact number of species in this genus in iran is in dispute (attar 2000; mehregan 2008; mehregan and kadereit 2008; assadi 2009; attar and mirtadzadini 2009; mehregan and assadi 2009; attar and djavadi 2010), approximately 270 cousinia species have been reported of which, almost 200 species are considered endemic (djavadi et al. 2007; zare et al. 2013). cousinia species could be taxonomically categorized into 70 sections (rechinger 1986), with section cynaroideae being the largest section of the genus containing 89 species (tscherneva 1962; rechinger 1972, 1979; hubermorath 1975; attar and djavadi 2010; rastegar et al. 2017, 2018). this section includes species consisting of decurrent leaves and appendiculate bracts (tscherneva 1962; rechinger 1972, 1979; hubermorath 1975), which are recognized as the irano-turkestanian elements (djamali et al. 2012; dehghani et al. 2017). iran has 77 taxa, 66 of which are endemic, thus appearing to be the variety center of the section (attar and ghahreman 2006; attar and djavadi 2010). the extensive morphological variability in the genus renders the taxonomy of cousinia complicated and controversial (de candolle 1837; boissier 1875; winkler 1892, 1897; tscherneva 1962; rechinger 1972, 1979; huber-morath 1975; haffner 2000; susanna et al. 2003; mehregan 2011; mehregan and assadi 2016; mabberley 2018; atazadeh et al. 2020). there is a controversy over the number of species within a single section, too; for instance, mehregan and kadereit (2008), in a taxonomic revision of the section cynaroideae, reduced the number of species occurring in iran to 31, while attar and djavadi (2010) reported 77 cousinia species in this section as present within the country. the sect. platyacanthae rech. f. has six species in flora iranica and is considered the sister group of the sect. cynaroideae (lopez-vinyallonga et al. 2009), five of the six species of which are endemic in iran (rechinger 1972). species identification and delineation as well as classification of the sections and the species relationships within the genus cousinia generally remain debatable, even after molecular investigations (susanna et al. 2003; ghaffari et al. 2006; lopez-vinyallonga et al. 2009; mehregan and assadi 2016; galtier 2019). therefore, the present study aims to: 1) identify and delineate the species based on differentiating taxonomical features within the two sections cynaroideae and platyacanthae; 2) study the species relationships based on morphological and molecular features (internal transcribed spacer (its) marker); 3) study sectional delimitation and its monophyly, and 4) estimate the divergence time of the studied species within the cynaroideae and platyacanthae sections for the first time. molecular information has been commonly utilized to create a system for phylogenetic classification. specifically, the its regions are considered as nuclear dna regions described by parental inheritance patterns and can be changed faster compared with the coding regions, which results in higher levels of disparity among those narrowly-related individuals. therefore, the its regions were used herein to study the interspecific and intergeneric relationships along with developmental styles and patterns in genetic variation (baldwin 1992; alvarez and wandel 2003; felniner and rossello 2007). molecular studies in the two sections cynaroideae and platyacanthae have not been fully performed until now. therefore, present study has attempted to investigate 50 species of both studied sections based on molecular features (its) for the first time and specifically collect new information on molecular phylogeny, evolution, divergence time, and species relationships. these findings can enhance our knowledge of the true evolutionary pathway of the genus cousinia. materials and methods plant material morphological studies were conducted on 150 plant specimens, of which 138 belonged to 46 species of cynaroideae and 12 belonged to 4 species of platyacanthae sections (table 1). one specimen of each species was used for the its marker. the voucher specimens were deposited in the herbarium of tehran university (tuh). arctium umbrosum bung (accession number: ay373745; ay373712) and arctium lappa l. (accession number: eu923773; eu923887) were obtained as out groups based on studies by susanna and garcia-jacas (2007) and lopez-vinyallonga et al. (2009). its sequences for all of the species except for the out groups were newly generated. 41molecular phylogeny and morphometric analyses in the genus cousinia ta bl e 1. in ve st ig at ed c ou si ni a sp ec ie s an d th ei r vo uc he r in fo rm at io n as w el l a s th e ac ce ss io n nu m be rs o f t ax a in p hy lo ge ny s tu di es . r . ta xa se ct io n lo ca lit y v ou ch er n um be r a cc es si on n um be r a bb re vi at io n 1 c ou si ni a ca ro lih en ri ci a tt ar & g ha hr em an c yn ar oi de ae b un ge k ur de st an 22 45 5 (t u h ) m h 99 27 48 ca ro lih en ri ci 2 c ou si ni a fu rs ei r ec h. f. c yn ar oi de ae b un ge k ur de st an -m ar iv an 18 31 4( t u h ) m h 99 27 34 fu rs ei 3 c ou si ni a m ill ef on ta na r ec h. f. c yn ar oi de ae b un ge k ur de st an -m ar iv an 20 22 7( t u h ) m h 97 12 23 m ill ef on ta na 4 c ou si ni a co nc in na b oi ss . & h au ss kn . c yn ar oi de ae b un ge k ur de st an 20 56 2( t u h ) m h 99 27 35 co nc in na 5 c ou si ni a su bi nfl at a b or nm . c yn ar oi de ae b un ge k er m an sh ah (t u h ) m k 00 51 81 su bi nfl at a 6 c ou si ni a ha m ad an en si s r ec h. f. c yn ar oi de ae b un ge h am ad an m al ay er 46 29 0( t u h ) m k 00 51 82 ha m ad an en si s 7 c ou si ni a ba rb ey i c . w in kl . c yn ar oi de ae b un ge b oy er -a hm ad 22 49 4( t u h ) m k 00 51 64 ba rb ey i 8 c ou si ni a de na en si s a tt ar & d ja va di c yn ar oi de ae b un ge b oy er -a hm ad 22 49 5( t u h ) m h 99 27 39 de na en si s 9 c ou si ni a sa rd as ht en si s r ec h. f. c yn ar oi de ae b un ge c ha ha r m ah al & b ak ht ia ri 20 07 3( t u h ) m k 00 51 84 sa rd as ht en si s 10 c ou si ni a da la hu en si s a tt ar & g ha hr em an c yn ar oi de ae b un ge k er m an sh ah m ah id as ht 19 92 9( t u h ) m h 99 27 47 da la hu en si s 11 c ou si ni a gr an di s c . a . m ey . c yn ar oi de ae b un ge a za rb ai ja n 21 34 3( t u h ) m h 99 27 38 gr an di s 12 c ou si ni a gr an tii r ec h. f. c yn ar oi de ae b un ge a za rb ai ja n 22 49 0( t u h ) m k 00 51 83 gr an tii 13 c ou si ni a ga ha re ns is a tt ar & d ja va di c yn ar oi de ae b un ge lo re st an sh ul ab ad 38 25 9( t u h ) m k 00 51 66 ga ha re ns is 14 c ou si ni a ke re dj en si s b or nm . & g au ba c yn ar oi de ae b un ge te hr an 21 80 7( t u h ) m h 99 27 32 ke re dj en si s 15 c ou si ni a za rd ku he ns is a tt ar & g ha hr em an c yn ar oi de ae b un ge c ha ha r m ah al & b ak ht ia ri 21 88 7( t u h ) m h 99 07 88 za rd ku he ns is 16 c ou si ni a lo rd eg an en si s m eh re ga n c yn ar oi de ae b un ge c ha ha r m ah al & b ak ht ia ri 46 30 1( t u h ) m k 00 51 73 lo rd eg an en si s 17 c ou si ni a el w en de ns is b or nm . c yn ar oi de ae b un ge h am ad an -a lv an d m ou nt ai ns 20 56 6( t u h ) m h 99 27 41 el w en de ns is 18 c ou si ni a kh or ra m ab ad en si s b or nm . c yn ar oi de ae b un ge lo re st an 21 85 1( t u h ) m h 99 27 37 kh or ra m ab ad en si s 19 c ou si ni a ph yl lo ce ph al a b or nm . & g au ba c yn ar oi de ae b un ge lo re st an k ho rr am a ba d 46 29 2( t u h ) m k 00 51 68 ph yl lo ce ph al a 20 c ou si ni a m ac ro ce ph al a c . a . m ey . c yn ar oi de ae b un ge a rd eb il m es hk in s ha hr 42 92 5( t u h ) m h 99 03 19 m ac ro ce ph al a 21 c ou si ni a lu re st an ic a a tt ar & d ja va di c yn ar oi de ae b un ge lo re st an 21 82 4( t u h ) m h 99 27 46 lu re st an ic a 22 c ou si ni a ir an ic a c . w in kl . & s tr au ss . c yn ar oi de ae b un ge a ra k 21 88 1( t u h ) m k 00 51 74 ir an ic a 23 c ou si ni a pa rs an a g ha hr em an c yn ar oi de ae b un ge h am ad an 20 55 3( t u h ) m k 00 51 69 pa rs an a 24 c ou si ni a ko rn hu be ri h ei m er l c yn ar oi de ae b un ge h am ad an 22 37 2( t u h ) m k 00 51 85 ko rn hu be ri 25 c ou si ni a ec ba ta ne ns is b or nm . c yn ar oi de ae b un ge h am ad an 22 37 1( t u h ) m h 98 87 70 ec ba ta ne ns is 26 c ou si ni a ve rb as ci fo lia b un ge c yn ar oi de ae b un ge k ho ra sa nm as hh ad 43 01 3( t u h ) m k 00 51 79 ve rb as ci fo lia 27 c ou si ni a di sf ul en si s b or nm . c yn ar oi de ae b un ge lo re st an k ho rr am a ba d 27 58 9( t u h ) m h 99 27 42 di sf ul en si s 28 c ou si ni a sh ul ab ad en si s a tt ar & g ha hr em an c yn ar oi de ae b un ge lo re st an sh ul a ba d 21 87 4( t u h ) m h 99 27 44 sh ul ab ad en si s 29 c ou si ni a sa ha nd ic a a tt ar & d ja va di c yn ar oi de ae b un ge a za rb ai ja n 46 27 2( t u h ) m k 00 51 75 sa ha nd ic a 30 c ou si ni a gi lli at ii r ec h. f. c yn ar oi de ae b un ge a za rb ai ja n 21 96 7( t u h ) m k 00 51 70 gi lli at ii 31 c ou si ni a al gu rd in a r ec h. f. c yn ar oi de ae b un ge a za rb ai ja n t ab ri z 30 53 3( t u h ) m k 00 51 65 al gu rd in a 32 c ou si ni a cy na ro id es c . a . m ey c yn ar oi de ae b un ge a rd eb il 22 58 1( t u h ) m k 00 51 67 cy na ro id es 33 c ou si ni a ko ts ch yi b oi ss . c yn ar oi de ae b un ge a za rb ai ja n 46 24 4( t u h ) m k 00 51 71 ko ts ch yi 34 c ou si ni a na na a tt ar c yn ar oi de ae b un ge a ra k 14 34 7( t u h ) m k 00 51 72 na na 35 c ou si ni a sh eb lie ns is g ha hr em an c yn ar oi de ae b un ge a za rb ai ja n t ab ri z 20 58 0( t u h ) m k 00 51 77 sh eb lie ns is 36 c ou si ni a ca lo ce ph al a ja ub . & s pa ch c yn ar oi de ae b un ge a za rb ai ja nm ia ne h 46 27 6( t u h ) m h 99 27 49 ca lo ce ph al a 42 neda atazadeh et al. dna extraction, amplification and sequencing garden-fresh leaves were dried in powder of silica gel. cetyltrimethyl-ammonium bromide (ctab) with activated charcoal protocol was used to extract genomic dna (murray and thompson 1980). the quality of the extracted dna was examined by running on 0.8% agarose (sheidai et al. 2013). its region (its1, 5.8s, its2) was amplified using 0.2 μm primer its1 (5 -́ tccgtaggtgaacctgcgg-3 ;́ bioron, germany), and primer its4 (5´tcc gct tattga tat gc -3´) (chen et al. 2010). pcr reactions were accomplished in a 25 μlvolume containing 10 mm tris-hcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 20 ng genomic dna; and 3 u of taq dna polymerase (bioron, germany). the mentioned reactions were amplified in a techne thermocycler (germany) by applying the process as follows: 5 min at 94 °c, followed by 35 cycles for 30 s at 94 °c, 1 min at 52 °c, and 1 min at 72 °c, followed by one ultimate extension for 10 min at 72 °c. the process was run on 2% agarose gel to picture the amplification result, followed by the staining with ethidium bromide. a 100-base pair (bp) molecular-sized ladder (fermentas, germany) was used to determine fragment size. data analysis morphological analysis in all, 19 morphological characteristics were investigated (quantitative and qualitative) and coded (table 2). after, the obtained data was standardized (mean = 0, variance = 1) and applied to perform multivariate analyses. the species categorization was performed using the ward (minimum spherical cluster method), upgma (unweighted paired group using average), pcoa (principal coordinate analysis), and mds (multidimensional scaling) methods (podani 2000). paleontological statistics software (past), ver. 2.17 (hammer et al. 2012) was used for analysis. moreover, morphological characteristics were coded for the maximum parsimony (mp) tree, and then created after tree bisection reconnection (tbr) branch swapping by applying them along with bootstrapping 1000 times in paup (phylogenetic analysis using parsimony) software, ver. 4 (swofford 2002). molecular analysis to assess homology, its sequences were aligned with muscle implemented in mega 7 (tamura et al. r . ta xa se ct io n lo ca lit y v ou ch er n um be r a cc es si on n um be r a bb re vi at io n 37 c ou si ni a be hb ou di an a r ec h. f. & e sf an d. c yn ar oi de ae b un ge g ha zv in 27 62 9( t u h ) m h 99 27 36 be hb ou di an a 38 c ou si ni a ki rr in di ca b or nm . & r ec h. f. c yn ar oi de ae b un ge il am 19 71 1( t u h ) m k 00 51 63 ki rr in di ca 39 c ou si ni a m ob ay en ii g ha hr em an & a tt ar c yn ar oi de ae b un ge k er m an sh ah es la m ab ad 20 56 9( t u h ) m k 00 51 80 m ob ay en ii 40 c ou si ni a sa na nd aj en si s r ec h. f. c yn ar oi de ae b un ge h am ad an 46 28 7( t u h ) m h 99 27 43 sa na nd aj en si s 41 c ou si ni a lu ro ru m b or nm . c yn ar oi de ae b un ge k er m an sh ah m ah id as ht 20 56 8( t u h ) m k 00 51 76 lu ro ru m 42 c ou si ni a ku rd is ta ni ca a tt ar c yn ar oi de ae b un ge k ur de st an m ar yv an 32 32 (t u h ) m k 00 51 78 ku rd is ta ni ca 43 c ou si ni a bo rn m ul le ri c . w in kl . c yn ar oi de ae b un ge es fa ha n 22 53 2( t u h ) m h 99 27 50 bo rn m ul le ri 44 c ou si ni a fa rs is ta ni ca b or nm . c yn ar oi de ae b un ge k er m an 28 63 6( t u h ) m h 99 27 33 fa rs is ta ni ca 45 c ou si ni a la ct ifl or a r ec h. f. c yn ar oi de ae b un ge lo re st an 46 29 9( t u h ) m k 00 51 56 la ct ifl or a 46 c ou si ni a al ig ud ar ze ns is a tt ar & g ha hr em an c yn ar oi de ae b un ge lo re st an -a lig ud ar z 27 61 3( t u h ) m h 99 27 45 al ig ud ar ze ns is 47 c ou si ni a pl at ya ca nt ha b un ge pl at ya ca nt ha e r ec h. f. k ho ra sa n 43 21 2( t u h ) m k 00 51 87 pl at ya ca nt ha 48 c ou si ni a fr ey ni i b or nm . pl at ya ca nt ha e r ec h. f. se m na n s ha hr ud 27 67 5( t u h ) m k 00 51 86 fr ey ni i 49 c ou si ni a bi en er ti bu ng e pl at ya ca nt ha e r ec h. f. k ho ra sa nn ey sh ab ur 28 68 2( t u h ) m h 99 27 40 bi en er ti 50 c ou si ni a re sh in ge ro ru m b or nm . pl at ya ca nt ha e r ec h. f. k ho ra sa nto rb at e ja m 39 72 9( t u h ) m h 99 70 00 re sh in ge ro ru m 43molecular phylogeny and morphometric analyses in the genus cousinia 2012). the test was conducted by comparing the maximum likelihood (ml) values for the known topology in the presence and absence of the molecular clock constraints under tamura and nei (1993). the similar rate of evolution of the investigated sequences was rejected by setting the significance level at 5%, and consequently, the relaxed molecular clock model was utilized in further analyses (minaeifar et al. 2016). the hky model was identified as the best substitution model as implemented in mega 7 (tamura et al. 2011). bootstrap analysis (bs) (felsenstein 1985) was completed to attain support estimates for the nodes in the ml tree. its sequences were also analyzed by tcs networking as implemented in the popart (population analysis with reticulate trees) program (http://popart.otago. ac.nz). estimation of species time of divergence beast v1.6.1 (drummond et al. 2012a, b) was applied for the bayesian mcmc inferred analyses of the nucleotide sequence data. beauti (bayesian evolutionary analysis utility version) v1.6.1 (drummond et al. 2012a, b) was used to generate initial xml files for beast. a yule speciation process (‘a pure birth’ process) was utilized as a tree prior for all tree model analyses. for the mcmc posterior analyses, the length of chain was 10,000,000. after 100 trees burn-in processing, 10,000 trees were utilized for the analyses. the beauti xml file was run in the beast v1.6.1 program, and the maximum clade credibility (mcc) chain generations were repeated five times for each molecular clock model with separate runs to confirm suitable convergence and sufficient mixing. the mcc tree was generated under the relaxed clock model (hky substitution). its substitution rates were applied between 1.72 × 10–9 to 8.34 × 10–9 (mean = 4.13 × 10–9), according to lopez-vinyallonga et al. (2009). tracer v1.5 software (rambaut and drummond 2007) was utilized for the production of the model parameters to assay the sampling and convergence results obtained from beast. treeannotator v1.6.1 software (drummond et al. 2012a, b) was utilized to annotate the phylogenetic results generated by beast as a form of single ‘target’ tree. on the target trees are shown summary statistics of posterior probabilities of the nodes: the 95% highest posterior density (hpd) limits of the node heights, rates, and the posterior estimates. table 2. morphological characters and their codes. characters codes 1 2 3 4 5 head diameter x<3 3≤x≤6 x>6 flowers number x<80 80≤x≤150 x>150 bracts number x<80 80≤x≤120 x>120 appendages length of median bracts x<9 9≤x≤15 x>15 appendages width of median bracts x< 5 5≤x≤15 x>15 corolla length x< 20 20≤x≤25 x>25 habitat woodland alpine steppe leaves indumentum present absent stem leaves interruptedly decurrent continuously decurrent nondecurrent uppermost leaves distant from the head close to the head surrounding the head appendages present absent inner bracts indumentum smooth scabrous position of median bracts imbricated spreading recurved spreading-recurved imbricatedspreading shape of appendages of median bracts sagitate triangular rhombic ovate lanceolate margin of appendages of median bracts smooth 1-2 spins spinose receptacle bristles smooth scabrous corolla color yellow pink purple white ratio limb to anther tube longer shorter as long as anther tube color yellow pink purple white 44 neda atazadeh et al. figtree v1.3.1 (rambaut 2009) program was also applied for the annotated beast mcc tree production analyses. the posterior probability was fix to 0.5, which is equal to the bootstrapping value in paup (phylogenetic analysis using parsimony analysis) analyses (hong and jury 2011). results morphometry pca analysis of morphological features showed that the first two pca components included about 79% general alteration. morphological features like the shape and length of the appendages of the median bracts, diameter of the heads, number of the flowers, and length of the corolla were the most variable morphological features among the investigated plants. in fact, these morphological features are of taxonomic value in the two sections cynaroideae and platyacanthae. an mp tree (figure 1) of morphological characteristics can delimit the two studied sections cynaroideae and platyacanthae because of difference in traits, like stem leaves (sect. cynaroideae: decurrent; sect. platyacanthae: nondecurrent) and the appendages of the median bracts (sect. cynaroideae: present; sect. platyacanthae: absent). in the mp tree, within the sect. platyacanthae, c. platyacantha bunge and c. freynii bornm. were located close to each other due to similarity in all characters except for the color of the corolla (c. platyacantha: white; c. freynii: purple). likewise, c. reshingerorum bornm. and c. bienerti bunge exhibited morphological similarity in traits like number of flowers, length of the corolla, color of the anther tube, median and the inner bracts, receptacle bristles, and the diameter of the head and ratio of the limb/tube. in the sect. cynaroideae, c. grandis c. a. mey. and c. grantii rech. f. were located close to each other because of similar morphological traits such as the shape of the appendages of the median bracts (ovate) and leaves indumentum (glabrous). the same applies for c. ecbatanensis bornm. c. kornhuberi heimerl, c. elwendensis bornm., c. parsana ghahreman, c. denaensis attar & djavadi and c. khorramabadensis bornm.; these species have similar morphological characters like the color of the corolla (white) and the position of the median bracts (spreading). c. millefontana rech. f., c. fursei rech. f., c. sardashtensis rech., c. carolihenrici attar & ghahreman, c. dalahuensis attar & ghahreman and c. concinna boiss. & hausskn. were also close to each other because of the similarity in morphological features such as the position of the median bracts (imbricated). the same applies for c. calocephala jaub. & spach and c. behboudiana rech. f. & esfand. because of their similar morphological traits, like the position of the median bracts (recurved) and the color of the corolla (yellow). its sequence analysis the pair-wise genetic distances determined for the studied cousinia species arranged from 0.01 (the lowest value between c. ecbatanensis and c. kotschyi boiss.) to 0.50 (the highest value between c. shebliensis ghahreman and c. behboudiana). these values showed the degree of sequence variability within species. the ml tree (figure 2) and tcs network (figure 3) of the studied species based on its sequences produced similar results. in these trees, the outgroups (a. umbrosum and a. lappa) were basically separated from the other species. based on these results, the its marker did not delimit the two studied sections. in the obtained genetic trees, the species of the sect. platyacanthae, such as c. platyacantha, c. reshingerorum, c. bienerti and c. freynii, were placed among the species of the sect. cynaroideae. these trees exhibited a close genetic affinity between c. platyacantha, c. reshingerorum and c. bienerti, which is in agreement with their morphological similarities. however, c. freynii is located far from the others in the genetic tree but close to them in the morphological tree. figure 1. maximum parsimony tree of the studied cousinia species based on morphological data. (species are according to table 1). values above branches are bootstrap value. 45molecular phylogeny and morphometric analyses in the genus cousinia in the ml tree based on its sequences, c. verbascifolia was placed far from the other species. it differs morphologically from the other studied species in the color of corolla (light pink), bracts (numerous), the appendage of the median bracts (triangular), presence of long spine found at the apex, and the spinulose at the margin. both mp and ml trees exhibited an affinity between c. kotschyi and c. ecbatanensis. these species are similar in morphological traits such as shape of the appendages in the median bracts (lanceolate) and position of the median bracts (spreading). similarly, c. elwendensis and c. denaensis are related to c. ecbatanensis. they have morphological similarities in the color of the corolla (white) and the position of the median bracts (spreading). the its-based phylogenetic tree showed a close affinity between c. millefontana and c. concinna, which have similar morphological characters such as the position of the median bracts (imbricated). c. keredjensis bornm. & gauba is well separated from the two species mentioned above (c. millefontana and c. concinna) and also differs in its morphological traits, like the color of the corolla (white), the number of flowers (ca. 125), and the number of bracts (ca. 130). the same applies between c. sahandica attar & djavadi and c. sanandajensis rech. f., as well as for c. nana attar and c. cynaroides c. a. mey. disagreement was observed between the other studied species of cousinia based on morphological and genetic characters. the tcs network exhibited the process of speciation and the number of nucleotide substitution in its sequences among the studied species. the highest number of nucleotide substitutions in its occurred in c. khorramabadensis (9). the bayesian tree (figure 4) obtained with beast based on its sequences estimated the divergence times of the studied species within the cynaroideae and platyacanthae sections to be approximately 3.5 mya. discussion taxonomy, molecular phylogeny, and the species relationships of the genus cousinia are complicated and unresolved, mainly because of disagreement between morphological and molecular phylogenetic studies (sausana et al. 2003; lopez-vinyallonga et al. 2009; mehregan and assadi 2016). moreover, several overlapping morphological characteristics at the species level hinder species identification and delineation (attar and djavadi 2010; minaeifar et al. 2016; atazadeh et al. 2020). susanna et al. (2003), performed an extensive investigation on the evolution and generic delineation in the arctium-cousinia complex, based on two very important characters: pollen type and chromosome number. they divided all studied species into two major lineages: the arctioid clade (including: arctium, sehmalhallsenia) with arctiastrum pollen type and x= 18 and the cousinioid clade (including: cousinia subg. cousinia) with cousinia pollen type and x= 11, 12, 13. they also showed that the palynological and chromosome number results figure 2. maximum likelihood tree of the studied cousinia species based on its data. (species are according to table 1). 46 neda atazadeh et al. are incongruent with morphological and molecular data in the arctium-cousinia complex and considered to be homoplasious morphological characters. these results were later confirmed by lopez-vinyallonga et al. (2009). in the present study, the studied cousinia species were delimited and useful taxonomic features, including the shape and length of the appendages of the median bracts, position of the median bracts, diameter of the heads, number of flowers and bracts, and the color and length of the corolla were identified. the species relationships obtained based on the morphological features within sect. platyacanthae are in agreement with those reported in previous studies (asaadi and mehregan 2017). for instance, within sect. platyacanthae, c. platyacantha and c. freynii were placed close to each other because of the similarity found in all features except color of the corolla (c. platyacantha: white; c. freynii: purple). asaadi and mehregan (2017) also noticed their close affinity to each other. likewise, c. reshingerorum and c. bienerti exhibited morphological likenesses in traits such as number of flowers, length of the corolla, color of the anther tube, the median and inner bracts, receptacle bristles, diameter of the head, and the ratio of the limb/tube. asaadi and mehregan (2017) also showed the close morphological affinity among these species. the morphological results in sect. cynaroideae, were in complete agreement with the obtained results by attar and djavadi (2010). the relationships among the studied cousinia species in the genetic trees were generally incongruent with those in the obtained morphological tree. the present study revealed that the studied sections within the genus cousinia, including cynaroideae and platyacanthae, are not monophyletic. this is fully consistent with the results reported by susanna et al. (2003) and lopez-vinyallonga et al. (2009), who also showed that morphological traits are highly incongruent with molecular data in arctium-cousinia complex and considered homoplasious morphological characters. various reasons are suggested for the disagreement between “gene tree” and “species trees”, including the figure 3. tcs network of studied cousinia species based on its sequences (species are according to table 1). 47molecular phylogeny and morphometric analyses in the genus cousinia figure 4. beast chronogram of studied cousinia species based on its sequences (species are according to table 1, numbers at the base of tree represents mya). 48 neda atazadeh et al. high number of taxa in cousinia (susanna et al. 2003; lopez-vinyallong et al. 2009; mehregan and assadi 2016), interspecific hybridization, the occurrence of the intermediate forms (mehregan and kadereit 2009) and homoploid hybrid speciation, of which there is little proof to prove them (lopez-vinyallong et al. 2009), and incomplete lineage sorting (zhang et al. 2015). the divergence time of the studied species within the cynaroideae and platyacanthae sections based on its sequences was estimated to be around 3.5 mya. this result is in agreement with lopez-vinyallonga et al. (2009), who also showed that the major radiation of the genus cousinia has been estimated to have started ca. 8.7 mya. according to our findings and previous authors (susanna et al. 2003; lopez-vinyallong et al. 2009) phylogeny, evolutionary pathway and species relationships of the genus cousinia are unclear and complicated. the genus cousinia with its relatively young geological age (ca. 8.7 mya) and high number of taxa is unusual exposed to speciation. djamali et al. (2012) showed that cousinia consistently existed in the glacial age. they recorded an ~200,000-year pollen from lake urmia, northwest iran. in contrast, the dispersal of its pollen grains was restricted. in the current results, all of the studied species except for c. calocephala had restricted geographical distributions and were isolated by geographical boundaries, which reduced the genflow. therefore, geographical processes can be determining factors in the speciation of this genus. this result is entirely consistent with the results reported by lopez-vinyallonga et al. (2009), as they also revealed that the dominant factor in speciation of the genus cousinia is allopatric geographic speciation. these may partly justify the complexity and incongruence of the relationships in the studied species of the genus cousinia. as a general conclusion, based on the molecular studies of the observed specimens, it is suggested that both cynaroideae and platyacanthae sections are synonymous. to make a definitive decision on this, further molecular studies are necessary. author contributions all authors contributed to the study conception and design. material preparation, data collection and analysis were performed by neda atazadeh, masoud sheidai and farideh attar. the first draft of the manuscript was written by neda atazadeh and all authors commented on previous versions of the manuscript. all authors read and approved the final manuscript. references alvarez i, wendel jf. 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(2015). assessing the impact of phylogenetic incongruence on taxonomy, floral evolution, biogeographical history, and phylogenetic diversity. am j bot. 102(4): 566-580. caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 4 2021 firenze university press cytogenetic analyses in three species of moenkhausia eigenmann, 1903 (characiformes, characidae) from upper paraná river (misiones, argentina) kevin i. sánchez1,*, fabio h. takagui2, alberto s. fenocchio3 genetic variations and interspesific relationships in lonicera l. (caprifoliaceae), using scot molecular markers fengzhen chen1, dongmei li2,* , mohsen farshadfar3 the new chromosomal data and karyotypic variations in genus salvia l. (lamiaceae): dysploidy, polyploidy and symmetrical karyotypes halil erhan eroğlu1,*, esra martin2, ahmet kahraman3, elif gezer aslan4 cytogenetic survey of eight ant species from the amazon rainforest luísa antônia campos barros1, gisele amaro teixeira2, paulo castro ferreira1, rodrigo batista lod1, linda inês silveira3, frédéric petitclerc4, jérôme orivel4, hilton jeferson alves cardoso de aguiar1,5,* molecular phylogeny and morphometric analyses in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f. neda atazadeh1,*, masoud sheidai1, farideh attar2, fahimeh koohdar1 a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) melika tabasi1, masoud sheidai1,*, fahimeh koohdar1, darab hassani2 genetic diversity and relationships among glaucium (papaveraceae) species by issr markers: a high value medicinal plant lu feng1,*, fariba noedoost2 morphometric analysis and genetic diversity in rindera (boraginaceae-cynoglosseae) using sequence related amplified polymorphism xixi yao1, haodong liu2,*, maede shahiri tabarestani3 biosystematics, fingerprinting and dna barcoding study of the genus lallemantia based on scot and remap markers fahimeh koohdar*, neda aram, masoud sheidai karyotype analysis in 21 plant families from the qinghai–tibetan plateau and its evolutionary implications ning zhou1,2, ai-gen fu3, guang-yan wang1,2,*, yong-ping yang1,2,* some molecular cytogenetic markers and classical chromosomal features of spilopelia chinensis (scopoli, 1786) and tachybaptus ruficollis (pallas, 1764) in thailand isara patawang1,*, sarawut kaewsri2, sitthisak jantarat3, praween supanuam4, sarun jumrusthanasan2, alongklod tanomtong5 centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates) simona ceraulo, vanessa milioto, francesca dumas* repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil marina souza cunha1,2,*,#, silvana melo1,3,#, filipe schitini salgado1,2, cidimar estevam assis1, jorge abdala dergam1,* karyomorphology of some crocus l. taxa from uşak province in turkey aykut yilmaz*, yudum yeltekin variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l. egizia falistocco1,*,+, gianpiero marconi1,+, lorenzo raggi1, daniele rosellini1, marilena ceccarelli2, emidio albertini1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(1): 165-171, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1411 caryologia international journal of cytology, cytosystematics and cytogenetics citation: kristen d. felt, makayla b. lagerman, samantha maurer, lu qian, oluwasefunmi oluwafemi, noemi pedraza-aguado, emily l. stowe, leocadia v. paliulis (2022) segregation of the univalent x chromosome in the wide-footed treehopper enchenopa latipes (say 1824). caryologia 75(1): 165-171. doi: 10.36253/caryologia-1411 received: september 22, 2021 accepted: march 23, 2022 published: july 6, 2022 copyright: © 2022 kristen d. felt, makayla b. lagerman, samantha maurer, lu qian, oluwasefunmi oluwafemi, noemi pedraza-aguado, emily l. stowe, leocadia v. paliulis. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid lvp: 0000-0002-8244-7548 segregation of the univalent x chromosome in the wide-footed treehopper enchenopa latipes (say 1824) kristen d. felt, makayla b. lagerman, samantha maurer, lu qian, oluwasefunmi oluwafemi, noemi pedraza-aguado, emily l. stowe, leocadia v. paliulis* biology department, bucknell university, lewisburg, pennsylvania, usa *corresponding author. e-mail: le.paliulis@bucknell.edu abstract. in metaphase i, autosomal bivalents align on the metaphase plate, while naturally-occurring univalent sex chromosomes can display a number of different behaviours depending on cellular conditions. here we describe the behaviour of the univalent x chromosome in the wide-footed treehopper enchenopa latipes (say 1824). we confirm the chromosome number and sex determination method for this species, and that males possess a univalent x chromosome. we show that the univalent x chromosome forms a bipolar attachment to the spindle in metaphase i, and then segregates intact toward one spindle pole in late anaphase i (long after autosomes have initiated poleward movement). movement of the univalent toward one pole is associated with loss of microtubule connections toward the opposite spindle pole. keywords: chromosomes, bivalent, univalent, x chromosome, meiosis, auchenorrhynca, enchenopa latipes. introduction the aim of meiosis is to divide the chromosome number of the cell by two, creating haploid gametes. reduction of chromosome number requires the formation of bivalents. to form a bivalent, the dna in each chromosome is replicated. the replicated chromosomes pair, and are held together through sister-chromatid cohesion. after dna replication, the homologues or partner sex chromosomes connect and undergo recombination, completing construction of the bivalent (moore and orr-weaver 1998). sister kinetochores are fused together in meiosis i and act as a single attachment site, allowing one half-bivalent to attach to microtubules coming from one spindle pole (a syntelic attachment), while the homologous half-bivalent associates with the opposite pole (moore and orr-weaver 1998). fusion of sister kinetochores ensures that sister chromatids will not separate prematurely in anaphase i. in “typical” meiosis i, homologues are guaranteed to separate from one another because they are initially connected, and because sis166 kristen d. felt et al. ter kinetochores are fused together. cells then undergo a second meiotic division, with the sister kinetochores now facing in opposite directions and associating with opposite spindle poles (an amphitelic attachment) in metaphase ii, and separating sister chromatids in anaphase ii (moore and orr-weaver 1998). initial formation of a bivalent is, in general, required for successful creation of four gametes, each with half of the chromosomes of the original parent. correct chromosome distribution depends on homologues linking with one another, but what if homologues fail to link? or, what if there is no partner at all? a number of organisms have sex chromosomes that do not have a pairing partner for meiosis i, and thus remain univalent in the first meiotic division. while errors that create univalent autosomes lead to erratic chromosome behaviours for the univalent, such as frequent and rapid oscillations between spindle poles and a failure to align at the metaphase plate, naturally-occurring univalent sex chromosomes appear to have a set of characteristic, stable behaviours depending upon conditions in the cell (fabig, et al. 2016; rebollo et al. 1998; nokkala 1986; bressa et al. 2001; rebollo and arana 1995; rebollo and arana 1997; ault 1984; bauer et al. 1961; dietz 1954; dietz 1969; john 1990). in some cases, the univalent’s sister kinetochores are fused together in meiosis i just like those of the autosomal bivalents nearby (fabig et al. 2016; ault 1984). in such systems, the univalent can form an attachment to a single spindle pole early in meiosis i and remain adjacent to that spindle pole through telophase i (fabig et al. 2016). in other cases, the univalent has sister kinetochores facing in opposite directions. it aligns on the metaphase plate with the bivalents. in anaphase i, when the autosomal half bivalents separate from one another, the univalent remains at the center of the spindle, and following separation of the autosomes, the univalent moves intact to one spindle pole (fabig et al. 2016). still other behaviors have been observed in univalentsand are well described in fabig et al. (2016). univalent x chromosomes are frequently seen in insects of the order hemiptera, suborder auchenorrhyncha. in fact, these insects are some of the first species in which an x chromosome was observed and realized to exhibit different behaviours than the other chromosomes in the cell. very early work on one species in the suborder auchenorrhyncha, the spittle bug philaenus spumarius, described the x chromosome to be an “odd” chromosome that “lags behind the others but goes undivided to one pole” (boring 1913). here we report on our study of the behaviour of the univalent x chromosome in another member of the suborder auchenorrhyncha, the treehopper, enchenopa latipes (say 1824). halkka and heinonen (1964) previously reported the karyotype and sex determination mechanism for the species to be 2n=19 in males with x0 (male)-xx (female) sex determination, but did not make any statement on the behaviour of the univalent x chromosome during meiosis. we confirm the previously-published report on chromosome number and sex determination mechanism, and use live-cell imaging and immunofluorescence staining to reveal that the x chromosome of e. latipes aligns with the autosomes in metaphase, forming an amphitelic attachment to the spindle. we also show that the x chromosome of e. latipes moves intact to one spindle pole after the autosomes have segregated, losing its connection to one spindle pole while retaining microtubule connections to the pole toward which it is moving. we also make conclusions about the conditions that lead to these characteristic behaviours. materials and methods collection and identification adult enchenopa latipes males were collected from a field site at the bucknell university farm (lewisburg, pa). treehoppers were identified and sexed according to dietrich et al. (2001) and kopp and yonke (1973). dna barcoding dna barcoding was done as described in the carolina biological supply company using dna barcodes to identify and classify living things kit (carolina 211385). cytochrome c oxidase subunit 1 was amplified using the primers and pcr beads supplied by carolina biological supply company and sequenced at genewiz using the m13forward and m13reverse primers. sequence was analyzed using sequencher v5.4.6 and trimmed to approximately 640 bp. alignments were produced using clustalomega (https://www.ebi.ac.uk/tools/ msa/clustalo/). orcein staining of spread chromosomes orcein stained chromosome spreads were prepared as described in felt et al. (2017). living cell preparations testes were removed from the abdomens of e. latipes males and transferred to a culture chamber (lin 167segregation of the univalent x chromosome in the wide-footed treehopper enchenopa latipes (say 1824) et al. 2018) under a layer of kel-f oil #10 (ohio valley specialty company, marietta, ohio). testes contents were spread thinly on a coverslip under oil, as described in lin et al. (2018). living meiosis i spermatocytes were imaged using a zeiss inverted microscope equipped with a 100x 1.25 na phase-contrast, oil-immersion objective and an infinity 1 camera with infinity analyze software or a nikon eclipse ts100 microscope equipped with a 100x, 1.25 na phase-contrast, oil-immersion objective and a spot rt monochrome camera (diagnostic instruments inc.) with spot basic 3.5.7 software. immunofluorescence fixation, immunostaining, and imaging of stained specimens were carried out as described in felt et al. (2017). results dna barcoding to confirm the identification of the insect specimens, we performed dna barcoding analysis on one kf919639.1 attttatttttggtatatgatctggaatattagggataataataagaattattattcgaa 60 hm416189.1 attttatttttggtatatgatctggaatattaggaataataataagaattattattcgaa 60 mz723494 attttatttttggtatatgatctggaatattagggataataataagaattattattcgaa 60 ********************************** ************************* kf919639.1 ttgaactgagtcagccgggccctttaattcaaaatgaccaaatctataatactgtagtga 120 hm416189.1 ttgaattaagtcagccgggtccttttattcaaaatgaccaaatttataatactgtagtga 120 mz723494 ttgaattaagtcaaccgggtccttttattcaaaatgaccaaatttataatactgtagtga 120 ***** * ***** ***** ***** ***************** **************** kf919639.1 cttcacatgcatttattataattttttttatagttatacccattataattgggggatttg 180 hm416189.1 cttcacatgcatttatcataattttttttatagttatacccattataattgggggatttg 180 mz723494 cttcacatgcatttatcataattttttttatagttatacccattataattgggggatttg 180 **************** ******************************************* kf919639.1 gaaattgattagtaccattaatagttggagcaccagatatagcttttcctcgtcttaata 240 hm416189.1 gaaattgactagtaccattaataattggagccccagatatagcttttcctcgtcttaata 240 mz723494 gaaattgattagtaccattaataattggagccccagatatagcttttcctcgtcttaata 240 ******** ************** ******* **************************** kf919639.1 atataagattttgattattacctccatcaatcttattacttctatctagatcagtggtag 300 hm416189.1 atataagattttgattattacctccatcaatcttattacttttatctagatcaatggtag 300 mz723494 atataagattttgattattacctccatcaatcttattacttttatctagatcaatggtag 300 ***************************************** *********** ****** kf919639.1 aatcaggtgcaggaactggatgaacagtataccctcctctttctagtaacattgctcatt 360 hm416189.1 aatcaggtgcaggtactggatggacagtatacccccctctttctagtaatattgctcatt 360 mz723494 aatcaggtgcaggtactggatggacagtatacccccctctttctagtaatattgctcatt 360 ************* ******** *********** ************** ********** kf919639.1 ctggggctagagtagatttagctattttttctctgcatttagctggtatttcatcaattt 420 hm416189.1 ctggggctagagtagatttagctattttttctctgcatttagctggtatttcatcaattt 420 mz723494 ctggggctagagtagatttagctattttttctctacatttagctggtatttcatcaattt 420 ********************************** ************************* kf919639.1 taggtgcaattaattttattacaactattataaatatacgttgtgatgaattaaatatag 480 hm416189.1 taggtgcaattaattttattacaactattataaatatacgttgtgatgaattaaatatag 480 mz723494 taggtgcaattaattttatcacaactattataaatatacgttgtaatgaattaaatatag 480 ******************* ************************ *************** kf919639.1 atcgtcttcctttatttgtttggtcagtaataatcacagcggttttacttttattgtccc 540 hm416189.1 atcgtcttcctttatttgtttggtcagtaataatcacagcggttttacttttattatccc 540 mz723494 atcgtcttcctttatttgtttggtcagtaataatcacagcggttttacttttattatccc 540 ******************************************************* **** kf919639.1 ttcccgttttagctggtgctatcactatattattaaccgatcgtaatataaatacttctt 600 hm416189.1 ttcccgtattagctggtgctattactatattattaaccgatcgtaatataaatacttctt 600 mz723494 ttcccgtattagctggtgctattactatattattaactgatcgtaatataaatacttctt 600 ******* ************** ************** ********************** kf919639.1 tctttgatccttctggtggaggagatcctattttataccaacatttattc 650 hm416189.1 tctttgatccttctgggggaggagatcccattttataccaacatttattt 650 mz723494 tctttgatccttctggggggggagaccccattttatatcaacatttattt 650 **************** ** ***** ** ******** *********** figure 1. clustalomega alignment of cytochrome oxidase 1 gene from enchenopa latipes specimens. the top two sequences represent specimens with the closest identity to our specimen from two independent barcoding studies of e. latipes based on blastn analysis. our specimen mz723494 is 98.46% identical to sequence hm4161189.1 and 95.84% to sequence kf919639.1. 168 kristen d. felt et al. individual and submitted the sequence to genbank. the sequence has accession number mz723494. the partial cox1 gene sequences were analysed using blastn and identified two sequences, one associated with kf919639, and a second associated with hm416189. the full sequence of mz723494 was used in clustal omega (madiera et al. 2019) to create the alignment (figure 1). the mz723494 isolate was 95.8% identical to the kf 919639 specimen and 98.5% identical to the hm416189 specimen (figure 1). karyotype analysis chromosome spreads from e. latipes were prepared and analysed to confirm chromosome number and sex determination mechanism. spreads of testes contents from ten individuals were used to determine the chromosome number. e. latipes has a chromosome number of 2n=19 in males, with nine bivalents and one univalent x chromosome (figure 2). sex determination and sex chromosome behaviour chromosome behaviour was observed in living metaphase i and anaphase i spermatocytes (figure 3). in metaphase i, the univalent x chromosome aligned on the metaphase plate along with all of the autosomal bivalents (figure 3; 0 min.). at anaphase i onset, the univalent x chromosome remained at the center of the spindle while the autosomes separated toward the spindle poles (figure 3; 5, 15, 25 min.). by late anaphase i, the x chromosome moved to one side of the spindle, approaching the bulk of autosomal half bivalents (figure 3; 45, 50 min.). immunofluorescence staining revealed microtubules associated with the x chromosome from both spindle poles in metaphase i spermatocytes (figure 4a). microtubule connections were also observed on both sides of the univalent x chromosome in early anaphase i (figure 4b). in late anaphase i spermatocytes, the x chromosome had microtubules associated with one side of the univalent, but the other side had no apparent microtubule connections on the other side (figure 4c and 4d). the x chromosome was located near the spindle pole with the microtubule connection in late anaphase i spermatocytes (figure 4c and 4d), and was positioned on one side of the cleavage furrow (figure 4d). discussion our results confirm the results of halk ka and heinonen (1964), with a chromosome number of 2n=19 in males and an xx (female)-x0 (male) sex determination mechanism. our work also corroborates previous studies that reveal chromosome numbers between 2n = 18 and 2n = 22 for other species within the membracidae family (of which e. latipes is a member), most of which have x0 (male)/xx (female) sex determination (boring 1907; halkka 1959; halkka 1962; tian and yuan 1997; bhattacharya and manna 1973). as was previously observed, all males in this study have a univalent x chromosome that does not have a pairing partner in meiosis i. the autosomes and the sex chromosomes of e. latipes all align on the metaphase plate in metaphase i (figure 3; 0 min., figure 4; metaphase). this demonstrates that the univalent x chromosome has a bipolar attachment to the spindle (reviewed in fabig et al. 2016), that is confirmed through our immunof luorescence data (figure 4a, 4b). our observations of anaphase in living cells (figure 3) and in fixed, stained specimens (figure 4) revealed that segregation of the univalent x chromosome is delayed relative to the autosomes, and that movement of the x chromosome is associated with loss of microtubule connections to one spindle pole and retention of connections to the pole toward which the chromosome moves. delayed or lagging segregation is frequently observed in cells that have bipolarly-attached univalent x chromosomes, including primary spermatocytes of other hemipteran insects, and the primary figure 2. orcein-stained chromosome spread generated from meiosis i spermatocyte of e. latipes. the spread shows 9 bivalents. x chromosome is indicated with arrow. bar=5μm. 169segregation of the univalent x chromosome in the wide-footed treehopper enchenopa latipes (say 1824) spermatocytes in the male caenorhabditis elegans (fabig et al. 2020; felt et al. 2017; fabig et al. 2016; john and claridge 1974; rao 1956; rebollo et al. 1998; rebollo and arana 1998). we have confirmed the previously-published chromosome number and sex-determination mechanism of the treehopper enchenopa latipes (halkka and heinonen 1964). we have also shown that the univalent x chromosome aligns at the spindle equator in metaphase i alongside the bivalent autosomes, and forms a bipolar attachment to the spindle. we finally show that the univalent x chromosome moves intact to one of the spindle poles in late anaphase, after all of the autosomes have initiated segregation, by losing microtubule connections to one spindle pole and retaining connections to the pole toward which is moving. figure 3. delayed segregation of the intact univalent x chromosome. the x chromosome aligns with the autosomes on the metaphase plate (0 min) and remains at the centre of the spindle after the autosomal half bivalents have initiated segregation to their associated spindle poles (5, 15, 25 min). in late anaphase, the x chromosome moves intact toward the upper spindle pole. bar=5μm. 170 kristen d. felt et al. hemipteran insects like e. latipes have holocentric chromosomes in mitosis (halkka 1959; melters et al. 2012; kuznetsova and aguin-pombo 2015). hemipterans of the suborder auchenorryncha (like e. latipes) appear to restrict kinetic activity of each bivalent so that bivalents behave as if they have localized kinetochores (halkka 1959; kuznetsova and aguin-pombo 2015). this allows one set of sister chromatids to move to one spindle pole while the homologous set moves to the opposite spindle pole in a traditional (non-inverted) meiosis (melters et al. 2012). in our previous examination of the behaviour of univalent x chromosomes, we have found that systems that have holocentric chromosomes in mitosis, a noninverted meiosis, and a univalent x chromosome show the same pattern of x-chromosome segregation in male meiosis i as we have observed in e. latipes (fabig et al. 2016; felt et al. 2017). this univalent-segregating behaviour is observed in different phyla of animals (fabig et al. 2016; felt et al. 2017; fabig et al 2020), suggesting that the characteristics of the meiotic system, rather than phylogeny, dictate univalent behaviour in meiosis. the question for the future will be to find the mechanistic underpinnings for these characteristic chromosome behaviours. acknowledgements we thank art forer for discussions essential to the completion of this work. funding details kdf was funded by a bucknell university graduate research fellowship and a robert p. vidinghoff memorial summer internship through the bucknell university biology department. mbl and lq were funded by the national science foundation (grant number nsf due-1317446). np-a was funded by the biology department, bucknell university. oo was funded through a stem scholars grant, bucknell university. els was funded by the biology department, bucknell university. lvp was funded by research funds awarded through bucknell university. references ault jg. 1984. unipolar orientation stability of the sex univalent in the grasshopper (melanoplus sanguinipes). chromosoma. 89:201-205. bauer h, dietz r, and röbbelen c. 1961. die spermatocytenteilungen der tipuliden. iii. das bewegungsverhalten der chromsomen in translokationheterozygoten von tipula oleracea. chromosoma. 12:116-189. bhattacharya ak, manna gk. 1973. morphology, behaviour, and metrical studies of the germinal chromosomes of ten species of membracidae (homoptera). cytologia. 38:657-665. boring am. 1907. a study of the spermatogenesis in twenty-two species of the membracidae, jassidae, figure 4. segregation of the x chromosome in anaphase i e. latipes spermatocytes results from reduction and subsequent loss of microtubule connections on one side of the univalent. immunofluorescence staining of microtubules (green) and dapi staining (purple) of e. latipes spermatocytes in metaphase i (a), early anaphase i (b), and late anaphase/telophase i (c, d). in metaphase i, the x chromosome aligns on the metaphase plate, with microtubules connecting the univalent to both spindle poles. in early anaphase, the half bivalents move toward their associated poles while the x univalent remains at the centre of the spindle. it retains microtubule 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(rosaceae) comprises about from 67 (lu and spongberg 2003) to at least 111 species in china (phipps et al.1990). chinese native species were traditionally assigned to three subgenera (three sections in the same names by yü and kuan (1963)): aria (pers.) host and micromeles decne. with simple leaved species, sorbus with pinnately leaved species (phipps et al. 1990). polyploidy together with hybridization and apomixis is an important evolutionary force in shaping plant diversification in sorbus (robertson et al. 2010; ludwig et al. 2013; lepší et al. 2019). the number of species recognized in the genus varied between authors mainly depending on the way that the 124 yuming qian et al. numerous polyploidy apomicts were treated (aldasoro et al. 2004; mcallister 2005; sennikov and kurtto 2017). knowledge of chromosome numbers is highly important in sorbus in understanding the species delimitation and relationship. modern taxonomic studies (mcallister 2005; sennikov and kurtto 2017) and descriptions of new species (rich et al. 2014; lepší et al. 2015; somlyay et al. 2017) are accompanied to count of chromosome numbers or dna ploidy levels based on flow cytometry. polyploidy was reported only in subgenus sorbus from china, which account for more than half of the subgenus’ species richness and are distributed mainly in the mountains of southwest area, especially the qinghai-tibet plateau (mcallister 2005; li et al. 2021). for subgenus aria, which is disjunctly distributed across europe and asia, with polyploidy species reported from only europe (sennikov and kurtto 2017). for the asian native subgenus micromeles, the only chromosome number available is s. alnifolia (2n = 2x = 34; sax 1931; baranec and murín 2003). though aldasoro et al. (2004) suggested that the great variability in leaf outline of s. alnifolia might reflect the presence of agamospermous individuals in populations, no evidence is available up to now. agamospermy as a form of asexual reproduction is prevalent in sorbus (robertson et al. 2010; hajrudinović et al. 2015) and other genera in maloideae such as amelanchier medik. (burgess et al. 2014), cotoneaster medik. (rothleutner et al. 2016) and crataegus l. (talent and dickinson, 2007) etc. the aim of this study is to investigate the chromosome number, karyotype, idiogram and other detailed measurements of eight chinese endemic taxa from subgenus aria and micromeles, together with an asian distributed species s. alnifolia, to find out whether there are polyploid and to analyze the species relationship. materials and methods plant materials nine taxa, including two species in sorbus subgenus aria: s. hemsleyi (c.k. schneid.) rehd. and s. yuana spongberg, and seven taxa in subgenus micromeles: s. alnifolia (siebold and zucc.) k. koch var. alnifolia , s. alnifolia (siebold and zucc.) k. koch var. angulata s.b. liang, s. alnifolia (siebold and zucc.) k. koch var. lobulata rehd., s. dunnii rehd., s. folgneri (c.k. schneid.) rehd., s. lushanensis x. chen and j. qiu, s. ochracea (hand.-mazz.) j.e. vidal, were investigated. one species, s. ochracea, was collected from the kunming botanical garden, and the remaining eight taxa were collected from wild populations between 2012 and 2016. voucher specimens are deposited at the herbarium of nanjing forestry university (nf; table 1). chromosome counts root tips were immersed in a mixed solution of 0.05% aqueous colchicine and 0.002 mol/l 8-hydroxy quinolone (1:1) at 0–4 °c for 2 h, and then fixed in carnoy’s fixative (3:1 alcohol:glacial acid, v/v) for at least 24 table 1. localities and voucher specimens of sorbus taxa examined in the present study. taxa locality collector/voucher specimen s. hemsleyi wawushan scenic spot, sichuan province, e102°57’06.82”, n29°39’54.35”, 2230 m, 25 september 2016 xin chen & zhongren xiong/0745 s. yuana shennongjia forestry district, hubei province, e110°19’17.76”, n31°34’21.30”, 2173 m, 20 october 2016 yun chen & yang zhao/0817 s. alnifolia var. alnifolia changbai mountains, jilin province, e127°49’24.85”, n42°02’04.85”, 867m xin chen & dan chen/2-1 s. alnifolia var. angulata lushan mountain, shandong province, e118°03’09.00”, n36°17’45.31”, 1047 m, 9 october 2015 xin chen & jing qiu/0140 s. alnifolia var. lobulata laoshan mountain, shandong province, e120°37’26.58”, n36°10’40.04”, 942 m, 28 october 2014 xin chen & wan du/0042 s. dunnii huangshan mountain, anhui province, e117°26’18.07”, n29°11’36.31”, 1603 m, september 2013 xin chen & dan chen/4-7 s. folgneri badong, hubei province, e110°17’19.75” n30°41 ’36.86”, 1543 m, 17 october 2016 yun chen & yang zhao/0791 s. lushanensis lushan mountain, jiangxi province, e116°00’46.29”, n29°32’58.59”, 1310 m, 2 october 2015 xin chen, weiqi liu & mingwei geng/0157 s. ochracea kunming botanical garden, yunnan province, e102°44’17.54”, n25°8 ’23.65”,1936 m, 5 august 2020 qin wang/0256-2 125chromosome counts and karyotype analysis of nine taxa in sorbus subgenera aria and micromeles (rosaceae) h at room temperature. the root tips were hydrolyzed in 1 mol/l hcl at 60 °c for 10 min, then were washed with distilled water for 2-3 min. the fixed roots were stained with carbol fuchsin for 3-4 h and were squashed on glass slides for observation. no less than five cells per individual and three to five plants per taxon were examined. photos were taken under a nikon eclipse ci-s microscope. karyotype analysis for the numerical characterization of the karyotypes, the following parameters were measured and calculated using karyotype software (altinordu et al. 2016): short arm length (s) and long arm length (l); mean length of the chromosome (cl); total haploid length of the chromosome set (thl); longest chromosome/shortest chromosome (lt/st); ratio of mean long to short arm length (mar); centromeric index (ci); coefficient of variation of the centromeric index (cvci) and coefficient of variation of chromosome length (cvcl); mean centromeric asymmetry (mca); the karyotype asymmetry index (ask%); the total form percent (tf%); the index of karyotype symmetry (syi%); the intra chromosomal asymmetry index (a1); the interchromosomal asymmetry index (a2); the degree of karyotype asymmetry (a); the dispersion index (di) and the asymmetry index (ai). karyotype formula was determined by chromosome morphology based on centromere position according to levan classification: median point (m, ar = 1.00), median region (m, ar = 1.01–1.70), submedian (sm, ar = 1.71–3.00), subterminal (st, ar = 3.01–7.00) and terminal region (t, ar > 7.00). satellite chromosomes were abbreviated as ‘sat’ (levan et al. 1964). idiograms were drawn using karyotype based on length of chromosome size. statistical analysis was carried out by using spss 26.0. results all nine investigated sorbus taxa are diploids with 2n = 2x = 34. new counts were reported for eight chinese endemic taxa, s. alnifolia var. angulata, s. alnifolia var. lobulata, s. dunnii, s. folgneri, s. hemsleyi, s. lushanensis, s. ochracea and s. yuana. for the remaining s. alnifolia var. alnifolia, the previously reported chromosome numbers was confirmed here. karyotype characters of the nine taxa were reported for the first time (table 2; figure 1, 2). the ta bl e 2. k ar yo ty pe fe at ur es o f t he n in e st ud ie d so rb us ta xa . su bg en us sp ec ie s 2n k ar yo ty pe fo rm ul a v c l (µ m ) t h l (µ m ) lt /s t m a r st eb bi ns ’s ty pe c v c i c v c l m c a a sk % t f% sy i% a 1 a 2 a d i a i a ri a s. h em sle yi 34 29 m +5 sm 0. 93 -1 .8 3 23 .4 3 1. 97 1. 33 2a 11 .5 1 18 .5 6 12 .9 2 56 .7 43 .3 76 .3 5 0. 22 0. 19 0. 13 7. 51 2. 14 s. y ua na 34 32 m +2 sm 1. 15 -1 .7 8 23 .4 4 1. 55 1. 27 1a 8. 36 11 .2 9 11 .0 6 55 .5 9 44 .4 1 79 .9 0. 19 0. 11 0. 11 5. 08 0. 97 m ic ro m el es s. a ln ifo lia v ar . a ln ifo lia 3 4 32 m (2 sa t) +2 sm 1. 48 -2 .4 9 32 .9 1 1. 68 1. 34 1a 9. 31 14 .2 13 .8 57 .0 2 42 .9 8 75 .3 8 0. 23 0. 14 0. 14 5. 73 1. 32 s. a ln ifo lia v ar . a ng ul at a 34 26 m +8 sm (2 sa t) 1. 04 -1 .8 6 24 .0 2 1. 78 1. 49 2a 13 .8 2 14 .7 9 18 .1 3 59 .4 1 40 .5 9 68 .3 2 0. 29 0. 15 0. 18 6. 45 2. 04 s. a ln ifo lia v ar . l ob ul at a 34 32 m (2 sa t) +2 sm 1. 27 -2 .5 6 31 .4 1 2. 01 1. 46 2b 9. 92 17 .7 2 17 .6 6 58 .8 41 .2 70 .0 6 0. 29 0. 18 0. 18 7. 14 1. 76 s. d un ni i 34 24 m (2 sa t) +1 0s m 2. 14 -3 .5 1 50 .0 4 1. 64 1. 51 2a 14 .5 7 14 .5 8 18 .4 9 59 .5 3 40 .4 7 67 .9 7 0. 30 0. 15 0. 18 5. 75 2. 12 s. fo lg ne ri 34 30 m (2 sa t) +4 sm 0. 91 -1 .9 6 24 .4 5 2. 15 1. 36 2b 9. 71 15 .6 8 14 .1 0 56 .9 9 43 .0 1 75 .4 7 0. 24 0. 16 0. 14 6. 95 1. 52 s. lu sh an en si s 34 30 m (2 sa t) +4 sm 1. 27 -2 .2 6 29 .2 1. 78 1. 39 2a 12 .0 5 16 .4 5 14 .9 4 57 .9 3 42 .0 7 72 .6 1 0. 25 0. 16 0. 15 6. 72 1. 98 s. o ch ra ce a 34 28 m (2 sa t) +6 sm 1. 26 -2 .5 29 .2 2 1. 98 1. 36 2a 11 .4 6 19 .9 2 13 .7 1 57 .1 1 42 .8 9 75 .1 1 0. 23 0. 20 0. 14 9. 38 2. 28 126 yuming qian et al. size of the chromosomes varied from 0.91 μm (0.91–1.96 μm) in s. folgneri to 3.51 μm (2.14–3.51 μm) in s. dunnii. the total haploid chromosome length (thl) changed from 23.43 μm in s. hemsleyi (the thl value of s. yuana is 23.44 μm, nearly the same as s. hemsleyi) to 50.04 μm in s. dunnii. two species, s. folgneri and s. hemsleyi have both very small (≤ 1 µm) and small (> 1 µm and ≤ 4 µm) chromosome. the remaining taxa had only small chromosome. two satellites were obser ved in seven subgenus micromeles taxa, whereas no satellites in two subgenus aria species. karyotypes of the analyzed species exhibit a predominance of metacentric chromosomes with 2–10 submetacentric chromosomes detected in different taxon. figure 1. somatic chromosomes at the metaphase stage in root tip cells of sorbus taxa. as. alnifolia; bs. alnifolia var. angulata; cs. alnifolia var. lobulata; ds. dunnii; es. folgneri; fs. hemsleyi; gs. lushanensis; hs. ochracea; is. yuana. scale bar = 5 μm. 127chromosome counts and karyotype analysis of nine taxa in sorbus subgenera aria and micromeles (rosaceae) the karyotype asymmetry was assessed based on stebbins classification and 11 different quantitative indices (table 2). according to the symmetry classification of stebbins, s. alnifolia var. alnifolia and s. yuana were classified as category 1a, s. alnifolia var. lobulata and s. folgneri as category 2b, the other five taxa as category 2a. for the two species in subgenus aria, s. hemsleyi and s. yuana, the latter presented the most symmetrical karyotype of all the analyzed species as shown in scatter diagram (figure 3), which with the highest values in syi% and tf% and the smallest values in the other nine asymmetrical indices (table 2). two species in subgenus aria were more symmetrical than those taxa in subgenera micromeles based on two indices a1 and mca. in general, s. hemsleyi was more asymmetrical than s. yuana with two more submetacentric chromosomes and with values of asymmetrical parameters among the taxa in subgenus micromeles (table 2; figures 3). for subgenera micromeles, the seven analyzed taxa figure 2. haploid idiograms of sorbus taxa. red arrows indicate satellites. scale bar = 5 μm. 128 yuming qian et al. displayed considerable differences in karyotypic parameters (table 1). the two species: s. dunnii with the smallest values in syi% and tf% and the highest values in ask%, cvci, mca, a and a1; s. ochracea with the highest values in cvcl, a2, ai and di. the most asymmetrical karyotype was observed in s. ochracea according to the scatter diagram based on ai and di (figures 3b). however, the two species, s. dunnii and s. ochracea, presented the most asymmetrical karyotypes respectively according to different indices as shown in scatter diagram based on a1 and a2 (table 2; figures 3a, c, d). discussion the chromosome base number in sorbus is x = 17, and it is common to all members of maloideae, rosaceae. four ploidy levels (di-, tri-, tetraand pentaploid with figure 3. scatter plots of studied taxa based on karyotype parameters. aa1 versus a1; bai versus di; ccvci versus cvcl; dmca versus cvcl. 129chromosome counts and karyotype analysis of nine taxa in sorbus subgenera aria and micromeles (rosaceae) 2n = 34, 51, 68 and approximately 87, respectively) were reported in the genus (nelson-jones et al. 2002; bailey et al. 2008). only diploids in subgenera aria and micromeles were found from china in this and previous studies (sax 1931; baranec and murín 2003), though a large number of polyploidy species (tri-, tetra and pentaploids) appeared in subgenus aria in europe (sennikov and kurtto 2017). polyploidy is an important evolutionary mechanism in sorbus and it is particularly widespread in subgenus sorbus native to china (mcallister 2005; li et al. 2021). whether there are polyploids in subgenera aria and micromeles in china, especially in the mountainous area in southwest where a great quantity of polyploidy species were reported from subgenus sorbus, ploidy-level determination of more species and on more populations are required. sorbus subgenus aria is considered the most primitive and micromeles is more derived (yü and kuan 1963; phipps et al. 1990). however, the taxonomic delimitation between aria and micromeles is complex. these two subgenera are easy to distinguish morphologically: aria species with a persistent upper part of the hypanthium, and micromeles species with a deciduous calyx and distinct annular scar at the apex of fruit (yü and kuan 1963). however, micromeles is included within aria by robertson et al. (1991) and aldasoro et al. (2004) based on comprehensive morphological characteristics. a merge (campbell et al. 2007) or separate (zhang et al. 2017) of the two is supported by molecular evidence in different phylogenetic studies of maloideae. not only the subgeneric concept changed, the delimitation of species varied greatly between authors. for chinese native subgenera aria and micromeles, 20 species out of the total 31 species and 7 varieties recognized in flora of china (lu and spongberg 2003) were accepted by aldasoro et al. (2004) in the latest revision of subgenera aria and torminaria. in this study, the two subgenera could be distinguished easily by the existence of satellites. both the two species of subgenus aria with much small chromosomes had more symmetrical karyotype, as showed by mca and a (table 1). not the same as s. yuana, which with all the indices indicating its primitive, s. hemsleyi showed status of taxonomically complicated with values of some parameters among species of subgenus micromeles. s. hemsleyi was assigned to micromeles by yü and kuan (1963) and was assigned to aria by phipps et al. (1990). the inconsistent in values of asymmetry indices just strengthened its complex classification status or a support factor for the merge of the two subgenera need to be further studied. for the limited sampling (two species in aria and seven taxa in micromeles), the comparison of karyotype data could not solve the taxonomic problems related to the two subgenera. whether or which karyotype parameters in sorbus are useful for distinguishing subgenus and species, additional karyotype analysis of a larger number of species are needed. conclusion the chromosome numbers, karyotypes, idiograms and karyotype asymmetry degrees of nine chinese native taxa in sorbus subgenera aria and micromeles were investigated in this study. the chromosome numbers (2n = 2x = 34) of eight chinese endemic taxa were firstly reported. in general, the karyotypes of species in subgenus aria were quite symmetric than that in subgenus micromeles. acknowledgements we are grateful to zhongren xiong, yun chen, yang zhao, jing qiu, wan du, mingwei geng and qin wang for collecting samples; to dan chen, haiying peng and weiqi liu for their help during chromosome preparations. we also acknowledge the priority academic program development of jiangsu higher education institutions, jiangsu province, china (papd) for financial support. references aldasoro j.j., aedo c., garmendia f.m., de la hoz f.p., navarro c. 2004. revision of sorbus subgenera aria and torminaria (rosaceae-maloideae). systematic botany monographs. 69: 1–148. altinordu f., peruzzi l., yu y., he x.j. 2016. a tool for the analysis of chromosomes: karyotype. taxon. 65 (3): 586–592. bailey j.p., kay q.o.n., mcallister h., rich t.c.g. 2008. chromosome numbers in sorbus l. 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(bioanim) – center for scientific visualization, si – na vidmu 65, 4201 zgornja besnica, slovenia *corresponding author. e-mail: peter.firbas@gmail.com abstract. the aim of this study was to evaluate the cytotoxic and genotoxic potential of the wastewater (ww), the effectiveness of the treatment used by the wastewater treatment plant (wwtp) with sequential batch reactors (sbr) technology, and whether its final treated effluent (fte) can compromise the water quality of the river at the location where it is discharged. we focused our research on six examples. for analytical chemistry and allium metaphase (m) test all six samples were collected. of these, three are socalled biotechnological patterns (ww, ww after mechanical step treatment and fte), and three are natural river environmental patterns. for the micronucleus (mn) test, fish specimens were collected from three sites in the river kamniška bistrica. the first two sites locations are up and down the fte outlet. results from these areas were compared to the third site (not polluted) reference site, the so-called natural negative control sector drinov rob. complementary study with analytical chemistry and biological tests shows that the treatment effect sbr in the domžale-kamnik central wwtp carried effectively proved to be efficient for the removal of the cytogenotoxic substances in treated effluent and consequently in aquatic environment. the upgraded and improved domžale-kamnik central wwtp has a very effective aerobic tertiary treatment stage. biological rate of achievement by such sbr technology has shown excellent results. we could not find any parameters than would show the final treatment effluent (fte) water to exceed the maximum permissible doses (mpds). keywords: chemical analysis, phytotoxicity, genotoxicity level, environmental monitoring, water quality. introduction the wastewater (ww) level is increasing with intensive anthropogenic activities like industrial, agricultural and urban development (kalia et al. 2018). ww can be characterized as complex chemical mixtures of individual organic and inorganic substances (fijalkowski et al. 2017). all such 120 peter firbas, tomaž amon chemicals can be potential sources of pollution and may result in different ecotoxicological effects (lyubenova et al. 2012), that can affect human health (pecol 2018). however well constructed and well working wastewater treatment plant (wwtp) can solve these problems. even better solution is if we combine this with various techniques like ecoremediations, such as constructed wetlands (firbas and amon 2013), processes in ecological drainage ditches (kumwimba et al. 2017), vermicomposting process the collective action of earthworms and microbes (bhat et al. 2018), associated with stabilization pond (hara and marin-morales 2017), and the most various attractive coremediation technologies. just physical and chemical measurements cannot tell us the whole picture of the degree of the toxicity of the pollutant (firbas 2015; firbas and amon 2017). the concentration of the toxic agent is not always proportional to the danger to human health. environmental monitoring detected potential environmental genotoxic xenobiotics and is based on the use of vascular (higher) plants and aquatic vertebrates (grisolia et al. 2005; oriaku et al. 2011). both onion plant and fish are sensitive indicators for assessment of environmental pollution (pathiratne et al. 2015; batista et al. 2016; hemachandra and pathiratne 2017). aquatic vertebrates accept foreign substances, e.g. xenobiotics through the epithelium, which accumulate in the body and may induce various potential toxic effects (walker et al. 2012). these xenobiotics produce multiple consequences at organism, population, community and ecosystem level, affecting organ function, reproductive status, population size, species survival, and thus biodiversity (bolognesi and hayashi 2011). in order to monitor the health of aquatic organism, biomarkers have been used as effective tools in environmental risk assessment (ghisi et al. 2016). the micronucleus (mn) assay is one of the most widely used genotoxicity biomarkers in aquatics organisms, and is used for in situ monitoring of water ecosystems assessing clastogenic and aneugenic events in different cell types (alsabti and metcalfe 1995; kirsch-volders et al. 2011). mn are cytoplasmic chromatin-containing bodies formed when centric and/or acentric chromosome fragments or whole chromosomes that are not included in the main nuclei after cell division. onion a. cepa l. is widely recognized and useful as excellent genetic model to detect environmental xenobiotics (leme and marin-morales 2009; firbas 2011; bakare et al. 2012;karaismailoglu 2015; verma et al. 2016; karaismailoglu 2017; bonciu et al. 2018; makar et al. 2020, şuţan et al. 2020). this plant can absorb xenobiotics across the leaf cuticle through stomatal and root epidermis. root growth inhibition and adverse effects on chromosomes, for example chromosome break provide on indication of likely cytotoxicity and/or genotoxicity. (dietrich et al. 2001; bonciu et al. 2018). the relatively large (2n = 16) and karyotype diverse chromosomes are very appropriate for the detection of morphological changes. in vivo allium m test (firbas and amon 2013; 2014) used for study of the specific morphology (structure) exclusively of metaphase chromatids and chromosome damage. furthermore, the chromosome and chromatid morphology is easily altered by chemical and natural compounds. by using biological (genotoxic) tests, we can ascertain those responses of the tested onion plant a.cepa, which result in eventual damage to its genetic material (chromosomes) regardless of the tolerance limits that can be caused by various contamination samples within an environment. in regard to the universality of the living organisms genetic codes, the research results are transferable (applicable) to human beings (nefic et al. 2013). several higher plant systems were evaluated bioassays with plant root meristematic cells such: coriandrum satuvum l. (pramanik et al. 2018), bidens laevis l. (pérez et al. 2011), drimia polyantha (blatt. &mccann) (daphedar and taranath 2018), lactuca sativa l. (moreira et al, 2020), helianthus annuus (karaismailoglu et al. 2013), elodea canadensis (zotina et al. 2015), nigella sativa (el-ghameryand mousa 2017) and trigonella foenum graecum l. (mahapatra et al. 2019) possessed suitable cytological characteristics for cytotoxicity and genotoxicity testing. the measured biological effects of some water samples appear related to the physical characteristics. therefore, genotoxicity assays should be included, along with conventional chemical analysis, in water quality monitoring programs (radić et al. 2010; singh et al. 2014; etteieb et al. 2016). so many ecotoxicological studies focus on the assessment of physical and chemical environmental parameters and biological responses of organisms (baldantoni et al. 2018; wijeyarante and wadasinghe 2019). the method of combining bioassays with the analytical chemistry and monitoring as well as screening represents a powerful approach to identify organic and inorganic pollutants the main toxic components in complex mixtures of treated wastewater (etteiab et al., 2016; firbas and amon, 2017). in this paper we studied the genotoxicity level (gl) sources (malakahmad et al. 2017) of the river kamniška bistrica both upstream and downstream of the final treated effluent (fte) of the waste water treating plant for the cities domžale-kamnik. the gl levels were determined in two ways. firstly with the fish periph121use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater eral blood erythrocytes mn test in the nine freshwater autochthonous salmonid and cyprinid fish species and allium metaphase (m) root type cell test and secondly with the allium metaphase (m) root type cell test. this combination represents a good assessment of both physical (e.g. temperature, oxygen, ph, …) and chemical (pesticides, metals, …) parameters. the work described here has been done in the period 2017-2019 and is based on the data from the renovated domžale-kamnik central wwtp (it was renovated in 2016). however in our article (firbas in amon 2017) we monitor this wwtp from 2013-2014 that is before its renovation. materials and methods description of the water system of the river kamniška bistrica the river kamniška bistrica (kb) is a left tributary of the river sava, which is a right tributary to danube river. it is 32.8 km long and has a torrential character from its spring in the alps to its confluence with the sava river. the river is spread over 535 km2 of mountain and plain area (figure 1). because of the construction and housing activities the river has been regulated into a moderately narrow channel running through the towns kamnik and domžale. in other areas the river is mainly untouched. some sources mention that there were probably close to 200 km of canals excavated and buried again in different times in history (vahtar 2006). today, there are over 40 km of active mill streams along the river kamniška bistrica and most of them are still in use. the flow of the river varies from 2 to 10 m3 per second. in one year the river receives 7 million m3 treated water from the domžale-kamnik central wwtp (hvala et al 2002). domžale-kamnik central wwtp, slovenia before the 2016 upgrade is central the domžalekamnik central wwtp (latitude 46°07’n; longitude 14°36’e) is a conventional two-stage activated sludge plant (asp) designed to remove organic matter from the wastewater, built in 1980 (hvala et al. 2002). the capacity of the plant is 200,000 pe (population equivalent) with an average daily inflow of approximately 20.000 m3/day. the plant influent consists of 45% municipal and 55% industrial wastewater (hvala et al. 2002). upgrade in year 2016 include construction of a new aerobic biological rate of achievement of tertiary treatment by sbr technology and the construction of the input object for the reception of large quantities of waste water and appropriate mechanical pre-treatment, which will increase operational safety. after upgrading the wwtp fourth largest system for wastewater treatment in slovenia, and ensuring the quality parameters of treated water for discharge into the watercourse, the river kamniška bistrica. the capacity of the upgraded figure 1. study map of models wwtp including allium m test and pisces mn test. samples and fishes were taken from river kamniška bistrica. legends: cbif: cart board industry factory with wwtp, wwtp: wastewater treatment plant; ww: wastewater; wwtw: wastewater treatment work; fte: final treated effluent; php: physical-chemical parameter; chp: chemical parameter. 122 peter firbas, tomaž amon wwtp is 149,000 pe, which means that it will accept the waste water of all residents in the reception area and other waste water (http://www.ccn-domzale.si/index. php/en/wastewater-treatment/plant-upgrade-project. accesed 13. august 2020). collection of water samples water type definitions natural samples: river fresh water. the study area, which is kamniška bistrica river spatially lies between latitude 46°19’n and 46°04’n and longitude 14°34’e and 14°37’e (figure 1). biotechnological samples: samples for all different stages of ww treatment. wastewater: complex mixtures of municipal, industrial (pharmaceutical, textile, food processing, dyes-paints, timber industry, laundry textile), and rain water so called inflow. inflow to domžalekamnik central wwtp has a heterogeneous composition composed of municipal, industrial and rain water. ww treatment after mechanical stage: sand trapping and solid separation. final treated effluent: treated water from domžale-kamnik central wwtp so called outflow. sampling locations in relation to the wwtp position sampling locations and biological tests according to the location of the wwtp are summarized according to firbas and amon (2017): 100 m or more upstream before the inlet into the wwtp (allium m test, pisces mn test), influent waste water in the wwtp (allium m test), wastewater after solid separation (allium m test), effluent water from sbr (allium m test), 100 m or more downstream after the outlet from the wwtp (allium m test, pisces mn test), the detailed description of sampling locations (figure 1) i. kamniška bistrica – drinov rob (as a negative control in the natural environmental pattern) ii. kamniška bistrica – v illage študa (950 m upstream central wwtp – discharge point) iii. ww inflow iv. ww after solid separation (ww treatment after mechanical stage) v. fte outflow from sbr vi. kamniška bistrica – village bišče (1750 m downstream central wwtp) terms of monitoringand sampling first sampling: allium m test and physical analytics 16. 02. 2017; electric fishing for in situ mn test 16. 02. 2017; sample ww and fte is 24 h on average from 02/14/2017, 8:00 to 15/02/2017, 8:00. second sampling: allium m test and physical analytics 18. 10. 2017; electric fishing for in situ mn test 24. 10. 2017; sample ww and fte 24 h on average from 17/18 oct. third sampling: in vivo allium m test, electric fishing for in situ mn test and physical analytics 25. 05. 2018;sample ww and fte is 24 h on average from24/25 may. fourth sampling: in vivo allium m test, electric fishing in situ mn test and physical analysis13. 08. 2019; sample ww and fte is 24 h on average from21/22 august. fifth sampling: in vivo allium m test, electric fishing in situ mn test and physical analysis 23. 07. 2020; sample ww and fte is 24 h on average from 22/23 july. we sampled on three experimental sites. on each of those the samples were taken four times. we have done the measurements february 2017, in october 2017, in may 2018, and the fourth august 2019, and fifth (last) sampling was done in july 2020. three experimental sites were chosen, namely area študa and area bišče, which were compared with sites drinov rob of no sewage influence, as the control area (figure 1). physical and chemical analyses for the measuring of water physical and chemical analyses we collected six samples. here three are natural river samples and three biotechnological samples. samples of river water and biotechnological samples were collected from the sites, stored in bottles with thermostable boxes and transferred to the laboratory. chemical parameters including: the metal/metalloid and organic component varied depending on the industrial profile. physical parameters including: temperature, alkalinity (ph), electrical conductivity (ec), total suspended solid (tss), dissolved oxygen (do), nutrients (nitrogen, phosphor),kjeldhal nitrogen (kn), chemical oxygen demand (cod) and biochemical oxygen demand (bod5)were determined in accordance with standard analytical methods (apha, 2012). water physical parameters such as temperature, ph, ec, do and tss were measured in situ using hach electrodes; tss by (with) gravimetry; nitrate n and nitrite n by segmented flow analysis (sfa); ammonia 123use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater by ion selective electrode (ise) or spectrophotometry; and kjeldahl n (with the composite method digestion – destillation – titration). total phosphorus, total nitrogen and cod were determined with spectrophotometry and bod5 with volumetry (vol) in incubation bottles. concentrations of metals (zn, al, cu, cd, co, cr, ni, ag, pb and fe) were analyzed using inductively coupled plasma-mass spectroscopy (icp-ms). benzene, toluene, ethylbenzene, xylene (btex) were analyzed using gass chromatography (db-5 60x0.53x3 columns) – flame ionizator detector(gc-fid).absorber organic halogens (aox) and cyanide were analyzed using liquid chromatography with tandem mass spectrometry (lcms/ms). chloroalcane (c10-c13) were analyzed using gass chromatography with electron capture detector (gc-ecd). nonylphenol and nonylphenol ethoxylate were analyzed using lc-ms/ms. di(2-ethylhexil) phthalate (dehp) were analyzed using gc (5-ms columns 30m x 0.25mm x 0.25µm) with electron capture detector (ecd). index mineral oil were analyzed using gc with flame ionization detector (fid). test organisms used as bioindicators plant: onion a. cepa, (stuttgarter riesen). small onion bulbs of the same uniform size, weighing about 3 – 3.5 g, were denuded by removing the loose outer scales and scraped so that the root primordia were immersed into the different tested liquids. fish: salmonids: grayling (thymallus thymallus), brown trout (salmo trutta fario); cyprinids: european minnow (phoxinus phoxinus), european bullhead (cottus gobio), common bardel (barbus barbus), mediteranean bardel (barbus meridionalis), european chub (leuciscus cephalus), perch (perca fluviatilis), nase (hondrostoma nasus nasus). seven cyprinids and two salmonids fish species inhabiting european freshwater ecosystems, were evaluated for their as in situ pollution biomarkers using the micronucleus test in peripheral blood erythrocytes. as the indicators fish species were used because of their ecological significance. fish species 150 200 g were collected from natural environment in the three locations. onion plant allium m assay the tests were done with the allium m or allium chromosome damage (csd) test and show the degree of genotoxicity by observing the aberrations of the exclusively metaphase chromosomes of the plant a. cepa that are evoked by genotoxic substances in the polluted water (firbas and amon, 2013; 2014). five onion bulbs are left to grow in the sample water for 72 hours. then first the macroscopic morphological parameters are observed – the length of roots (showing the general toxicity), their shape, number, color and degree of malformation. the genotoxicity level (gl) on the microscopic observation is a general term referring to alternation to the gross structure or content of chromosome damage by exposure to toxic agents (malakahmad et al. 2017). gl is defined by the percentage between all the metaphase cells and the cells with their chromosomes damaged. 200 random chosen metaphase cells (with well-recognized chromosomes) originate from the sample composed of ten root apex cells taken from five onion bulbs two roots from each onion bulb (firbas and amon, 2013). chromosome preparations were set up from root meristems containing actively growing cell by the following method: developing root with bulbs were pretreated with 0.1 % aquatic solution of colchicine for 3 hours at 21 °c. after washing in distilled water for 20 min the terminal developing roots of 2 mm length were fixed for 1h in methanol:propionic acid mixture (3:1 or 1:1). then they were macerated and stained in order to obtain a cellular suspension. this sample was stained with 0.5 % aceto-carmine for 4-5 minutes at 60 °c without hydrolysis, and squashed in aceto-carmine (firbas and al-sabti, 1995). the optical microscope used in the investigation was the olympus – bx 41 (japan) with the photo system pm 10 sp, typical magnifications used were 400 x and anisole-immersion 1.000 x. onion (a. cepa) has 16 (2n = 16) monocentric chromosomes. the possible aberrations seen at metaphase are: chromosome break, chromatide break and centromere break (firbas and amon 2014). the cell is called aberrant if at least one chromosome gets damaged. sometimes 4 to 8, or even up to all 16 chromosomes in the chromosome set are damaged, with dicentric and ring chromosomes (firbas 2015). electrofishing collected methods salmonid and cyprinid fish inhabiting european freshwater ecosystems were evaluated for their use as in situ pollution biomarkers using the micronucleus test in peripheral blood erythrocytes. fish were collected with electrofishing (ef) which is a common professional survey method used to sample fish population to determine abundance, density, and species composition. when performed correctly, ef results in no permanent harm to fish, which are returned to their natural environment only two minutes after being caught. 124 peter firbas, tomaž amon fish mn assay for the mn assay, peripheral blood samples were obtained from the gill region. blood was smeared immediately on clean grease free microscope slides, air dry for 6 hours and the fixed in absolute methanol for 18-20 minutes, the prepared slides were left to air dry at room temperature. the blood smears were stained with 5 % giemsa in sorenson buffer solution for 7 minutes. after washing with distilled water and left to air dry at room temperature, the slides are then ready for microscopy. since giemsa solution stains the nuclear material much darker than the cytoplasmic material, the mn were readily visible with anisol 1.000 x next to the normal nuclei and a micronucleus of the erythrocyte cells. erythrocytes, 10.000 ± 200 per specimen, were analyzed to determine the frequency of cells with one or two micronuclei and then calculated to the number of mn per 1000 erythrocytes (mn/1000 e). statistics calculation values are the mean of five replicates with standard deviation (±sd). statistically established significant differences among the investigated samples are confirmed by the statistical calculation of paired data analysis using the two-way fisher’s exact test, which gives the p-value property between pairs of data calculated for a 2x2 contingency table (agresti 1992). in the 2x2 frequency tables the statistical results as shown by the p-values sometimes do not show the significance, the addition of allium and micronucleus test clearly show the difference with other methods leads us to suggest that the picture could be more complex (firbas and amon 2013; 2017). results chemical parameters the chemical analysis of the fte and maximum permissible concentrations (mpc) standard are presented in table 1. we could not find any parameters that would show the fte water worse in quality than the original river water. the fte does not additionally burden the river kamniška bistrica. physical parameters in addition to standard parameters (cod, bod5, tss) we included also the fundamental physical parameters nitrate (no3—n), nitrite (no2—n), kjeldhal— n, ammonia (nh4—n), phosphate (po4—p), electrical conductivity and ph. we measured the river kamniška bistrica at three locations from its location drinov rob to the location called bišče (figure 1). it is this sector where the treatment water from the wwtp enters the river kamniška bistrica. this wwtp reduces pollutants to a level that nature can successfully process further. detailed analysis of water physical parameters here are presented in table 2, 3, 4, 5 and 6. general toxicity phytotoxicity the results of the general toxicity are shown in combined tables 2, 3, 4, 5, 6 and figure 2. the location drinov rob is taken as the negative control. general toxicity of the river samples they have more or less the same root length of the test plants (p > 0.05). all three of the river water samples shows longer roots and lesser general toxicity than the ww or fte (p < 0.05). the fte show longer roots and lesser general toxicity than the untreated wastewater (p < 0.01). table 1.chemical analysis of the final treated effluent (fte) and maximum permissible concentrations (mpc) standard. the domžale-kamnik central wwtp with an upgrade of a new aerobic biological rate of achievement of tertiary treatment by sbr technology treating has been shown to be very effective. parameter and unit mpc* fte zn/zink (mg/l) 2.0 0.0414 total cyanide (mg/l) 0.5 0.010 aox/absorber organic halogens (mg/l) 0.5 0.150 chloralcanes c10-c13 (mg/l) 0.04 0.0035 di(2-ethylhexil) phthalate (dehp) (mg/l) 0.13 0.0004 nonylphenol and nonylphenol ethoxylate (mg/l) 0.03 0.00023 al/aluminium (mg/l) 3.0 0.047 cu/cupper (mg/l) 0.5 0.010 cd/cadmium (mg/l) 0.025 0.001 co/cobalt (mg/l) 0.03 0.0010 cr/cromium total (mg/l) 0.5 0.010 ni/nicel (mg/l) 0.5 0.010 ag/silver (mg/l) 0.1 0.010 pb/lead (mg/l) 0.5 0.010 fe/iron (mg/l) 2.0 0.150 index minerals oils (mg/l) 5.0 0.100 btx – benzene, tuolene, xylene (mg/l) 0.1 0.03 benzene (mg/l) 0.1 0.03 toluene (mg/l) 0.1 0.03 ethylbenzene (mg/l) 0.1 0.03 m,pxylene (mg/l) 0.1 0.03 o-xylene (mg/l) 0.1 0.03 *mpc: maximum permissible concentration – legistation (official leaf republic of slovenia: 64/2012). 125use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater genotoxicity level the results of the genotoxicity level (gl) expressed in percentage points (%), are shown in the combined tables 2 to 6. the treated outflowing water is significantly less genotoxic than the inflowing (polluted) water. so the gl decreased from 34.5 % lowers on 11 % in the year 2017 (p = 6.0-8 < 0.00001). the wastewater first undergoes the mechanical treatment. afterwards the genotoxic level is significantly lower (shown by less damaged chromosomes) while the cytotoxic level remains the same (the lengths of tested allium bulbs roots are not significantly changed). the kamniška bistrica river in k2 sector (drinov rob) shows zero (figure 3) or not more than one damaged chromosome in a chromosome set (figure 4). incoming wastewater is highly genotoxicity. typically at least 4-10 chromosomes (figure 5), sometimes even all chromosome get damaged (figure 6). also dicentric and ring chromosomes can appear. the treated wastewater from the domžale-kamnik central wwtp and kamniška bistrica river in k4 sector (študa and bišče) shows a much lesser degree of genotoxicity – typically two chromosomes, rarely four are damaged. micronucleus (mn) the results of the mn studies are shown in tables 2 to 5. peripheral blood erythrocytes with mn are shown in figure 7. three experimental sites were chosen, namely, drinov rob, študa and bišče. sampling was carried out in february 2017, october 2017, may 2018, and table 2. united parameters physical quantities;cytological effects of the investigated samples survey – the genotoxicity level (damage to chromosomes) and general toxicity phytotoxicity (root length inhibition) on the test onion plant a. cepa and piscesmicronuclei (mni)frequencies in peripheral blood erythrocytes of river fish. first sampling february 2017 parameter unit sample sites k2 – drinov rob (negative control) k4 – študa ww – wwtp after mechanical step fte – wwtp k4 – bišče physical analysis water temperature 0c 4.9 6.1 9.4 10.1 7.5 ph 8.2 8.0 7.6 7.7 7.6 6.7 electrical conductivity µs/cm 226 372 470 dissolved oxygen mg/l 11.78 10.38 10.98 to subside 1 h ml/l 0 0 0 0 to subside 2 h ml/l 0 0 14 3 0.1 0 tss 1µm mg/l 2 4 236 167 13.4 3 ammonia nh4—n mg/l 33 53 29 kjeldahl n mg/l 2.5 2.5 50 71 32 2.7 nitrate no3—n mg/l 0.8 1.3 2.2 7.4 2.0 nitrite no2—n mg/l 0.75 0.50 total n mg/l 34.0 6.3 total p mg/l 0.5 0.5 3,67 7.9 0.38 cod mg/l 5.0 6.0 551 595 33 7.4 bod5 mg/l 3.0 3.0 347 240 5 3.0 allium metaphase (m) test phytotoxicity mm 34±2.7 33±1.5 11.0±1.2 11.0±1.1 31±1.7 34±2.4 genotoxicity % 2.50±0.3 4.50±0.5 34.50±2.6 21.50±1.3 11.0±0.9 4.50±0.5 micronucleus (mn) pisces test leuciscus cephalus ‰ 0.90 ±0.33 0.98 ±0.61 thymallus thymallus ‰ 1.29 ±0.35 0.82 ±0.34 phoxinus phoxinus ‰ 0.78 ±0.45 0.74 ± 0,31 salmo trutta fario ‰ 0.19 ±0.02 2.00 ±0.65 cottus gobio ‰ 0.44 ±0.11 4.50 ±1,50 1.36 ± 0.77 legend. kb: kamniška bistricariver, k2, k4: fishing area, ww: wastewater, fte: final treated effluent, wwtp: central domžale-kamnik wastewater treatment plant, %: chromosome damage (csd) per 100 cells, gl: genotoxicity level, ‰:micronucleiper 1000 erythrocytes. 126 peter firbas, tomaž amon august 2019. all fishes, regardless of species composition, show lower values (0.32-1.1 mn/1000 erythrocytes (e) in the bišče sector than in the študa sector (0.78-2.01 mn/1000 e), which is the most relevant for the species of european bullhead (cottus gobio). the lowest mn abundance (0.0-0.2-0.34 mn/1000 e) is in the sector drinov rob. the increased appearances of micronuclei in fish blood erythrocytes shows that the fish is living in water of higher genotoxic level. a significant increase in the number of mn in specimens of cottus gobio at the kamniška bistrica river is the result of the discharge of the waste water of the cart board industry factory (cbif) in the sector študa. we have monitored the frequencies of mn from 2017 to 2020 and saw that the frequency falls from year to year what points to cleaner water. from the results of these researches we conclude that the fte from the domžale-kamnik central wwtp does not adversely affect the quality watercourse of the kamniška bistrica river. kamniška bistrica is already partially contaminated above the outflow (the študa sector) since the measured values are 2 to 5 times higher than in the sector drinov rob. measurements of the genotoxicity level with allium m test and physical parameters that support biological parameter implement a good basis for the risk assessment studies and environmental quality standard – ecological status (eqs-es) (firbas 2015; firbas and amon 2017). in this article, we add the results of the pisces mn tests (table 7). table 3. united parameters physical quantities;cytological effects of the investigated samples survey – the genotoxicity level (damage to chromosomes) and general toxicity phytotoxicity (root length inhibition) on the test onion plant a. cepa and pisces micronuclei (mni) frequencies in peripheral blood erythrocytes of river fish. second sampling october 2017 parameter unit sample sites k2 – drinov rob (negative control) k4 – študa ww – wwtp after mechanical step fte – wwtp k4 – bišče physical analysis water temperature 0c 7.0 12.2 16.2 17.5 12.7 ph 8.2 8.1 7.6 7.6 7.1 7.9 electrical conductivity µs/cm 218 415 1301 461 dissolved oxygen mg/l 11.39 11.68 10.99 to subside 1 h ml/l 0 0 0 0 to subside 2 h ml/l 0 0 13 1,9 0.05 0 tss 1µm mg/l < 2 < 2 380 177 5.0 2 ammonia nh4—n mg/l 0.05 < 0.015 31.90 38 0.33 < 0.015 kjeldahl n mg/l 24.40 55.1 2.29 nitrate no3—n mg/l 0.79 1.1 0.70 4.99 2,4 nitrite no2—n mg/l 0.32 0.13 total n mg/l 47 6.2 total p mg/l < 0.05 < 0.05 7.00 7.5 0.28 < 0.05 cod mg/l < 5.0 7.3 638 599 26.1 < 5.0 bod5 mg/l < 3.0 < 3.0 240 6 < 3.0 allium metaphase (m) test phytotoxicity mm 34.0 ±2.9 33.0 ±2.2 11.0 ±1.2 13.0 ±1.4 31.0 ±2.9 34.0 ±2.7 genotoxicity % 2.50 ±1.1 4.50 ±1.3 34.50 ±2.1 19.50 ±1.3 9.0 ±1.3 4.50 ±1.1 micronucleus (mn) pisces test leuciscus cephalus ‰ 4.31 ±1.55 2.98 ±0.51 thymallus thymallus ‰ 1.77 ±0.23 0.7 ±0,21 chondrostoma nasus ‰ 1.00 ±0.20 1.36 ±0.34 salmo trutta fario ‰ 0.20 ±0.1 cottus gobio ‰ 0.32 ±0.1 2.34 ±0.39 legend. kb: kamniška bistricariver, k2, k4: fishing area, ww: wastewater, fte: final treated effluent, wwtp: central domžale-kamnik wastewater treatment plant, %: chromosome damage (csd) per 100 cells, gl: genotoxicity level, ‰:micronuclei per 1000 erythrocytes. 127use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater discussion the present work has been done in order to evaluate the genotoxic effects of ww and fte on different sites in river kamniška bistrica using the physical and chemical analysis on allium m and pisces mn assays. ww treatment is an important process of considerable significance for the environment. in the eco-genotoxicology the samples are subjected to the physical and chemical analysis as well as to the genotoxical tests (radić et al. 2010; matsumoto et al. 2006; grisolia et al. 2009; okonkwo et al. 2011; bakare et al. 2012; polard et al. 2011; akpoilih 2012; firbas and amon 2017; francisco et al. 2019; kaur et al. 2020). these two methods are complementary. as the water leaves the wwtp it flows into the river and again becomes the integral part of the ecosystem (walia et al. 2013). our results show how important is the effectiveness of this wwtp along with the continual monitoring including the study of all necessary parameters (bolognesi and hayashi 2011; radić et al. 2010; raisuddin and jha 2004; herrero et al. 2012; tabres et al. 2011; bagatini et al. 2009; galindo and moreira 2009). domžale-kamnik central wwtp after the upgrade in 2016 represents the superb state of the technology of modern wastewater treatment in the world and highquality clean waste water and achieves a high cleaning effect. the modernization also included additional system comprises three main process blocks: (i) a new inlet that complements the existing mechanical stage, table 4. united parameters physical quantities;cytological effects of the investigated samples survey – the genotoxicity level (damage to chromosomes) and general toxicity phytotoxicity (root length inhibition) on the test onion plant a. cepa and piscesmicronuclei (mni) frequencies in peripheral blood erythrocytes of river fish. third sampling may 2018. parameter unit sample sites k2 – drinov rob (negative control) k4 – študa ww – wwtp after mechanical step fte – wwtp k4 – bišče physical analysis water temperature 0c 7.8 12,1 15.7 17.8 14.5 ph 8.2 8.08 8.2 7.2 7.8 electrical conductivity µs/cm 193 287 338 dissolved oxygen mg/l 11.53 10.66 9.92 to subside 1 h ml/l 0 0.1 < 0.1 to subside 2 h ml/l 0 0.2 15 0 < 0.1 tss 1µm mg/l 14.3 200 37.6 ammonia nh4—n mg/l 0.04 31 39 1.34 0.016 kjeldahl n mg/l (< 2.5) 0.28 (< 2.5) 0.37 54.2 (< 2.5) 0.59 nitrate no3—n mg/l 0.7 0.97 1.48 nitrite no2—n mg/l total n mg/l < 0.05 0.97 42 5.6 0,094 total p mg/l 6.95 7.0 0.603 cod mg/l < 5 7.6 445 467 47 10.8 bod5 mg/l < 3 < 3 240 295 5.0 < 3 allium metaphase (m) test phytotoxicity mm 36 ±3.1 34 ±2.8 11.0 ±1.2 12.0 ±1.1 33 ±2.3 34 ±2.5 genotoxicity % 2.90 ±1.2 5.50 ±1.3 28.0 ±2.2 12.30 ±1.1 7.0 ±1.2 4.50 ±1.2 micronucleus (mn) pisces test leuciscus cephalus ‰ 0.81 ±0.23 perca fluviatilis ‰ 0.78 ±0.19 barbus meridionalis ‰ 1.78 ±0.45 1.1 ±0.11 barbus barbus ‰ 0.75 ±0.21 salmo trutta fario ‰ 0.15 ±0.10 0.82 ±0.19 cottus gobio ‰ 0.95 ±0.19 0.32 ±0.21 legend. kb: kamniška bistricariver, k2, k4: fishing area, ww: wastewater, fte: final treated effluent, wwtp: central domžale-kamnik wastewater treatment plant, %: chromosome damage (csd) per 100 cells, gl: genotoxicity level, ‰:micronuclei per 1000 erythrocytes. 128 peter firbas, tomaž amon (ii) a new aerobic biological stage with advanced sbr technology with anaerobic selector for partial biological phosphorus removal and a new de-ionization process, (iii) the existing anaerobic biological stage with the production of biogas and cogeneration (http://www.ccndomzale.si/index.php/en/wastewater-treatment/plantupgrade-project). as the environmental discharge standards are getting more advanced, the traditional (continuous flowbased) ww treatment process faces severe challenges. it has become inevitable to include tertiary treatment units for nutrient removal from ww. sbrs due to its operational flexibility and excellent process control possibility are being extensively user for the treatment of ww which nowadays is fast becoming contaminated with newer and more complex pollutants (dutta and sarker, 2015). some wwtp with sbr may use additional step such as nitrogen or phosphorous removal as well as biological nutrient removal. this third and last step in the basic wastewater management system is mainly comprised of removing phosphates and nitrates (saito et al. 2004). nitrogen and phosphorous have become the key factors leading to eutrophication of receiving water. while achieving simultaneous nitrogen and phosphorous removal, biological methods play an important role in treating municipal and/or industrial wastewater such as sbrs (jungles et al. 2014). the character of w ws eff luents varies greatly, dependent on the nature of the specific industry involved, both in terms of the likely bod5 loading of table 5. united parameters physical quantities;cytological effects of the investigated samples survey – the genotoxicity level (damage to chromosomes) and general toxicity phytotoxicity (root length inhibition) on the test onion plant a. cepa and pisces micronuclei (mni) frequencies in peripheral blood erythrocytes of river fish. fourth sampling august 2019 parameter unit samples sites k2 – drinov rob (negative control) k4 – študa ww – wwtp after mechanical step fte – wwtp k4 – bišče physical analysis water temperature 0c 8.5 19.1 19.3/19.6* 21.4/21.6* 15.9 ph 8.2 8.2 8.0/8.2* 7.7 7.2/7.3* 7.6 electrical conductivity µs/cm 211 413 499 dissolved oxygen mg/l 11.85 9.15 8.69 to subside 1 h ml/l 0 0 0 to subside 2 h ml/l 0 0 13 0,7 < 0.05 0 tss 1µm mg/l 2 4,1 290 72.5 < 2 18.3 ammonia nh4—n mg/l 0.015 0.032 29.6 26.8 < 0.3 0.13 kjeldahl n mg/l nitrate no3—n mg/l 0.71 1.1 2.7 nitrite no2—n mg/l total n mg/l 6.48 0.84 total p mg/l 0.05 0.05 50 5.4 7.7 0,06 cod mg/l 5 12.1 575 243 < 30 7.8 bod5 mg/l 280 < 5 allium metaphase (m) test phytotoxicity mm 36 ±3.1 34 ±3.0 12 ±0.9 12 ±0.7 34 ±2.5 36 ±2.7 genotoxicity % 3.0 ±1.3 5.50 ±1.2 29.0 ±1.9 11.0 ±1.1 6.50 ±1.3 4.50 ±1.2 micronucleus (mn) pisces test leuciscus cephalus ‰ 0.75 ±0.31 0.50 ±0.23 thymallus thymallus ‰ 1,21 ±0.22 1.14 ±0.19 phoxinus phoxinus ‰ 0.85 ±0.11 barbus meridionalis ‰ 1.06 ±0.29 salmo trutta fario ‰ 1.22 ±0.10 cottus gobio ‰ 0.31 ±0.17 0.96 ±0.21 1.52 ±0.27 legend. kb: kamniška bistricariver, k2, k4: fishing area, ww: wastewater, fte: final treated effluent, wwtp: central domžale-kamnik wastewater treatment plant, %: chromosome damage (csd) per 100 cells, gl: genotoxicity level, ‰:micronuclei per 1000 erythrocytes. 129use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater any organic components and the type of additional contaminants which may also be present. accordingly, the chemical industry may offer wws with high cod and rich various toxic compounds is another high bod5 that effluent contains (evans and furlong 2011). parameters of the cod, bod5 and tss properties are the key parameters for the standardized monitoring of the cleaning process in the wwtp and at the same time they show the how much the environmental picture gets modified after mixing with the effluent of the wwtp. the correlation between cod, bod5 and tss properties is linearly proportional to the results obtained from the genotoxicity tests (firbas and amon 2013). test systems need to be developed on the basis of criteria that allow a realistic assessment of gl and are of major ecological importance in environmental screening and monitoring at the cell, organism, population and ecosystem levels. currently, clastogenic and/or aneugenic bio-marker so called mn and csd are most frequently and trustworthy for genotoxicity testing in aquatic environments. many toxic and potentially toxic chemical substances, some of natural origin and others due to human activities, are released into the environment daily (obiakor et al. 2012). it has been shown that the chemical analysis alone is not enough to assure that the effluent water is really clean. to protect human and ecosystem health, it is necessary to perform also the biological analysis and to develop sensitive assays and to identify responsive cells and species and their life stages (raisuddin and jha 2004). table 6. united parameters physical quantities; cytological effects of the investigated samples survey – the genotoxicity level (damage to chromosomes) and general toxicity phytotoxicity (root length inhibition) on the test onion plant a. cepa and pisces micronuclei (mni) frequencies in peripheral blood erythrocytes of river fish. fifth sampling july 2020 parameter unit sample sites k2 – drinov rob (negative control) k4 – študa ww – wwtp after mechanical step fte – wwtp k4 – bišče physical analysis water temperature 0c 8,3 15.0 15.0 ph 7,9 8,2 7,5 7,7 7,8 7,7 electrical conductivity µs/cm 210 362 812 908 871 438 dissolved oxygen mg/l 11,02 9,3 9,07 to subside 1 h ml/l 0 0 16 1,2 0 0 to subside 2 h ml/l 0 0 15 1,5 0 0 tss 1µm2 mg/l ammonia nh4—n mg/l 0,1 0,06 19,7 10,6 0,3 0,06 kjeldahl n mg/l <2,5 2,5 37,9 30,1 2,5 2,5 nitrate no3—n mg/l 0,58 0,94 0,36 0,34 6,06 1,8 nitrite no2—n mg/l total n mg/l total p mg/l 0,026 0,075 6,6 3,9 0,97 0,12 cod mg/l <5 11,1 534 125 22,2 9,1 bod5 mg/l <3 <3 255 85 4,5 <3 allium metaphase (m) test phytotoxicity mm 39 34 12 13 35 38 genotoxicity % 2,5 6,50 30,0 10,5 6,50 4,50 micronucleus (mn) pisces test cottus gobio ‰ 0.9±0.33 0,7±0.24 leuciscus cephalus ‰ 0,7±0.22 phoxinus phoxinus ‰ 0,5±0.13 barbus meridionalis ‰ 1,1±0.35 thymallus thymallus ‰ 0,5±0.12 salmo trutta fario ‰ 0,3±0.11 legend. kb: kamniška bistricariver, k2, k4: fishing area, ww: wastewater, fte: final treated effluent, wwtp: central domžale-kamnik wastewater treatment plant, %: chromosome damage (csd) per 100 cells, gl: genotoxicity level, ‰:micronuclei per 1000 erythrocytes. 130 peter firbas, tomaž amon the in situ quantification of mn fish erythrocytes has shown to be an adequate bio-marker in the evaluation of aquatic ecosystems quality (al-sabti and metcalfe 1995; minissi et al. 1996). given the nucleated nature of erythrocytes in fish the mn test has gained high relevance in bio-monitoring of aquatic environments, also including assessment of water quality (palacio-betancur et al. 2009). aquatic vertebrates and invertebrates are directly exposed to many pollutants dissolved or suspended in the surface water (bolognesi and hayashi 2011; smital and kurelec 1997; bolognesi and fenech 2012; beršiene et al. 2012; walker et al. 2012). salmonids figure 2. examples of series of onions cultivated 72 h in different biotechnological and environmental river samples. a) drinov rob, b) študa, c) wastewater, d) ww after mechanical step, e) final treated effluent, f) bišče. 131use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater t. thymallus, s. trutta fario, and cyprinidsp. phoxinus, c. gobio, b. barbus, b. meridionalis, l. cephalus, p. fluviatilis, h. nasus nasus that are inhabiting european freshwater ecosystem were evaluated for their use as in situ using the micronucleus test (rodriguez-cea et al. 2003; minissi et al. 1996). in situ surveys of wild freshwater ecosystems with different levels of pollution showed that cyprinids fish in moderately pollution sites do not present higher micronuclei averages than those caught in clean rivers system, whereas micronuclei are induced by thymallus thymallus, salmo trutta fario and cottus gobio inhabiting moderately polluted sites. our results demonstrated the suitability of cottus gobio for in situ monitoring of freshwater ecosystems using the pisces mn test. some researchers have reported the sensitivity of this species, including in the detection of genotoxicity effects: frequency of micronucleated erythrocytes (e) in blood of phoxinus phoxinus in the laboratory conditions and in environment is 0.3-0.7 mn/1000 e (bolognesi and hayashi m 2011; ayllon and garcia-vaszquez 2000). in blood of leuciscus cephalus that lives in partially contaminated river waters one finds levels of micronucleated erytrocytes 0.7-2.9 mn/1000 e (piccoli et al. 2010). the low to high frequency (0.5-5 mn /1000 e) mn in the erythrocytes (e) is known for chondrostoma nasus in an uncontaminated environment 0.5 mn/1000 e and 4 mn/1000 e in a contaminated environment (koca and koca 2008). the barbus barbus also has 0.5 mn/1000 e in uncontaminated environment and 3 mn/1000 e in a contaminated environment (boettcher et al 2010). the frequency of mn in the erythrocytes of salmo truta fario specimens was increased after exposure to a configure 3. diploid monocentric metaphase chromosome from the root meristem cells of the onion (allium cepa l.), containing 2n of 16, with basic chromosome number x=8 (2n=16). figure 4. one chromosome is damaged. figure 6. whole chromosome set is damaged. figure 5. different number chromosome damage in metaphase cells obtained from the meristem root-type cells of onion (a. cepa l.). 132 peter firbas, tomaž amon figure 7. normal mononucleated erythrocytes, and micronucleated erythrocytes in peripheral blood.a) salmo trutta fario, b) thymallus thymallus, c) barbus barbus, d) barbus meridionalis, e) phoxinus phoxinus, f ) leuciscus cephalus, g) cottus gobio, h) hondrostoma nasus, i) perca fluviatilis. 133use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater centration 25 ppm ems (ethyl methanesulfonate) under laboratory condition to 1,8-2,7 mn/1000 e and concentration 780 pg/ml pcb (polychlorinated biphenyl) induced 1,5-1,7 mn/1000 e (belpaeme et al. 1996). the relevance of this mn test is also confirmed by the work by de flora et al. (1993), schultz et al. (1993), al-sabti and metcalf (1995), marlasca et al. (1998), ayllonand  garcia-vazquez(2000), ayllón et al. (2000), ayllón et al. (2001), raisuddin and jha (2004), bagdonas and vosyliene (2006), kim and hyun (2006), baršieneet al. (2006), ali et al. (2008), palacio-betancur et al. (2009),boettcher et al. (2010), bolognesi and hayashi (2011) and llorente et al. (2012). the frequency of occurrences of csd in root cells (csd/200 cells) in the plant of common onion (allium cepa l.) in uncontaminated laboratory and natural conditions is 2.0-2.5 damaged chromosome cells/200 cells (%) and is evaluated as a negative control (firbas and amon 2014). very little of allium test studies are focused on clastogenic and/or aneugenic effects, thus, chromosome damage and chromosome number changes in the chromosome set. the use of colchicine during chromosome preparation destroys microtubules, but influencing the chromosome movement, increased frequency of metaphase with arranged condensed chromosomes and reduced transition from metaphase to anaphase, allowing a better observations of the exclusively metaphase chromosome (ray et al. 2013; firbas and amon 2014; kundu and ray 2017). however this research strategy is provided by the allium metaphase (m) test. chromosome preparation is a key and crucial step in all cytogenetic techniques (kirov et al. 2014), and cytogenetic assay are classical method to detect chromosome damage (aberration, anomalies). the metaphase chromosome anomalies as detected in the allium m test procedure are not excluded to occur also in the human chromosomes when exposed to similar pollutants. plant cytogenetic, using allium m test, identifies same chromosome damages as they are identified in human cytogenetic such as: chromosome and chromatid damages, dicentric chromosomes, aneuploidy, euploidy and translocation (stimpson et al. 2013; firbas and amon 2014; firbas 2015; polsikovsky et al. 2018). mentioned chromosome aberrations cause clinical defects on human body (schauer 1981; pardee et al. 2007; gibbs 2008; duesberg 2005; duesberg 2007; gardner 2009). the study of dna damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis (fenech 2000), the genotoxic disease syndrome (kurelec 1993) and evaluated genotoxic effect in autoimmune diseases by the micronucleus test assay (torres-bugarín et al. 2015). the allium m test—pisces mn test means that these two independent testing system technologies show the same results in our research (firbas and amon 2017). allium m test—pisces mn test is a reliable, preferred and accurate method for the monitoring of the wwtp and water quality in aquatic ecosystem. both tests help us to monitor the chromosome damages caused by the water pollution. the test system allium m test—pisces mn test has two restrictions: (i) mn can produce also a whole and undamaged chromosome and (ii) in the allium m test cytostatic colchicine can mask the occurrence of table 7.correlation between chromosome damage (csd) of the allium m test and pisces mn test of the genotoxicitylevel (gl) with parallel physical parameters (bod5, no3 – n). the relationship is directly related to the environmental quality standard-ecological status (eqs-es) and environmental risk assessment (era). mn pisces mn test (υ mn/1000 e) csd allium m test υ csd /100 c level to endanger (risk assessment) environmental samples eqs-es* bod5 (o2 mg/l) no3 – n (mg/l) < 2 natural mutagenicity test organisms high quality drinking water > 0.5 > 1.5 0.09/0.20 2 -5 no risk spring (drinking) water i. quality class rivers and lakes wery good 1.6 – 2.4 3.2 – 7.0 0.30/0.50/1.26 4 – 10 low low/midle i.ii. quality class rivers and lakes good 2.0 – 5.4 6.5 – 9.5 1.21/2.01/5.50 9 – 21 midlle midle/high ii. quality class rivers and lakes, moderation < 8.5 >9.6 22 – 39 high wastewater (municipal waste) treated wastewater, fte weacly 15 – 20 9 – 12 40 – 55 critical wastewater (industrial, leachate, intensity chemical) badly 100 – 500 > 20 134 peter firbas, tomaž amon c-mitosis. in both cases here we talk about the malfunction of the mitotic process, however, this is not a chromosomal or chromatid lesion. for testing the genotoxicity of the collected water samples, two assays were used: chromosome aberration assay in metaphase mitotic cell (kumar and panneerselvam 2007; panneerselvam et al. 2012; ragunathan and panneerselvam 2007) and mn assay in interphase cell (bolognesi et al. 2006). the csd and mn studies have shown to be highly reliable and preferred in genotoxicity testing. the aim of this study was to determine of river water quality by conducting an experiment involving biomonitoring of water constituents of genotoxicity in fish and onion inhabiting these sites. in summary, ww treatment process in one of the most important environmental conservation processes that should be encouraged worldwide. conclusion as an environmental essay how healthy is a water (firbas 2016) and/or living environment is definitely an opinion-forming issue. its message is an integral part of biological science, thus opening up a world which enables us to determine the quality of any living environment by using current biological observation. this concerns the different lengths of a tested onion plant’s roots, and any injuries to the chromosomes within their cells and micronucleus (mn) in blood erythrocytes indigenous fish in relation to their environment. physical and chemical analysis alone do not provide any reliable answer to the question of how healthy the water is. however complementary research in association with biological and chemical studies are needed in order to obtain a fully comprehensive picture, because it is difficult to identify a wide variety of effects (chemical pollution) within the environment. the biological method reveals an integrated impact on the growth and development of living cells or organisms, and detects the presence of harmful substances within the limits and capabilities of analytical methods. by using biological (genotoxicity) tests, we see that the outcomes of both plant and animal testing show damage of their genetic material (chromosomes) regardless of the tolerance limits that can be caused by various contamination sample concentrations within an environment. in regard to the universality of the living organisms “genetic codes”, the research results are transferable (applicable) to human beings. it is time for us to act in a responsible way, thus ensuring a healthy environment which based on high quality drinking water. the different feedback from the tested plant’s root growth is a general quality indicator of an environmental sample. their straight growth is an indicator of how adequate is the environment they are growing in. looking at the cellular level of the tested plant’s root-tip growth, especially when monitoring the cells and determining the ratio between the undamaged and damaged chromosomes, and the presence or absence of micronuclei in the blood of erythrocytes gives us a very detailed picture of the environmental living quality, respectively answering the question as to how healthy the living environment is. co-dependence of pollution within an environment is substantial evidence that some genotoxic stuff causes chromosomal 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damage analysis, to assess the quality of urban wastewater treatment and water of the kamniška bistrica river (slovenia) peter firbas1, tomaž amon2 the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bo shi1,*, majid khayatnezhad2, abdul shakoor3,4 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 107-113, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-122 citation: p. mahditabar bahnamiri, a. mahmoudi otaghvari, n. ahmadian chashmi, p. azizi (2020) electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran. caryologia 73(1): 107-113. doi: 10.13128/caryologia-122 received: januray 9, 2019 accepted: february 23, 2020 published: may 8, 2020 copyright: © 2020 p. mahditabar bahnamiri, a. mahmoudi otaghvari, n. ahmadian chashmi, p. azizi. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 1 department of biology, faculty of basic sciences, university of mazandaran, babolsar, iran 2 department of soil science, university of guilan, rasht, iran *corresponding author. e-mail: p.mahditabar@gmail.com, botany1347@gmail.com, najme.ahmadian@gmail.com abstract. in this research we studied the electrophoretic of seed storage proteins in the genus hypericum l. from iran. the plant samples were collected from various phytogeographical regions of iran to study the seed storage proteins. the study was performed to determine the boundary among different species of genus hypericum using sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). all samples belong to three species of h. perforatum, h. tetrapterum and h. androsaemum. a total of 22 protein bands were observed in the studied species. the results show that h. perforatum, h. tetrapterum are closely related based on seed storage proteins. a closely relationship and high protein similarity (j=0.66) were found between h. perforatum, h. tetrapterum. electophoretic results compared with earlier molecular and morphological studies. the highest number of bands was observed in kordkoy1 population (pop12) and gardane heyran population (pop20) of h. perforatum and the lowest in gorgan/ naharkhoran population (pop 25) of h. androsaemum. our results showed the species of hypericum were placed intermixed. the aim of this study to delimit species in the genus hypericum and used these seeds storage protein for the correct identification. keywords. hypericum, north of iran, species relationships, sds-page. introduction the genus hypericum (guttiferae, hypericoideae) is perennial, belonging to the hypericaceae family, having 484 species in forms of trees, shrubs, and herbs, distributed in 36 taxonomic sections (crockett and robson 2011). the species of the family are distributed worldwide in the temperate zones but are absent in extreme environmental conditions such as deserts and poles. iranian species of this genus grow mainly in north, northwest and center of iran and form floristic elements of hyrcanian mountainous areas, irano-turanian, mediterranean and zagros elements. they generally prefer steep slopes of rocky and calcareous cliffs and margin of mountainous forests (robson 1968; azadi 1999). robson (1968) introduced 21 species in the 108 parisa mahditabar bahnamiri et al. area covered by flora iranica. robson (1977) and assadi (1984) reported h. fursei n. robson and h. dogonbadanicum assadi as two endemics of north and south west of iran. in flora of iran, azadi (1999) identified 19 species, 4 subspecies arranged in 5 sections (comprising campylosporus (spach) r. keller, hypericum, hirtella stef., taeniocarpum jaub. & spach. and drosanthe (spach) endl.), and two doubtful species including h. heterophyllum vent. and h. olivieri (spach) boiss. hypericum species are generally known locally in iran with the names “hofariqun” which ebn sina (or bo ali sina) called it (rechinger, 1986). plants of the genus hypericum have traditionally been used as medicinal plants in various parts of the world. hypericum perforatum l. is the source to one of the most manufactured and used herbal preparations in recent years, especially as a mild antidepressant, and thus is the most studied hypericum species (mozaffarian, 1998). according to brutovská et al. (2000), h. perforatum is probably originated from autopolyploidization of an ancestor closely related to diploid h. maculatum. the chemical composition of h. perforatum oil has been the subject of many researcher in recent past (cakir et al. 1997; baser et al. 2002; osinska 2002; schwob et al. 2002; mockute et al. 2003; smelcerovic et al. 2004). the methanolic extract from the aerial parts of hypericum plants typically contain hypericins, hyperforins and phenolic compounds (osinska 2002). proteins and enzymes, characterized as primary gene products, are important parameters in biochemical taxonomy. storage proteins separated by electrophoretic methods are thought to undergo the process of evolution with relative slowness due to their “non-essential nature’’ (margoliash and fitch 1968), while enzymes are thought to be extremely sensitive to selection pressures in evolution and thus to survival of the organism (mcdaniel 1970). analysis of proteins and isozymes is a tool for supplementing the evidence obtained by comparative morphology, breeding experiments and cytological analysis. seed protein electrophoresis for the study of phylogenetic relationship in capsicum l. was performed by panda et al. (1986). although phenotypic traits are important for diversity studies, they need to be supported by molecular markers to give robust genetic diversity estimates (esfandani-bozchaloyi et al. 2018a, 2018b, 2018c, 2018d). genetic diversity studies in capsicum using morphological, cytological and biochemical marker systems (kaur and kapoor 2001; gopinath et al. 2006) are also conducted. the data on agronomic, morphological and physiological plant traits are generally used to estimate the magnitude of genetic diversity present in the germplasm. however, such data may not provide an accurate indication of genetic diversity because of environmental influences upon the expression of observed traits and also the time consuming and laborious field evaluation procedures. the introduction of biochemical techniques like sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page), isozyme markers has been particularly helpful in deducing systematic relationships between groups where morphological and cytological data were not corollary. sds-page is an economical, simple and extensively used technique for describing the seed protein diversity of crop germplasm (fufa et al. 2005; iqbal et al. 2005). furthermore, seed proteins, used as genetic markers convey greater precision to measures of genetic diversity because they are the primary products of structural genes (srivalli et al. 1999). seed protein electrophoresis for the study of phylogenetic relationship in capsicum annuum was performed by panda et al. (1986) and of diploids and tetraploid hybrids of capsicum was initiated by srivalli et al. (1999). there is no report of sds-page in hypericum species in iran. the present study was conducted on the genetic diversity of hypericum genotypes from different locations which will be useful for breeding programmes and also for conservation of germplasm. to use genetic resources adequately, it is necessary to understand the extent and pattern of genetic diversity. therefore, an attempt with the present investigation was undertaken to evaluate the extent of variability existing in 29 geographical populations belonging to three species of hypericum of north region of iran through seed protein analysis to provide a scientific basis for future selection and crop improvement program. the objective of this study was to assess the level of seed electrophoretic patterns of hypericum taxa in iran and used it for the correct taxonomy of the genus. material and methods plant material extensive field visits and collections were undertaken during 2016-2017 throughout the north of iran. in present study 63 plant samples from 29 geographical populations belonging to three species of hypericum in iran were collected from field: h. perforatum l., h. tetrapterum fries. and h. androsaemum l. different references were used for the correct identification of species (rechinger, 1999 , azadi, 1999). the details of the voucher specimens and their localities are given in table 1 and fig 1. all materials were examined with a stereomicroscope (nikon-smz1) and all voucher specimens 109electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran are deposited at the university of mazandaran herbarium (humz). protein extraction & electrophoresis an amount of 0.1 g of mature seeds were selected from each population and crushed in liquid nitrogen at low temperature. after obtaining a fine powder, proteins were extracted under cool conditions with 3 ml of trisglycin buffer (ph 8). the resulting samples were centrifuged twice for 5 min at 11000 g. the protein electrophoresis was based on laemmlis procedure (1970), using a discontinuous vertical slab gel. the separating gel comprises 12 ml of 30 % acrylamid stock solution, 2.5 ml tris-hcl 1.5 m (ph 8.8), 100 μl sds 10 %, 2.3 ml water, 7 μl temed, and 60 μl aps (ammonium per sulfate). after polymerization of the separating gel, the stacking gel with 530 μl of 30 % acrylamide stock solution, l ml tris 0.5 m (ph 6.8), 40 ml sds 10 %, 2.37 ml water, 5μl temed, and 40 μl aps was polymerized on the separating gel. the electrophoresis was carried out at a constant voltage of 100v for 7-15h. gels were stained in coomasie brilliant blue for 1-2 h and overnight distained with acetic acid and methanol (laemmli 1970). we used jaccard similarity coefficient. standard proteins (b-galactosisase, ovalbumin, lactate dehydrogenase, lactoglobulin-b, lysozyme and bovine serum albumin) were used to evaluate the molecular weight of the unknown proteins. the protein density was determined by bradford protocol(bradford, 1976). protein banding profile analysis number and location of each protein band were identified and their rf (relative factor) and molecular weight were estimated. in statistical analysis, each protein band was considered as a qualitative character and coded as 1 (presence) versus 0 (absence). for grouping of the plant specimens,ward (minimum spherical characters) were used (podani 2000). pca (principal components analysis) biplot was used to identify the most variable characters among the studied populations (podani 2000). past version 2.17 (hammer et al. 2012) results a total of 22 protein bands were observed for these taxa. (fig. 2 and table 2). all studied taxa had bands 76.12 kd and 45 kd except for gorgan/naharkhoran population (pop 25) of h. androsaemum. the highest number of bands was observed in kordkoy1 population (pop12) and gardane heyran population (pop20) of h. perforatum and the lowest in gorgan/naharkhoran population (pop 25) of h. androsaemum (fig. 2 and table 2). in order to find out the most variable protein bands in the studied taxa, a principal component analysis was implemented. primitive analysis showed that three factors were responsible for 62.37 % of total studied variation in the taxa. in the first factor, with almost 37.81 % of the total variation, bands 6.12, 9.87, 34.87, 51.12 kd had the highest correlation. in the second factor, with about 14.63 % of the observed variation, bands 27.37, 30.19, 76.77 and 81.67 kd had the highest positive correlation. in the third factor, with 9.92 % of the total variation, bands 21.54, 78.14, 93.16 kd had the highest correlation. both clustering and pca analyses of the hypericum species studied produced similar groupings and therefore only ward clustering characters are presented here (fig. 3). two major clusters were formed in ward clustering (fig.3). ward clustering, of the studied populations did not entirely delimit the studied species and revealed that plants in these species are intermixed. in ward dendrogram, a higher degree of intermixture occurred between h. perforatum, h. tetrapterum and h. androsaemum. also ward dendrogram revealed that although population of the species h. perforatum is more distinct than the other two species, but it showed a high degree of intraspecific genetic variability as they are positioned in different places of the dendrogram. figure 1. distribution map of hypericum populations studied. 110 parisa mahditabar bahnamiri et al. table 1. voucher details of hypericum species examined in this study from iran. population no. species population cod locality / voucher number 1 h. perforatum l. hp1 mazandaran , ramsar, 1723 humz 2 h. perforatum l. hp2 mazandaran , ramsar/javaherde1,1724 humz 3 h. perforatum l. hp3 mazandaran , ramsar/javaherde2/daryache ghoo,1725 humz 4 h. perforatum l. hp4 mazandaran , savadkoh/alasht,1726 humz 5 h. perforatum l. hp5 mazandaran , babolkenar1,1727 humz 6 h. perforatum l. hp 6 mazandaran , babolkenar2,1728 humz 7 h. perforatum l. hp 7 mazandaran , galogah1,1729 humz 8 h. perforatum l. hp 8 mazandaran , galogah2,1730 humz 9 h. perforatum l. hp9 mazandaran , aliabad katool,1731 humz 10 h. perforatum l. hp10 golestan , gorgan,1732 humz 11 h. perforatum l. hp 11 golestan , ziarat,1733 humz 12 h. perforatum l. hp 12 mazandaran ,kordkoy1,1734 humz 13 h. perforatum l. hp13 mazandaran , kordkoy2,1736 humz 14 h. perforatum l. hp14 mazandaran , kelachay,1737 humz 15 h. perforatum l. hp15 guilan , langrood,1738 humz 16 h. perforatum l. hp16 guilan , lahijan/bam lahijan,1739 humz 17 h. perforatum l. hp17 guilan , somesara,1740 humz 18 h. perforatum l. hp18 guilan , asalem,1741 humz 19 h. perforatum l. hp19 guilan , heyran,1742 humz 20 h. perforatum l. hp20 guilan , gardane heyran,1743 humz 21 h. perforatum l. hp21 mazandaran , nowshahr/sisangan,1744 humz 22 h. perforatum l. ht22 guilan , astara,1745 humz 23 h. tetrapterum fries. ht23 mazandaran , savadkoh/alasht,1746 humz 24 h. tetrapterum fries. ha24 guilan , asalem,1747 humz 25 h. androsaemum l. ha25 golestan , gorgan/naharkhoran,1748 humz 26 h .androsaemum l. ha26 guilan , astara,1759 humz 27 h. androsaemum l. ha27 mazandaran, ramsar/bam ramsar,1750 humz 28 h. androsaemum l. ha28 mazandaran , aliabad katool/ kabodval,1751 humz 29 h. androsaemum l. ha29 mazandaran , amol/sangchal,1752 humz figure 2. sds-page electrophoresis profiles of the studied population of hypericum. note: populations abbreviations are according to table 1. 111electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran discussion in the present study, 29 geographical populations belonging to three species of hypericum of north region of iran was examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). electrophoretic data of the seed storage proteins presented in this study have shown that the dendrogram obtained from the studied species is not capable of the species recognition. we found findings the seed storage was often incongruent with the result of faghir, et al. (2018) and mahmoudi otaghvari & al, (2015) and bayat & al., (2015) for pollen data. in previous studies, the micromorphology of pollen grains was performed in several species and their importance in plant taxonomy was emphasized (faghir, et al. 2018; mahmoudi otaghvari & al,2015; bayat & al., 2015). faghir, et al. (2018) pollen grains of ten species and two subspecies of the genus hypericum in iran belonging to four sections were studied using light and scanning electron microscopy. palynological analysis of selected species of the genus hypericum revealed important pollen morphological characters, especially pollen outline, numbers and types of apertures, colpus length; presence and absence of operculum; exine sculpturing type, pore shape, size and arrangements. these traits can be used for infrageneric classification, especially at sectional and species levels (faghir, et al. 2018). different techniques including morphological, biochemical and especially molecular markers let scientable 2. band number and molecular weight for each studied population of hypericum. (1band is present in the seed sample, 0band is absent in the seed sample). band no 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 mw(kd) 99 95 94 92 85 82 76 68 65 62 59 55 51 45 40 37 34 27 22 18 12 8 pop 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 0 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 4 1 0 0 0 1 1 1 1 1 1 0 0 0 1 1 1 1 0 0 1 1 1 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 7 0 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 0 0 1 1 1 8 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 0 0 1 1 1 9 0 1 1 1 1 1 1 0 0 0 0 1 1 1 0 1 1 0 0 1 1 1 10 0 0 0 0 1 1 1 0 0 0 0 1 1 1 0 0 1 0 0 1 0 0 11 1 1 1 0 1 1 1 0 0 0 0 1 1 1 0 0 1 0 0 1 0 0 12 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 14 1 1 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 15 1 1 1 0 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 16 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 17 0 0 0 0 1 1 1 0 0 0 0 0 1 1 1 0 1 1 1 0 0 0 18 0 0 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 0 1 1 0 0 19 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 0 0 0 1 1 1 1 1 0 0 0 1 1 1 1 0 1 0 1 1 0 0 22 1 0 1 1 1 1 1 1 0 0 0 1 1 1 1 0 1 0 1 1 1 1 23 0 0 0 1 1 1 1 1 0 0 0 1 1 1 1 0 0 0 0 1 1 1 24 1 1 1 1 1 1 1 0 1 1 0 0 1 1 1 0 0 0 1 1 1 0 25 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 26 0 0 0 0 1 1 1 0 0 0 1 0 1 1 1 0 1 0 1 1 1 0 27 0 1 1 1 0 0 1 0 1 1 1 0 1 1 1 0 1 1 1 1 1 0 28 1 1 1 1 0 0 1 0 1 1 1 0 1 1 1 1 1 1 1 1 1 0 29 0 0 0 1 0 0 1 0 0 0 0 0 0 1 1 0 1 1 0 1 1 0 112 parisa mahditabar bahnamiri et al. tists to study genetic variability of plants. as molecular markers present reproducible results regardless of environmental conditions, they have gained nowadays considerable attention for studies relating to the genetic diversity (farooq and azam 2002). according to morshedloo et al. (2015) genetic variability among ten wild populations of h. perforatum growing in different climatic regions of iran via issr markers. they observed the studied populations were classified into four main groups which was, to the some extent, in accordance with their geographical origins. also they recovered, issr markers revealed relatively a high level of genetic variability among iranian h. perforatum populations suggesting that the issr technique is efficient and powerful for assessment of genetic diversity at the intraspecific level. the present study also provide the way for use of molecular systematics within genus hypericum. the taxa are not clearly separated on the basis of electrophoretic data of seed storage proteins. the results have revealed that h. perforatum, h. tetrapterum were closely related. a high similarity index is a reflex of genomic identity (j=0.66). the dendrogram showed close relationship and high protein similarity (j=0.66) between h. perforatum, h. tetrapterum. this is the first of its kind report on electrophoretic data of the seed storage proteins of three species of hypericum of north region of iran. references azadi, r. 1999. guttiferae. in: assadi, m. 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(cucurbitaceae). caryologia 75(1): 89-97. doi: 10.36253/ caryologia-1358 received: july 5, 2021 accepted: march 27, 2022 published: july 6, 2022 copyright: © 2022 sanjay kumar, asikho kiso. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid sk: 0000-0003-4463-9159 new reports of somatic chromosome number and symmetric or asymmetric karyotype estimation of sechium edule (jacq.) sw. (cucurbitaceae) sanjay kumar1,*, asikho kiso2 1 department of botany, banaras hindu university, varanasi, up 221005, india 2 department of botany, nagaland university, lumami, nagaland798627, india *corresponding author. e-mail: skumar.bot@bhu.ac.in abstract. sechium edule (jacq.) sw. distributed well in eastern himalayan region, fourteen genotypes collected randomly from kigwema village, kohima (nagaland) at an average altitude of 1538 masl (meter above sea level) with 25.61°n latitude and 94.35°e longitudes. somatic chromosome numbers are diploid in nature. chromosome numbers are in agreement and comparable with earlier reports except 2n=2x=22. the chromosome number 2n=2x=22 could not be traced out in the present material collected for study. two (2) new chromosome numbers 2n=2x=32 and 2n=4x=52 were recorded and expected to be diploid and tetraploid in nature respectively. it was expected that tetraploid (2n=4x=52) number of chromosome might be originated from the earlier reports of diploid chromosome number 2n=2x=26. both chromosome numbers 2n=2x=32 and 2n=4x=52 are reported for the first time by the authors in the present study. range of chromosome length was recorded in between 0.501 – 1.343. the range of chromosome length are in agreement with earlier reports of 0.700 – 0.900 approximately. range of chromosome length suggested minute (<1µm) and small (1-3µm) size of chromosomes with small differences and variations in chromosome length (cvcl). inter-chromosomal indices (a2 and rec), intra-chromosomal index (particularly, stebbin’s classification) and both inter and intra chromosomal indices (di and gi) estimated symmetric nature of the karyotype. keywords: sechium edule (jacq.) sw., mitosis, somatic chromosome number, karyotype, inter and intra chromosomal symmetry/asymmetry estimation. introduction genus sechium p. browne was first published in a monograph on cucurbitaceae in 1881. literature survey on historical background suggested that it was a monospecific genus with a single species and represented as sechium edule (jacq.) sw. (cogniaux 1881; dedonato and cequea 1994). historically, genus sechium was originally recorded from jamaica (browne 1756). genus sechium had been recorded as sicyos edulis and chocho edulis simultaneously in initial classification (adanson 1763). jacquin (1788) changed the genus 90 sanjay kumar, asikho kiso chocho into chayota and re-designated as chayota edulis. later on, chayota edulis re-designated as sechium edule by swartz (1800). at present, the genus is known as a combination of both jacquin and swartz i.e. sechium edule (jacq.) sw. the most accepted term for sechium is ‘chayote’ worldwide. the presence of single species for the genus (monospecific genus) had worn-out, when more species were reported for the genus by various authors from different regions during 1900s and some of them are s. edule sub spp. edule, s. edule sub spp. sylvestre, s. chinatlense, s. compositum and s. hintonii (goldblatt1990; singh 1990; mercado et al. 1993; mercado and lira 1994). recently, a new species called sicyos angulatus l. for indian flora and sechium mexicana for mexico have been reported respectively (thakur 2016; lira and nee 1999). the reported species were morphologically very similar and never verified for the presence of a new species in the genus. cytologically, genus sechium was attempted and studied for the presence of some more species, if any. many authors reported different chromosome numbers for the genus sechium with base chromosome number x=12, 13, 14, and 15. the base chromosome number x=11 has also been reported for the genus by singh (1990). genus sechium categorized into s. edule sub spp. edule, s. edule sub spp. sylvestre, s. chinatlense, s. compositum, s. hintonii, s. mexicana and sicyos angulatus respectively on the basis of earlier reports of base chromosome number. the base chromosome numbers suggest that it remains unresolved and needs thorough examination cytologically. so, the present aim of the paper is to attempt and extend the information of new chromosome number count to the genus sechium, if any. materials and methods genus sechium is a shrub climber of cucurbitaceae family. fruit samples of genus were collected randomly from kigwema village, kohima, nagaland (india) at an average altitude of 1538 meter above sea level (masl), latitude (25.61°n) and longitude (94.35°e). mitosis was studied from the secondary root tips of germinating fruits. root tips of 2-3 cm in length were pre-treated with α-bromonaphthalene at 6±2 °c for 3-4 h followed by overnight fixation (3:1 ethanol-acetic acid) and preservation (70% ethanol). the root tips were hydrolyzed with 1 n hcl for 10-15 min at about 50-60 °c. the root tips were squashed in 2% acetocarmine. three somatic chromosome preparations under 100x (emersion oil) were photographed using digital motic ba 210 microscope and recorded for further analysis. statistical analysis total chromosome length (µm) were measured for the genotypes with the scale bar of 10µm using imagej sof tware and further computation was attempted through windows ms-excel and with the help of standard formulas for inter and intra chromosomal differences among the chromosome complement of genotypes (see box 1). results and discussion the genotypes are diploid in nature in somatic chromosome count except genotype 2 (fig. 1d). two diploids with different somatic chromosome number 2n=2x=26 (fig. 1b) and 2n=2x=32 (fig.1c) and a tetraploid 2n=4x=52 (fig.1d) were recorded for the genotype. the chromosome numbers 2n=2x=32 (diploid) and 2n=4x=52 (tetraploid) are the first report for the sechium edule. other somatic chromosome numbers are in agreement and comparable with earlier reports except 2n=2x=22 which could not be traced out in the present study materials (sugiura 1940; sobti and singh 1961; giusti et al. 1978; mercado and lira 1994). the presence of differences, if any, in cultivated or wild forms of the sechium, possibly could have been originated through the chromosomal evolutionary factors in due course of time with the help of primary, secondary, agmatoploidy, symploidy, dysploidy or pseudoaneuploidy evolutionary factors and needs to be verified through cytological and molecular techniques. similarly, sechium edule suggested ploidy nature 2n=4x=52 (genotype 2) of the species. ploidy is not reported earlier in the sechium edule and hence, the first report for the species. the findings of ploidy nature in sechium suggested towards the whole genome content change, diversification, evolutionary changes and speciation in the genus. it could be correlated that a new species might be established from the pre-existing species through reproductive or genetic isolation from the progenitors. the speciation through evolution and diversification required various events of primary (deletion, duplication, inversion, and translocation), secondary (fusion, fission, rearrangements) and disploid (ascending or descending) alterations of chromosome numbers. in the present paper, origin, diversification, genetic isolation or possibility of interbreeding between and among sechium needed to be explored (fig. 1a – p). in past, few reports are available on the origin and evolution of cultivated cucurbits and suggested the mexico, central america and guatemala as the centre of 91chromosome number and karyotype study of sechium edule (jacq.) sw. from india variation for the crop. earlier, sechium edule was considered mono-specific (genus with single species) and native to new world, but now it includes as many as eight species and cultivated throughout tropical and subtropical regions of the world but not explored extensively (newstrom 1990). at present, first report on new chromosome number gives a hope for the presence of some more species in the genus, sechium. statistical analysis results on genotype chromosomes presented in table 1. total chromosome length (σtcl) or chromosome volume (cv) was recorded maximum for the genotype 3 (29.618) which is very close to the genotype 2 with 2n=4x=52 (28.884) but the somatic chromosome number differs in both suggested the differences in the size of the chromosomes (martonfiova, 2013). chromosome length range (clr) was recorded in between 0.501 – 1.343 for the present genotypes. seven genotypes have chromosome length more than 1 µm which indicates the heterogeneity of chromosome length for chromosome complement. two types, minute and small size chromosomes were recorded based on the classification minute (<1µm), small (1-3µm), medium (3-5µm) and large (>5µm) suggested by kutarekar and wanjari (1983). earlier, range of chromosome length was reported in between 0.700 – 0.900 for sechium edule. the range of chromosome length are in agreement with earlier reports approximately and comparable (sanjappa 1979; cadenainiguez et al. 2007). value of relative chromatin (vrc) was recorded high and indicated towards the heterochromatic nature of genotypes. high heterochromatic nature could be correlated with the less advanced type of karyotype with more number of metacentric chromosomes alongwith small differences in size of largest and smallest chromosome of karyotype (beevy and kuriachan 1996). coefficient of variation of chromosome length (cvcl) was recorded high for each chromosome and hence σ cvcl for genotypes indicated variation in the chromosome length of karyotype complement (thakur and sinha 1973). a2 value was computed for each chromosome and recorded near to zero and summation value σa2 for each genotype presented. a2 value close to zero indicates the conservation of chromosome size in the karyotype with low variation among the chromosome length and asymmetry remains the constant i.e. a2, approximately, indicated towards the symmetric nature of the karyotype (carvalheira et al.1991). similarly, rec index value ranges from 0-100. the value was recorded for individual chromosome in karyotype and summation value σ rec for the each genotype presented is high. high value for rec index suggested the maximum resemblance among the chromosomes with symmetric nature of the karyotype. rec index value measures the resemblance between the chromosomes and the average degree of symmetry over the whole karyotype (huziwara 1962). rec index = 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇∑𝑇𝑇𝑒𝑒𝑒𝑒𝑒𝑒𝑇𝑇ℎ𝑇𝑇𝑜𝑜𝑒𝑒𝑇𝑇𝑜𝑜ℎ𝑜𝑜ℎ𝑟𝑟𝑇𝑇𝑟𝑟𝑇𝑇𝑟𝑟𝑇𝑇𝑟𝑟𝑒𝑒 ÷ 𝐿𝐿𝑇𝑇𝑒𝑒𝑒𝑒𝑒𝑒𝑟𝑟𝑇𝑇𝑜𝑜ℎ𝑟𝑟𝑇𝑇𝑟𝑟𝑇𝑇𝑟𝑟𝑇𝑇𝑟𝑟𝑒𝑒 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑒𝑒𝑇𝑇𝑟𝑟𝑇𝑇𝑒𝑒𝑟𝑟𝑇𝑇𝑜𝑜𝑜𝑜ℎ𝑟𝑟𝑇𝑇𝑟𝑟𝑇𝑇𝑟𝑟𝑇𝑇𝑟𝑟𝑒𝑒𝑟𝑟 x 100 (greilhuber and speta 1976) a2 index = 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆ℎ𝑆𝑆𝑆𝑆𝑟𝑟𝑆𝑆𝑟𝑟𝑆𝑆𝑟𝑟𝑆𝑆𝑟𝑟𝑆𝑆𝑆𝑆𝑟𝑟𝑆𝑆ℎ 𝑀𝑀𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆ℎ𝑆𝑆𝑆𝑆𝑟𝑟𝑆𝑆𝑟𝑟𝑆𝑆𝑟𝑟𝑆𝑆𝑟𝑟𝑆𝑆𝑆𝑆𝑟𝑟𝑆𝑆ℎ (romero – zarco1986) coefficient of variation (cvcl) = 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆ℎ𝑆𝑆𝑆𝑆𝑟𝑟𝑆𝑆𝑟𝑟𝑆𝑆𝑟𝑟𝑆𝑆𝑟𝑟𝑆𝑆𝑆𝑆𝑟𝑟𝑆𝑆ℎ 𝑀𝑀𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆ℎ𝑆𝑆𝑆𝑆𝑟𝑟𝑆𝑆𝑟𝑟𝑆𝑆𝑟𝑟𝑆𝑆𝑟𝑟𝑆𝑆𝑆𝑆𝑟𝑟𝑆𝑆ℎ x 100 (lavania and srivastava 1999; paszko 2006) disparity index (di) = 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ𝑟𝑟𝐿𝐿𝑟𝑟𝐿𝐿𝐿𝐿𝐿𝐿𝑟𝑟𝐿𝐿 − 𝑆𝑆ℎ𝐿𝐿𝑟𝑟𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ𝑟𝑟𝐿𝐿𝑟𝑟𝐿𝐿𝐿𝐿𝐿𝐿𝑟𝑟𝐿𝐿 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ𝑟𝑟𝐿𝐿𝑟𝑟𝐿𝐿𝐿𝐿𝐿𝐿𝑟𝑟𝐿𝐿 + 𝑆𝑆ℎ𝐿𝐿𝑟𝑟𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ𝑟𝑟𝐿𝐿𝑟𝑟𝐿𝐿𝐿𝐿𝐿𝐿𝑟𝑟𝐿𝐿 x 100 (mohanty et al. 1991) value of relative chromatin (vrc) = ∑ total length of chromosome / n (dutta and bandyopadhyaya 2014) where n=somatic chromosome count gradient index (gi) = 𝑆𝑆ℎ𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜ℎ𝑜𝑜𝑜𝑜𝑟𝑟𝑜𝑜𝑜𝑜𝑜𝑜𝑟𝑟𝑜𝑜 𝐿𝐿𝑜𝑜𝐿𝐿𝐿𝐿𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜ℎ𝑜𝑜𝑜𝑜𝑟𝑟𝑜𝑜𝑜𝑜𝑜𝑜𝑟𝑟𝑜𝑜 x 100 (lavania and srivastava 1992) chromosome volume = πr2h where h = total length of chromosome (toijam et al. 2013) box 1 92 sanjay kumar, asikho kiso c a f e d b figure 1. somatic chromosome count, sechium edule (jacq.) sw.; a) g1, 2n=2x=26; b) g2, 2n=2x=26; c) g2, 2n=2x=32; d) g2, 2n=4x=52; e) g3, 2n=2x=30; f) g4, 2n=2x=26. 93chromosome number and karyotype study of sechium edule (jacq.) sw. from india i j h g k l figure 1 (continued). somatic chromosome count, sechium edule (jacq.)sw.; g) g5, 2n=2x=28; h) g6, 2n=2x=28; i) g7, 2n=2x=28; j) g8, 2n=4x=24; k) g9, 2n=2x=24; l) g10, 2n=2x=28. 94 sanjay kumar, asikho kiso intra chromosomal asymmetry index could contain more number of acrocentric or telocentric chromosomes than the metacentric and submetacentric chromosome which could be the result of change in position of centromere. the change in centromere position brings the rearrangement in the chromosomes and may lead to increase in karyotype asymmetry percent. intra chromosomal asymmetry depends on exact identification of the centromere and the chromosomal morphology but not only the chromosome size. the extreme symmetry (ideal karyotype a) or asymmetry (ideal karyotype c) of karyotype is meager in nature (stebbin 1971). however,the present analysis indicates an extreme symmetric karyotype (1a) among the genotypes except genotypes g2 and g11 and may be classified as ideal karyotype b of stebbin’s classification and suggested that karyotypes of the two genotypes deviated from symmetric to asymmetric and are in agreement with the hypothesis of stebbins classification (1971). according to the hypothesis asymmetric karyotypes are being originated from the symmetrical karyotypes over a period of time and due course of evolution. similar work has been reported earlier and in agreement that primitive members with symmetrical karyotypes give rise to advance members with the asymmetrical karyotype (levitzky 1931; kumar and kumar 2014). n o p m figure 1 (continued). somatic chromosome count, sechium edule (jacq.) sw.; m) g11, 2n=2x=26; n) g12, 2n=2x=30; o) g13, 2n=2x=26; p) g14, 2n=2x=28. 95chromosome number and karyotype study of sechium edule (jacq.) sw. from india the presence of asymmetric karyotype could be the result of chromosome structural changes particularly centric fusion or fission which leads to symploid or agmatoploid chromosome rearrangements in due course of plant species evolution. the centric fusion and fission could also be suggested as cause of frequent disploidy or pseudoaneuploidy among plant species (eroglu et al. 2013). both dispersion index (di) and gradient index (gi) are considered as combination of inter-intra chromosomal index and used for the evaluation of karyotype symmetry. both represents the nature of evolutionary process occurring or occurred in genus or species and indicates the trend of evolution had taken place in genus, species or cytotypes (lavania and srivastava 1992). comparatively, lower di value especially below 30 and higher gi value more than 30 suggested symmetrical nature of the karyotype for the genotypes except g2 with 2n=32 and 52 and supports stebbins hypothesis. both di and gi showed high degree of symmetry which may lead to the lesser degree of chromosomal variation and evolution (stebbins 1971). at present, 2n=2x=32 and 2n=4x=52 chromosome numbers were not reported earlier and, hence first report in the present paper. new chromosome number may suggest towards the whole genome content change, diversification, evolution and speciation in the genus. a very few or negligible reports are available on genus sechium from india (sanwal et al. 2008; kapoor et al. 2014; jain et al. 2015; jain et al. 2017). genus sehium remains very poorly known cytologically, therefore, proper chromosome count is important for understanding the interrelationship among different sechium species. conclusion somatic chromosome number and kar yomorphometric estimations are in the agreement of earlier reports except two new reports of chromosome number in genotype 2 of the sechium edule. acknowledgement author, sk is thankful to ak for collection of material and squash preparation of the sechium edule (jacq.) sw. the authors gratefully acknowledged the nagaland table 1 karyotype symmetry/asymmetry estimation of sechiumedule. g en ot yp e (s ) bcn (2x) σtcl or cv interchromosomal index intrachromosomal index inter+intra chromosomal index clr vrc σcvcl σa2 σrec largest/smallest chromosome ratio arm ratio proportion stebbin’s classification di gi g1 2n=2x=26 16.733 0.501-0.764 0.643 815.648 3.145 84.225 1.52 < 2:1 1a 20.79 65.575 g2 2n=2x=26 20.919 0.583-1.043 0.804 414.048 4.132 77.128 1.78 < 2:1 1a 28.29 55.896 g2 2n=2x=32 18.099 0.295-0.855 0.565 752.794 7.517 66.138 3.13 > 2:1 1b 70.289 31.929 g2 2n=4x=52 28.884 0.342-0.803 0.555 802.814 8.850 69.098 2.34 > 2:1 1a 40.174 42.643 g3 2n=2x=30 29.618 0.696-1.343 0.987 467.969 4.669 73.496 1.92 < 2:1 1a 31.731 51.824 g4 2n=2x=26 22.890 0.642-1.113 0.880 417.482 4.162 79.029 1.73 < 2:1 1a 26.837 57.681 g5 2n=2x=28 23.470 0.628-1.086 0.838 408.486 4.071 77.177 1.72 < 2:1 1a 26.721 57.826 g6 2n=2x=28 26.090 0.651-1.209 0.931 447.191 4.458 77.043 1.85 < 2:1 1a 30.00 53.846 g7 2n=2x=28 20.320 0.583-0.876 0.725 312.523 3.115 82.819 1.50 < 2:1 1a 20.082 66.552 g8 2n=2x=24 16.274 0.521-0.904 0.678 305.383 3.042 76.735 1.73 < 2:1 1a 26.877 57.632 g9 2n=2x=24 16.899 0.541-0.863 0.704 272.606 2.713 80.907 1.54 < 2:1 1a 22.934 62.688 g10 2n=2x=28 19.276 0.591-0.805 0.688 279.181 3.612 85.503 1.36 < 2:1 1a 15.329 73.416 g11 2n=2x=26 23.784 0.600-1.202 0.914 454.258 4.528 76.089 2.00 > 2:1 1b 33.407 49.916 g12 2n=2x=30 26.747 0.620-1.097 0.891 484.374 4.829 81.258 1.76 < 2:1 1a 27.781 56.517 g13 2n=2x=26 20.009 0.568-0.946 0.769 291.900 2.901 81.349 1.66 < 2:1 1a 24.966 87.925 g14 2n=2x=28 21.956 0.603-0.939 0.784 322.102 4.983 83.46 1.55 < 2:1 1a 21.789 64.217 bcn, basic chromosome number; 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(cucurbitaceae): a new adventives species for the flora of india. current science 111 (5): 789. thakur g k, sinha b m b. 1973.cytological investigation in some cucurbits. journal of cytology and genetics 7(8): 122-130. toijan h, borah s p, bhaben t, borthakur s k. 2013.karyomorphological studies in two species of allium l. journal of research in plant sciences 2(2): 213-221. caryologia. international journal of cytology, cytosystematics and cytogenetics 74(3): 3-8, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1134 caryologia international journal of cytology, cytosystematics and cytogenetics citation: ahmet l. tek, hümeyra yıldız, kamran khan, bilge ş. yıldırım (2021) chromomycin a3 banding and chromosomal mapping of 45s and 5s ribosomal rna genes in bottle gourd. caryologia 74(3): 3-8. doi: 10.36253/ caryologia-1134 received: november 12, 2020 accepted: semptember 24, 2021 published: december 21, 2021 copyright: © 2021 ahmet l. tek, hümeyra yıldız, kamran khan, bilge ş. yıldırım. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid alt: 0000-0002-3292-5142 kk: 0000-0001-7928-3290 hy: 0000-0003-2143-9242 chromomycin a3 banding and chromosomal mapping of 45s and 5s ribosomal rna genes in bottle gourd ahmet l. tek*, hümeyra yıldız, kamran khan, bilge ş. yıldırım department of agricultural genetic engineering, ayhan şahenk faculty of agricultural sciences and technologies, niğde ömer halisdemir university, 51240, niğde, turkey *corresponding author. e-mail: altek2@gmail.com abstract. ribosomal dnas and various banding patterns are landmarks in molecular cytogenetics providing useful information for karyotyping and addressing individual chromosomes. bottle gourd is the only cultivated species of the lagenaria genus with high genetic diversity. after cma3/dapi fluorochrome banding we investigated the gcand at-rich regions in interphase nuclei of five different local accessions. fluorescence in situ hybridization (fish) was conducted to determine the number and location of 45s and 5s rdnas in bottle gourd. our results showed four strong cma3 regions in interphase and on mitotic metaphase chromosomes. fish revealed four strong signals of 45s rdna at the termini of two metaphase chromosome pairs and terminal 5s rdna signals at another pair of chromosomes. the presence of four positive cma3 bands colocalizes with four 45s rdna signals in all bottle gourd accessions. our results allow distinguishing two out of eleven chromosome pairs of bottle gourd. keywords: bottle gourd, chromomycin a3, fluorescence in situ hybridization, local accessions, ribosomal dna, 5s, 45s. introduction bottle gourd, lagenaria siceraria, is a member of the cucurbitaceae family, also known as calabash or white-flowered gourd. it is a diploid crop species with 2n = 2× = 22 chromosomes (beevy and kuriachan 1996). the genome size of this plant is approximately 365 mbp (achigan-dako et al., 2008). dna markers revealed that chinese bottle gourd and turkish bottle gourd accessions have a close phylogenetic relationship within other cucurbit species (xu et al., 2011; yildiz et al., 2015). these findings have implications for the preservation of bottle gourd genetic diversity and advanced markerassisted breeding studies (xu et al., 2011; yildiz et al., 2015). the lagenaria genus consists of six species: l. siceraria (cultivated form), l. sphaerica, l. rufa, l. breviflora, l. sphaerica, and l. guineensis. all six species are naturally found in africa, the supposed center of genetic diversity for l. siceraria. 4 ahmet l. tek, hümeyra yıldız, kamran khan, bilge ş. yıldırım bottle gourd is the only cultivated species. in the tropics, it is one of the oldest crops (erickson et al., 2005). bottle gourd is cultivated for food, decoration, medicine, domestic utensils, musical instruments, containers, and fishing floats (morimoto and mvere, 2004, xu et al., 2011). some bottle gourd varieties are grown for their seeds which are rich in oil and essential amino acids (achigan-dako et al., 2008). additionally, bottle gourd seedlings are used as a rootstock for watermelon against adverse effects in soil such as low temperature, high ph, salinity, excessive water as well as soil-borne diseases, such as fusarium wilt (yetisir et al., 2007). for seedless watermelon production, bottle gourd pollen has recently been utilized to pollinate watermelons (sugiyama et al., 2014). therefore, bottle gourd is a crop of great economic interest and a detailed characterization at the chromosome and genome level is desirable. the plant genomes contain a significant amount of repetitive dna sequences. among them, ribosomal dnas (rdnas) encode the rna components of ribosomes. two structurally distinct gene families of rdnas exist in plant genomes, specifically known as 45s and 5s rdnas. non-transcribed spacers and tandem repeat units of the 18s-5.8s-26s ribosomal genes are present in the 45s rdna. the 5s rdna genes consist of a nontranscribed spacer and a conserved coding region of 120 bp (long and dawid, 1980). 45s and 5s rdna genes can be present at one or more positions within a set of chromosomes and be used as chromosomal markers (long and dawid, 1980; lombello and pinto-maglio, 2007; han et al., 2008; heslop-harrison and schwarzacher, 2011; li et al., 2016; santos-sanchês et al., 2019). cma3 (chromomycin a3), a gc-rich specific f luorochrome, and dapi (4’-6-diamidino-2-phenylindole), an at-rich specific fluorochrome, banding techniques can also be useful to differentiate between chromosomes (kim et al. 2002). the bottle gourd and its relatives have small and morphologically similar chromosomes, and fluorescent chromosome staining techniques such as cma3 and dapi might be helpful to distinguish them and proved to be useful for determining the phylogenetic relationships among plant species (schweizer, 1976; kim et al. 2002; yamamoto et al., 2007; volkov et al., 2017; maragheh et al., 2019). fluorescence in situ hybridization (fish) has been widely used for constructing chromosomal maps, for chromosome identification, for studying the dynamic organization of chromatin in interphase nuclei as well as for studying chromosome homology and karyotype evolution (lysak et al., 2006; probst, 2018; santos et al., 2020). previously, rdna mapping in bottle gourd was investigated by waminal and kim (2012) and li et al. (2016), although definite information is not available about origin of accessions used for comparison. these authors found four signals of 45s and two signals of 5s on metaphase chromosomes. there is no report for the cma3/dapi staining of the metaphase chromosome of bottle gourd. we hypothesize that a combination of cma3/dapi staining and fish can be used for the determination of any possible chromosomal variability from different geographical origins. given the high genetic diversity of bottle gourds, we tested a set of local varieties from turkey for potential variability of rdnas loci and differentiation of their metaphase chromosomes by cma3/dapi staining. materials and methods plant material bottle gourd (lagenaria siceraria) seeds were obtained from a local population in sandıklı, afyonkarahisar, turkey and used as a main accession for the research if not indicated otherwise. additionally, four different accessions were used from provinces of hatay, niğde/merkez, niğde/ulukışla, and niğde/bor (turkey). these accessions with different seed morphology and high germination rates were chosen to cover different localities to determine variability. moistened seeds were placed on sterile filter paper and germinated in petri dishes using double distilled water. mitotic chromosome preparation the mitotic chromosome preparation was performed according to the published protocols (tek et al. 2011) with the following modifications. young root (~1 cm) tips were cut off with a razor blade, treated with 2 mm 8-hydroxyquinoline at room temperature, and subsequently, fixed in 3:1 methanol: glacial acetic acid for 24 h at -20 °c. after fixation, root tips were washed three times in distilled water and 30 mm potassium chloride, digested in an enzyme mixture, ph 4.5, containing 4% cellulase and 2% pectinase at 37 °c for 90 min. digested root tips were washed in distilled water. following a fixation step, slides were prepared by the flame-dry (tek et al. 2011). slides with suitable cells were selected using a phase-contrast microscope. chromomycin a3/dapi staining chromosome staining with cma3 and dapi was performed as described (schweizer 1976). briefly, slides 5mapping ribosomal genes in bottle gourd were stained for 20 min with cma3 (0.5 mg/ml) in mcllvaine’s buffer (ph = 7.0). subsequently, slides were incubated at 37 ºc for 2 days with dapi (0.5 µg/ml) (hasterok et al., 2006). probe preparation to determine 45s and 5s rdna sites, plasmid clones pta71, and pta794 were used, respectively (gerlach and bedbrook, 1979; gerlach and dyer 1980). both rdnas were labeled with digoxigenin-11-dutp using a nick translation kit (roche) according to the manufacturer’s instructions. fluorescence in situ hybridization and signal detection fish was conducted according to tek et al., (2011) with modifications. a hybridization mixture containing the denatured probe dna in 50% formamide, 10% dextran sulfate, 2×ssc was applied. the slides with chromosomes were denatured in 70% formamide with 2×ssc at 80 °c for 2 min before hybridization at 37 °c overnight. the rhodamine-conjugated anti-digoxigenin antibody (roche) was used to detect both 45s and 5s signals in independent experiments. slides were checked using a fluorescence microscope at 63× magnification (carl zeiss axio imager.a2). photographs were captured with monochromatic charge-coupled device (ccd) camera (carl zeiss axiocam 702) operated with multichannel zen pro imaging software. results and discussion chromosomes of a local bottle gourd accession were examined for the presence of gcand at-rich heterochromatin regions by cma3/dapi and by fish for distribution of rdnas. gc-rich heterochromatin regions, as displayed by fluorochrome banding, proved to be correlated with rdna genes. in the interphase stage, four strong positive cma3 bands were observed (fig. 1a-c). four strong positive cma3 bands were also detected on the metaphase chromosomes (fig. 1d-f ). among these positive cma3 bands were clear differences observed on the size and brightness on the interphase as well as the mitotic metaphase chromosomes (fig. 1h, k, n, r). two of the bands are small and less bright, while the other two are large and bright. these observations are consistent among all five accessions analysed indicating the similarity of the structural cma3 bands. on the metaphase chromosomes, all four bright and strong cma3 bands were present in termini regions of two pairs of chromosomes. also fish with the 45s rdna probe yielded four signals. in plants, 45s rdna loci and cma3 positive heterochromatic blocks often coincide spatially (lombello and pinto-maglio, 2007; maragheh et al., 2019; santos-sanchês et al., 2019). 5s rdna sites were not detected by cma3/dapi banding. the same number and position of ribosomal gene (45s rdna) showed that cma3/ dapi positive bands may overlap with 45 rdna sites. similarly, our method, first with fish experiment and subsequently with cma3/dapi banding procedure on the same interphase nuclei, does allow conclusive evidence of overlap between 45s rdna signals and cma3/ dapi positive bands (fig. 2a-c, d-f ). when we investigate cma3 bands in terms of size and intensity, a pair of chromosomes have big and another pair of chromosomes consistently have small signals in both metaphase and interphase stages whereas we did not detect clear differences on 45s rdna signals using fish (fig. 2a-c, d-f ). nevertheless, these differences are more prominent as presented in fig. 1 lombello and pinto-maglio, (2007) worked on the bitter gourd (momordica charantia), which is a member of the same family as bottle gourd. they found no band on the chromosomes with dapi staining. santos-sanchês et al., (2019) conducted similar work on different melon accessions and reported a dapi positive band on the metaphase chromosomes. chromosomes stained with cma3 revealed four bands in terminal regions of chromosomes in this species. our results of cma3/dapi staining are in line with those obtained by lombello and pinto-maglio, (2007) and santossanchês et al., (2019). to detect the position and number of 45s and 5s rdnas, fish with digoxigenin-labeled probes was applied. in interphase nuclei, four strong red signals of 45s rdna were observed (fig. 2a-c). the size and intensity of all four red signals were similar, which is in contrast to our findings from the cma3 bands. four strong 45s rdna signals were also detected on the short arm ends of two mitotic metaphase chromosome pairs (fig. 2g-i). two strong signals of 5s rdna were observed in interphase nuclei (fig. 2j-l) as well as on metaphase chromosomes (fig. 2m-o). there was no prominent difference in the size and intensity of 45s and 5s rdna signals. the 5s rdna signals appeared on the short arm of termini of a metaphase chromosome pair (fig. 2o). the two rdna families are usually not positioned on the same chromosomes, with some exceptions (waminal and kim, 2012; li et al., 2016). in conclusion, rdna loci and cma3 bands in accessions of lagenaria siceraria, as in other species provide useful markers to distinguish at least two chromosome pairs individually. also, our data 6 ahmet l. tek, hümeyra yıldız, kamran khan, bilge ş. yıldırım demonstrate that a relatively low level of intraspecific chromosomal diversity is present among morphologically different bottle gourd accessions. author contributions alt conceived the study and designed the experiments. hy, kk and alt performed the experiments. hy, kk, bşy and alt conducted data analysis. hy, kk, bşy and alt wrote the paper. all authors read and approved the final manuscript. figure 1. chromomycin a3/dapi (cma3/dapi) staining in lagenaria siceraria nuclei and mitotic chromosomes (2n = 2× = 22) from five different local accessions. interphase nuclei (a-c, g-i, j-l, m-o, p-s), prometaphase chromosomes (d-f ), gc-rich loci stained with cma3 (b, e, h, k, n, r; green signals) and dapi merge image (c, f, i, l, o, s) are shown on the chromosomes. images are shown from the local accessions obtained from provinces of sandıklı (a-f ), hatay (g-i), niğde/ulukışla (j-l), niğde/merkez (m-o), niğde/bor (p-s). scale bar = 5 μm. figure 2. localization of 45s and 5s rdna in lagenaria siceraria nuclei and mitotic chromosomes (2n = 2× = 22). dapi stained interphase chromosomes (a-c), metaphase chromosomes (g-i), 45s rdna loci labeled with digoxigenin (b, h; red signals of rhodamine), 45s rdna loci merge image (c, i) is shown on the chromosomes. interphase nuclei (d-f ), gc-rich loci staining with cma3 (e; green signal). dapi stained interphase chromosomes (j-l), metaphase chromosomes (m-o), 5s rdna loci labeled with digoxigenin (k, n; red signals of rhodamine), 5s rdna loci merge image (l, o) is shown on the chromosomes. scale bar = 5 μm. 7mapping ribosomal genes in bottle gourd funding t his work was pa r t ia l ly suppor ted by t he indepth-cost action ca16212 and scholarship program from ayhan şahenk foundation. hy and bşy recognize the scholarship from yök 100/2000 plant genetics and agricultural biotechnology. acknowledgments we thank prof. ingo schubert for critical reading and valuable suggestions on the manuscript and the lab members for technical assistance. references achigan-dako eg, fuchs j, ahanchede a, blattner fr (2008) flow cytometric analysis in lagenaria siceraria (cucurbitaceae) indicates correlation of genome size with usage types and growing elevation. plant syst evol 276:9–19. https://doi.org/10.1007/s00606008-0075-2 beevy ss, kuriachan p (1996) chromosome numbers of south indian cucurbitaceae and a note on the cytological evolution in the family. j cytol genet 31:65– 71 erickson dl, smith bd, clarke ac, et al (2005) an asian origin for a 10,000-year-old domesticated plant in the americas. proc natl acad sci usa 102:18315– 18320. https://doi.org/10.1073/pnas.0509279102 gerlach wl, bedbrook jr (1979) cloning and characterization of ribosomal rna genes from wheat and barley. nucleic acids res 7:1869–1885. https://doi. org/10.1093/nar/7.7.1869 gerlach wl, dyer ta (1980) sequence organization of the repeating units in the nucleus of wheat which contain 5s rrna genes. nucleic acids res 8:4851– 4865. https://doi.org/10.1093/nar/8.21.4851 han yh, zhang zh, liu jh, et al (2008) distribution of the tandem repeat sequences and karyotyping in cucumber (cucumis sativus l.) by fluorescence in situ hybridization. cytogenet genome res 122:80–88. https://doi.org/10.1159/000151320 hasterok r, wolny e, hosiawa m, et al (2006) comparative analysis of rdna distribution in chromosomes of various species of brassicaceae. ann bot 97:205– 216. https://doi.org/10.1093/aob/mcj031 heslop-harrison jsp, schwarzacher t (2011) organisation of the plant genome in chromosomes. plant j 66:18– 33. https://doi.org/10.1111/j.1365-313x.2011.04544.x kim es, punina eo, rodionov av (2002) chromosome cpd(pi/dapi)and cma/dapi-banding patterns in allium cepa l. genetika 38:489–496 https://doi. org/10.1023/a:1015250219322 li k-p, wu y-x, zhao h, et al (2016) cytogenetic relationships among citrullus species in comparison with some genera of the tribe benincaseae (cucurbitaceae) as inferred from rdna distribution patterns. bmc evol biol 16:85. https://doi.org/10.1186/s12862016-0656-6 lombello ra, pinto-maglio caf (2007) cytomolecular studies in momordica charantia l. 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(asteraceae) species that around the van lake in turkey pelin yilmaz sancar1,*, semsettin civelek1, murat kursat2 molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant jinxin cheng1,*, dingyu hu1, yaran liu2, zetian zhang1, majid khayatnezhad3 genetic variations and interspesific relationships in salvia (lamiaceae) using scot molecular markers songpo liu1, yuxuan wang1, yuwei song1,*, majid khayatnezhad2, amir abbas minaeifar3 development of female gametophyte in gladiolus italicus miller (iridaceae) ciler kartal1, nuran ekici2,*, almina kargacıoğlu1, hazal nurcan ağırman1 colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae) harshita dwivedi*, girjesh kumar statistical evaluation of chromosomes of some lathyrus l. taxa from turkey hasan genç1, bekir yildirim2,*, mikail açar3, tolga çetin4 use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater treatment and water of the kamniška bistrica river (slovenia) peter firbas1, tomaž amon2 the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bo shi1,*, majid khayatnezhad2, abdul shakoor3,4 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(4): 111-119, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1296 caryologia international journal of cytology, cytosystematics and cytogenetics citation: simona ceraulo, vanessa milioto, francesca dumas (2021) centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates). caryologia 74(4): 111-119. doi: 10.36253/caryologia-1296 received: april 22, 2021 accepted: november 27, 2021 published: march 08, 2022 copyright: © 2021 simona ceraulo, vanessa milioto, francesca dumas. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates) simona ceraulo, vanessa milioto, francesca dumas* department of scienze e tecnologie biologiche, chimiche e farmaceutiche (stebicef), university of palermo, palermo, italy *corresponding author. e-mail: francescadumas@unipa.it abstract. line-1 sequences have been linked to genome evolution, plasticity and speciation; however, despite their importance, their chromosomal distribution is poorly known in primates. in this perspective, we used fluorescence in situ hybridization (fish) to map line-1 probes onto two representative platyrrhine species, aotus nancymaae (cebidae) and alouatta belzebul (atelidae), both characterized with highly rearranged karyotypes, in order to investigate their chromosomal distribution and role and to better characterize the two genomes. we found centromeric enrichment of line-1 sequences on all biarmed and acrocentric chromosomes co-localized with heterochromatin c-positive bands. this distribution led us to hypothesize that line 1 sequences may have a role in the centromere architecture and karyotype organization of platyrrhine genomes. keyword: transposable elements, c-banding, molecular cytogenetics probes, genome evolution. introduction through classic and molecular cytogenetics, many primates have been shown to have variable karyotypes; many kinds of probes have been mapped, including single locus probes (dumas and sineo 2010, 2012), and bacterial artificial chromosomes (bac) (dumas and sineo 2014; dumas et al. 2015) and whole chromosome paints have been used (dumas et al. 2007; dumas et al. 2012), showing a high rate of intrachromosomal and intrachromosomal rearrangements. in particular, among platyrrhini (new world primates) living in tropical and neotropical regions, the genera alouatta (howler monkeys) (cebidae) and aotus (owl monkey) (cebidae) have very derived karyotypes. originally, one or only a few species were recognized in these two genera: in aotus there was just one, while later up to eleven species were described, with many of them showing different karyomorphs and having diploid numbers ranging between 2n=46 to 56; alouatta went from five rec112 simona ceraulo, vanessa milioto, francesca dumas ognized species up to 15, with diploid numbers ranging between 2n=43 to 58. furthermore, both species show an extra sex chromosome system due to a translocation between an autosome and the y chromosome. among the two genera, chromosome painting has been applied to two aotus species, aotus nancymaae, and aotus lemurinus greisemebra (stanyon et al. 2004; stanyon et al. 2011) and six alouatta species, including alouatta belzebul (consigliere et al. 1996, 1997; de oliveria et al. 2002), showing high genome variability. bac-fish has also been performed on aotus and alouatta showing intrachromosomal rearrangements (dumas et al. 2015; scardino et al. 2020a). although, these species have been studied through molecular cytogenetics with different kinds of probes, repetitive sequences have been poorly studied and, among them, only rdna and telomeric probes have been mapped often (mazzoleni et al. 2017; 2018, ceraulo et al. 2021a). the study of these sequence probes’ distribution can help locate useful cytogenetic markers for evolutionary and phylogenetic studies. repetitive elements have been extensively investigated in order to clarify their possible role in genome evolution and organization (ahmed and liang 2012; biscotti et al. 2015; dumas et al. 2016; mazzoleni et al. 2017, 2018; milioto et al. 2019; paço, et al. 2019; scardino et al. 2020b). in primates, repetitive sequences constitute about 50% of their genome and are linked to chromosome evolution (mathews et al. 2003; jurka 2007; xing et al. 2007; kvikstad and makova 2010). a class of repetitive sequences called long interspersed elements of the family 1 (line-1) are retrotransposable; the biological roles of this repetitive dna fraction have been linked to many mechanisms implicated in the genome structure, evolution and disease (zhu et al. 2011; paço et al. 2019). in addition, their involvement in genome architecture such as in dna packaging, centromere stability and plasticity, gene expression, and epigenetic mechanisms has been shown (kim and han 2015; klein and o’neill 2018; ahmed et al. 2020). these sequences have also been supposed to be promoters of genomic evolutionary changes and of biological diversity among vertebrates, with an important role in speciation (böhne et al. 2008; belyayev 2014, klein and o’neill 2018). with advances in dna technologies, the approaches useful for identifying them have changed, with the main approaches being the use of restriction enzyme digestion of dna, in situ hybridization and bioinformatic analysis of dna sequencing data. in mammals and primates, line-1 were studied through different approaches including the use of restriction enzymes (seuanez et al., 1989) or whole genome screening in simians (ohshima et al. 2003). in humans and anthropoids, line-1 sequence comparisons have been made (ovchinnikov et al. 2001, 2002, mathews et al. 2003) showing that line-1 amplification may change rapidly during primate evolution giving different families with variable forms among new world monkeys, high line activity has also been shown in the saimiri and saguinus lineages (callitrichini), and reduced activity has been found in the ateles lineage (boissinot et al. 2004, sookdeo et al. 2018). so far, however, the distribution of these repetitive sequences through fish with line-1 probes has been studied in few platyrrhine species, belonging to the callithricini subfamily of the cebidae family (serfaty et al. 2017, ceraulo et al. 2021b). the objective of this study was to overcome this lack and analyze the distribution of these line-1 sequences by fish onto two more representative platyrrhine genomes, aotus nancymaae (atelidae) and alouatta belzebul (cebidae), with the aim of contributing to the understanding of their role and dynamics as well as the evolution of these highly derived groups of species. materials and methods following the standard protocol (scardino et al. 2020b), metaphases were obtained from primary fibroblast cell line cultures for aotus nancymaae and alouatta belzebul. l1 probe preparation dna extraction from the cell culture pellet derived from the fibroblast cell line was done according to the basic dna extraction protocol from invitrogen. line1 retrotransposon was amplified through polymerase chain reaction (pcr) using the following primers: l1r, 5’-attctrttc cat tgg tct a-3’ and l1f 5’-cca tgc tcatsgat tgg -3’ (waters et al. 2004). 200 ng of genomic dna was amplified in 50 μl reactions in an applied biosystems pcr simpliamp thermal cycler (thermo fisher scientific): five units of taq dna polymerase were incubated together with the template dna, 500 nm of each primer, 200 μm each of datp, dctp, dttp and dgtp in 10 mm tris-hcl, ph 8.3, 1.5 mm mgcl2, 50 mm kcl. cycling parameters were 30 cycles of 94°c, 30 s; 52.5°c, 30 s; 72°c, 30 s, following a 2 min denaturation at 94°c. products were visualized on 1% agarose gel. the pcr amplification products were labelled through nick translation using 11-dutp-fluorescein. 113centromeric enrichment of line-1 retrotransposon in alouatta belzebul and ateles nancymaae fish, karyotyping and chromosome staining fluorescence in situ hybridization (fish) was performed following previously described protocols (scardino et al. 2020a, b; milioto et al. 2019; vizzini et al. 2021); c-banding was done sequentially, post-fish, according to a protocol which includes denaturation with formamide (fernandez et al. 2002). the karyotype was reconstructed using both g-banding and inverted dapi banding, in agreement with previous published karyotypes: aotus nancymaae (stanyon et al. 2004, 2011; ruiz herrera et al. 2005; dumas et al. 2016) and alouatta belzebul (consigliere et al. 1998; de oliveira et al. 2002; stanyon et al. 2011). dapi images were inverted with a photo editing program (adobe photoshop); inverted gray bands correspond to dark g bands. the chromosomes with the line-1 probe signals were identified using inverted dapi. after fish, the metaphases were analyzed under a zeiss axio2 epifluorescence microscope. images were captured using a coupled zeiss digital camera, and the chromosomes were classified according to the nomenclature proposed by levan et al. (1964). results metaphases of the two analyzed species, obtained through cell culture and chromosome harvesting, were stained post-fish using dapi. the species studied here have the diploid number of 2n=50 and 2n=54, respectively, in alouatta belzebul and aotus nancymaae (fig 1, 2). the former has 11 pairs of metacentric and submetacentric chromosomes (1-11) and 13 acrocentric chromosomes pairs (12-24) plus xy; the second species has 18 pairs of metacentric and submetacentric chromosomes (1-18) plus the xx, and 7 acrocentric/subacrocentric chromosome pairs (19-26); c-banding showed signals at the centromeric position of both biarmed and acrocentric chromosomes pairs (fig 1, 2) with peculiar amplified c bands on the bigger subtelocentric chromosomes, in agreement with previous analysis (torres et al. 1998). in all the analyzed species, the line-1 probe mapping revealed bright signals at the centromeric position of almost all chromosomes, with a variable signal amplification. on subtelocentric chromosomes, signals were very bright on the p arms (fig. 1, 2). chromosome x was rich in line-1 at the centromere. we did not find l1 signals in areas away from centromeres. we speculated that l1 elements should be in a lower copy number in chromosome regions far from centromeres, below the detection efficiency of fish, or our probe was not able to hybridize the variable or degraded l1 elements. discussion in general, many mammalian species have deposition of this line-1 element in euchromatic regions in g-positive bands (parish et al. 2002; waters et al. 2004), while in other few species it occurs in heterochromatic regions, especially in the centromeric region (waters et al. 2004). however, the pattern of line-1 distribution at the centromere is not a common phenomenon among the mammalian genome (waters et al. 2004; dobigny et al. 2004, 2006; acosta et al. 2008; vieira-dasilva et al. 2016; de sotero-caio et al. 2017); indeed, these elements are not often incorporated at major core centromeres, with the exception of the x chromosome euchromatic regions where they are usually abundant along the chromosomal length (waters et al. 2004; acosta et al. 2008). on the other hand, massive accumulations of repetitive elements at the centromeres was previously shown in many other mammals, such as bats and rodents (sotero-caio et al. 2017; paco et al. 2015; paço et al. 2019), and in some primates (carbone et al. 2012; serfaty et al. 2017, ceraulo et al., 2021b). in particular, among primates species, line-1 have been previously identified through fish into platyrrhine genomes, in saguinus midas and saguinus bicolor (serfaty et al. 2017), in saguinus mystax, leontocebus fuscicollis, leontopithecus rosalia (ceraulo et al., 2021b) (tamarins of the cebidae family). this result is in agreement with sequence data analysis that showed active line on cebidae species (boissinot et al. 2004) and, more recently, also in atelidae (sookdeo et al. 2018). at the beginning, from an analysis of just ateles paniscus (boissinot et al. 2004), the extinction of line 1 in atelidae was proposed, but a larger phylogenetic sampling permitted researchers to show their presence (sookdeo et al. 2018). in our work, we found line-1 elements by fish in the two species analyzed of both the cebidae and atelidae families, at centromeric position in agreement with previous cytogenetic molecular data (serfaty et al. 2017, ceraulo et al., 2021b) and supporting also previous molecular data (sookdeo et al. 2018). line-1 probes displayed a non-random distribution by accumulating primarily in cma3 positive bands at centromeres or pericentromeric regions, co-localizing with c-positive heterochromatin bands (fig 1, 2); the co-localization of line-1 with c-positive bands was previously identified 114 simona ceraulo, vanessa milioto, francesca dumas figure 1. examples of alouatta belzebul metaphases in dapi blue (a), fish with line-1 probe in green (b), sequential c-inverted banding (c), dapi and line-1 overlap (d); the reconstructed karyotype from another metaphase of the species after sequential staining and probe mapping (e). 115centromeric enrichment of line-1 retrotransposon in alouatta belzebul and ateles nancymaae figure 2. examples of aotus nancymaae metaphases in dapi blue (a), fish with line-1 probe in green (b), and g-inverted banding (c), dapi and line-1 in overlap (d); the reconstructed karyotype from the same metaphase after sequential staining and probe mapping (e). 116 simona ceraulo, vanessa milioto, francesca dumas not only in primates but also in other taxa (kapitonov et al. 1998; serfaty et al. 2017). the finding of the centromeric enrichment of lines in all analyzed platyrrhine species permitted us to hypothesize that this accumulation might have occurred in the common ancestor of all platyrrhini, contributing to their current karyotype features. these very intense, amplified and bright signals at centromeres possibly indicate that line-1, together with the alpha satellite dna, are presumably responsible for the architecture of almost all biarmed and acrocentric chromosomes in platyrrhini. traditionally, alpha-satellite dna has been identified as the main dna component of primate centromeres; it consists of multiple repeat units forming a larger repeat unit, and the larger units are repeated tandemly (koga et al. 2014), with exception of marmosets where the larger repeat unit is not present (cellamare et al. 2009). the presence of line-1 at the centromere position is not a surprise; indeed, in the pericentromeric region of the human genome, for example, in addition to satellite dna, additional elements, mainly retrotransposon elements, have also been shown (ahmed et al. 2020). moreover, the presence of different and diverse sequences at the centromere have also been shown in the platyrrhine genome, indicating that this region has very variable components in new world monkeys (valeri et al. 2021). furthermore, although transposable elements and satellite dna present differences in their structure, genomic organization, spreading mechanisms and evolutionary dynamics, several studies have highlighting their relatedness, with transitions from transposable elements to alpha-satellite dna, and vice versa, through a process known as the dna remodeling mechanism (mestrovich et al. 2015; paco et al. 2019). the possible link between transposable elements and alphasatellite dna, could be show in the line signal amplification at the centromere position of subtelocentric/ acrocentric chromosome p arms, localizing with heterochromatin c-positive bands in ateles chromosomes; these regions are the same where often it is found telomeric signal probes and rdna probes amplification in many primates, as previous works have demonstrated (mazzoleni et al. 2017, 2018, ceraulo et al., 2021a); thus, this observation presumably indicates that these line1, alpha-satellite and other repetitive sequences could be involved to this dna remodeling mechanism. however, considering that line have also been linked with chromosomal rearrangements (böhne et al. 2008; de soterocaio et al. 2017, klein and o’neill 2018), we should take into consideration that these sequences could have had a role in the process leading to the increased rates of chromosomal evolution responsible for the highly rearranged karyotypes of many taxa; for example, the link between evolutionary reshufflings and li accumulation have been hypothesized in other platyhirrine species saguinus mystax, leontocebus fuscicollis, leontopithecus rosalia (ceraulo et al., 2021b) and from data obtained in rodent genera, where the most derived species display a higher level of line-1 accumulation on both autosomal and sex chromosomes then the more conserved ones (with accumulation usually only in the sex chromosomes) (dobigny et al. 2004; rebuzzini et al. 2009; vieiradasilva et al. 2016). in order to clarify this hypothesis regarding the link between evolutionary reshufflings and li accumulation, more samples should be analyzed in the future in a comparative perspective considering derived and conserved primate taxa. conclusion chromosomal studies through fish mapping onto the analyzed species’ genomes permitted the localization of the line sequences at the centromere position of biarmed and acrocentric chromosomes, co-localizing with c-positive bands. in a comparative perspective, the presence of these sequences at centromeres in many platyrrhine species led us to propose that line-1 could have had a role in the architecture and organization of the present features of platyrrhine karyotypes. in the future, studies on more samples at the species and population level could help in understanding their origin, evolutionary dynamics and function in the karyotypes of primates. funding the study was supported by an ffr 2020 grant from the university of palermo to fd. acknowledgments special thanks to polina perelman from the institute of 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(caprifoliaceae), using scot molecular markers fengzhen chen1, dongmei li2,* , mohsen farshadfar3 the new chromosomal data and karyotypic variations in genus salvia l. (lamiaceae): dysploidy, polyploidy and symmetrical karyotypes halil erhan eroğlu1,*, esra martin2, ahmet kahraman3, elif gezer aslan4 cytogenetic survey of eight ant species from the amazon rainforest luísa antônia campos barros1, gisele amaro teixeira2, paulo castro ferreira1, rodrigo batista lod1, linda inês silveira3, frédéric petitclerc4, jérôme orivel4, hilton jeferson alves cardoso de aguiar1,5,* molecular phylogeny and morphometric analyses in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f. neda atazadeh1,*, masoud sheidai1, farideh attar2, fahimeh koohdar1 a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) melika tabasi1, masoud sheidai1,*, fahimeh koohdar1, darab hassani2 genetic diversity and relationships among glaucium (papaveraceae) species by issr markers: a high value medicinal plant lu feng1,*, fariba noedoost2 morphometric analysis and genetic diversity in rindera (boraginaceae-cynoglosseae) using sequence related amplified polymorphism xixi yao1, haodong liu2,*, maede shahiri tabarestani3 biosystematics, fingerprinting and dna barcoding study of the genus lallemantia based on scot and remap markers fahimeh koohdar*, neda aram, masoud sheidai karyotype analysis in 21 plant families from the qinghai–tibetan plateau and its evolutionary implications ning zhou1,2, ai-gen fu3, guang-yan wang1,2,*, yong-ping yang1,2,* some molecular cytogenetic markers and classical chromosomal features of spilopelia chinensis (scopoli, 1786) and tachybaptus ruficollis (pallas, 1764) in thailand isara patawang1,*, sarawut kaewsri2, sitthisak jantarat3, praween supanuam4, sarun jumrusthanasan2, alongklod tanomtong5 centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates) simona ceraulo, vanessa milioto, francesca dumas* repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil marina souza cunha1,2,*,#, silvana melo1,3,#, filipe schitini salgado1,2, cidimar estevam assis1, jorge abdala dergam1,* karyomorphology of some crocus l. taxa from uşak province in turkey aykut yilmaz*, yudum yeltekin variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l. egizia falistocco1,*,+, gianpiero marconi1,+, lorenzo raggi1, daniele rosellini1, marilena ceccarelli2, emidio albertini1 404 not found caryologia. international journal of cytology, cytosystematics and cytogenetics 74(4): 129-134, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1354 caryologia international journal of cytology, cytosystematics and cytogenetics citation: aykut yilmaz, yudum yeltekin (2021) karyomorphology of some crocus l. taxa from uşak province in turkey. caryologia 74(4): 129-134. doi: 10.36253/caryologia-1354 received: june 29, 2021 accepted: november 30, 2021 published: march 08, 2022 copyright: © 2021 aykut yilmaz, yudum yeltekin. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyomorphology of some crocus l. taxa from uşak province in turkey aykut yilmaz*, yudum yeltekin department of molecular biology and genetics, faculty of science and arts, uşak university, 64200, uşak, turkey *corresponding author. e-mail: aykut.yilmaz@usak.edu.tr abstract. the increasing number of new taxa for each day and the presence of the samples exhibiting variable characters depend on this situation make very problematic the genus crocus as taxonomic and phylogenetic. for this reason, the many studies based on pcr, dna barcoding and cytogenetics are applied to provide contribution for taxonomic problems and phylogenetic relationships of the genus crocus. in this study, detailed karyotypic investigation of four taxa (c. pallasii goldb. subsp. pallasii, c. olivieri j.gay subsp. olivieri, c. fleischeri j.gay and c. uschakensis rukśans) belonging to uşak province in turkey was carried out and compared with the studies made previously. the somatic chromosome numbers of studied taxa were found to be 2n=14 for c. pallasii subsp. pallasii, 2n=8 for c. olivieri subsp. olivieri, 2n=20 for c. fleischeri and 2n=20 for c. uschakensis. c. uschakensis has only satellite on the short arm of chromosome 7. some differences with previous studies in aspect of chromosome number and morphology were determined in this study. furthermore, there is no enough literature information on crocus uschakensis and it was provided with this study based on detailed chromosomal investigation. keywords: crocus, cytogenetics, c. pallasii subsp. pallasii, c. olivieri subsp. olivieri, c. fleischeri and c. uschakensis. introduction the genus crocus l. belonging to the family iridaceae is represented by about 200 species and show distribution from western europe and north west africa to western china (mathew 1982; harpke et al. 2016; saxena 2016; roma-marzio et al. 2018). especially, the mediterranean region extending eastward into the irano-turanian region is the place containing the majority of the species in the genus crocus (saxena 2016). turkey is one of the most important countries with species number and endemism rate for the crocus taxa. the genus crocus is systematically very problematic. the variable characters caused by environmental factors due to extensive variety of habitats is the one of the most important reasons for taxonomic problems. furthermore, intermediate characters caused by introgression as a result of hybridization is observed frequently in closely related species (harrison and lar130 aykut yilmaz, yudum yeltekin son 2014; kerndorff et al. 2016; yılmaz 2021b). the number of the taxa belonging to the genus crocus have recently doubled with the detailed field studies particularly in turkey (addam et al. 2019). while uslu et al. (2012) states that there are almost 70 crocus taxa which is their 31 endemic to turkey, gedik et al. (2017) states that turkey is represented by 132 taxa which is their 108 endemic. however this caused increase the taxonomic problems at the infraspecific level. as a result, it is proposed that subspecies status can not be maintained and anymore must be categorized as species (harpke et al. 2016; addam et al. 2019). another important situation which increase taxonomic problems in the genus crocus is the changes observed in the chromosome number. studies on the karyotypes of crocus taxa show chromosome number changes from 2n=6 to 2n=70 within the genus (brighton et al. 1973; uslu et al. 2012; harpke et al. 2013). furthermore, it was observed that some species from different localities show variation in chromosome number (uslu et al. 2012; karamplianis et al. 2013). all of these makes problematic the genus and doubtful the species identification within the genus. in addition to cytogenetic studies, many molecular studies based on different pcr methods and dna barcoding containing nuclear and cpdna sequences show reality of this situation (petersen et al. 2008; harpke et al. 2013; erol et al. 2014; yılmaz 2021 a,b). in this study, the crocus taxa (c. pallasii subsp. pallasii, c. olivieri subsp. olivieri, c. fleischeri and c. uschakensis) from uşak province were detailed examined based on their somatic chromosome numbers, karyotypic descriptions, length ranges, haploid complements and other morphometric parameters such as ic (centromeric index), a1 (intrachromosomic asymmetric index), a2 (interchromosomic asymmetric index). one of the most important reasons for the choosing this region in the study is that there is not enough information about crocus taxa in uşak which is one of the regions with the highest species diversity, in addition to cytological data. materials and methods plant samples examined in this study were collected from uşak province in turkey. there are four taxa containing c. pallasii subsp. pallasii, c. olivieri subsp. olivieri, c. fleischeri and c. uschakensis in this study (table 1). root tips for plant samples belonging to each taxa were used to provide somatic metaphase chromosomes. firstly, root tips that are convenient for working were put into small glass bottles and then pretreated in α-monobromobromonaphthalene for 14-16 h at 4°c. after the first treatment, root tips were fixed with carnoy solution for overnight. fixed root tips were transferred to bottles with 70% alcohol and stored at 4°c until use. after the all treatments, hydrolysis with 1 n hcl solution was done at 60°c between 14-16 min. prior to staining, root tips were washed with distilled water. they were stained with 2% aceto-orcein for two hours and then squashed with 45% acetic acid to obtain metaphase chromosomes. preparations containing the best metaphase chromosomes were photographed using leica dm lb2 microscope with camera. the measurements detailed based on small-long arm length and arm ratio were made for each taxa represented by the least five plates. chromosomes for each taxa examined were classified according to the nomenclature of levan et al. (1964) and stebbins (1971). in addition to somatic chromosome number, karyotypic description and length ranges, karyotype asymmetry parameters including centromeric index (ic), intrachromosomic asymmetry index (a1) and interchromosomic asymmetry index (a2) were calculated according to romero zarco (1986). results and discussion all samples examined were provided from uşak province in turkey (table 1). this study aims to analyze the karyotypes of four crocus taxa and to determine the relationships among the taxa studied in addition to crocus taxa examined previously according to chromosome number and other morphometric parameters. at the same time, an important crocus species: c. uschakensis which is not well known and not have sufficient literature information was examined for the first time in detail. the following cytological features belonging to four crocus taxa examined were observed in this study. table 1. species names, localities and chromosome numbers of studied species. species locations somatic chromosome number c. pallasii subsp. pallasii 5-10 km after kaşbelen/uşak 2n=14 c. olivieri subsp. olivieri kent forest/uşak 2n=8 c. fleischeri 5-10 km after kaşbelen/uşak 2n=20 c. uschakensis 5-10 km after kaşbelen/uşak 2n=20 131karyomorphology of some crocus l. taxa from uşak province in turkey c. pallasii subsp. pallasii plant samples for c. pallasii subsp. pallasii were collected from kaşbelen around in uşak province. the chromosome number of c. pallasii subsp. pallasii was determined as 2n=14 (table 1, figure 1-2). karyotypic description consists of 10 metacentric and 4 submetacentric chromosomes (4sm+10m) (table 2). c. pallasii subsp. pallasii evaluated within the series crocus show wide distribution from serbia and macedonija to turkey (karamplianis et al. 2013). chromosome number have been reported in previous studies as 2n=14 and 2n=16 for this taxon (šopova 1972; brighton et al. 1973; brighton 1977; randelovic et al. 2007; candan et al. 2009). furthermore, it was determined the both chromosome numbers in the study based on three different populations of c. pallasii subsp. pallasii by karamplianis et al. (2013). the results provided from the population belonging to samos island show similarfigure 1. somatic chromosomes of (a) c. pallasii subsp. pallasii; (b) c. olivieri subsp. olivieri; (c) c. fleischeri; (d) c. uschakensis. 132 aykut yilmaz, yudum yeltekin ity with this study according to chromosome number (2n=14) and karyotypic description (4sm+10m). c. pallasii subsp. pallasii had the smallest chromosomes set (1.93–5.17 μm) in addition to lowest haploid complement value (22.01 µm) among the studied taxa (table 2). a1 had the lowest value (0.27) among all the studied taxa, while a2 had the second highest value (0.36) after c. uschakensis. the highest centromeric index value with 41.97 was observed in c. pallasii subsp. pallasii (table 2). c. olivieri subsp. olivieri crocus olivieri subsp. olivieri is distributed in turkey, macedonia, southeast romania, south bulgaria, albania and greece (yüzbaşıoğlu 2012). c. olivieri subsp. olivieri chromosome number was found to be 2n=8 (table 1, figure 1-2). all chromosomes belonging to this taxon examined were submetacentic. chromosome counts for c. olivieri subsp. olivieri from different locations were done previously and reported as 2n=6 (mather 1932; brighton et al. 1973; uslu et al. 2012). in this aspect, chromosome number of this taxon from uşak location show differences from other study results. chromosome length range was between 7.30–12.80 µm (table 2). in other words, the longest chromosomes set was observed in c. olivieri subsp. olivieri, although it has the least chromosome number in comparison to other taxa examined. furthermore the second highest haploid complement value with 39.51 µm was found in this taxon (table 2). the lowest centromeric index value with 26.43 and highest a1 value (0.65) were determined in c. olivieri subsp. olivieri (table 2). c. fleischeri chromosome number of c. fleischeri was found to be 2n=20 (table 1, figure 1-2). crocus fleischeri is distributed south and west anatolia regions of turkey. chromosome number of this taxon which is endemic was previously reported as 2n=20 (mathew 1984; candan et al. 2009). furthermore, it is stated by candan et al. (2009) that all of the chromosomes are submetacentric except 3 chromosomes being metacentric. in this study, karyotypic description of this taxon consist of 8 metacentric and 12 submetacentric chromosomes (12sm+8m) (table 2). chromosome length range and haploid complement value for this taxon have the lowest value after c. pallasii subsp. pallasii with 2.42–4.22 µm and 34.35 µm respectively (table 2). chromosomal asymmetry index, a1 and a2 were determined as 0.36 and 0.17, respectively. the lowest a2 figure 2. idiograms of (a) c. pallasii subsp. pallasii; (b) c. olivieri subsp. olivieri; (c) c. fleischeri; (d) c. uschakensis. (bar : 2 µm). 133karyomorphology of some crocus l. taxa from uşak province in turkey value was observed in c. fleischeri with 0.17 (table 2). c. uschakensis c. uschakensis is an endemic species and there is not enough information about this taxon. this work represents the first detailed chromosomal study on c. uschakensis. rukśans (2014) states that they observed this taxon on low mountains belonging to north of uşak. similarly, we observed and collected this taxon on north parts of uşak province. chromosome number of c. uschakensis was found to be 2n=20 (table 1, figure 1-2). which consist of 6 metacentric, 12 submetacentric and 2 acrocentric chromosomes (2a+12sm+6m) (table 2). furthermore, satellite was observed on the short arm of chromosome 7. chromosome length range was between 2.87–7.82 µm (table 2). in other words, the longest chromosomes set was observed in c. uschakensis after c. olivieri subsp. olivieri which has the least chromosome number among taxa examined. the longest haploid complement was determined in c. uschakensis with 50.93 µm. the second lowest centromeric index value with 34.49 and highest a2 value with 0.37 was observed in this taxon (table 2). the variation in chromosome counts for two species were observed in this study. while the chromosome number for c. pallasii subsp. pallasii have been reported as 2n=14 and 2n=16 in previous studies, it was determined 2n=14 in present study. similarly, another different chromosome count was found in c. olivieri subsp. olivieri as previously reported as 2n=6, whereas in the present study it was found as 2n=8. it is observed wide range of variation on the chromosomes counts (from 2n=6 to 70) and morphology of the species belonging to the genus crocus (brighton et al. 1973; uslu et al. 2012; harpke et al. 2013). the most probably reasons of variations in the chromosome number and morphology of the species are geographical differences, environmental factors caused by locations of taxa, hybridization, polyploidization and aneuploidy. geographical differences and variations in environmental factors caused by geographical differences could be reason of chromosome count differences in c. olivieri subsp. olivieri. similarly, karamplianis et al. (2013) examining the c. pallasii subsp. pallasii in three different populations states that chromosome numbers for this taxon change as 2n=14 and 2n=16. other an important taxon, c. uschakensis were examined in detailed according to chromosome number and other morphometric parameters. furthermore, in addition to contribution for literature based on its karyotype informations determined, it was firstly evaluated the relationships of c. uschakensis with other crocus taxa. besides the first detailed karyotype analysis, satellite chromosome was determined in c. uschakensis. turkey with 132 taxa which is their 108 endemic is found in very rich region according to species number and diversity in the world (gedik et al. 2017) and accepted as the center of species diversity for the genus crocus (erol et al. 2012; candan and özhatay 2013). the genus crocus in the world in comparison to turkey according to their species number and diversity, it can be said that turkey is the center of genetic variation for the genus. furthermore, the high endemism rate for the crocus species make very important the turkey in aspect of studies on the genus. in this study, four species belonging to uşak province located in the western anatolia region which is the richest region of turkey according to species diversity were examined caryologically. one of the most important gains of this study is to obtain literature information on crocus uschakensis which is an endemic species, in addition to determining the species diversity of the region. acknowledgments the authors would like to thank uşak university directorate of scientific research projects (bap-2021/ mf003) for providing financial support and also special thanks to ahmet kahraman for helping to identify the plant material. table 2. karyotypic descriptions, length ranges and other morphometric parameters of studied crocus species. species karyotypic description length range (µm) haploid complement (µm) ic a1 a2 c. pallasii subsp. pallasii 4sm+10m (1.93 – 5.17) 22.01 41.97 0.27 0.36 c. olivieri subsp. olivieri 8sm (7.30 – 12.80) 39.51 26.43 0.65 0.24 c. fleischeri 12sm+8m (2.42 – 4.22) 34.35 38.80 0.36 0.17 c. uschakensis 2a+12sm+6m (2.87 – 7.82) 50.93 34.49 0.47 0.37 134 aykut yilmaz, yudum yeltekin reference addam k, bou-hamdan m, sabbagh n, takkoush j, hout k. 2019. crocus baalbekensis k. addam and m. bou hamdan sp. nov and its three forms (iridaceae), new endemic species and forms from lebanon, joined the lebanese flora. moj eco environ sci. 4(2): 75–83. brighton ca, mathew b, marchant cj. 1973. chromosome counts in the genus crocus (iridaceae). kew bulletin. 28(3):451–464. brighton ca. 1977. cytology of crocus sativus and its allies (iridaceae). plant syst evol. 211:149–154. candan f, şik l, kesercioğlu t. 2009. cytotaxonomical studies on some crocus l. taxa in turkey. african j biotech. 8(18):4374–4377. candan f, özhatay n. 2013. crocus chrysanthus s. lato in turkey. ann bot fennici. 50:423–430. erol o, can l, şık l. 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(2012). crocus l. in: güner, a., aslan, s., ekim, t., vural, m. & babaç, m.t. (eds.), türkiye bitkileri listesi (damarlı bitkiler). nezahat gökyiğit botanik bahçesi ve flora araştırmaları derneği yayını. i̇stanbul, pp. 530–534. caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 4 2021 firenze university press cytogenetic analyses in three species of moenkhausia eigenmann, 1903 (characiformes, characidae) from upper paraná river (misiones, argentina) kevin i. sánchez1,*, fabio h. takagui2, alberto s. fenocchio3 genetic variations and interspesific relationships in lonicera l. (caprifoliaceae), using scot molecular markers fengzhen chen1, dongmei li2,* , mohsen farshadfar3 the new chromosomal data and karyotypic variations in genus salvia l. (lamiaceae): dysploidy, polyploidy and symmetrical karyotypes halil erhan eroğlu1,*, esra martin2, ahmet kahraman3, elif gezer aslan4 cytogenetic survey of eight ant species from the amazon rainforest luísa antônia campos barros1, gisele amaro teixeira2, paulo castro ferreira1, rodrigo batista lod1, linda inês silveira3, frédéric petitclerc4, jérôme orivel4, hilton jeferson alves cardoso de aguiar1,5,* molecular phylogeny and morphometric analyses in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f. neda atazadeh1,*, masoud sheidai1, farideh attar2, fahimeh koohdar1 a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) melika tabasi1, masoud sheidai1,*, fahimeh koohdar1, darab hassani2 genetic diversity and relationships among glaucium (papaveraceae) species by issr markers: a high value medicinal plant lu feng1,*, fariba noedoost2 morphometric analysis and genetic diversity in rindera (boraginaceae-cynoglosseae) using sequence related amplified polymorphism xixi yao1, haodong liu2,*, maede shahiri tabarestani3 biosystematics, fingerprinting and dna barcoding study of the genus lallemantia based on scot and remap markers fahimeh koohdar*, neda aram, masoud sheidai karyotype analysis in 21 plant families from the qinghai–tibetan plateau and its evolutionary implications ning zhou1,2, ai-gen fu3, guang-yan wang1,2,*, yong-ping yang1,2,* some molecular cytogenetic markers and classical chromosomal features of spilopelia chinensis (scopoli, 1786) and tachybaptus ruficollis (pallas, 1764) in thailand isara patawang1,*, sarawut kaewsri2, sitthisak jantarat3, praween supanuam4, sarun jumrusthanasan2, alongklod tanomtong5 centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates) simona ceraulo, vanessa milioto, francesca dumas* repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil marina souza cunha1,2,*,#, silvana melo1,3,#, filipe schitini salgado1,2, cidimar estevam assis1, jorge abdala dergam1,* karyomorphology of some crocus l. taxa from uşak province in turkey aykut yilmaz*, yudum yeltekin variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l. egizia falistocco1,*,+, gianpiero marconi1,+, lorenzo raggi1, daniele rosellini1, marilena ceccarelli2, emidio albertini1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(1): 89-96, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1017 caryologia international journal of cytology, cytosystematics and cytogenetics citation: s. aiumsumang, s. phimphan, c. suwannapoom, p. chaiyasan, w. supiwong, a. tanomtong (2021) a comparative chromosome study on five minnow fishes (cyprinidae, cypriniformes) in thailand. caryologia 74(1): 89-96. doi: 10.36253/caryologia-1017 received: july 10, 2020 accepted: february 10, 2021 published: july 20, 2021 copyright: © 2021 s. aiumsumang, s. phimphan, c. suwannapoom, p. chaiyasan, w. supiwong, a. tanomtong. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. a comparative chromosome study on five minnow fishes (cyprinidae, cypriniformes) in thailand surachest aiumsumang1,3, sumalee phimphan1, chatmongkon suwannapoom2, patcharaporn chaiyasan3, weerayuth supiwong4,*, alongklod tanomtong3,5 1 biology program, faculty of science and technology, phetchabun rajabhat university, phetchabun 67000, thailand 2 department of fishery, school of agriculture and natural resources, university of phayao, muang, phayao 56000, thailand 3 department of biology, faculty of science, khon kaen university, khon kaen 40002, thailand 4 applied science program, faculty of multidisciplinary study, khon kaen university, nong khai campus, muang, nong khai 43000, thailand 5 toxic substances in livestock and aquatic animals research group, department of biology, faculty of science, khon kaen university, khon kaen 40002, thailand *corresponding author. e-mail: supiwong@hotmail.com abstract. the cytogenetic comparisons of five minnow species from thailand were presented here, i.e., devario regina, d. laoensis, rasbora paviana, r. aurotaenia and esomus metalicus. the mitotic chromosomes were prepared directly from renal cells. conventional staining and ag-nor banding techniques were applied to stain the chromosomes. the results revealed that all minnow fishes studied possessed the same diploid chromosome number (2n) as 50 chromosomes. the fundamental numbers (nf) of d. laoensis, d. regina, r. paviana, r. aurotaenia and e. metalicus are 100, 100, 98, 98, and 98 respectively. their karyotypes composing of metacentrics-submetacentrics-acrocentrics-telocentrics were as follows: 6-12-32-0 in d. regina, 6-10-34-0 in d. laoensis, 8-16-24-2 in r. paviana, 8-16-24-2 in r. aurotaenia and 8-10-30-2 in e. metalicus. the ag-nor banding technique provides the nucleolar organizer regions (nors) at subtelomeric region of the short arm chromosome in the a submetacentric or acrocentric chromosomes that are located differently in the different chromosome pairs among species. keywords: karyotype, minnow, fish chromosome, cyprinid fishes, minnow fishes. introduction devario laoensis, d. regina, esomus metalicus, rasbora aurotaenia, and r. paviana are some species of minnows, belonging to the family cyprinidae (subfamily danioninae-danionini). they are tropical freshwater fish of minor commercial importance, which are native in thailand. their distribu90 surachest aiumsumang et al. tions include the mekong, chao phraya, and meklong basins (froese and pauly 2012) and they can be easily found in large and small rivers, ponds, ditches, lakes, paddy field, and swamps. it rarely occurs in low oxygen waters (brittan 1954, 1971, 1998). they could be used to assess if they were sensitive to change in environmental problems and aquatic pollution (blazer 2002, frame and dickerson 2006, raskovic et al. 2010, yenchum 2010, reddy, rawat 2013). the current spurt in the fish cytogenetical studies has its origin in the standardization of newer techniques and the realization of an immense applied value of the cytogenetic data of fishes. the study on fish chromosomes has received considerable attention in recent years because of their importance in classification, evolution, heredity, systematic (gold et al. 1990, ueda et al. 2001, barat et al. 2002, barat and sahoo 2007), fish breeding, rapid production of inbred lines including cytotaxonomy (kirpichnikov 1981) and prove the ploidy status in some sturgeons (zhou et al. 2013). the several methods namely, conventional staining, c-banding, ag-nor banding, and fluorescence in situ hybridization (fish) have been used by ichthyologists for gathering of cytogenetic information of fish (sola et al. 2000, kavaco et al. 2005, zhou et al. 2013), yet each of these methods provides a different aspect of the karyotype characteristics. for example, agnor staining shows the regions containing the actively transcribed ribosomal rna genes (rdna). nors characterization can be a cytogenetic marker for cytotaxonomic studies and has been used for studying on phylogenetic relationships among the cyprinids (amemyia and gold 1988, gatetti jr 1998, almeida-toledo et al, 2000). however, cytogenetic studies conducted on this group (devario, esomus and rasbora) are quite scarce. there are some karyotype reports, including rasbora trilineata and r. heteromorpha: 2n=48 (post 1965), r. buchanani: 2n=50 (manna and khuda-bukhsh 1977), r. daniconius: 2n=50 (khuda-bukhsh et al. 1979), r. sumatrana: 2n=50 (donsakul and magtoon 1995), r. caudimaculata, r. myersi, r. paviei and r. retrodorsalis: 2n=50 (donsakul and magtoon 2002), r. aurotaenia: 2n=50 (seetapan and moeikum 2004), r. trilineata, r. heteromorpha, r. daniconius, r. borapetensis and r. einthovenii: 2n=50 (donsakul et al. 2005), r. agilis, r. dorsicellata and r. rubrodorsalis: 2n=50 (donsakul et al. 2009), e. metallicus: 2n=50 (neeratanaphan et al. 2017) and r. einthovenii: 2n=50 (yeesaem et al. 2019) (table 1). the studies on the karyotypes help to investigate the genetic structure of aquatic animal species in each habitat, thus it can determine what species are related to each other in an accurate manner. this may help to facilitate the hybridization between them in the future for strain improvement (sofy et al. 2008). in the present study, we conducted chromosomal analyses using conventional staining and ag-nor banding techniques. the examined karyotypes of five minnow species from thailand belonging to three different genera (devario, esomus, and rasbora); d. laoensis, d. regina and r. paviana were reported chromosomes characterized for the first time. the obtained results will provide useful cytogenetic information for further studies on taxonomy and evolutionary relationship of fishes. matherial and methods chromosome preparation individuals from both sexes of five analyzed minnows were collected from various river basins in thailand (table 1 and fig. 1). the fishes were transferred to table 1. collection sites of the analyzed species show the sample number. species number of specimens in site sampling mae khong basin sirindhorn peat swamp forest ping basin yom basin pa-sak basin chi basin chao phraya basin songkhram basin remark with fig. 1. devario regina 05 ♀ 06 ♂ 06 ♀ 08 ♂ site 1 d. laoensis 03 ♀ 05 ♂ site 2 rasbora paviana 05 ♀ 08 ♂ 03 ♀ 04 ♂ 05 ♀ 07 ♂ 04 ♀ 05 ♂ site 3 r. aurotaenia 08 ♀ 07 ♂ 05 ♀ 08 ♂ site 4 esomus metalicus 04 ♀ 05 ♂ 10 ♀ 10 ♂ site 5 91a comparative chromosome study on five minnow fishes (cyprinidae, cypriniformes) in thailand laboratory aquaria and kept under standard conditions for three days before the experiments. chromosomes were prepared in vivo as follows (supiwong et al. 2014). the colchicine was injected into the fish’s intramuscular and/or its abdominal cavity at a dose of 0.1 ml/100 g of body weight and then left for 1-2 hours. the kidney was cut into small pieces then squash mixed with 0.075 m kcl. after discarding all large piece tissues, 8 ml of cell sediments were transferred to a centrifuge tube and incubated for 30 minutes. the kcl was discarded from the supernatant after centrifugation at 1,200 rpm for 8 minutes. cells were fixed in fresh cool carnoy’s fixative (3 methanol: 1 glacial acetic acid) allows to preserve the internal structure of the cells for better staining of the chromosomes (pradeep et al. 2011) to which up to 8 ml were gradually added before being centrifuged again at 1,200 rpm for 8 minutes, at which time the supernatant was discarded. the fixation was repeated until the supernatant was clear and the pellet was mixed with 1 ml fixative. the mixture was dropped onto a clean and cold slide by micropipette followed by air-drying technique. chromosome staining conventional staining was carried out using 20% giemsa’s solution for 15 minutes (phimphan et al. 2017). ag-nor banding was performed by adding 4 drops of 50% silver nitrate and 2% gelatin on slides (howell and black 1980). the slides were then sealed with cover glasses and incubated at 60°c for 5 minutes. after that, the slides were soaked in distilled water until the cover glasses were separated. then, they were stained with 20% giemsa’s solution for 1 minute. chromosome check and image processing twenty clearly observable metaphase cells with a well-spread chromosome of each male and female were selected. images were captured under a light microscope nikon eclipse by a digital ccd camera (nikon dsfi1). the chromosomes were classified based on the position of a centromere as metacentric (m), submetacentric (sm), acrocentric (a), telocentric (t) according to the arm ratios (chaiyasut 1989). results five minnow fishes were similar in the diploid number of 2n= 50, with the karyotype composed of m6+sm12+a32 in d. regina. the mean values calculated from twenty mitotic metaphases showed the relative length (rl) of chromosomes complement ranging from 0.041±0.010 to 0.033±0.004. the nor was found on the short arm of chromosome pair 15 (fig. 2a). the chromosome complements of d. laoensis consisting of m6+10sm+34a. the mean value of relative length ranged from 0.0.44±0.005 to 0.030±0.002. the nor was presented on the short arms of chromosome pair 11 (fig. 2b). karyotype of r. paviana composes of 8m+16sm+24a+2t. the present investigation in this fish species revealed that the mean value of rl from 0.048±0.001 to 0.032±0.004. ag-nor banding result showed that nor-bearing chromosomes locate at subtelomeric on the short arm of chromosome pair 9 (fig. 2c). the karyotypic analysis result revealed that the chromosome complements of r. aurotaenia consisting of 8m+16sm+24a+2t. the parameters of all chromosomes were measured and it showed the mean value of rl from 0.0.054±0.003 to 0.033±0.002. the result of silver-staining exhibited the nors show that it locates) at short arm of chromosome pair 23 (fig. 2d). the karyotype of e. metalicus consisting of 8m+10sm+30a+2t. the mean value of rl from 0.0.51±0.001 to 0.025±0.002. the nor was presented on the short arms of chromosome pair 7 (fig. 2e). figure 1. collection sites of cyprinid fishes studied herein. 1=devario regina, 2=devario laoensis; 3=rasbora paviana, 4=rasbora aurotaenia, 5=esomus metalicus. 92 surachest aiumsumang et al. discussion the details of each metaphase chromosome spread and karyotype of five minnow fishes, including d. regina, d. laoensis, r. paviana, r. aurotaenia, and e. metalicus are shown in figure 2. the present study is the first report on the chromosomal characteristics of d. laoensis, d. regina, and r. paviana determined using conventional staining and ag-nor banding techniques. the diploid chromosome number of all species provided 50 chromosomes, which is shared by most of the cyprinid species previously analyzed (post 1965, manna and khuda-bukhsh 1977, khuda-bukhsh et al. 1979, donsakul and magtoon 1995, donsakul and magtoon 2002, seetapan and moeikum 2004, donsakul et al. 2005, donsakul et al. 2009, neeratanaphan et al. 2017, yeesaem et al. 2019) (table 2). the nfs of d. laoensis and d. regina are 100 equally, while those of r. paviana, r. aurotaenia, and e. metalicus are equal to 98 in both sexes. to compare with previous studies, they are differences from seetapan and moeikum (2004) who reported the nf=92 in r. aurotaenia and neeratanaphan et al. (2017) showed the nf of e. metallicus as 100. the differences in nf values are caused by the difference in the number of monoarm chromosomes. this phenomenon may be resulting from the intra-specific variation between populations of those species. this finding is in agreement with other species such as r. daniconius (khuda-bukhsh et al. 1979, donsakul et al. 2005), r. einthovenii (donsakul et al. 2005, yeesaem et al. 2019), and r. rebrodorsalis (donsakul and magtoon 2002, donsakul et al. 2009). the nf of these genera varied from 74 to 100 (table 2). all species were analyzed herein display without morphologically differentiated sex chromosomes. this character is the same as in previous studies of this family (arai 2011). although five minnows analyzed herein have the same diploid number, there are differences in karyotype complements as follows (fig. 2). d. regina has six metacentric (m) (pairs 1-3), 12 submetacentric (sm) (pairs 4-9) and 32 acrocentric (a) (pairs 10-25) chromosomes. the mean values were calculated from twenty mitotic metaphases showed the centromeric index (ci) of chromosome complements ranging from 0.548±0.004 to 0.808±0.005. the karyotype formula of d. regina could be deduced as 2n(50) = 6m+12sm+32a. d. laoensis has six metacentric (pairs 1-3), 10 submetacentric (pairs 4-8) and 34 acrocentric (pairs 9-25) chromosomes. the mean values of ci ranged from 0.553±0.005 to 0.798±0.002. the karyotype formula of this species is 2n(50) = 6m+10sm+34a. r. paviana consisted of eight metacentrics (pairs 1-4), 16 submetacentrisc (pairs 5-12), 24 acrocentrics (pairs 13-24) and two telocentrics (t) (pair 25). the mean values of ci ranged between 0.526±0.002 and 1.000±0.000. the proposed karyotype of this species was 2n(50) = 8m+16sm+24a+2t. r. aurotaenia shows eight metacentrics (pairs 1-4), 16 submetacentrics (pairs 5-12), 24 acrocentrics (pairs 13-24) and two telocentrics (pair 25) chromosomes. the mean values of ci in this species ranged from 0.569±0.003 to 1.000±0.000. the karyotype of this species was 2n(50) = 8m+16sm+24a+2t, which differs from the previous study by seetapan and moeikum (2004) that reported the karyotype of this species consisting of 2n(50) = 14m+26sm+2st+8a. in e. metalicus, the karyotype composed of eight metacentric (pairs figure 2. metaphase chromosome plates and karyotypes of the devario regina (a.), d. laoensis (b.), rasbora paviana (c.), r. aurotaenia (d.) and esomus metalicus (e.), by conventional staining. the arrows indicate nor banding by ag-nor staining technique (inserted box). all species share the karyotype composed of 50 chromosomes. scale bar indicates 5 µm. 93a comparative chromosome study on five minnow fishes (cyprinidae, cypriniformes) in thailand 1-4), 10 submetacentric (pairs 5-9), 30 acrocentric (pairs 10-24), and two telocentric (pair 25) chromosomes. the mean values of ci ranged between 0.558±0.003 and 1.000±0.000. the karyotype of e. metalicus showed 2n(50) = 8m+10sm+30a+2t. these results are inconsistent with previous cytogenetic data (neeratanaphan et al. 2017). this fact suggests that some pericentric inversions have occurred in the karyotype differentiation of this species. besides), the occurrence of chromosomal rearrangements has been considered a relatively common evolutionary mechanism inside the cyprinidae family reviewed (arai 2011). family cyprinidae are diploid chromosome ranges from 48–50 in the tribes labeonini and smiliogastrini while the tribe poropuntiini and danionini are more conserved as 2n = 50 (phimphan et al. 2020). karyotype diversification processes in species are subjected to multiple factors, whether intrinsic (genomic or chromosomal particularities) or extrinsic (historic contingencies) factor. among these, restricted gene flow between populations is an important factor for the fixation of karyotype changes. for example, after the occurrence of an inversion, it can be lost in the polymorphic state or, under the proper conditions, spread in the population until it is fixed. inversions maintain areas of imbalance between alleles in loci within or influenced by these rearrangements, leading to an adaptive condition, primarily along environmental gradients. this could occur, particularly concerning possible historical expansion and adaptation to new environments for a review hoffmann (2008). as mention above, the chromosomal study is very important and clearly exhibits the benefits. table 2. cytogenetic reported of the genera devario, esomus and rasbora. species 2n nf1 nf2 karyotype formula nor reference devario laoensis 50 100 66 6m+10sm+34a 2 present study d. regina 50 100 68 6m+12sm+32a 2 present study esomus metallicus 50 100 86 14m+22sm+14a neeratanaphan et al. (2017) 50 98 68 8m+10sm+30a+2t 2 present study rasbora agilis 50 100 100 24m+26sm donsakul et al. (2009) r. aurotaenia 50 92 90 14m+26sm+2a+8t seetapan and moeikum (2004) 50 98 74 8m+16sm+24a+2t 2 present study r. borapetensis 50 88 88 24m+14sm+12t donsakul et al. (2005) r. buchanani 50 100 96 30m+18sm+2a manna and khuda-bukhsh (1977) r. caudimaculata 50 98 96 20m+26sm+2a+2t donsakul and magtoon (2002) r. daniconius 50 80 74 18m+6sm+6a+20t khuda-bukhsh et al. (1979) r. daniconius 50 92 90 32m+8sm+2a+8t donsakul et al. (2005) r. dorsicellata 50 92 92 18m+24sm+8t donsakul et al. (2009) r. einthovenii 50 94 86 6m+30sm+8a+6t donsakul et al. (2005) 50 100 84 16m+18sm+16a 2 yeesaem et al. (2019) r. heteromorpha 48 post (1965) 48 74 72 14m+10sm+2a+22t donsakul et al. (2005) r. myersi 50 90 84 20m+14sm+6a+10t donsakul and magtoon (2002) r. paviei 50 100 84 10m+24sm+16a donsakul and magtoon (2002) r. paviana 50 98 74 8m+16sm+24a+2t 2 present study r. retrodorsalis 50 88 86 26m+10sm+2a+12t donsakul and magtoon (2002) r. rubrodorsalis 50 82 82 16m+16sm+18t donsakul et al. (2009) r. sumatrana 50 94 92 26m+16sm+2a+6t donsakul and magtoon (1995) r. trilineata 48 post (1965) r. trilineata 50 94 92 26m+16sm+2a+6t donsakul et al. (2005) abbreviations: diploid chromosome number (2n), fundamental number m, sm, a =2, t=1 (nf1), fundamental number m, sm, =2, a, t=1 (nf2), metacentric (m), submetacentric (sm), acrocentric (a), telocentric (t), nucleolar organizer region (nor). 94 surachest aiumsumang et al. moreover, the karyological and nors characteristics in cyprinid fishes were reported in some species. the present study is the first report on the nor phenotypes in five minnow species studied. the single pair of nor-bearing chromosomes were observed at subtelomeric regions on the short arm chromosomes in all species analyzed. however, there are differences in chromosome types and pair numbers as follows. the nors were observed on acrocentric chromosome pair 15 in d. regina whereas those were found on acrocentric chromosome pair 11 in d. laoensis. in the genus rasbora, the nors located on the submetacentric chromosome pair 9 in r. paviana and distinct revealed on the acrocentric chromosome pair 23 in r. aurotaenia. for e. metalicus, nor-baring chromosomes were found on the submetacentric chromosome pair 7 (fig. 2). to compare with the same genus in previous report, r. einthovenii has single pair of nor on chromosome pair 4 (yeesaem et al. 2019). moreover, the single pair of nor bearing chromosomes can be observed in other cyprinids such as aspius aspius (ràb et al. 1990), osteochilus waandersi (magtoon and arai 1993), barbonymus gonionotus (khuda-bukhsh and das 2007), puntioplites proctozysron (supiwong et al. 2012), puntius brevis (nitikulworawong and khrueanet 2014). also, the subtelomeric region of chromosome pair showed clearly observable nors in most cyprinid fishes. however, nor variation can be revealed in among populations of the same species as found in garra rufa. this variation is caused by geographically isolated populations (arzu and ergene 2009). normally, most fishes have only one pair of small nors on chromosomes. only some fishes have more than two nors, which may be caused by the translocation between some parts of the chromosomes that have nor and another chromosome (sharma et al. 2002). our present study showed that the species analyzed had a nor site on a single chromosome pair at a subtelomeric position. this is considered a simple condition in fish (almeida-toledo 1985). in the present study, five minnows belong to genera of which have closely related species. the obtained results have shown that this fish group shares the same 2n. however, there are differences in karyotype complements and nor-bearing chromosome markers. these seem to be that cytogenetic methods can be used for the systematics of this fish family. acknowledgments the authors are grateful to phetchabun rajabhat university and toxic substances in livestock and aquatic animals research group khon kaen university and unit of excellence 2020 on biodiversity, natural resources management, university of phayao (uoe63005) and the post-doctoral training program from research affairs and graduate school, khon kaen university, thailand (grant no. 59255) for financial supports. references amemiya ct, gold jr. 1988. chromosomal nors as taxonomic and systematic characters in north american cyprinid fishes. genetica 76: 81–90. https://doi. org/10.1007/bf00058806 almeida-toledo lf. 1985. the nucleolar organizer regions in fish. science culture 37: 448–453. 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(online) | doi: 10.36253/caryologia-1375 caryologia international journal of cytology, cytosystematics and cytogenetics citation: egizia falistocco, gianpiero marconi, lorenzo raggi, daniele rosellini, marilena ceccarelli, emidio albertini (2021) variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l.. caryologia 74(4): 135-143. doi: 10.36253/ caryologia-1375 received: august 06, 2020 accepted: november 30, 2021 published: march 08, 2022 copyright: © 2021 egizia falistocco, gianpiero marconi, lorenzo raggi, daniele rosellini, marilena ceccarelli, emidio albertini. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l. egizia falistocco1,*,+, gianpiero marconi1,+, lorenzo raggi1, daniele rosellini1, marilena ceccarelli2, emidio albertini1 1 department of agricultural, food and environmental sciences, university of perugia, perugia, italy 2 department of chemistry, biology and biotechnology, university of perugia, perugia, italy *corresponding author. e-mail: egizia.falistocco@unipg.it + contributed equally to this work abstract. apomixis is a rather widespread phenomenon in plants. it is defined as the asexual formation of a seed from the maternal tissues of the ovule, avoiding the processes of meiosis and fertilization. some species are facultative apomicts and form seeds by means of sexual and apomictic pathways to different extents. this is the case of poa pratensis, the kentucky bluegrass, which reproduces by aposporous pseudogamous facultative apomixis. this grass is one of the most studied apomictic systems, however some aspects, such as the male meiotic behavior, have not been so far investigated. in this study the process of microsporogenesis in genotypes of p. pratensis with a different mode of reproduction was investigated. the analysis revealed an almost regular meiosis in the sexual plants whereas apomictic genotypes exhibited different levels of meiotic irregularities, mainly due to cell fusion and irregular segregation in i and ii division. our data did not reveal evident connections between the extent and types of abnormalities and the components of apomixis, apomeiosis and parthenogenesis. the meiotic behavior of the examined plants was discussed in the light of their origin. keywords: poa pratensis l., kentucky bluegrass, apomixis, microsporogenesis, meiotic abnormalities. introduction the sexual seed formation is based on two fundamental mechanisms, meiosis and fertilization. the combination of these events produces new nuclear compositions so that sexual reproduction is a means not only of generating new but also variable individuals. however, in some flowering plants, seeds form asexually, from maternal tissues by a process known as apomixis (bicknell and koltunow 2004). apomixis, is a complex trait resulting from the circumvention of female meiotic reduction (a process known as apomeiosis) and fertilization (parthenogenesis). in gametophytic apomixis, a mega136 egizia falistocco et al. gametophyte (embryo sac) is originated from an unreduced cell, and subsequently a clonal embryo develops by parthenogenesis from a 2n egg (matzk et al. 2005). several species need fertilization for endosperm development while others do not (barcaccia and albertini 2013). the three components of apomixis, namely apomeiosis, parthenogenesis and autonomous endosperm formation, have been uncoupled experimentally, as documented in numerous genera such as taraxacum (van dijk et al. 1999; van dijk, 2003), erigeron (noyes and rieseberg 2000), poa (albertini et al. 2001), hypericum (barcaccia et al. 2006; schallau et al. 2010), cenchrus (conner et al. 2013), and hieracium (catanach et al. 2006; henderson et al. 2017). most plants of apomictic species are facultative apomicts and form seeds by means of sexual and apomictic pathways to different extents. this means that plants that reproduce by apomixis also retain the ability to reproduce sexually to varying degrees (nogler 1994; tucker 2003). the phenomenon of apomixis is far from rare and its pattern of distribution suggests that it evolved many times during plant evolution. among flowering plants it occurs with high frequency in certain families such as asteraceae, rosaceae, ranunculaceae and poaceae (bicknell and koltunow 2004). most apomictic plants produce viable pollen, this implies that within apomictic populations the formation of viable pollen represents a possibility for the fertilization of unreduced eggs. however, alterations of microsporogenesis in apomictic individuals have not been so far extensively investigated. poa pratensis l., kentucky bluegrass, is an important fodder and turf grass which mainly reproduces by aposporous pseudogamous apomixis, i.e. unreduced aposporous embryo sacs develop through parthenogenesis to viable apomictic seeds if the unreduced polar nuclei fuse with a sperm cell from the male gametophyte (pseudogamy). the species is highly variable when reproduction mode, chromosome number and phenotypic traits are considered. in this species apomixis is facultative, with a frequency ranging from 0 to 100%, while chromosome numbers from 2n=18 to 150 have been reported (matzk et al. 2005). among the native monocot apomictic systems, p. pratensis is one of most explored. for several years, selected genotypes from wild italian populations have been investigated with the aim of understanding the genetic control and mechanisms that regulate apomixis (mazzucato 1995; albertini et al. 2001; porceddu et al. 2002; raggi et al. 2015; marconi et al. 2020). these studies provided a solid background for the present investigation that aimed at analyze the meiotic behavior of plants of p. pratensis exhibiting a different mode of reproduction and to find possible relationships between apomixis and its components and the alterations of microsporogenesis. materials and methods plant material genotypes of p. pratensis with different reproductive systems were examined: i) a sexual genotype s1/17 derived from a cross between two completely sexual genotypes selected from german cultivars (matzk 1991); ii) an apomictic (aposporic and parthenogenetic) rs7-3 (mazzucato 1995) and l4 (marconi et al. 2020) plants, both from italian natural populations and iii) several plants belonging to two f1 segregating populations produced by crossing s1/1-7 x rs7-3 (barcaccia et al. 1998) and s1/1-7 x l4 (marconi et al. 2020). reproductive mode and chromosome number of the above reported materials employed in this study were investigated in previous studies (barcaccia et al. 1998; albertini et al. 2001; porceddu et al. 2002; marconi et al. 2020 and references therein) and are summarized in table 1. plants were grown at the experimental field of the dept. of agricultural, food and environmental sciences in perugia (n 43°10’15.3’’, e 12°39’58.7’’). meiotic analysis for meiotic investigations inflorescences not completely emerged from the flag leaf were employed. for each plant, four-five inflorescences were collected and immediately fixed in absolute ethanol-acetic acid 3:1 (v/v) for 24 hours, then they were transferred to 70% ethanol and stored at 4°c until analysis. cytological preparations were made by squashing the anthers of a single flower on a glass slide with some drops of 0.5% acetocarmine (merck life science, italy), intensified by ferric oxide. for each plant 150-200 pollen mother cells (pmcs) for each meiotic stage were analyzed. slides were observed under a microphot nikon microscope. images were recorded with a digital photocamera sony icx282aq and then processed using adobe photoshop 5.0. the alterations observed in each meiotic phase were expressed as percentage of the meiocytes examined. for pollen viability analysis, pollen samples were collected from each plant, and stained with a mixture of acetocarmine and glycerol (1:1) (ramanpreet and gupta 2019). the pollen viability was expressed as percentage of fully stained pollen grains over a total of at least 1.000 137variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis pollen grains for each sample. the percentage of the meiotic anomalies recorded at i and ii division and pollen viability were graphically displayed. results meiotic analysis of the parental genotypes the sexual plant s1/1-7, with 2n=36, is considered a tetraploid with four additional chromosome pairs (matzk 1991; barcaccia et al. 1998; porceddu et al. 2002); it exhibited an almost regular meiotic behavior with only few exceptions consisting of cell fusion and meiocytes linked by cytoplasmic connections at prophase i (4.3%). pollen viability was almost complete reaching the 98.4% (fig. 1a). in the apomictic parental plant rs7-3 (2n=64), previously described as a probable octoploid having four additional chromosome pairs (mazzuccato 1995; porceddu et al. 2002), few abnormalities at different meiotic stages were observed. these included meiocytes at metaphase i with univalents (4.0%), anaphase i with lagging chromosomes (7.0%) and irregular segregation in the second division (15.0%). a high percentage of viable pollen was recorded (98.0%, fig. 1b). the apomictic l4 plant, hexaploid with 2n=42 (marconi et al. 2020), revealed numerous abnormalities during both first and second division. at prophase i meiocytes linked by cytoplasmic connections (4.5%) were observed (fig. 2a), whereas lagging chromosomes (19.0%) and irregular segregation (24.0%) were detected at anaphase i and ii, respectively. dyads and triads frequently occurred (15.0%) at the end of meiosis. the viable pollen produced by this plant was reduced to 51.0% with the grains displaying a quite heterogeneous size (fig. 1c). meiotic analysis of f1 progenies four plants among those obtained from the cross s1/1-7 x rs7-3 were analyzed: sexual pg-f122, aposporic and parthenogenetic pg-f115 and pg-f146, and aposporic pg-f15. as showed by porceddu and colleagues (2002) all these plants have a chromosome number 2n=50. the sexual plant pg-f122 showed an almost regular microsporogenesis with few exceptions consisting of anaphase i with laggards (8.6%), and few triads at telophase ii (1.3%). the pollen viability was extremely high (99.0%, fig. 1d). in pg-f115 events of cellular aggregation at prophase i (1.9%, fig. 2b) and numerous cells at anaphase i with lagging chromosomes (50.0%) were found. in the second division, the irregular congression and segregation of chromosomes was observed in numerous meiocytes (15.0%). as a consequence, a considerable number of triads and dyads (13.0%) was produced at the end of meiosis. the pollen displayed variability in size but appeared fully stained (fig. 1e). genotype pg-f146 showed irregularities along the entire microsporogenetic process. these consisted in meiocytes at prophase i aggregated by cytoplasmic connections (1.4%), univalents at metaphase i (9.4%), lagging chromosomes at anaphases i (20.0%), and irregular orientation of chromosomes at metaphase ii and anaphases ii (24.0%). a conspicuous number of triads and dyads table 1. name, progeny, mode of reproduction, somatic chromosome number (2n) with relative reference. name progeny mode of reproduction 2n reference for chromosome number determination s1/1-7 sexual 36 porceddu et al. 2002 rs7-3 apomictic* 64 porceddu et al. 2002 l4 apomictic* 42 marconi et al. 2020 pg-f122 s1/1-7 ´ rs7-3 sexual 50 porceddu et al. 2002 pg-f115 s1/1-7 ´ rs7-3 apomictic* 50 porceddu et al. 2002 pg-f146 s1/1-7 ´ rs7-3 apomictic* 50 porceddu et al. 2002 pg-f15 s1/1-7 ´ rs7-3 aposporic only 50 porceddu et al. 2002 apo143 s1/1-7 ´ l4 aposporic only 39-42 marconi et al. 2020 apo40 s1/1-7 ´ l4 parthenogenetic only 44-48 marconi et al. 2020 apo98 s1/1-7 ´ l4 parthenogenetic only 39-42 marconi et al. 2020 *aposporic and parthenogenetic. 138 egizia falistocco et al. figure 1. percentage of meiotic abnormalities at i and ii division and pollen viability recorded on parental genotypes s1/1-7 (a), rs7-3 (b) and l4 (c); on progenies from the cross s1/1-7 x rs7-3: pg-f122 (d), pg-f115 (e), pg-f146 (f), pg-f15(g) and from the cross s1/1-7 x l4: apo 143 (h), apo 40 (i) and apo 98 (j). 139variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis was recorded at the end of meiosis (20.0%). a considerable variability of the pollen size was observed; however, pollen viability of this plant was complete (fig. 1f ). the aposporic recombinant pg-f15 displayed a quite anomalous microsporogenesis. at prophase i, fusion and aggregation of meiocytes (4.7%) were observed, with fusions that in some cases involved meiocytes at prophasic stages (fig. 2c). in addition, cells with chromosomes sepafigure 2. aspects of alterations of microsporogenesis and pollen grains in some of the examined genotypes. meiocytes at prophase i connected by cytoplasmic channels in l4 (a); fusion of three cells at the beginning of prophase i in pg-f115 (b); fusion of two meiocytes at different stage of prophase i in pg-f15 (c); cell at the diakinesis stage with chromosomes separated into three groups (d); absence of chromosome segregation at anaphase i in pg-f15 (e); spreading of chromosomes in both cells of a meiocyte at anaphase ii in pg-f15 (f); triad formed at the end of microsporogenesis in apo 40 (g); dyad formed at the end of microsporogenesis in apo 40 (h); pollen grains produced by apo 40 exhibiting a large size variability (i). the bar represents 10 μm. 140 egizia falistocco et al. rated into two or three groups were observed (fig. 2d). at anaphase i, numerous pmcs (33.0%) showed lagging chromosomes or absence of segregation (fig. 2e). in the second division the spreading of chromosomes in one or both cells of meiocytes was frequently observed (15.0%, fig. 2f), as well as dyads and triads at the end of meiosis (12.0%). despite these abnormalities, also in this case the pollen appeared fully viable (fig. 1g). among the progeny of the cross s1/1-7 x l4, the aposporic apo143 and parthenogenetic apo40 and apo98 were analyzed. the chromosome numbers of these genotypes, 2n=44-48 for apo40 and 2n=39-42 for apo98 and apo143, was previously estimated by means of flow cytometry (marconi et al. 2020). in apo143 most of the anthers resulted empty and the few meyocites that was possible to analyse showed anomalies consisting of linked cells (4.5%) at prophase i and lagging chromosomes at anaphase i (5.0%). the few pollen grains produced, were not viable (fig. 1h). the meiotic irregularities detected in apo40 mainly affected the first division where about 6.0% of pmcs at prophase i were connected by cytoplasmic channels and 34.0 % of meiocytes at metaphase i showed univalents. anomalies of the second division were due to the absence of segregation of chromosomes at anaphase ii (8.0%) and formation of triads and dyads (9.0%) at the completion of meiosis (fig. 2g, h). the pollen showed a remarkable variability in size but all grains appeared fully stained (fig. 1i, fig. 2i). in apo48 a high number of cells at anaphase i with laggards were recorded (48.0%). the scarcity of meiocytes in the second division suggests a possible degeneration of pmcs before the dyad stage. a remarkable number of cells that entered the second division displayed irregular segregation of chromosomes (26.0%) and the pollen was not viable (fig. 1j). discussion in this work the meiotic behavior of sexual, apomictic and f1 recombinant genotypes of p. pratensis was investigated. in sexual plants an almost regular microsporogenesis was obser ved, whereas the apomictic genotypes displayed meiotic abnormalities at different degrees, mostly consisting in events of cell fusion and irregular segregation of chromosomes during i and ii division. data did not reveal relationships between the amount and types of such abnormalities and the reproductive mode of the apomictic genotypes. however, the origin of the parental genotypes s1/1-7, rs7-3 and l4 may offer useful indications for interpreting their meiotic behavior and those of their progenies. in fact, the sexual s1/1-7 plant was derived from a cross between two completely sexual genotypes selected from german cultivars (matzk 1991; barcaccia et al. 1998). the achievement and persistence of sexuality by means of meiosis and fertilization is guaranteed by regular processes of microsporogenesis and gametogenesis. the fact that s1/1-7 was obtained by crossing two completely sexual genotypes contributed to preserving its fertility. conversely, the apomictic rs7-3 and l4 were collected in the wild and did not undergo any anthropogenic pressure (mazzuccato 1995; marconi et al. 2020). these genotypes differ in chromosome number, meiotic behavior and pollen fertility, and also their progenies, obtained by crossing them with s1/1-7 as female parent, showed variability in the same traits. given that both crosses have the same female parent, it is possible to evaluate the contribution of each male parent to the characteristics of the corresponding offspring. since all plants from the cross s1/1-7 x rs7 had the same chromosome number 2n=50 (porceddu et al. 2002) this evidence demonstrates that a regular chromosome segregation occurred and that fertilization took place between normal haploid female and male gametes with n=18 and n=32, respectively. on the contrary, two different ranges of chromosome number (2n=39-42 and 2n=44-48) were detected in plants from s1/1-7 x l4 (marconi et al., 2020). this suggests that l4 produced functional gametes with a different chromosome number and that they accomplished fertilization. it has been suggested that the meiotic events are controlled by a large number of genes, some controlling the meiotic phases and others post-meiotic events and gametogenesis. the mutation of any of these genes can cause anomalies affecting the gamete fertility (ma 2005). most of the meiotic abnormalities observed in this study were common to all plants examined; for example, the irregular segregation of chromosomes, which is probably due to the defective spindle formation or its total absence (kaul and murthy 1985). generally, these alterations are not directly responsible for pollen viability but rather for pollen chromosome number; so that they are considered one of the principal sources of polyploid or aneuploid-polyploid pollen grains (stebbins 1963; podio et al. 2012). this may explain why, despite their meiotic disturbances, rs7-3 and the apomictic and recombinant progenies pg-f15, pg-f115 and pg-f146 showed a high level of pollen fertility. the considerable number of triads and dyads and the heterogeneous size of pollen detected in pg-f115 and pg-f146 are a further evidence that these meiotic alterations influence the chromosome constitution of pollen grains (stanley and linskens 1974). the genotype l4 as well as genotypes apo143, apo40 and apo98 displayed a different situation. the 141variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis meiotic alterations detected in l4 could explain the production of aneuploid pollen grains, as above suggested, but do not clarify the cause of the reduction in pollen fertility of this plant. this, most likely, is the result of postmeiotic events, such as the alteration of the normal activity of genes controlling the steps following the completion of meiosis and gametogenesis (lalanne and twell 2002). the scarcity of pmcs detected in apo143 could be the consequence of mutations affecting the normal development of anthers and the formation of meiocytes. a certain degree of pmcs scarcity has been already reported in boechera (rojek et al. 2018) while genetic and molecular analyses in arabidopsis demonstrated that a high number of genes controls several aspects of anther development and that their mutations can seriously damage the anther cell differentiation, tapetum function and microspore development (ma 2005; sanders et al. 1999). studying mutants in arabidopsis, yang and colleagues (2003) demonstrated that mutations of genes controlling the meiotic progression can result in programmed cell death with the consequence of the death of most meiocytes before cytokinesis. the dramatic reduction in the number of meiocytes observed in apo98 could be the result of a phenomenon of cell degeneration similar to the one described in arabidopsis. among those obtained from the cross s1/1-7 x l4, the parthenogenetic recombinant apo40 was the only genotype producing viable pollen. moreover, the microsporogenesis pathway of this plant was not affected by the scarcity of meiocytes that characterized apo143 and apo98. a possible explanation for the different meiotic behavior of the f1 plants from the cross s1/1-7 x l4 could be the different number of mutations that these plants inherited from the male parent; the different chromosome number characterizing these genotypes could support this hypothesis. further considerations can be done taking into account the cytological data and the reproduction mode of the plants obtained from the cross s1/1-7 x rs7. it can be observed that the apomictic and recombinant genotypes have similar behavior as the male parent, whereas the sexual pg-f122 reflects the meiotic characteristics of the female parent. this suggests that the meiotic behavior and the mode of reproduction are together inherited from one of the parents. however, the progeny from s1/1-7 x l4 does not provide enough evidence supporting this hypothesis because all the examined genotypes were apomictic recombinant. cellular aggregations due to cytoplasmic channels and cell fusion represent sporadic events in the examined plants. such aggregations involved only few cells (2-4) and did not damage the pollen fertility because they did not proceed to meiosis but degenerated, as demonstrated by the fact that they were not observed from prophase i onwards. cell fusion has been reported in several plant species and may result from suppression of cell wall formation during premeiotic mitoses (nirmala and rao 1996). instead, the cytoplasmic connections originate from the pre-existing system of plasmodesmata which form within anther tissues and subsequently become completely obstructed by the progressive deposition of callose (heslop-harrison 1966). in some cases, due to the scarce production of callose, the connections remain giving origin to meiocytes aggregation. the 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cytogenetic markers and classical chromosomal features of spilopelia chinensis (scopoli, 1786) and tachybaptus ruficollis (pallas, 1764) in thailand isara patawang1,*, sarawut kaewsri2, sitthisak jantarat3, praween supanuam4, sarun jumrusthanasan2, alongklod tanomtong5 centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates) simona ceraulo, vanessa milioto, francesca dumas* repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil marina souza cunha1,2,*,#, silvana melo1,3,#, filipe schitini salgado1,2, cidimar estevam assis1, jorge abdala dergam1,* karyomorphology of some crocus l. taxa from uşak province in turkey aykut yilmaz*, yudum yeltekin variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l. egizia falistocco1,*,+, gianpiero marconi1,+, lorenzo raggi1, daniele rosellini1, marilena ceccarelli2, emidio albertini1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(3): 99-106, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1113 caryologia international journal of cytology, cytosystematics and cytogenetics citation: harshita dwivedi, girjesh kumar (2021) colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae). caryologia 74(3): 99-106. doi: 10.36253/caryologia-1113 received: october 09, 2020 accepted: september 24, 2021 published: december 21, 2021 copyright: © 2021 harshita dwivedi, girjesh kumar. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid hd: 0000-0001-7197-3000 colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae) harshita dwivedi*, girjesh kumar plant genetics laboratory, department of botany, university of allahabad, prayagraj, 211002 india corresponding author. e-mail: harshitadwivedi88@gmail.com abstract. unreduced gametes are the key source for the natural polyploidization in plants, but rate of its formation is very low in nature. meiotic mutants are second source for the formation of 2n pollen. in this cytological investigation, the meiotic aberrations and its impact on post-meiotic products were analysed in autotetraploid trachyspermum ammi (l.) sprague (4n=36). the seedlings of t. ammi (l.) sprague were treated with 3 different concentrations of colchicine (0.2, 0.4 and 0.5%, w/v) for 3 different durations. six polyploid plants were induced which was confirmed on the basis of cytological analysis. colchicine, an anti-microtubular drug induced different meiotic and post-meiotic abnormalities such as chromosomal bridges, lagging chromosomes, scattering, precocious, fragments, dyads, triads, and polyads. the formation of several abnormal sporads clearly signifies the meiotic restitution. the tendency of univalents to scattered in the cytoplasm at metaphase was identified as a peculiar aberration asynapsis. pollen variability and fusion of pollen walls was reported and pollen fertility was calculated. the morphological analysis of the pollen allowed us to confirm the occurrence of 2n pollen. keywords: unreduced gametes, 2n pollen, polyploidy, colchicine, meiotic aberrations, trachyspermum ammi (l.) sprague. introduction ploidy induction is an effective method to induce novel quantitative and qualitative traits in plants which make them superior to their diploid ancestors. it is estimated that up to 70% of angiosperm species are polyploid, and this number is even higher if ancient polyploidization events are taken into account (bretagnolle and thompson 1995; ramsey and schemske 1998). various polyploid plants have been reported in natural ecosystems; among them some are important crop species such as potato, coffee, banana, peanut, tobacco, wheat, oats, sugarcane, and many fruits (bretagnolle and thompson 1995; stebbins 1950; udall and wendel 2006). meiosis is a well-coordinated event which is vital for accomplishment of microsporogenesis. the product of male meiosis in plants is a tetrad of four 100 harshita dwivedi, girjesh kumar haploid microspores that are temporarily joined by a callosic wall (brownfield and kohler 2011). after release from the tetrad each microspore undergoes two mitotic divisions to produce a pollen grain containing the two sperm cells required for double fertilization (mccormick 2004). whereas any disturbance to the meiotic event often has severe effects and leads to the abortion of the meiocytes or the developing gametophytes and thus sterility, a number of meiotic mutants that produce viable, unreduced gametes have been described in a range of plants (bretagnolle and thompson 1995; ramanna and jacobsen 2003). ga metes w it h somatic chromosome numbers referred to as unreduced (2n) gametes. according to bretagnolle and thompson (1995), the production of unreduced gametes is considered the main cause of polyploid induction in nature. the unreduced (2n) gametes can be formed due to both preand post-meiotic genome duplication events, as well as meiotic restitution (bretagnolle and thompson 1995; ramsey and schemske 1998). the heterozygosity within 2n gametes depends on its cytological mechanism which is subdivided as first division restitution (fdr), second division restitution (sdr) and indeterminate meiotic restitution (imr) (ramanna and jacobsen, 2003). an easier diagrammatic representation of restitution mechanism was given by ferris et al. (1992). a fdr 2n gamete comprises nonsister chromatids, whereas a sdr 2n gamete comprises two sister chromatids (tang 2002). the third (imr) type was observed in lily, carried characteristics similar to both sdr and fdr (lim et al. 2001). in this intermediate type, both univalents and bivalents are formed during metaphase i. crossing 2n pollen with a normal female gamete has been shown to be one of the most effective methods to produce triploid individuals (nilsson-ehle 1936; müntzing 1936). according to dewitte et al. (2012), meiotic mutants are a second source of 2n gametes. yet, it has been considered that the rate of occurrence of 2n pollen in nature is very low hence the production rate of polyploids was less than 0.1% (liu et al. 2019). to increase the occurrence rate of 2n pollen, novel methods for inducing polyploid production in plants via the use of spindle inhibitors, such as dinitroanilines or colchicine, have been successfully explored and used in some species (vaughn and lehnen 1991; hancock 1997; zlesak et al. 2005; dhooghe et al. 2009). amongst several antimicrotubular drugs, colchicine is traditionally and preferably being used as doubling agent (kumar and dwivedi 2017). a widespread data has been accumulated over the last 60 years which specifies that the drug colchicine manifest defects in meiotic prophase. it reduces the frequency of chiasmata (driscoll and darvey 1970, shepard et al. 1974) and impairs synaptonemal complex formation (tepperberg et al. 1997). the large number of world’s population rely either solely or largely on traditional herbal remedies for health care (dwivedi 2016). one of these important medicinal plants is ajwain (trachyspermum ammi (l.) sprague, 2n = 2x = 18) which is a rich source of various neutraceutical components, due to which it occupies a significant economic position in pharmaceutical industries (dwivedi and kumar 2018). many of the medicinal and aromatic plants do not have stable production in their growing areas and are usually gathered in accordance with conventional methods to meet demands (dalkani et  al. 2012). therefore to meet the growing pharmaceutical demands, attention need to be paid to medicinal plants with stable quality. 2n gametes are an effective and efficient way to transmit genetic diversity to the plants, including both valuable qualitative and quantitative traits (peloquin et al. 1999). recently, plant breeders have become interested in the practical use of 2n gametes in breeding program due to the new tools available for 2n gamete manipulation and insights into the genetic background of their formation (dewitte et al. 2012). however artificial manipulation of ploidy in ajwain has been previously achieved by few researchers (kumar and dwivedi 2017 and noori et al. 2017) yet the potential role of unreduced gametes to create genetic variability in this crop is unmapped. hence, through this study we had made an effort to understand the effect of in  vitro colchicine-induced disruption in meiotic products, mechanism of formation of 2n pollen and its repercussions on the crop fertility. material and methods plant material fresh and healthy seeds of t. ammi var. aa-1 were procured from national research centre for seed spices, ajmer, rajasthan, india. seeds were sown in earthen pots in triplicates during october to november season to raise the seedlings. agro-climatic conditions of experimental site the present experiment has been performed in the area of roxburgh botanical garden, department of botany, university of allahabad, prayagraj, u.p., india, during the rabi season. the exact experimental location is 25°27”43.01’n, 81°51”10.42’e. prayagraj is situated 98 m 101colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi sprague above mean sea level. prayagraj is in the sub-tropical climatic zone; the average rainfall is 1027 mm and relative humidity is 59%. induction of autotetraploidy for the present work colchicine (c22h25npo6) manufactured by himedia laboratory pvt. ltd., mumbai, india was used. the treatment was given to the seedlings within 2–3 days of germination (before third leaf emergence). emerging shoot apices at cotyledonary stage of each of the 20 seedlings/pot were treated with aqueous solution of 0.2, 0.4, and 0.6% concentration by cotton plug method for one, two and three consecutive days with an alternate recovery time period of 12 hours in each case. a set of control was also maintained of 20 plants/ pot. meiotic preparation small sized floral buds were fixed in carnoy’s fixative viz. glacial acetic acid: ethyl alcohol (1 : 3, v/v) and then transferred to 70% alcohol after 24 hours and stored at 4°c until use. meiotic slides were prepared by using anther squash technique with 2% standard acetocarmine stain, observed and microphotographed under nikon phase contrast research microscope (nikon eclipse, e200, japan) at 40x magnification. pollen viability assessment pollen viability was estimated using a glyceroacetocarmine i.e. stained cytoplasm with nucleus were considered as fertile whereas unstained and shriveled pollen grains without nuclei were considered as sterile. statistical analysis all the statistical analyses of morphological and cytological observations have been done by using spss 16.0 software to measure mean values of variables. a pair wise comparison of means was made using duncan’s multiple range test (dmrt) at p≤0.05 significance level. results the diploid plant of trachyspermum ammi (l.) sprague of var. aa-1, used in the present study, had 2n=18 (n=9) as confirmed in mitotic as well as meiotic studies (figure 1.1). the meiotic behaviour of six induced polyploid plants of ajwain were analysed and found that the number of bivalents was more than 9 at early prophase (figure 1.2). moreover, the pmcs of colchicine treated plants showed various chromosomal aberrations such as unorientation, laggard (figure 1.9 and 1.11), bridge (figure 1.9), scattering (figure 1.4 and 1.10), precocious movement (figure 1.7 and 1.8), fragment (figure 1.3 and 1.10), etc. the range of these aberrations was higher in metaphase i and ii as compared figure 1. 1. a pmc showing 9 bivalents at diakinesis (diploid), 2-12. meiotic stages of autotetraploid plants (4n=36); 2. normal pmc at early prophase, 3. fragment at anaphase i, 4. scattering at metaphase i, 5 and 6. meiotic configurations of asynaptic plants, 5. 2iv+5ii+10i, 6. 5ii+26i, 7 and 8. precocious movement at metaphase i, 9. bridge formation alongwith laggard at sticky anaphase i, 10. scattering and fragment at anaphase i, 11. laggard at anaphase i, 12. fragment at anaphase ii. scale: 10µm 102 harshita dwivedi, girjesh kumar to both divisions of anaphase. the dividing phases of plants were less affected by lower doses of colchicine (24 and 36 hours treatment of 0.2% concentration) while it was higher in case of 0.4% and 0.6% concentrations. the highest total meiotic aberration (tma) was observed at 0.6% concentration (10.51±1.22%) however it was range from 7.50±0.79% to 9.77±0.69% at 0.2% concentration (table 1). the tendency of univalents to scattered in the cytoplasm at metaphase exhibited the peculiar phenomenon of asynapsis which was witnessed at 0.4% concentration of colchicine (figure 1.5 and 1.6). owing to the outcomes of these meiotic aberrations, abnormal post-meiotic products have been reported among which triads showed predominance over the dyads and polyads. an increasing trend for polyads formation was recorded with respect to colchicine i.e. 4.19±0.42% to 8.20±0.51% in a dose dependent manner (table 2). the arrangement of these sporads was quite different from the normal tetrad. figure 2.3 showed dyad with one small microcyte unlike the dyad shown in figure 2.4. in some instances, the polyads were recorded in which the sporads were not joined by callosic wall (figure 2.8 and 2.9) unlike the normal tetrad stage. such sporads can produce gametes of different ploidy levels. as a consequence of these abnormal sporads, meiotic index (mi) was decreased along with increasing the concentrations of colchicine. the mi was table 1. effect of colchicine on different meiotic phases of trachyspermum ammi (l.) sprague. concentration (%) duration (hours) plant no. meiotic abnormalities (mean±s.e.) meta i/ii ana i/ii tma control p1-p10 0.2 24 p-1 4.22±0.58a 3.28±0.26ab 7.50±0.79a 36 p-2 5.07±0.80ab 4.26±0.26ab 9.33±1.68ab 36 p-3 5.50±0.39ab 4.27±0.38ab 9.77±0.69ab 36 p-4 6.68±0.34b 2.97±0.29a 9.65±0.41ab 0.4 24 p-5 5.47±0.17ab 4.50±0.14bc 9.97±0.28ab 0.6 12 p-6 5.72±0.47ab 4.79±0.76c 10.51±1.22b *mean±s.e., values followed by the superscript differ at p<0.05 between treatments by the dmrt. table 2. effect of colchicine on post-meiotic products, meiotic index and pollen fertility of trachyspermum ammi (l.) sprague. concentration (%) duration (hours) plant no. post-meiotic abnormalities (mean±s.e.) mi (%) pf (%)dyads triads polyads control p1-p10 1.75±0.24a 98.72±0.33f 98.99±0.22f 0.2 24 p-1 7.80±0.43b 21.35±0.09b 4.19±0.42b 64.86±0.30e 59.30±0.20e 36 p-2 8.16±0.36b 21.49±0.33b 5.69±0.43c 62.11±0.15c 58.80±0.17d 36 p-3 8.20±0.64b 21.92±0.42bc 6.43±0.47c 64.05±0.07d 58.32±0.22cd 36 p-4 8.70±0.42bc 22.96±0.47cd 6.15±0.41c 63.40±0.22d 58.29±0.25c 0.4 24 p-5 8.80±0.40bc 23.64±0.26d 6.52±0.60c 57.20±0.19b 41.20±0.14b 0.6 12 p-6 9.75±0.43c 24.03±0.52d 8.20±0.51d 55.10±0.20a 39.10±0.09a tmatotal meiotic abnormalities, mi-meiotic index, pfpollen fertility. *mean±s.e., values followed by the superscript differ at p<0.05 between treatments by the dmrt. figure 2. 1. normal tetrad of diploid plant, 2. normal tetrad of autotetraploid plant, 3. dyad with a microcyte, 4. dyad, 5. triad, 6. pentad, 7. polyads, 8. a polyad with fused microspore in two groups, 9. a polyad completely fused microspore. scale: 10µm 103colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi sprague recorded as 64.86±0.30% at 24 hour duration of 0.2% concentration whereas 55.10±0.20% at 0.6% concentration of colchicine (table 2). on account of these aberrant post meiotic products, the process of microsporogenesis is significantly affected and consequently resulted variability in pollen grains (figure 3). the pollen grains exhibited variability in terms of their shape and size. the shape was observed to be remarkably transformed which is represented in figure 3. the fertility of pollen grains were gradually reduced (table 2) and ranged from 59.30±0.20% (at 0.2% concentration) to 39.10±0.09% (at 0.6% concentration). figure 4.1 and 4.2 represents the pollen cytomixis in which the chromatin material was transferred through both wide and narrow channels to the proximate pollens. onset of passing event was evident by the assemblies of pollens where the dissolution of walls was observed (figure 4.2). direct fusion of pollen grains is represented in figure 4.1. the size of pollen grain of diploids was registered as 4.42 0.04 μm × 2.67 ± 0.12 μm while 7.09±0.17 μm × 5.31±0.20 μm in case of autotetraploids. the pollens of polyploids were near about two times larger as compared to diploids thus it was considered as unreduced pollen (2n) (figure 3.2 and and 4.1-4.4). discussion the repercussions of colchicine induced meiotic aberrations resulted to genetically unstable polyploid plants. the proper spindle formation and its precise ongoing activity during whole meiotic event are essential for the accomplishment of fertile progeny. however, levan (1939) stated that colchicine causes a temporary inactivation of the spindle mechanism without damaging any other life processes of the chromosomes. thus, testing the stability of induced polyploids is critical and should be studied at various stages as the plant material matures (harbard et al. 2012). meiotic prophase i is characterized by chromosome cohesion, pairing, and recombination (ma, 2006). these bivalents are the physical sites of crossover between homologous chromosomes and are only established if pairing and recombination occur normally. the mutations concerning to meiotic prophase i that often result in univalents (paired sister chromatids) rather than bivalents (ross et al. 1997; bai et al. 1999; bhatt et al. 1999; couteau et al. 1999; caryl et al. 2000; grelon et al. 2001; de muyt et al. 2009). the unequal segregation of univalents in meiosis i followed by an equal second division, results in the formation of aneuploid cells which abort during development, however in some mutants, a few functional gametes are produced (coufigure 4. 1. fusion of wall between two pollen grains, 2. fusion of walls in a group of pollen grains, 3 and 4. pollen variability. scale: 10µm. figure 3. 1. a diploid pollen (2n), 2-4. different autotetraploid pollen grains (4n), 2. a fertile tetraploid pollen 3. a sterile budding pollen grain, 4. a fertile pollen showing variability in shape. scale: 10µm. 104 harshita dwivedi, girjesh kumar teau et al. 1999; azumi et al. 2002; ravi et al. 2008). in both meiosis i and ii, bridge formation accompanied with the fragment/s was exhibited at the higher concentration of colchicine which attributed to the presence of paracentric inversion (sybenga, 1996). chromosome bridges have often been studied by observing cancer cells containing chromosomes damaged by spontaneous telomere loss (fouladi et al. 2000; lo et al. 2002; acilan et al. 2007). these damaged chromosomes enter the breakage–fusion–bridge cycle (zheng et al. 1999). bajer (1964) mentioned that chromatin bridges are responsible for retardation of kinetochore movement, or, frequent bridges formation may result in nuclear restitution. in the present study, the phenomenon of asynapsis has been observed which showed the inability of univalents to assemble at the equatorial plate and widely scattered in cytoplasm at metaphase stage suggests lack of pairing at early prophase. according to gottschalk and kaul (1980 a, b), the absence or failure of synapsis is termed as asynapsis. the asynaptic mutant induced by colchicine was previously described in soybean by kumar and rai (2007); proposed that this mutation might have affected specific genes for chromosome pairing. the sporads are highly specialized cells which are able to produce four haploid cells after a series of genetically controlled steps (caetano-pereira et al. 1999). the elimination or addition of one or more chromosomes (as a consequence of laggard, precocious, bridge, fragments, etc.) is responsible for the formation of anomalous postmeiotic products such as monads, dyads, triads and polyads resulted to the unreduced gametes (golubovskaya 1989) or sterile (bosco et al. 1999) pollen grains. according to kiihl et al. (2011), the meiotic phases that precede cytoplasm cleavage might have caused failure in the cytokinesis process resulting into monads, dyads, triads and polyads. there are three main types of abnormal spindles orientation i.e. parallel, fused and tripolar that have been reported to produce 2n pollen (mok and peloquin 1975; veilleux 1985; bretagnolle and thompson 1995; ramsey and schemske 1998; zhang et al. 2009; zhang and kang 2010; silva et al. 2011). the parallel spindles resulted in two fdr 2n pollens while fused spindles are accountable to form a dyad and then two fdr 2n pollens. the tripolar spindles develop a mother cell to produce one fdr 2n pollen and two 1n pollen (zhang and kang 2013). however, ramanna and jacobson (2003) stated that fdr is typical in synaptic mutants or distant hybrids, in which homologous chromosome pairing (bivalent formation) is completely absent, although other mechanisms, such as cytokinesis failure or spindle abnormalities during metaphase ii, can also lead to an equivalent of fdr. fdr gametes contain an equal number of parental chromosomes due to the equatorial division of sister chromatids. therefore, fdr pollen are key entities in producing heterozygous hybrids, because of the highly heterozygous 2n gametes formed (bretagnolle and thompson 1995). on the contrary, sister chromosomes move to the same daughter cell in case of sdr gametes, thus due to the absence of crossing-over, it exhibit maximum homozygosity (hermsen 1984; veilleux 1985; peloquin et al. 1999). owing to the abnormal spindle activity, the unreduced pollen of t. ammi genetically represents the fdr mechanism. naturally, the establishment of new polyploid genotypes is infeasible without the existence of 2n gametes. thus its role has great significance in evolution, as the cytological mechanisms of 2n gamete formation demonstrate that it might be the source of variable genetic combinations. since, our investigation was performed only to determine the cytological behavior of unreduced gametes; further researches are required to understand the extent of heterozygosity of 2n gametes and its influential role to create variability in ajwain crop. acknowledgments authors are thankful to nrcss, ajmer, rajasthan, india for supplying seeds of t. ammi var. aa-1. one of the authors (harshita dwivedi) thanks the university grant commission (ugc) for financial assistance and the head of the department of botany, university of allahabad, prayagraj, for providing the necessary facilities. sincere thanks are also due to all the members of plant genetics laboratory for their encouragement and support. funding this work was supported by the university grants commission. references acilan c, potter dm, saunders ws (2007). dna repair pathways involved in anaphase bridge formation. gen chrom cancer. 46:522–531. azumi y, liu d, zhao d, li w, wang g, hu y, ma h. 2002. homolog interaction during meiotic prophase i in arabidopsis requires the solo dancers gene encoding a novel cyclin-like protein. embo journal. 21:3081–3095. 105colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi sprague bai x, peirson bn, dong f, xue c, makaroff ca. 1999. isolation and characterization of syn1, a rad21-like gene essential for meiosis in arabidopsis. the plant cell. 11:417–430. bajer a. 1964. cine-micrographic studies on dicentric chromosomes. chromosoma 15:630–651. bhatt am, lister c, page t, fransz p, findlay k, jones gh, dickinson hg, dean c. 1999. the dif1 gene of arabidopsis is required for meiotic chromosome segregation and belongs to the rec8/rad21 cohesion gene family. the plant journal. 19:463–472. bosco sf, tusa n, conicella c. 1999. microsporogenesis in a citrus interespecific tetraploid somatic hybrid and its fusion parents. heredity. 83:373-377. bretagnolle f, thompson j. 1995. gametes with the somatic chromosome number: mechanisms of their formation and role in the evolution of autopolyploid plants. new phytol. 129:1–22. brownfield l, köhler c. 2011. unreduced gamete formation in plants: mechanisms and prospects. journal of experimental botany. 62:1659–1668. caetano-pereira cm, pagliarini ms, brasil em. 1999. cell fusion and chromatin degeneration in an inbred line of maize. genet mol biol. 22:69-72. caryl ap, armstrong sj, jones gh, franklin fch. 2000. a homologue of the yeast hop1 gene is inactivated in the arabidopsis meiotic mutant asy1. chromosoma. 109:62–71. couteau f, belzile f, horlow c, grandjean o, vezon d, doutriaux mp. 1999. random chromosome segregation without meiotic arrest in both male and female meiocytes of a dmc1 mutant of arabidopsis. the plant cell. 11:1623–1634. dalkani m, hassani a, darvishzadeh r. 2012. determination of the genetic variation in ajowan (carum copticum l.) populations using multivariate statistical techniques. rev ciênc agron. 43:698–705. de muyt a, pereira l, vezon d, chelysheva  l, gendrot g, chambon a,  et al. 2009. a high throughput genetic screen identifies new early meiotic recombination functions in arabidopsis thaliana. plos genetics. 5:e1000654. dewitte a, van laere k, van huylenbroeck j. 2012. use of 2n gametes in plant breeding. ed. dr. ibrokhim abdurakhmonov. isbn: 978-953-307-932-5. dhooghe e, grunewald w, leus l, van labeke mc. 2009. in vitro polyploidisation of helleborus species. euphytica. 165:89–95. driscoll, c. and darvey, n. 1970. chromosome pairing: effect of colchicine on an isochromosome. science 169: 687-688. dwivedi h, kumar g. 2018. induced syncyte formation via cytomixis in trachyspermum ammi (l.) sprague (apiaceae). caryologia. 71:420-427. dwivedi h. 2016. cytogenetic impact of environmental stresses in trachyspermum ammi l. 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(asteraceae) species that around the van lake in turkey pelin yilmaz sancar1,*, semsettin civelek1, murat kursat2 molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant jinxin cheng1,*, dingyu hu1, yaran liu2, zetian zhang1, majid khayatnezhad3 genetic variations and interspesific relationships in salvia (lamiaceae) using scot molecular markers songpo liu1, yuxuan wang1, yuwei song1,*, majid khayatnezhad2, amir abbas minaeifar3 development of female gametophyte in gladiolus italicus miller (iridaceae) ciler kartal1, nuran ekici2,*, almina kargacıoğlu1, hazal nurcan ağırman1 colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae) harshita dwivedi*, girjesh kumar statistical evaluation of chromosomes of some lathyrus l. taxa from turkey hasan genç1, bekir yildirim2,*, mikail açar3, tolga çetin4 use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater treatment and water of the kamniška bistrica river (slovenia) peter firbas1, tomaž amon2 the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bo shi1,*, majid khayatnezhad2, abdul shakoor3,4 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(4): 29-38, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1106 caryologia international journal of cytology, cytosystematics and cytogenetics citation: luísa antônia campos barros, gisele amaro teixeira, paulo castro ferreira, rodrigo batista lod, linda inês silveira, frédéric petitclerc, jérôme orivel, hilton jeferson alves cardoso de aguiar (2021) cytogenetic survey of eight ant species from the amazon rainforest. caryologia 74(4): 29-38. doi: 10.36253/caryologia-1106 received: october 02, 2020 accepted: september 24, 2021 published: march 08, 2022 copyright: © 2021 luísa antônia campos barros, gisele amaro teixeira, paulo castro ferreira, rodrigo batista lod, linda inês silveira, frédéric petitclerc, jérôme orivel, hilton jeferson alves cardoso de aguiar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid hjaca: 0000-0001-7738-1460 gat: 0000-0002-7106-5798 lacb: 0000-0002-1501-4734 lis: 0000-0003-4172-4489 fp: 0000-0002-2015-1258 jo: 0000-0002-5636-3228 cytogenetic survey of eight ant species from the amazon rainforest luísa antônia campos barros1, gisele amaro teixeira2, paulo castro ferreira1, rodrigo batista lod1, linda inês silveira3, frédéric petitclerc4, jérôme orivel4, hilton jeferson alves cardoso de aguiar1,5,* 1 universidade federal do amapá, campus binacional, oiapoque, 68980-000, brazil 2 programa de pós-graduação em biologia celular e estrutural, universidade federal de viçosa, viçosa, brazil 3 programa de pós-graduação em biologia animal, universidade federal de viçosa, viçosa, brazil 4 cnrs, umr ecofog, agroparistech, cirad, inra, université de guyane, université des antilles, campus agronomique, kourou, france 5 programa de pós-graduação em biodiversidade tropical, universidade federal do amapá, macapá, brazil *corresponding author. e-mail: hilton@unifap.br abstract. the scarce information regarding ant diversity in the state of amapá and lack of cytogenetic data of species from the amazon region can hide ant biodiversity information that may be detectable with affordable cytogenetic techniques. in this study, we describe the karyotypes of eight ant taxa collected from amazonian localities in french guiana and brazil. chromosome numbers ranged from 2n = 18 to 2n = 68. the following chromosome numbers were observed for each species: azteca sp. group chartifex 2n = 28; dolichoderus bidens (linnaeus, 1758) 2n = 18; gnamptogenys tortuolosa (smith, 1858) 2n = 44; camponotus renggeri emery, 1894 n = 20; pseudomyrmex unicolor (smith, 1855) 2n = 68 and n = 34; apterostigma sp. pilosum complex 2n = 46; odontomachus bauri emery, 1892 2n = 44, and wasmannia auropunctata (roger, 1863) 2n = 32. the karyotypes of p. unicolor, g. tortuolosa, and o. bauri are reported here for the first time. our data enabled comparisons between chromosomal data of some species from amazon and atlantic rainforests. we also highlight the methods used for the ant chromosome classification. keywords: karyotype, chromosome evolution, biodiversity, formicidae, neotropics, taxonomy. introduction the classical cytogenetic approach utilizes a single dye, orcein or giemsa (liehr 2017), without previous trypsin-treatment, for the study of chromosomes and has also been denoted as beta karyology by white (reviewed by petitpierre 2009). the low cost of classical cytogenetics allows more extensive sampling and plays a vital role in the discovery and understanding of diver30 luísa antônia campos barros et al. sity in different organisms (zacharopoulou et al. 2017; di-nizo et al. 2017; cioffi et al. 2018). in hymenopteran cytogenetics, chromosomes can be obtained from live larvae using a stereomicroscope and chemicals, even from distant localities such as those in amazonia. the technique provided by imai et al. (1988) enables the use of artisanal procedures with rustic material such as empty pill packs to keep the ganglia in hypotonic solution and syringes for their dissociation on the slides. important taxonomic insights may be achieved from karyotype information and, according to schubert (2011), efforts must be made to avoid losing such data. the resolution of sampling issues is particularly important in population-level approaches for understanding taxonomic problems (petitpierre 2011; cioffi et al. 2018; chèvre et al. 2018). to date, classical cytogenetic studies are routinely performed for many organisms (petitpierre 2009; liehr 2017), thus supporting the accuracy and validity of their results. karyotype configuration can be useful for species delimitation, as karyotypes with structural and/ or numerical differences may not pair properly during meiosis (king 1993). this kind of chromosomal variation can affect fertility in heterozygotes and, in extreme cases, lead to sterility caused by gamete aneuploidy. remarkable examples of chromosome number distinctness in closely related species or within the same species have been reported. for instance, in the cervidae species muntiacus muntjak (zimmermann, 1780), females possess 2n = 6 chromosomes and males possess 2n = 7, and muntiacus reevesi (ogilby, 1839) has a distinct chromosomal organization of 2n = 46 (wurster and benirschke 1970). recent examples of intraspecific chromosomal variations in ants have been observed from different populations within the species. for instance, different cytotypes have been found in holcoponera striatula (mayr, 1884) (as gnamptogenys striatula) (2n = 32, 34), holcoponera moelleri forel, 1912 (as gnamptogenys moelleri) (2n = 34, 44) (teixeira et al. 2020), and mycetophylax morschi (emery, 1888) (2n = 26, 28, 30) (micolino et al. 2019). karyological information is currently available for approximately 800 species of ants distributed across the world (reviewed by lorite and palomeque 2010; cardoso et al. 2018; mariano et al. 2019). neotropical ant species have been targeted for cytogenetic studies since the first surveys conducted by crozier (1970) in south america, including brazil, and by goñi et al. (1983) in uruguay. pioneering studies in ant cytogenetics in brazil were performed by fadini and pompolo (1996) and mariano et al. (2000) and, since then, there has been a steady increase in the number of cytogenetic researches in ants using different approaches. thus far, more than 180 ant taxa have been cytogenetically studied in the neotropics, most of them from the atlantic rainforest in brazil (reviewed by mariano et al. 2019). in the amazonian region, karyological information is limited to that obtained from species restricted to french guiana and brazil (reviewed by aguiar et al. 2020). in this study, we describe the karyotypes of eight ant species from the amazon rainforest using a comparative approach with available population data, as our contribution toward understanding the evolutionary pattern of ant diversity in the neotropics. materials and methods ant colonies were collected by active search in french guiana at kourou and sinnamary, and in brazil at oiapoque, state of amapá and açailândia, state of maranhão (table 1). adult voucher specimens were deposited into the ant collection at the laboratório de mirmecologia do centro de pesquisas do cacau (cpdc/brazil) in bahia, brazil, under records #5802, #5803, and #5816. mitotic chromosomes were obtained from the cerebral ganglia of the larvae after meconium elimination, as described by imai et al. (1988). the chromosome number and morphology of metaphases were analyzed using conventional 4% giemsa staining. chromosomes were arranged in order of decreasing size and based on the ratio of the chromosome arm lengths (r = long arm/ short arm), i.e., on the centromeric position, according to the classification proposed by levan et al. (1964). the chromosomes were measured and classified as m = metacentric (r = 1–1.7), sm = submetacentric (r = 1.7–3), st = subtelocentric (r = 3–7), and a = acrocentric (r > 7). chromosomes were organized using corel photopaint x3 and measured using image pro plus. reflexions on the nomenclature used to classify ant chromosomes imai (1991) proposed a detailed chromosomal nomenclature based on heterochromatin location; however, a classification based on this type of chromatin is impractical because large (detectable) heterochromatic blocks are not present in many ant groups. additionally, the use of chromosome measurements diminishes subjectivity and enables karyotype comparisons between populations or species. analysis of the karyotypes of acromyrmex spp. (reviewed by barros et al. 2021) using the nomenclature of levan et al. (1964) allowed for the detection of dissimilarities in the karyotypic formula caused by the 31cytogenetic survey of eight ant species from the amazon rainforest variations in short arm size due to differential heterochromatin growth. among the atta spp., differences were not detected even with chromosome classification using chromosomal measurements (barros et al. 2014), but variations could be identified by karyomorphometric comparison with the leaf-cutting ant amoimyrmex striatus (roger, 1863) (cristiano et al. 2013). amoimyrmex striatus, in addition to two other species, currently belongs to the new genus amoimyrmex (cristiano et al. 2020). the nomenclature of levan et al. (1964) is typically used for chromosomal classification of different organisms such as plants (winterfeld et al. 2018; sadeghian et al. 2019), spiders (araújo et al. 2020), beetles (şendoğan and alpagut-keskin 2016), bees (lopes et al. 2021), wasps (tavares and teixeira 2021), velvet worms (reviewed by duarte et al. 2020), and fishes (brandão et al. 2018). recent ant cytogenetic studies have focused on measurements of chromosomes (barros et al. 2010, 2014, 2016; cristiano et al. 2013, 2017, santos et al. 2016, micolino et al. 2019, 2020; teixeira et al. 2020). we suggest the use of the standardized chromosomal nomenclature employing measurements described by levan et al. (1964) in formicidae as well as in hymenoptera, thereby allowing for comparisons between the species and populations. we also suggest the use of less condensed chromosomes and care with centromeric location (primary constriction) to diminish subjectivity in chromosome measurements. this chromosome classification based on measurements will also facilitate access to data on ant cytogenetics by researchers working on other organisms and could likely contribute to a better understanding of ant chromosomal diversity and evolution. results and discussion we analyzed the chromosomes of eight ant species, eight genera, and six subfamilies. our analysis presents the first karyological records for pseudomyrmex unicolor (smith, 1855), gnamptogenys tortuolosa (smith, 1858), and odontomachus bauri emery, 1892. three species have already been described for the atlantic rainforest, and showed karyotypic similarities. unique karyotypes were detected in two different species complexes, suggesting genera revision. subfamily dolichoderinae azteca sp. group chartifex presented 2n = 28, 10m + 4sm + 6st + 8a (figure 1a). previously, karyological data for only five taxa from the genus azteca were available; four of these taxa were characterized as 2n = 28 and one, azteca alfari emery, 1893, as 2n = 26 (reviewed by mariano et al. 2019). the karyotype of azteca chartifex emery, 1896 from french guiana is 2n = 28, 10m + 18a (mariano et al. 2019). if we group the chromosomes of azteca table 1. ant species collected from the amazon rainforest and analyzed using classical cytogenetics. collection sites, sample sizes (numbers of colonies/individuals), diploid (2n) and haploid (n) chromosome numbers, and karyotype formula. ant species locality col./ind. 2n (n) karyotype formula 2n / (n) subfamily dolichoderinae azteca sp. group chartifex la montagne des singes, kourou, fg 1/8 28 10m + 4sm + 6st + 8a dolichoderus bidens (linnaeus, 1758) chácara du rona, oiapoque-ap, br 2/11 18 14m + 4sm subfamily ectatomminae gnamptogenys tortuolosa (smith, 1858) * sinnamary, fg 1/4 44 12m + 17sm + 15st subfamily formicinae camponotus renggeri emery, 1894 campus agronomique, kourou, fg 1/4 (20) (2sm + 17st + 1a) subfamily myrmicinae apterostigma sp. pilosum complex la montagne des singes, kourou, fg 2/6 46 6m + 18sm + 16st + 6a wasmannia auropunctata (roger, 1863) chácara du rona, oiapoque-ap, br 1/5 32 16m + 10sm + 6st subfamily ponerinae odontomachus bauri emery, 1892 * açailândia-ma, br 1/7 44 6sm + 24st + 14a subfamily pseudomyrmecinae pseudomyrmex unicolor (smith, 1855) * campus agronomique, kourou, fg 2/5 68 56m + 12sm (56m+12sm) (34) (28m + 6sm) abbreviations: * first cytogenetic report; br = brazil, fg = french guiana; brazilian states: ap = amapá, ma = maranhão. 32 luísa antônia campos barros et al. sp. group chartifex from the present study into two categories, partially in accordance with imai et al. (1988), as with az. chartifex, the karyotypic formula is 14m + 14a. this seems to indicate differences in chromosome morphology between the two taxa, which corroborates the morphological data. data from molecular cytogenetic studies may contribute to corroborate these two taxa. colonies of dolichoderus bidens (linnaeus, 1758) were found in carton nests built on the abaxial surface of leaves of the family musaceae. the behavior of the workers was particularly aggressive. there are several records of d. bidens in french guiana (franco et al. 2019) and a single record in the neighboring brazilian state of amapá, in serra do navio, the center of the state (kempf 1959). to date, there has been no report of d. bidens inhabiting areas between these regions, which are approximately 400 km apart. dolichoderus bidens showed a karyotype of 2n = 18, 14m + 4sm (figure 1b) in our study. heterochromatic blocks around the centromeric/pericentromeric area of the chromosomes were identified (figure 2a). until now, the karyotype of d. bidens was only available for specimens collected in the atlantic rainforest of ilhéus, bahia (santos et al. 2016). our results for the specimens collected from the amazon rainforest showed similarities between these two rainforest populations, with subtle variations due to measurement divergences. in contrast, in a recent study, dolichoderus imitator emery, 1894 showed remarkable karyotypic differences between the population from the amazon rainforest (2n = 46) and that from the atlantic rainforest (2n = 38) (santos et al. 2016; aguiar et al. 2020). subfamily ectatomminae gnamptogenys tortuolosa, which is included in the neotropical sulcata group, presented 2n = 44, 12m + 17sm + 15st (figure 1c). as observed previously by imai (1991), using standard giemsa staining, all chromosomes showed heterochromatic blocks restricted to the pericentromeric region and the short arms of subtelocentric pairs (figure 2b). cytogenetic data for 14 taxa of gnamptogenys are available, including representatives of the mordax, striatula, and rastrata neotropical groups (reviewed by teixeira et al. 2020). this is the first chromosomal record for the sulcata group. the high chromosome number (2n >12, according to imai et al. 1994) and the high number of subtelocentric pairs with heterochromatin in the short arms suggest that centric fission rearrangements could have played an important role during the evolution of g. tortuolosa, as other spefigure 1. karyotypes of ant species from subfamilies: dolichoderinae (a) azteca sp. group chartifex (2n = 28), (b) dolichoderus bidens (2n = 18); ectatomminae (c) gnamptogenys tortuolosa (2n = 44); and formicinae (d) camponotus renggeri (n = 20). box in (c) show size heteromophism of the long arm of pair 22 in g. tortuolosa, with one homologous submetacentric and the other subtelocentric chromosome. scale bars = 5 µm. 33cytogenetic survey of eight ant species from the amazon rainforest cies of gnamptogenys do not typically have a large number of chromosomes (teixeira et al. 2020). mariano et al. (2015) have proposed that centric fissions are important in the evolution of this genus and, although there are scarce cytogenetic data concerning the sulcata group, these fissions appear to play an important role in this group. heteromorphism involving the long arm of chromosome pair 22 was observed in g. tortuolosa, which resulted in differences in the morphology of homologous chromosomes, with one chromosome being submetacentric and the other subtelocentric (figure 1c, box). the two chromosome variants are different in size and, therefore, processes that duplicate or delete chromatin could have been involved in the origin of this heteromorphism. subfamily formicinae the nest of camponotus renggeri emery, 1894 collected during the present study was found on fallen rotten wood. in oiapoque, north of the state of amapá, brazil, we also observed underground nests, as previously reported by ronque et al. (2016). it is important to note that it is rarer to find c. renggeri nests in rainforest areas than in savannah regions, including the amazonian savannahs (aguiar, barros personal observation). the colony of c. renggeri from the amazon rainforest showed n = 20, 2sm + 17st + 1a (figure 1d). colonies from other localities, such as the amazonian savannah located at macapá and the savannahs of cerrado in the states of mato grosso (aguiar et al. 2017) and goiás (vieira and santana 2020), also showed n = 20 chromosomes. the presence of a secondary constriction on the short arm of a subtelocentric chromosome of medium size (pair 5) suggests the presence of rdna clusters. two chromosome-rdna bearer pairs, a submetacentric pair and a subtelocentric pair of medium size, have previously been reported for this species (aguiar et al. 2017). this is in contrast to that observed in the sister species camponotus rufipes (fabricius, 1775) and camponotus (myrmothrix) spp., which show a single submetacentric rdna-bearer pair (aguiar et al. 2017). several chromosomal polymorphisms are associated with camponotus (myrmothrix) spp., but no variation was observed among the males analyzed in this study. subfamily myrmicinae wasmannia auropunctata roger (1863) presented 2n = 32, 16m + 10sm + 6st (figure 3a). its karyotype showed the same chromosome number and similar morphology to that of the atlantic rainforest population (souza et al. 2011). although souza et al. (2011) used a different chromosome classification method (imai 1991), without the use of chromosome measurements, the karytoype is similar to that obtained in this study, being possible to recognize all chromosome pairs. a chromosomal polymorphism was detected in ants from french guiana (aguiar et al. 2020, see figure 5b, since the karfigure 2. metaphases showing heterochromatic blocks (arrowheads) via 4% giemsa staining in (a) dolichoderus bidens (2n = 18) on centromeric and pericentromeric regions and (b) gnamptogenys tortuolosa (2n = 44) on pericentromeric regions of all chromosomes and short arms of subtelocentric pairs. scale bar = 5 µm. 34 luísa antônia campos barros et al. yotype is incorrectly written in table 1); however, ants collected at oiapoque, brazil (about 200 km away) did not show karyotype variations. the comparison between the karyotypes of specimens from these two localities provided insights into the polymorphism observed in french guiana. a submetacentric chromosome, which corresponds to the largest chromosome of the karyotype in ants from french guiana, is absent in specimens from oiapoque, so we can infer that this particular chromosome originated from a chromosomal rearrangement that need to be further investigated. the apterostigma sp. pilosum complex was characterized as 2n = 46, 6m + 18sm + 16st + 6a (figure 3b). the chromosome number among apterostigma ranges from 2n = 20 to 2n = 46 (mariano et al. 2019). the genus apterostigma contains six taxa that have been cytogenetically analyzed, but only half of the species have been taxonomically described. the apterostigma pilosum complex is composed of nine similar species and is considered to be taxonomically difficult to resolve (lattke 1997). some species were placed in synonymy of apterostigma mayri forel, 1893 by weber (1958). apterostigma mayri and apterostigma sp. pilosum complex showed distinct chromosome numbers of 2n = 24 and 2n = 46, respectively, although both are included within the pilosum complex (lattke 1997). the karyotypes with a lower chromosome number show more meta/submetacentric chromosomes when compared to species with higher chromosome numbers, including members of the apterostigma sp. pilosum complex. this suggests that centric fission rearrangements seem to be a part of the chromosomal evolution of the genus apterostigma. a taxon figure 3. karyotypes of ant species from subfamilies: myrmicinae (a) wasmannia auropunctata (2n = 32), (b) apterostigma sp. pilosum complex (2n = 46); ponerinae (c), (d) odontomachus bauri (2n = 44); and pseudomyrmecinae (f), (g) pseudomyrmex unicolor (2n = 68, n = 34). the boxes show polymorphism for subtelocentric chromosome pair 22 in o. bauri: (c) homozygous individual with small arms and (d) heterozygous individual with a distinctive large subtelocentric chromosome. scale bars = 5 µm 35cytogenetic survey of eight ant species from the amazon rainforest from french guiana showed a distinct and intermediate number of chromosomes (2n = 32) (mariano et al. 2011) compared to that in the apterostigma sp. described here. cytogenetic data highlight the need for revision of the pilosum complex and the genus apterostigma. subfamily ponerinae odontomachus bauri showed 2n = 44, 6sm + 24st + 14a (figure 3c-d). this species is included in the haematodus group, and all the studied species have the same chromosome number, 2n = 44 (reviewed in santos et al. 2010). however, variations in chromosomal morphology exist among species and provide insights into the mode of karyotypic evolution in this group (aguiar et al. 2020). differential heterochromatin growth after centric fission events may have played a role in the chromosomal evolution of the haematodus group according to imai et al. (1994). the o. bauri karyotype, according to the morphological variations due to heterochromatin growth on short arms, is derived within the haematodus group (see aguiar et al. 2020) and corroborates the molecular phylogenetic position (larabee et al. 2016). the long arm of the second subtelocentric pair of o. bauri collected from the amazon rainforest showed a size polymorphism that was observed in individuals of the same colony. homozygous individuals harbored two smaller subtelocentric chromosomes (figure 3c, box). only heterozygous individuals showed a distinctive large subtelocentric chromosome (figure 3d, box). no individuals with two large subtelocentric chromosomes were observed. this type of chromosome size polymorphism has been observed in several ant species (e.g., barros et al. 2013; teixeira et al. 2020) and can originate from unequal crossing-over or translocations that cause visible chromosomal deletions or duplications (schubert and lysak 2011; barros et al. 2013). subfamily pseudomyrmecinae pseudomyrmex unicolor has been reported from serra do navio in the state of amapá (kempf 1959); however, it was also reported by different researchers in french guiana (franco et al. 2019) highlighting the scarcity of myrmecological studies in the state of amapá. pseudomyrmex unicolor was characterized as having 2n = 68, 56m + 12sm and n = 34, 28m + 6sm (figure 3e, f); a similarly high chromosome number is present in pseudomyrmex gracilis (fabricius, 1804) (2n = 70) obtained from the atlantic rainforest. cytogenetic information is available for seven pseudomyrmex spp. ranging from 2n = 24 to 2n = 70 (sposito et al. 2006). despite having high chromosome numbers, only metacentric and submetacentric chromosomes were detected in p. unicolor. polyploidy does not appear to be an important factor in the chromosomal evolution of ants (lorite and palomeque 2010) and, thus far, there is no evidence indicating polyploidization among pseudomyrmex spp. (tsutsui et al. 2008; ardila-garcia et al. 2010). the presence of heterochromatin blocks on the short arms of chromosomes of p. unicolor suggests that the “heterochromatic growth” after centric fissions (imai et al. 1994) occurred during the chromosomal evolution of this species. although this process is not well understood (hirai et al. 1994), it may involve distinct mechanisms that enlarge the size of heterochromatic blocks on the chromosomes, such as slippage saltatory amplification, which contributes to an increase in the amount of dna; unequal crossing-over, which extends the heterochromatin among homologous regions; and also distribution by ectopic recombination among nonhomologous chromosomes (hirai 2020). the dispersion of rdna on terminal regions indicates the involvement of different mechanisms (hirai 2020). the increase in heterochromatin after chromosome fission has been previously suggested as a mechanism of chromosomal evolution in leaf-cutting ants of the genus acromyrmex (barros et al. 2016). final remarks as there are few ant cytogenetic studies at the population level, we conducted the karyotypic analysis of some ant species from the amazon rainforest and carried out a comparative analysis with the populations of the atlantic rainforest to detect karyotypic similarities and dissimilarities between them. despite its simplicity, classical cytogenetics can reveal chromosomal variations that may affect the ability of a species to generate fertile progeny. this study highlights the need for a taxonomic revision of the apterostigma pilosum complex and the azteca chartifex group. structural variations provide insights into the chromosomal evolution responsible for the polymorphisms detected in this study in w. auropunctata and o. bauri, as well as the heteromorphism in g. tortuolosa. geolocation information ant colonies were collected from the following locations in french guiana: la montagne des sing36 luísa antônia campos barros et al. es, kourou (5.07225, -52.69407), campus agronomique, kourou (5.17312, -52.65480), and sinnamar y (5.28482, -52.91403). colonies were collected in brazil at oiapoque, amapá (3.84151, -51.84112), and açailândia, maranhão (-4.84200, -47.29667) (table 1). acknowledgments we are grateful to sr. rona (in memoriam) for providing us access to the field collection and denilce meneses lopes for use of the equipment at the laboratório de citogenética de insetos at universidade federal de viçosa. pcf and rbl thank the conselho nacional de desenvolvimento científico e tecnológico (cnpq) for their scholarships. gat and lis acknowledge the scholarships provided by coordenação de aperfeiçoamento de pessoal de nível superior (capes). financial support for this study was provided by “investissement d’avenir” grants managed by the french agence nationale de la recherche (driihm ref. anr-11-labx-0010 and ceba ref. anr-10-labx-25-01), by the po-feder 2014-2020, région guyane (bing, ref. gy0007194), and by the programa de auxílio ao 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rosellini1, marilena ceccarelli2, emidio albertini1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(1): 127-133, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1149 caryologia international journal of cytology, cytosystematics and cytogenetics citation: f. dotti do prado, a. abrigato de freitas mourão, f. foresti, j. augusto senhorini, f. porto-foresti (2021) first cytogenetic characterization of the amazon catfish leiarius marmoratus (gill, 1870) and its hybrid with pseudoplatystoma reticulatum (eigenmann & eigenmann, 1889). caryologia 74(1): 127-133. doi: 10.36253/caryologia-1149 received: december 03, 2020 accepted: april 26, 2021 published: july 20, 2021 copyright: © 2021 f. dotti do prado, a. abrigato de freitas mourão, f. foresti, j. augusto senhorini, f. porto-foresti. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid fdp: 0000-0002-4189-0375 ff: 0000-0002-0862-0445 fpf: 0000-0001-8845-3845 first cytogenetic characterization of the amazon catfish leiarius marmoratus (gill, 1870) and its hybrid with pseudoplatystoma reticulatum (eigenmann & eigenmann, 1889) fernanda dotti do prado1, andrea abrigato de freitas mourão2, fausto foresti3, josé augusto senhorini4, fábio porto-foresti1,* 1 faculdade de ciências, universidade estadual paulista júlio de mesquita filho, av. eng. luiz edmundo carrijo coube, 17033-360, bauru, são paulo, brazil 2 universidade paulista – unip, r. luís levorato, 140, 17048-290, bauru, são paulo, brazil 3 instituto de biociências, universidade estadual paulista júlio de mesquita filho, distrito de rubião júnior, 18618-970, botucatu, são paulo, brazil 4 centro nacional de pesquisa e conservação da biodiversidade aquática continental – cepta, rodovia sp-2, 01 (pref. euberto nemésio pereira de godoy), 13.630-970, pirassununga, são paulo, brazil *corresponding author. email: fp.foresti@unesp.br abstract. this study reports the first cytogenetic characterization of the amazonian catfish leiarius marmoratus (“jandiá”) and its f1 (first generation) hybrid “cachandiá” with pseudoplatystoma reticulatum (“cachara”). a diploid number of 56 chromosomes and a single argyrophilic nucleolus organizer region (ag-nor) in the short arm of two sub-telocentric chromosomes were observed for both l. marmoratus and p. reticulatum, but with differences in the karyotype formula and the size of the chromosome pair with nors. the hybrid showed 2n = 56 chromosomes with an intermediate karyotype when compared to the parental species. a single ag-nor was maintained in the hybrid but located in two chromosomes with marked differences in size and presenting intraindividual variation in nor activity (nucleolar dominance). for l. marmoratus and the hybrid, heterochromatic bands were predominately distributed in the terminal, centromeric, and sub-centromeric regions of some chromosomes and 5s rdna sites located in two distinct sub-telocentric chromosomes, similar to the previously described for p. reticulatum. the data suggested that the hybrid karyotype might be insufficient for a precise discrimination of hybrids, however, ag-nor can be used as a chromosome marker to differentiate “cachandiá” from l. marmoratus and p. reticulatum. the current study also provides insights into the chromosomal features of l. marmoratus and contributes with novel cytogenetic information of this native amazonian catfish included in the pimelodidae family. keywords: pimelodidae, hybrid karyotype, cachandiá, pintado da amazônia, yaque, ag-nor. 128 fernanda dotti do prado et al. introduction the long-whiskered catfish leiarius marmoratus belongs to the pimelodidae family, (teleostei: siluriformes) (lundberg and littmann 2003) and is an endemic species that naturally occurs along the amazon and orinoco river basins. this fish is commonly known as “ jandiá”, “ jundiá amazônico”, “peixe-onça” in brazil (porto-foresti et al. 2013), and “yaque” in other andine countries (mateo et al. 2008). widely used in aquariums and local fisheries, l. marmoratus is also cultivated in brazilian aquaculture to produce interspecific hybrids with the “cachara” catfish (campos 2010, hashimoto et al. 2012; hashimoto et al. 2016). the “cachara” correspond to other native south american pimelodidae fish classified as pseudoplatystoma fasciatum (sensu latu) in the amazon area or p. reticulatum (sensu strictu) in southern regions of south america as the paraguay and parana river basins (buitrago-suarez and burr 2007). the hybrids between l. marmoratus and p. reticulatum are usually named as “cachandiá”, “cachadia” or “jundiara” (kubitza et al. 2011, porto-foresti et al. 2013) and are commercialized in the southern regions of brazil as “pintado da amazônia”, “pintado amazônico” or simply “pintado” (kubitza et al. 2011). morphological data indicated that, spite with intermediate characteristics, these hybrids can externally resemble more to p. reticulatum (coelho et al. 2021). although the hybridization practice can provide economic advantages during the production as low cannibalism and fast growth rates, accidental escapes or intentional releases of hybrids in the wild environment represents a serious problem, since they can present partial or total fertility and cause genetic introgression with native populations (yabu et al. 2018). despite the large biodiversity of fish found in the tropics, information is still lacking for several species and there is no cytogenetic data for any species of leiarius including l. marmoratus. in this study, we performed the first cytogenetic characterization of l. marmoratus and its hybrid “cachandiá” with p. reticulatum, and thereby provide new biological information of this important group of fishes. material and methods seven juveniles of l. marmoratus previously breeded in captivity in cepta (centro nacional de pesquisa e conservação de peixes continentais, pirassununga, sp, brazil) and eight juveniles of the“cachandiá”hybrid (♀ p. reticulatum × ♂ l. marmoratus) were cytogenetically analyzed in this study. hybrids were artificially produced through hormonal induction of parental species with carp pituitary extract. mitosis was stimulated as described by oliveira et al. (1988), fishes were anesthetized with benzocaine and then euthanized and deposited in the fish collection at laboratório de genética de peixes unesp (universidade estadual paulista júlio de mesquita filho) (bauru, sp, brazil). chromosome preparation and cytogenetic analysis were performed based on kidney cell suspensions basically according to foresti et al. (1993). all fishes were previously identified with nuclear and mitochondrial species-specific molecular markers (porto-foresti et al. 2013) confirming them as pure l. marmoratus and the hybrid “cachandiá”. chromosomal preparations of p. reticulatum were obtained from prado et al. (2012) and new metaphases were used for the study of the karyotype formulae and argyrophilic nucleolus organizer regions (ag-nors) silver staining of the nor was obtained following the technique of howell and black (1980). c-banding technique was applied according to sumner (1972). fluorescent in situ hybridization (fish) was performed using 5s rdna probes based on genomic dna of another pimelodidae species, pseudoplatystoma corruscans. the probe was obtained by pcr using the primers 5sa (5´-tcaaccaaccacaaagacattggcac-́ 3) and 5sb (5´-tagacttctgggtggccaaaggaatca-́ 3) (pendás et al. 1994). the pcr was performed in a total volume of 25 µl and contained 150 µm of dttp, dgtp, and dctp; 100 µm of datp; 1.5 mm mgcl2; 1x taq buffer (20 mm tris-hcl, ph 8.4 and 50 mm kcl); 0.5 unit (u) of taq polymerase (invitrogen); 0.2 µm of each primer; and 10–50 ng of genomic dna. metaphases were hybridized as described by pinkel et al. (1986). the probe was digoxigenin-11-dutp labelled and hybridization signals were developed using antidigoxigenin-rhodamine. cells in metaphase were posteriorly stained with 4’,6-diamidino-2-phenylindole (dapi). karyotype images were captured digitally with a fluorescence microscope (olympus bx50) and processed for contrast and luminosity using adobe photoshop cs5 software. chromosomal morphology was determined based on arm ratio, according to levan et al. (1964), chromosomes were classified as metacentric (m), sub-metacentric (sm), sub-telocentric (st) and acrocentric (a), and arranged in decreasing size order for the karyotype organization. for the hybrid, chromosomes were not organized by pairs, but named with individual numbers (from 1 to 56) according to the morphology and also arranged in decreasing size order. 129first cytogenetic characterization of the amazon catfish and its hybrid results l. marmoratus showed a diploid number of 56 chromosomes organized as 20 m + 12 sm + 10 st + 14 a (fundamental number = 98) (fig. 1a). ag-nors were located in the terminal region of the short arm of the sub-telocentric chromosome pair number 20 (fig. 1a). p. reticulatum presented 2n= 56 chromosomes distributed in a karyotype of 20 m + 12 sm + 12 st + 12 a (fundamental number = 112) (fig. 1c) and a single ag-nor stained in the short arm of the sub-telocentric pair number 18 (fig. 1c). for both species, the ag-nor region was heteromorphic (fig. 1a, 1c) and corresponding with a conspicuous secondary constriction when stained with giemsa (fig. 1a, 1c). the hybrid presented a diploid number of 56 chromosomes, organized in a karyotype formula intermediate to the parental species, with 20 m + 12 sm + 11 st + 13 a (nf = 99) (fig. 1b). two non-homologous subtelocentric chromosomes (39 and 42) of different sizes possessed ag-nor signals in the terminal region of the short arm (fig. 1b). nucleolar dominance was verified for all hybrid individuals (table 1), counting a total of 154 metaphases presenting one active nor (fig. 2b) in contrast with 39 metaphases presenting ag-nor signals in two chromosomes (fig. 2a). results of nucleus analysis (table 1) also showed a majority of cells presenting only one active ag-nor (115) (fig. 3b) versus 37 nuclei figure 1. karyotype of leiarius marmoratus (a), the hybrid “cachandiá” (b) and pseudoplatystoma reticulatum (c) after giemsa staining. in the box, the nor-bearing chromosomes. figure 2. metaphases of the hybrid “cachandiá” after ag-nor staining. in (a), a metaphase presenting two chromosomes of different sizes with ag-nors and (b) a metaphase with nucleolar dominance and only one ag-nor. arrows indicates the norbearing chromosomes. figure 3. nucleous of the hybrid “cachandiá” after ag-nor staining. in (a), nucleous presenting two ag-nors and (b) nucleous with only one ag-nor. 130 fernanda dotti do prado et al. with two marks (fig. 3a). nucleolar dominance varied intraindividually, i.e., each individual presented both metaphases or nucleous with one or two active nors. heterochromatic bands of l. marmoratus were located in the pericentromeric and terminal areas of some chromosomes and the ag-nor sites (fig. 4a). for this species, 5s rdna sites were located at the pericentromeric region of the short arm of two sub-telocentric chromosomes, distinct from the ag-nor chromosome pairs that were identified by a secondary constriction (fig. 5a). for the hybrid, c-bands marked the terminal and pericentromeric areas of some chromosomes as well as the nor sites (fig. 4b) and 5s rdna hybridization signals were located in the terminal regions of two sub-telocentric chromosomes and were distinct from the nor pair (fig. 5b). discussion conventional cytogenetics remains a powerful tool to characterize ichthyofauna biodiversity and to elucidate features of populations and species at the chromosomal level (cioffi et al. 2018). the neotropical region presents one of the most diverse ichthyofauna in the world (reis et al. 2016), and the amazonian basin in special, harbors a rich variety of endemic fishes. in this region, pimelodidae catfishes are very diverse, with species presenting the most diverse variations on body size, colours and ecological roles in the aquatic environment (lundberg and littmann, 2003). despite that, a great amount of fish species has never been biologically or genetically studied. recent findings showed efforts to cytogenetically characterize pimelodidae species in the amazonian region, providing important data for this group of fishes, as the described for pimelodus (fonseca et al 2018) and the giant catfishes phractocephalus hemioliopterus (swarça et al. 2017) and brachyplatystoma filamentosum (gonçalvez et al. 2014). the present study describes the first cytogenetic description of l. marmoratus and contributes to characterize the rich biodiversity of amazonian fishes. l. marmoratus shared cytogenetic characteristics commonly observed in pimelodidae fishes as a diploid number of 56 chromosomes, a global pattern of heterochromatic bands distributed in terminal, peri and centromeric areas of the chromosomes, single ag-nor and 5s rdna sites (swarça et al. 2007, nirchio et al. 2013; swarça et al. 2017, girardi et al. 2018). p. reticulatum presented the same chromosomal characteristics than previously described by prado et al. (2012) and similar to other pseudoplatystoma species as p. corruscans (prado et al. 2012), p.metaense and p. orinocoense (nirchio et al. 2013). the same conserved pattern of 2n=56 chromosomes, single ag-nor and 5s rdna sites was verified, supporting the close relationships within this group of fishes. pimelodidae family is characterized by a majority of species with conservative karyotypes which can be explained by the hypothesis that more dispersive and migratory species usually presents more stable karyotypes (bertollo et al. 2017). this information corroborates the observed in this study for l. marmoratus and p. reticulatum, two large size catfishes presenting long distance reproductive migratory habits. table 1. number of metaphases and nucleus presenting one or two ag-nors for the hybrid “cachandiá”. ag-nors number metaphases 1 2 154 39 nucleus 1 2 115 37 figure 4. metaphases of leiarius marmoratus (a) and the hybrid “cachandiá” (b) after c-banding. arrows indicate the putative norbearing chromosomes. figure 5 metaphases of leiarius marmoratus (a) and the hybrid “cachandiá” (b) after hybridization in situ with 5s rdna. arrows indicates the 5s rdna sites (red). 131first cytogenetic characterization of the amazon catfish and its hybrid despite the conserved chromosomal characteristics, l. marmoratus and p. reticulatum showed variation in the karyotypic formula, with differences in the number of sub-telocentric and acrocentric chromosomes and the nor-bearing chromosomes with a remarkable difference in size between the species. variability in the karyotype formula without changes on the diploid number is a common feature in the pimelodidae family, also verified for other pseudoplatystoma species (porto-foresti et al. 2000; nirchio et al. 2003) and among the pimelodidae family (swarça et al 2000), which can be explained by structural chromosomal rearrangements as pericentric inversions during their evolution and speciation events (swarça et al. 2000; 2000) a polymorphism of ag-nor marks between the homologous chromosomes were detected for l. marmoratus and p. reticulatum in this study (fig. 1a, 1c boxes), which is a relatively common feature observed for several groups of fishes including characiformes (vicari et al. 2006), cypriniformes (supiwong et al. 2012), siluriformes (swarça et al. 2005; prado et al. 2012) and others fishes (kasiroek et al. 2017). differences in nor size have been attributed to structural events such as chromosomal breaks, duplications of the ribosomal dna clusters or differences in nor activity. association of nors with secondary constrictions in the same chromosome region is also a common feature in fishes (foresti et al. 1981, feldberg and bertollo 2014), also detected in this work for l. marmoratus and p. reticulatum. data for other pimelodidae species also related nor polymorphisms as verified for p. metaense and p. orinocoense (nirchio et al. 2013) with the nor-bearing chromosome heteromorphic in size and correspondent with ag-positive signals on the short arms of the chromosomes. the “cachandiá” hybrid presented the same diploid number, similar 5s rdna bands and similar patterns of heterochromatin than the verified for l. marmoratus and p. reticulatum. this chromosomal pattern followed the previously observed for the parental species, which were apparently maintained in the hybrid. however, different karyotype formulae and chromosomes with ag-nor were observed for the hybrid. hybrid chromosomes were organized in a karyotype intermediate to the parental species, formed by non-homologous chromosomes. the lack of homology could be clearly visualized by the chromosome number 11 (sub-telocentric) and the chromosome number 13 (acrocentric) (fig. 1b), without their respective homologous pair, and the presence of ag-nors in two non-homologous chromosomes with a marked difference in size (39 and 42). cytogenetic is an important tool to discriminate hybrids from theirs parental species (hashimoto et al. 2009) with applications for aquaculture and conservation. chromosome morpholog y visualized by the karyoty pe, ag-nors, hibridization of rdna genes or c-bands can be used as chromosome markers to identify species and hybrids and to elucidate chromosomal heritage in hybrids (hashimoto et al. 2009). in this study, the intermediate karyotype of the hybrid “cachandiá” is probable insufficient to establish a precise chromosoma l diagnosis since the dif ferences between the chromosome types were very subtle and might vary in classifications according to chromosomal condensation. conventional cytogenetic techniques as chromosomal morphology were also not sufficient to differentiate hybrids of p. reticulatum and p. corruscans (prado et al. 2012). otherwise, ag-nors were very specific for the hybrid, located in two non-homologous chromosomes different in size, allowing an accurate diagnosis of the hybrid. the presence of nors in chromosomes with distinct morphology have been previously detected for hybrids of pimelodus (hashimoto et al. 2009) and a similar situation was observed for species of cobitiis and their hybrids (grabowska et al. 2019). a considerable number of metaphases or nucleolus with only one active nor indicated dominant rdna expression of one parental species in the hybrid, fact already described for other hybrid of fishes (hashimoto et al., 2012; prado et al., 2012). data obtained in this study may be valuable for hybrid identification in brazilian aquaculture and suggested that ag-nor is a marker to identify the “cachandiá” hybrid by a simple and low-cost cytogenetic technique. chromosomal data also contributes with novel information for the amazonian catfish l. marmoratus to be future included in evolutionary and cytogenetic studies of pimelodidae fishes. acknowledgments this work was supported by the fundação de amparo à pesquisa do estado de são paulo (fapesp) and conselho nacional de desenvolvimento científico e tecnológico (cnpq). 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(caprifoliaceae), using scot molecular markers. caryologia 74(4): 11-20. doi: 10.36253/caryologia-1320 received: may 21, 2021 accepted: august 24, 2021 published: march 08, 2022 copyright: © 2021 fengzhen chen, dongmei li, mohsen farshadfar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. genetic variations and interspesific relationships in lonicera l. (caprifoliaceae), using scot molecular markers fengzhen chen1, dongmei li2,*, mohsen farshadfar3 1 peony research institute, heze university, heze , shandong 274000, china 2 college of horticulture science and engineering, shandong agricultural university, taian, shandong 271018, china 3 department of agriculture, payame noor university (pnu), tehran, iran *corresponding author: e-mail:majidkhayatnezhad126@gmail.com; spfood3200@163. com abstract. lonicera l. (caprifoliaceae) includes more than 200 species worldwide. the genus is mainly distributed in temperate to subtropical regions of the northern hemisphere: europe, russia, east asia and north america. some species are medicinal plants. dried lonicera flowers and buds are known as flos lonicera and have been a recognized herb in the traditional chinese medicine for more than 1500 years. it has been applied for treatment of arthritis, diabetes mellitus, fever, and viral infections. due to the importance of these plant species, we performed a combination of morphological and molecular data for this species. for this study, we used 85 randomly collected plants from six species in 6 provinces. amplification of genomic dna using 10 primers produced 103 bands, of which 95 were polymorphic (90.98%). the obtained high average pic and mi values revealed high capacity of scot primers to detect polymorphic loci among lonicera species. the genetic similarities of 6 collections were estimated from 0.67 to 0.90. according to the scot markers analysis, l. hypoleuca and l. iberica had the lowest similarity and the species of l. korolkowii and l. nummulariifolia had the highest similarity. the aims of present study are: 1) can scot markers identify lonicera species, 2) what is the genetic structure of these taxa in iran, and 3) to investigate the species inter-relationship? the present study revealed that scot markers can identify the species. keywords: gene flow, genetic admixture, lonicera, network, population structure. introduction genetic diversity is a basic component of biodiversity and its conservation is essential for long term survival of any species in changing environments (mills and schwartz 2005, tomasello et al. 2015). this is very important in fragmented populations because are more vulnerable due to the loss of allelic richness and increased population differentiation by genetic drift (decreases heterozygosity and eventual fixation of alleles) and inbreeding 12 fengzhen chen et al. depression (increases homozygosity within populations; frankham 2005). among different populations, genetic diversity is non randomly distributed and is affected by various factors such as geographic variations, breeding systems, dispersal mechanisms, life span, etc (khatamsaz 1995; ghahremaninejad and ezazi 2009).change in environmental conditions often leads to variation in genetic diversity levels among different populations and populations with low variability are generally considered less adapted under adverse circumstances (falk and holsinger 1991, olivieri et al. 2016). most of the authors agree that genetic diversity is necessary to preserve the long-term evolutionary potential of a species (falk and holsinger 1991). in the last decade, experimental and field investigations have demonstrated that habitat fragmentation and population decline reduce the effective population size. in the same way, most geneticists consider population size as an important factor for maintaining genetic variation (turchetto et al. 2016). lonicera l. (caprifoliaceae) includes more than 200 species (mabberley 2008) worldwide, with 19 species in the region of flora iranica (wendelbo 1965). the genus is mainly distributed in temperate to subtropical regions of the northern hemisphere: europe, russia, east asia, and north america (hsu and wang 1988; mabberley 2008). in the flora of iran, the genus lonicera is represented by nine species (khatamsaz 1995; ghahremaninejad and ezazi 2009) across the north, northwest and northeast of the country. some species are medicinal plants (zeng et al. 2017). dried lonicera flowers and buds are known as flos lonicera and have been a recognized herb in the traditional chinese medicine for more than 1500 years (li et al. 2015). it has been applied for treatment of arthritis, diabetes mellitus, fever, and viral infections (shang et al. 2011; li et al. 2015). the plants are erect shrubs, occasionally small trees. members of lonicera are characterized by opposite, narrowly elliptic to obovate leaves, white, yellow, reddish, or purplered corolla with capitate stigma (judd et al. 2007), and undulate calyx margin. in flora iranica, wendelbo (1965) classified 19 species of the lonicera into two subgenera (chamaecerasus and lonicera) and three sections, namely isoxylosteum, isika and coeloxylosteum. the four studied species belong to subgenus chamaecerasus and sections isika and coeloxylosteum. molecular data have been obtained in phylogenetic studies and species divergence researches (kazempour osaloo et al. 2003, 2005). these data can also provide supportive and extra criteria for systematic classification of the studied species that have been based only on the morphological characters (chase et al. 1993). the internal transcribed spacer (its) is the region of the 18s-5.8 s-26s nuclear ribosomal cistron (baldwin et al. 1995). the spacers contain the signals needed to process the rrna transcript (baldwin 1992, baldwin et al. 1995) and have often been used for inferring phylogeny at the generic and infrageneric levels in plants (e.g. baldwin 1992; baldwin et al. 1995; kazempour osaloo et al. 2003, 2005). theis et al. (2008) studied phylogenetics of the caprifolieae and lonicera (dipsacales) on the basis of nuclear and chloroplast dna sequences. their analysis indicates monophyly in lonicera and highlights instances of homoplasy in several morphological characters. molecular phylogenetics of lonicera in japan has been studied by nakaji et al. (2015) on the basis of chloroplast dna sequences. according to the results, circumscription of the higher taxonomic groups for the japanese species of lonicera proposed by hara in 1983 is fundamentally acceptable. lonicera is well known for its taxonomic complexity resulting from overlapping morphological characters. with the progress in plant molecular biolog y, numerous molecular marker techniques have been developed and used widely in evaluating genetic diversity, population structure and phylogenetic relationships. in recent years, advances in genomic tools provide a wide range of new marker techniques such as, functional and gene targeted markers as well as develop many novel dna based marker systems (wu et al. 2013). start codon targeted (scot) polymorphism is one of the novel, simple and reliable gene-targeted marker systems. this molecular marker offers a simple dna-based marker alternative and reproducible technique which is based on the short conserved region in the plant genes surrounding the atg (collard and mackill 2009) translation start codon (collard and mackill 2009). this technique involves a polymerase chain reaction (pcr) based dna marker with many advantages such as low-cost, high polymorphism and extensive genetic information (collard and mackill 2009, luo et al. 2011, wu et al. 2013). the present investigation has been carried out to evaluate the genetic diversity and relationships among lonicera species using new gene-targeted molecular markers, i.e. scot. this is the first study on the use of scot markers in lonicera genus; therefore, we performed molecular study of 85 specimens of 6 lonicera species. we try to answer the following questions: 1) is there infra and interspecific genetic diversity among studied species? 2) is genetic distance among these species correlated with their geographical distance? 3) what is the genetic structure of populations and taxa? 4) is there any gene exchange between lonicera species in iran? 13genetic variations and interspesific relationships in lonicera l. (caprifoliaceae), using scot molecular markers materials and methods plant materials a total of 85 individuals were sampled representing six geographical populations belong six lonicera species (sp1= lonicera caucasica; sp2= lonicera iberica m. bieb.; sp3= lonicera nummulariifolia jaub. et spach; sp4= lonicera bracteolaris boiss. & buhse; sp5= lonicera korolkowii stapf; sp 6= lonicera hypoleuca decne.) in east azerbaijan, guilan, mazandaran, tehran, khorasan and hormozgan provinces of iran during july-agust 2017-2019. for morphometric and scot analysis we used 85 plant accessions (nine to eighteen samples from each populations) belonging to six different species with different eco-geographic characteristics were sampled and stored in -20 till further use. voucher specimens are deposited in herbarium of azad islamic university (haiu). more information about geographical distribution of accessions are in table. 1. morphological studies nine to eighteen samples samples from each species were used for morphometry. in total 17 morphological (9 qualitative, 8 quantitative) characters were studied. data obtained were standardized (mean= 0, variance = 1) and used to estimate euclidean distance for clustering and ordination analyses (podani 2000). morphological characters studied are: corolla shape, bract shape, seed color, seed shape, bract color, leaf surface, calyx shape, basal leaf shape, pedicel length, calyx length, bract length, corolla length, basal leaf length, basal leaf width, corolla color, stem leaf length and stem leaf width. dna extraction and scot assay fresh leaves were used randomly from nine to eighteen plants in each of the studied populations. these were dried by silica gel powder. ctab activated charcoal protocol was used to extract genomic dna (doyle and doyle 1987). the quality of extracted dna was examined by running on 0.8% agarose gel. a total of 25 scot primers developed by collard and mackill (2009), 10 primers with clear, enlarged, and rich polymorphism bands were chosen (table 2). pcr reactions were carried in a 25μl volume containing 10 mm tris-hcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). the amplifications, reactions were performed in techne thermocycler (germany) with the following program: 5 min initial denaturation step 94°c, followed by 40 cycles of 1 min at 94°c; 1 min at 52-57°c and 2 min at 72°c. the reaction was completed by final extension step of 7-10 min at 72°c. the amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated by using a 100 bp molecular size ladder (fermentas, germany). data analyses morphological studies morphological characters were f irst standardized (mean = 0, variance = 1) and used to establish euclidean distance among pairs of taxa (podani 2000). for grouping of the plant specimens, the upgma (unweighted paired group using average) ordination methods were used (podani 2000). anova (analysis of variance) were performed to show morphological difference among the populations while, pca (principal components analysis) biplot was used to identify the most variable morphological characters among the studied populations (podani 2000). past version 2.17 (hammer et al. 2012) was used for multivariate statistical analyses of morphological data. table 1. voucher details of lonicera species and relative genera examined in this study from iran. sp. locality sample size latitude longitude altitude (m) voucher no. l. caucasica mazandaran, chalus 18 34°52’393” 46°25’92” 1133 hiau 201677 l. iberica m. bieb. east azerbaijan, kaleybar, road side 16 38°52’373” 47°23’92” 1144 hiau 201683 l. nummulariifolia jaub. et spach tehran, alamut 14 33°52’353” 48°27’92” 1330 hiau 201686 l. bracteolaris boiss. & buhse guilan, gole rodbar, road sid 9 34°09’55” 47°55’49” 1600 hiau 201689 l. korolkowii stapf khorasan, bojnurd 15 320702.32 504432.6 2300 hiau 201690 l. hypoleuca decne. hormozgan, bandar abbas, siyahu 13 38°52’373” 47°23’92” 1144 hiau 201695 14 fengzhen chen et al. molecular analyses scot bands obtained were coded as binary characters (presence = 1, absence = 0) and used for genetic diversity analysis. discriminatory ability of the used primers was evaluated by means of two parameters, polymorphism information content (pic) and marker index (mi) to characterize the capacity of each primer to detect polymorphic loci among the genotypes (powell et al. 1996). mi is calculated for each primer as mi = pic × emr, where emr is the product of the number of polymorphic loci per primer (n) and the fraction of polymorphic fragments (β) (heikrujam et al. 2015). the number of polymorphic bands (npb) and the effective multiplex ratio (emr) were calculated for each primer. parameter like nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism (p% = number of polymorphic loci/number of total loci) were determined (weising et al, 2005, freeland et al. 2011). shannon’s index was calculated by the formula: h’ = -σpiln pi. rp is defined per primer as: rp = ∑ ib, were “ib” is the band informativeness, that takes the values of 1-(2x [0.5p]), being “p” the proportion of each genotype containing the band. the percentage of polymorphic loci, the mean loci by accession and by population, uhe, h’ and pca were calculated by genalex 6.4 software (peakall & smouse 2006). nei’s genetic distance among populations was used for neighbor joining (nj) clustering and neighbor-net networking (huson & bryant 2006, freeland et al. 2011). mantel test checked the correlation between geographical and genetic distances of the studied populations (podani 2000). these analyses were done by past ver. 2.17 (hammer et al. 2012), darwin ver. 5 (2012) software. amova (analysis of molecular variance) test (with 1000 permutations) as implemented in genalex 6.4 (peakall and smouse, 2006), and nei,s gst analysis as implemented in genodive ver.2 (2013) (meirmans and van tienderen 2004) were used to show genetic difference of the populations. moreover, populations, genetic differentiation was studied by g’st est = standardized measure of genetic differentiation (hedrick 2005), and d_est = jost measure of differentiation (jost 2008). to assess the population structure of the lonicera accessions, a heuristic method based on bayesian clustering algorithms were utilized. the clustering method based on the bayesian-model implemented in the software program structure (pritchard et al. 2000; falush et al. 2007) was used on the same data set to better detect population substructures. this clustering method is based on an algorithm that assigns genotypes to homogeneous groups, given a number of clusters (k) and assuming hardy-weinberg and linkage equilibrium within clusters, the software estimates allele frequencies in each cluster and population memberships for every individual (pritchard et al. 2000). the number of potential subpopulations varied from two to ten, and their contribution to the genotypes of the accessions was calculated based on 50,000 iteration burn-ins and 100,000 iteration sampling periods. the most probable number (k) of subpopulations was identified following evanno et al. (2005). in k-means clustering, two summary statistics, pseudo-f, and bayesian information criterion (bic), provide the best fit for k (meirmans, 2012). table 2. scot primers used for this study and the extent of polymorphism. primer name primer sequence (5’-3’) tnb npb ppb pic pi emr mi scot-1 caacaatggctaccacca 15 14 93.74% 0.47 5.66 17.56 5.67 scot-3 caacaatggctaccaccg 13 12 92.31% 0.54 3.21 15.60 5.55 scot-6 caacaatggctaccacgc 7 7 100.00% 0.47 4.32 9.55 3.45 scot-11 aagcaatggctaccacca 11 9 82.89% 0.43 5.56 6.34 5.11 scot-14 acgacatggcgaccacgc 10 10 100.00% 0.56 4.86 9.55 3.22 scot-15 acgacatggcgaccgcga 9 8 84.99% 0.41 4.91 7.43 4.85 scot-16 ccatggctaccaccggcc 8 8 100.00% 0.44 4.34 11.55 6.44 scot-17 catggctaccaccggccc 16 16 100.00% 0.67 5.88 8.56 3.65 scot-18 accatggctaccaccgcg 13 13 100.00% 0.55 6.23 8.23 6.47 scot-19 gcaacaatggctaccacc 10 10 100.00% 0.59 6.25 9.7 5.87 mean 10 9 90.98% 0.56 5 9.5 5.9 total 103 95 abbreviations: tnb = the number of total bands, npb = the number of polymorphic bands, ppb (%) = the percentage of polymorphic bands, pi = polymorphism index, emr = effective multiplex ratio, mi = marker index, pic, polymorphism information content for each of cbdp primers. 15genetic variations and interspesific relationships in lonicera l. (caprifoliaceae), using scot molecular markers gene flow (nm) which were calculated using popgene (version 1.31) program (yeh et al., 1999). gene flow was estimated indirectly using the formula: nm = 0.25(1 fst)/fst. in order to test for a correlation between pairwise genetic distances (fst) and geographical distances (in km) between populations, a mantel test was performed using tools for population genetic analysis (tfpga; miller, 1997) (computing 999 permutations). this approach considers equal amount of gene flow among all populations. (ii) population assignment test based on maximum likelihood as performed in genodive ver. 2. (2013). the presence of shared alleles was determined by drawing the reticulogram network based on the least square method by darwin ver 5. (2012). results species identification and inter-relationship. morphometry anova showed significant differences (p <0.01) in quantitative morphological characters among the species studied. in order to determine the most variable characters among the taxa studied, pca analysis has been performed. it revealed that the first three factors comprised over 65% of the total variation. in the first pca axis with 47% of total variation, such characters as seed shape, calyx shape, calyx length, bract length and basal leaf shape have shown the highest correlation (>0.7), seed color, leaf surface, corolla length and basal leaf length, were characters influencing pca axis 2 and 3 respectively. different clustering and ordination methods produced similar results therefore, pca plot of morphological characters are presented here (fig. 1). in general, plant samples of each species were grouped together and formed separate groups. this result show that both quantitative and qualitative morphological characters separated the studied species into distinct groups. in the studied specimens we did not encounter intermediate forms. species identification and genetic diversity ten scot primers were screened to study genetic relationships among lonicera species; all the primers produced reproducible polymorphic bands in all 6 lonicera species. an image of the scot amplification generated by scot-14 and scot-6 primer is shown in figure 2. a total of 95 amplified polymorphic bands were generated across 6 lonicera species. the size of the amplified fragments ranged from 100 to 2000 bp. the highest and lowest number of polymorphic bands were 16 for scot-17 and 7 for scot-6, on an average of 9 polymorphic bands per primer. the pic of the 10 scot primers ranged from 0.41 (scot-15) to 0.67 (scot-17) with an average of 0.56 per primer. mi of the primers ranged from 3.22 (scot-14) to 6.47 (scot-18) with an average of 5.9 per primer. emr of the scot primers ranged from figure 1. pca plots of morphological characters revealing species delimitation in the lonicera species. 16 fengzhen chen et al. 6.34 (scot-11) to 17.56 (scot-1) with an average of 9.5 per primer (table 2). the primers with the high emr values were considered to be more informative in distinguishing the genotypes. the genetic parameters were calculated for all the 6 lonicera species amplified with scot primers (table 3). unbiased expected heterozygosity (h) ranged from 0.13 (l. caucasica) to 0.33 (l. hypoleuca), with a mean of 0.21. a similar pattern was observed for shannon’s information index (i), with the highest value of 0.34 observed in l. hypoleuca and the lowest value of 0.18 observed in l. caucasica with a mean of 0.28. the observed number of alleles (na) ranged from 0.201 in l. bracteolaris to 0.892 in l. caucasica. the effective number of alleles (ne) ranged from 1.00 (l. bracteolaris) to 1.138 (l. caucasica). amova test showed significant genetic difference (p = 0.01) among studied species. it revealed that 53% of total variation was among species and 47% was within species (table 4) moreover, genetic differentiation of these species was demonstrated by significant nei’s gst (0.66, p = 0.01) and d_est values (0.222, p = 0.01). these results revealed a higher distribution of genetic diversity among lonicera species compared to within species. two major clusters were formed in upgma tree (fig. 3). the first major cluster (a) contained two sub-clusters: l. nummulariifolia and l. korolkowii are separated from the other studied species and join the others with a great distance and comprised the first sub-cluster. the second sub-cluster was formed by l. caucasica; l. iberica and l. bracteolaris. the second major cluster also contained only 1 species of l. hypoleuca. in general, relationships obtained from scot data agrees well with species relationship obtained from morphological. this is in agreefigure 2. electrophoresis gel of studied ecotypes from dna fragments produced by scot-14, scot-6; sp1= l. caucasica; sp2= l. iberica m. bieb.; sp3= l. nummulariifolia jaub. et spach; sp4= l. bracteolaris boiss. & buhse; sp5= l. korolkowii stapf; sp 6= l. hypoleuca decne; l = ladder 100 bp, arrows are representative of polymorphic bands table 3. genetic diversity parameters in the studied lonicera species. sp n na ne i he uhe %p l. caucasica 18.000 0.892 1.138 0.18 0.141 0.13 28.63% l. iberica 16.000 0.244 1.032 0.26 0.23 0.18 55.53% l. nummulariifolia 14.000 0.314 1.044 0.26 0.18 0.23 39.38% l. bracteolaris 9.000 0.201 1.00 0.33 0.17 0.18 52.23% l. korolkowii 15.000 0.341 1.058 0.24 0.27 0.20 33.75% l. hypoleuca 13.000 0.567 1.062 0.34 0.324 0.333 64.73% abbreviations: n = number of samples, na= number of different alleles; ne = number of effective alleles, i= shannon’s information index, he = genetic diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations). table 4. analysis of molecular variance (amova) of the studied species. source df ss ms est. var. % φpt among pops 20 1991.364 70.789 12.154 53% 53%within pops 177 774.443 8.905 2.888 47% total 197 2555.807 14.060 100% abbreviations: df = degree of freedom, ss = sum of squared observations, ms = mean of squared observations, ev = estimated variance, φpt = proportion of the total genetic variance among individuals within an accession, (p < 0.001). 17genetic variations and interspesific relationships in lonicera l. (caprifoliaceae), using scot molecular markers ment with amova and genetic diversity parameters presented before. the species are genetically well differentiated from each other. these results indicate that scot molecular markers can be used in lonicera species taxonomy. the nm analysis by popgene software also produced mean nm= 0.186, that is considered very low value of gene flow among the studied species. mantel test with 5000 permutations showed a significant correlation (r = 0.177, p=0.0002) between genetic distance and geographical distance, so isolation by distance (ibd) occurred among the lonicera species studied. nei’s genetic identity and the genetic distance determined among the studied species (table not included). the results showed that the highest degree of genetic similarity (0.90) occurred between l. korolkowii and l. nummulariifolia. the lowest degree of genetic similarity occurred between l. hypoleuca and l. iberica (0.67). the low nm value (0.186) indicates limited gene flow or ancestrally shared alleles between the species studied and indicating high genetic differentiation among and within lonicera species. the species genetic structure we performed structure analysis followed by the evanno test to identify the optimal number of genetic groups. we used the admixture model to illustrate interspecific gene flow or/and ancestrally shared alleles in the species studied. structure analysis followed by evanno test produced δk =6 (table 5). the structure plot (figure. 4) produced more detailed information about the genetic structure of the species studied as well as shared ancestral alleles and/or gene flow among lonicera species. this plot revealed that genetic affinity between l. caucafigure 3. upgma tree of scot data revealing species delimitation in the lonicera species. branch support values are given as bootstrap (bp) value above branches. figure 4. structure plot of lonicera species based on scot data. 18 fengzhen chen et al. sica and l. iberica (similarly colored, no. 1, 2), as well as l. nummulariifolia and l. korolkowii (sp no. 3,5) due to shared common alleles. this is in agreement with upgma dendrogram presented before. the other species are distinct in their allele composition. discussion knowledge of the genetic variability and diversity within and among different populations is crucial for their conservation and management (e.g. mills and schwartz 2005; khayatnezhad and gholamin 2021; guo et al. 2021; ren et al. 2021). in the present study we used morphological and molecular (scot) data to evaluate species relationship in lonicera. morphological analyses of the studied lonicera species showed that they are well differentiated from each other both in quantitative measures (the anova test result) and qualitative characters (the pca plot result). in addition, pca analysis suggests that characters like bract length, stipule length, bract shape, calyx shape, petal shape, length and width of stem-leaf, length and width of petal could be used in species groups delimitation. four species and 12 populations of the genus lonicera have been studied in terms of pollen and seed micro-morphology and molecular phylogeny (amini et al. 2019). their results showed that micro-morphological and molecular data provide reliable evidence for differentiation of some populations from others. since lonicera systematically is a problem genus, it is necessary to use alternative methods to distinguish its taxa. statistical evaluation of taxa can be used for taxa delimitation. the present study intends to provide further evidence for taxonomists, so as to help them in separating these six species. genetic structure and gene flow pic and mi characteristics of a primer help in determining its effectiveness in genetic diversity analysis. sivaprakash et al. (2004) suggested that the ability of a marker technique to resolve genetic diversity may be more directly related to the degree of polymorphism. generally, pic value between zero to 0.25 imply a very low genetic diversity among genotypes, between 0.25 to 0.50 shows a mid-level of genetic diversity and value ≥0.50 suggests a high level of genetic diversity (tams et al. 2005; hou et al. 2021; huang et al. 2021; khayatnezhad and gholamin 2020b). in this research, the scot primers’ pic values ranged from 0.43 to 0.67, with a mean value of 0.56, which indicated a mid-ability of scot primers in determining genetic diversity among the lonicera species. in the study conducted by chen et al. (2012), 20 issr primers amplified 186 bands with 103 (54.63%) polymorphic bands and 58 sequence-related amplified polymorphism (sr ap) primer combinations amplified 591 bands with 347 (55.46%) polymorphic bands. both issr and srap analyses revealed a middle level of genetic diversity in lonicera macranthoides cultivars. smolik et al. (2006) found a level of similarity for 6 populations of lonicera periclymenum ranging from 82.3% to 86.6%, indicating their closely related nature. issr amplification was used by smolik et al. (2010) to analyze polymorphisms of microsatellite sequences in the honeysuckle genome and to evaluate genetic diversity among 14 polish and russian blue honeysuckle accessions. random amplified polymorphic dna (rapd) analysis was used by naugžemys et al. (2011) to assess the genetic relationships among 51 accessions of blue honeysuckle. the pairwise genetic distance (gdxy) values among studied accessions ranged from 0.054 to 0.479; the mean gdxy was 0.283. knowledge of the content of secondary metabolites in individual genotypes allows us to choose the best in lonicera breeding programs in order to increase the nutritional value and health benefits. in conclusion, the results of this study showed that to evaluate the genetic diversity of the lonicera genus, the primers derived from scot were more effective than the other molecular markers. also, lonicera ecotypes/ species were clearly separated from each other in the dendrogram and mds, indicating the higher efficiency of scot technique in lonicera species identification. acknowledgment the authors are grateful to all colleagues in the laboratory of plant physiology in school of horticulture for providing help and assistance. and thanks to dr. jianqiu han in shanghai institute of technoligy for modifying this paper. this study was supported by natural science table 5 . k-means clustering result of scot data. k ssd(t) ssd(ac) ssd(wc) pseudo-f bic 1 66.133 0 0 0 192.432 2 66.133 35.707 30.426 16.038 168.916 3 66.133 28.688 37.445 16.089 174.449 4 66.133 35.707 30.426 16.038 168.916 5& 66.133 40.09 26.043 15.394 165.722 6* 66.133 20.586 45.547 19.435 179.457 19genetic variations and interspesific relationships in lonicera l. 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(caprifoliaceae), using scot molecular markers fengzhen chen1, dongmei li2,* , mohsen farshadfar3 the new chromosomal data and karyotypic variations in genus salvia l. (lamiaceae): dysploidy, polyploidy and symmetrical karyotypes halil erhan eroğlu1,*, esra martin2, ahmet kahraman3, elif gezer aslan4 cytogenetic survey of eight ant species from the amazon rainforest luísa antônia campos barros1, gisele amaro teixeira2, paulo castro ferreira1, rodrigo batista lod1, linda inês silveira3, frédéric petitclerc4, jérôme orivel4, hilton jeferson alves cardoso de aguiar1,5,* molecular phylogeny and morphometric analyses in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f. neda atazadeh1,*, masoud sheidai1, farideh attar2, fahimeh koohdar1 a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) melika tabasi1, masoud sheidai1,*, fahimeh koohdar1, darab hassani2 genetic diversity and relationships among glaucium (papaveraceae) species by issr markers: a high value medicinal plant lu feng1,*, fariba noedoost2 morphometric analysis and genetic diversity in rindera (boraginaceae-cynoglosseae) using sequence related amplified polymorphism xixi yao1, haodong liu2,*, maede shahiri tabarestani3 biosystematics, fingerprinting and dna barcoding study of the genus lallemantia based on scot and remap markers fahimeh koohdar*, neda aram, masoud sheidai karyotype analysis in 21 plant families from the qinghai–tibetan plateau and its evolutionary implications ning zhou1,2, ai-gen fu3, guang-yan wang1,2,*, yong-ping yang1,2,* some molecular cytogenetic markers and classical chromosomal features of spilopelia chinensis (scopoli, 1786) and tachybaptus ruficollis (pallas, 1764) in thailand isara patawang1,*, sarawut kaewsri2, sitthisak jantarat3, praween supanuam4, sarun jumrusthanasan2, alongklod tanomtong5 centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates) simona ceraulo, vanessa milioto, francesca dumas* repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil marina souza cunha1,2,*,#, silvana melo1,3,#, filipe schitini salgado1,2, cidimar estevam assis1, jorge abdala dergam1,* karyomorphology of some crocus l. taxa from uşak province in turkey aykut yilmaz*, yudum yeltekin variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l. egizia falistocco1,*,+, gianpiero marconi1,+, lorenzo raggi1, daniele rosellini1, marilena ceccarelli2, emidio albertini1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(1): 55-63, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1067 caryologia international journal of cytology, cytosystematics and cytogenetics citation: anup kumar sarkar, ranita saha, rupak halder (2022) chromosomes damage by sewage water studies in the allium cepa l. and zea mays l.. caryologia 75(1): 55-63. doi: 10.36253/ caryologia-1067 received: august 29, 2020 accepted: march 20, 2022 published: july 6, 2022 copyright: © 2022 anup kumar sarkar, ranita saha, rupak halder. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid aks: 0000-0002-6777-740x chromosomes damage by sewage water studies in the allium cepa l. and zea mays l. anup kumar sarkar*, ranita saha, rupak halder department of botany, west bengal state university, berunanpukuria, malikapur, north 24 parganas, barasat, kolkata–700126, west bengal, india *corresponding author. e-mail: ak94sarkar@yahoo.com abstract. the effect of sewage water sample of the three locations khardah (22.7003° n, 88.3753° e), titagarh (22.7383° n, 88.3737° e), madhyamgram (22.6924° n, 88.4653° e), and control (distilled h2o) in the district of north 24 parganas (22.6168° n, 88.4029° e), west bengal, india on the damage of chromosomes in the onion (allium cepa l.) and maize plant (zea mays l.) were investigated by employing mitotic chromosomal aberration assay. physiochemical analysis of sewage water samples showed the ph is 5.10-5.30 in nature. few heavy elements: fe, mn and zn in the sample from khardah (22.7003° n, 88.3753° e) sewage water exceeded the indian standard 10500:2012 and who’s (2006) permissible limits. whereas cl, cu, pb, cr, and cd are more or less within limit of the standard condition. the obtained data exhibited a decline in reproductive capacity of cells and the occurrence of deviation from the normal mitotic cell division. the mitotic index (mi) decreased significantly (p < 0.05) in both the cases and is given as control (57.03 %) > madhyamgram (41.70 %) > titagarh (33.85 %) > khardah (31.57 %) in allium cepa l. and control (49.33 %) > titagarh (21.45 %) > madhyamgram (26.47 %) > khardah (24.05 %) in zea mays l. the chromosomal aberrations (cas): karyorrhexis, karyolysis, fragments, lagging chromosome, anaphase bridges are present in significant amount in the crops treated with sewage water sample than the one with control condition. heavy metals act as pollutants in the sewage water sample which has cytotoxic effect on cells, threat to water ecosystem and human health. keywords: sewage water, mitotic index, indian standard, heavy metal, cytotoxic, ecosystem. introduction rapid industrialization in the last four decades has resulted in the mushrooming of production units even in the vicinity of semiurban and rural areas of the country. several hazardous chemical industries discharge their untreated effluents into the atmosphere. water and soil along with the ecoenvironmental profile of the area are adversely disturbed. the endemic exposure to pollutants causes toxicity, morbidity, early mortality, genetic and cytogenetic damage and various other pathological symptoms in the exposed human and plant populations. 56 anup kumar sarkar, ranita saha, rupak halder this study was undertaken to evaluate the cytotoxic effects of effluents in sewage water of the three locations khardah (22.7003° n, 88.3753° e), titagarh (22.7383° n, 88.3737° e), and madhyamgram (22.6924° n, 88.4653° e) of north 24 parganas (22.6168° n, 88.4029° e), west bengal, india. for more than forty years officially accepted “allium test” is used widely for assessment of the environmental water pollution (fiskesjo 1985, 1997; ivanova et al. 2002, 2005; rank 2003). various investigators (al-sabti 1989; smakakink l et al. 1996; rank and nielsen, 1998; moraes and jordao, 2001) advocate different plant test systems which are useful for studying cy totoxicity of heav y metals. currently, the physio-chemical and cyto-toxicological evaluation of sewage water discharges from the three locations (t1, t2, and t3) by different ways has not been documented, thereby no information on their hazardous effect on agricultural field and the ecosystem is recorded. with this background, the present work was undertaken to investigate chromosomal damage (cytotoxic) impact of sewage effluents collected from three different locations on root tip meristematic cells of allium cepa l. and zea mays l. with special reference to analysis of physio-chemical parameters of the liquid waste. materials and methods bluish and blackish sewage water was collected from the three main drains of t1 = khardah (22.7003° n, 88.3753° e), t2 = titagarh (22.7383° n, 88.3737° e), and t3 = madhyamgram (22.6924° n, 88.4653° e), of north 24 parganas (22.6168° n, 88.4029° e), west bengal, india at the depth of six inches from three random points within the drain of each location. the sewage water samples were filtered four times by muslin cloth and then stored in a clean plastic jar for chemicals analysis and setting experiment along with distilled water as a control (t4) on two species namely, allium cepa l. and zea mays l. physicochemical parameters were analyzed from the three locations’ (t1, t2, and t3) sewage water samples (filtered four times) for a standard physicochemical property (chloride) according to is:3025 (part 32): 1988, ra 2003. the eight heavy metals, i.e., copper (cu), chromium (cr), nickel (ni), iron (fe), zink (zn), cadmium (cd), lead (pb), manganese (mn) were determined in mg/l, following the methods described in apha 22nd edition 3125b and who-2006 limits (olorunfemi et al. 2014). chromosome preparation was made from the treated root tips of both the species chromosome preparation was performed in both the species following the protocol adapted by sharma and sharma (1980). the root tips of treated and control sets of both species were fixed in carnoy’s fluid-i for overnight followed by treatment with 45% acetic acid for 10 minutes at room temperature. the resultant root samples were stained for 45 minutes with a mixture of 2% aceto-orcein:1n hcl (9:1) and warmed lightly at 60°c. the meristematic tip portion (~ 1mm size) of onion and maize roots were cut and placed on a clean grease free slide in a drop of acetic acid (45%) and squashed, later temporarily sealed with paraffin wax. slides were prepared from five randomly drawn root tips from each treatment of both the species. five random microscopic fields from each slide were scored under olympus with the prog-res capture pro 2.1 photo system. the mitotic indices were calculated for all the treated materials of each treatment. statistical analysis the experiment was organized according to a randomized complete design (rcd) with three replications. a two-way anova was performed for test of significance at p<0.05, employing f-test. data were expressed as mean ± standard error (sem) (gomez and gomez 1984). the mean mitotic index of each treatment was compared with those corresponding to control employing “t” test for significant difference, if any. results heavy metals and chloride determination in the sewage water samples the heavy metal and chloride analysis of the sewage water sample of the three locations have been shown in the table 1. the sewage water collected from different locations were acidic in nature on ph scale: 5.30 (t1), 5.15 (t2) and 5.10 (t3) during the middle of february, 2017.the sewage water sample from the three locations attained a higher range of iron concentration i.e., 0.340.52 mg/l as compared to the limit of 0.001-0.30 mg/l. the contents of copper, chromium, cadmium and lead were found less than the permissible limit (0.001 mg/l) in the sample of t1, t2, and t3 treatments, except copper (0.006 mg/l) in t3 treatment within limit compared with national (apha 22nd edition 3125 b and is: 10500: 57chromosomes damage by sewage water studies in the allium cepa l. and zea mays l. 2012) and who’s (2006) standards. th e result revealed that the concentration of manganese (0.321 mg/l) content is higher in the sample of khardah (22.7003° n, 88.3753° e) location while it was found in the range of limit (0.001-0.03 mg/l) in the sewage water sample of titagarh (22.7383° n, 88.3737° e), and madhyamgram (22.6924° n, 88.4653° e) respectively. general toxicity-root growth inhibition and deformity of allium cepa l. & zea mays l. test th ere was a signifi cant (p< 0.05) root growth inhibition of the two species in the wastewater samples of three locations compared with distilled water (figure 1). root length in distilled water was higher than that in wastewater samples for both of them (figure 2). sewage water trials were compared with control treatment (distilled water). th e mitotic index (mi) signifyingly decreased along with an increase in chromosomal aberrations (cas) of the root tips meristematic cells of onion and maize were found (table 2, figure 5). table 1. contents of heavy metals, chloride and ph in the experimental sewage water samples (t1, t2 and t3). sl. no. diff erent parameters in the water samples of the three locations limit requirements as per is 10500: 2012. maximum result of the three locations or treatments (t1, t2, & t3) who (2006) limit t1=khardah (22.70030 n, 88.37530 e) t2=titagarh (22.73830 n, 88.37370 e) t3=madhyamgram (22.69240 n, 88.46530 e) 1 copper (cu)mg/l 0.001 max:1.5 < 0.001 < 0.001 0.006 2 chromium (cr)mg/l 0.001 max: 0.05 < 0.001 < 0.001 < 0.001 0.05 3 nickel (ni) mg/l 0.001 max: 0.02 0.006 < 0.001 0.002 0.02 4 iron (fe) mg/l 0.001 max: 0.30 0.340 0.501 0.520 5 zink (zn)mg/l 0.001 max: 15.0 0.450 < 0.001 0.002 0.01 6 cadmium (cd)mg/l 0.001 max: 0.003 < 0.001 < 0.001 <0.001 0.003 7 lead (pb) mg/l 0.001 max: 0.01 < 0.001 < 0.001 < 0.001 0.01 8 manganese (mn) mg/l 0.001 max: 0.30 0.321 0.275 0.101 9 chloride (cl)(mg/l) n/a max: 1000 89.19 79.55 269.98 10 ph 5.30 5.15 5.10 6.5-9.5 11 colour bluish blackish blackish contents of heavy metals & chloride present in the three experimental fi elds (t1, t2, & t3) done by efrac (edward food research & analysis centre limited, subash nagar, p.o. nilgunj bazar, barasat, kolkata-700121, india. email: efraclab@cfrac.org, ph. no.91-3371122800. figure 1. eff ect of sewage water and control samples (t1, t2, t3, and t4) on germination % and disinhibition root length % in allium cepa l. and zea mays l. figure 2. percentage root growth of allium cepa l. and zea mays l. roots exposed to the test sewage water and control samples (t1, t2, t3 and t4). 58 anup kumar sarkar, ranita saha, rupak halder allium cepa l. test with sewage water samples allium cepa l. meristematic cells in the root tips after 72 hours exposure to the different sewage water treatments exhibited various chromosomal aberrations (cas) in comparison to distilled water (control) that included karyorrhexis, karyolysis, fragmentation, laggard, and anaphase bridge (figure 3). t1 has significantly decreased anaphase bridge (5.60 ± 1.27), laggard chromosome (5.60 ± 1.27), mitotic index (31.57%). t2 has extensively decreased fragmentation (2.00 ± 0.79) only. t3 has significantly decreased karyorrhexis (22.00 ± 1.76), karyolysis (19.80 ± 7.09) and aberration frequency (8.09%). the activities of different types of abnormal cells and aberration frequency % were seen to have a higher value in all samples (t1, t2, and t3) as compared to control (t4) in the onion (table 2). zea mays l. test with sewage water samples chromosomal aberrations (cas) induced in zea mays l. root tips meristematic cells after 72 hours exposure to different waste water treatments (t1, t2, and t3) in comparison with distilled water (t4) were summarized in figure 4. the treatment t1 has significantly decreased only mitotic index (24.05%). t2 has considerably decreased laggard chromosome (0.60 ± 0.61) and anaphase bridge (0.60 ± 0.61). t3 has drastically decreased karyorrhexis (53.60 ±16.32), fragmentation (2.80 ± 1.22) and aberration frequency (10.13%). no effect on karyolysis was found in t1, t2 and t3 samples. the activities of different types of abnormal cells and aberration frequency % was observed to have higher values in all sewage water samples (t1, t2, and t3) as compared to control (t4) in the maize (table 2). different types of abnormal cells the aberration shown at level of nucleus is of two types: (i) karyorrhexis and karylysis (ii) chromosomes. karyorrhexis it is the manner of destructive fragmentation of the nucleus of dying cells where the chromatin is irregularly distributed throughout the cytoplasm (figure 3a, e, i, and figure 4a, d, f). ta bl e 2. f re qu en ci es o f d iff er en t t yp es o f c el ls a fte r tr ea tm en t w ith d iff er en t w at er s am pl es ( t 1, t 2, t 3 a nd t 4) m ea n ± se & a n o va in b ot h sp ec ie s. species l oc at io ns to ta l c el ls d iv id in g c el ls c el ls o f di ff er en t s ta ge s a bn or m al c el ls m ito tic in de x (% ) a be rr at io n fr eq ue nc y (% ) pr op ha se m et ap ha se a na ph as e te lo ph as e k ar yo rr he xi s k ar yo ly si s fr ag m en te d l ag ga rd a na ph as e br id ge allium cepa l. k ha rd ah 60 8. 80 ± 13 1. 53 19 2. 20 ± 49 .4 6 17 3. 20 ± 50 .2 6 7. 40 ± 1. 69 8. 00 ± 1. 76 3. 60 ± 1. 86 29 .2 0± 5 .4 1 30 .4 0± 5 .9 7 3. 40 ± 0. 99 5. 60 ± 1. 27 5. 60 ± 1. 27 31 .5 7 11 .2 7 t ita ga rh 58 2. 60 ± 46 .4 4 19 7. 20 ± 57 .7 1 17 4. 80 ± 59 .9 0 6. 80 ± 2. 14 12 .0 0± 3 .0 5 3. 60 ± 1. 69 65 .4 0± 1 8. 11 29 .0 0± 9 .5 7 2. 00 ± 0. 79 8. 60 ± 2. 17 8. 60 ± 2. 17 33 .8 5 18 .0 2 m ad hy am gr am 62 0. 20 ± 90 .7 0 25 8. 60 ± 44 .1 7 23 8. 40 ± 39 .2 3 10 .6 0± 3 .2 0 6. 80 ± 2. 88 2. 80 ± 1. 83 22 .0 0± 1 .7 6 19 .8 0± 7 .0 9 2. 60 ± 1. 69 5. 80 ± 2. 77 5. 80 ± 2. 77 41 .7 0 8. 09 d is til le d w at er 6 78 .2 0± 6 1. 68 38 6. 80 ± 3 3. 06 35 7. 00 ± 2 7. 86 17 .6 0 ± 8. 92 7. 6± 1 .8 6 4. 60 ± 2. 68 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 57 .0 3 0. 00 a n o v a n s ** ** ** ** n s ** zea mays l. k ha rd ah 59 0. 40 ± 11 4. 33 14 2. 00 ± 34 .6 3 13 0. 80 ± 33 .3 3 9. 60 ± 2. 56 1. 40 ± 1. 69 0. 40 ± 0. 61 90 .8 0± 2 4. 85 0. 00 ± 0. 00 6. 40 ± 2. 31 0. 80 ± 0. 50 0. 80 ± 0. 50 24 .0 5 16 .6 0 t ita ga rh 75 7. 20 ± 40 .0 5 21 4. 60 ± 66 .9 9 13 2. 80 ± 29 .8 2 9. 80 ± 4. 81 1. 80 ± 1. 45 0. 80 ± 0. 93 15 6. 40 ±6 8. 61 0. 00 ± 0. 00 5. 40 ± 2. 90 0. 60 ± 0 .6 1 0. 60 ± 0. 61 28 .3 4 21 .4 5 m ad hy am gr am 56 4. 60 ±1 42 .0 5 14 9. 40 ± 53 .4 6 13 7. 20 ± 51 .7 4 7. 00 ± 2. 84 1. 80 ± 0. 93 3. 40 ± 1. 69 53 .6 0± 1 6. 32 0. 00 ± 0. 00 2. 80 ± 1. 22 0. 80 ± 0. 50 0. 80 ± 0. 50 26 .4 6 10 .1 3 d is til le d w at er 6 54 .0 0± 3 9. 87 32 2. 6 ± 64 .7 3 28 7. 40 ± 5 3. 67 29 .8 0± 1 3. 47 4. 00 ± 2. 08 1. 40 ± 2. 44 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 0± 0 0 49 .3 3 0. 00 a n o v a * ** ** ** * * ** a na ly si s of v ar ia nc e (a n o va ): si gn ifi ca nt e ffe ct s ar e in di ca te d by a s * = p < 0. 05 ( 5% ); ** = p < 0 .0 1 (1 % ) an d w hi le n on -s ig ni fic an t e ffe ct s ar e in di ca te d by n s. 59chromosomes damage by sewage water studies in the allium cepa l. and zea mays l. karyolysis enzymatic dissolution leads to complete suspension of the chromatin in a dying cell. after karyolysis the whole cells will be stained uniformly (figure 3b, f, j). chromosome laggard particular concentration of few turbagens which has affinity for thiol groups that induce various types of spindle disturbances at all stages of mitosis division in most of cells were confined to one or more number of chromosomes. the movement of chromosomes deviated from the main mass and were often seen to be lost. such aberrant chromosomes have been called “laggards” (figure 3k). it may be present in the location of spindle area or outside of it (figure 3k). anaphase bridge chromatin bridge is a mitotic event that forms when telomeres of sister chromatids combine together and fail to completely segregate into their respective daughter cells. this event mostly occurs during the anaphase stage that is why called anaphase bridge (figure 3c, g, k and figure 4c, e, g). chromosome fragmentation chromosome fragmentation results are indicators of a clastogenic action from the numerous breaks in the chromosome arms where there is loss of integrity of the chromosome. disintegration can range from partial to total breakup of the chromosome (figure 3d, h, l and figure 4b). discussion the research survey works (ma 1999; fatima and ahmed 2005, 2006b) on industrial eff luent samples that were taken from different parts of the city of aligarh and ghaziabad, up in india. it may be used as a bio indicator for aquatic atmosphere. in our experiment different elements and microorganism present in the wastewater samples of the three locations might have induced cytological effects on the roots of both species i.e., allium cepa l. and zea mays l. it may have direct or indirect risk on their life due to irriga-ta bl e 2. f re qu en ci es o f d iff er en t t yp es o f c el ls a fte r tr ea tm en t w ith d iff er en t w at er s am pl es ( t 1, t 2, t 3 a nd t 4) m ea n ± se & a n o va in b ot h sp ec ie s. species l oc at io ns to ta l c el ls d iv id in g c el ls c el ls o f di ff er en t s ta ge s a bn or m al c el ls m ito tic in de x (% ) a be rr at io n fr eq ue nc y (% ) pr op ha se m et ap ha se a na ph as e te lo ph as e k ar yo rr he xi s k ar yo ly si s fr ag m en te d l ag ga rd a na ph as e br id ge allium cepa l. k ha rd ah 60 8. 80 ± 13 1. 53 19 2. 20 ± 49 .4 6 17 3. 20 ± 50 .2 6 7. 40 ± 1. 69 8. 00 ± 1. 76 3. 60 ± 1. 86 29 .2 0± 5 .4 1 30 .4 0± 5 .9 7 3. 40 ± 0. 99 5. 60 ± 1. 27 5. 60 ± 1. 27 31 .5 7 11 .2 7 t ita ga rh 58 2. 60 ± 46 .4 4 19 7. 20 ± 57 .7 1 17 4. 80 ± 59 .9 0 6. 80 ± 2. 14 12 .0 0± 3 .0 5 3. 60 ± 1. 69 65 .4 0± 1 8. 11 29 .0 0± 9 .5 7 2. 00 ± 0. 79 8. 60 ± 2. 17 8. 60 ± 2. 17 33 .8 5 18 .0 2 m ad hy am gr am 62 0. 20 ± 90 .7 0 25 8. 60 ± 44 .1 7 23 8. 40 ± 39 .2 3 10 .6 0± 3 .2 0 6. 80 ± 2. 88 2. 80 ± 1. 83 22 .0 0± 1 .7 6 19 .8 0± 7 .0 9 2. 60 ± 1. 69 5. 80 ± 2. 77 5. 80 ± 2. 77 41 .7 0 8. 09 d is til le d w at er 6 78 .2 0± 6 1. 68 38 6. 80 ± 3 3. 06 35 7. 00 ± 2 7. 86 17 .6 0 ± 8. 92 7. 6± 1 .8 6 4. 60 ± 2. 68 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 57 .0 3 0. 00 a n o v a n s ** ** ** ** n s ** zea mays l. k ha rd ah 59 0. 40 ± 11 4. 33 14 2. 00 ± 34 .6 3 13 0. 80 ± 33 .3 3 9. 60 ± 2. 56 1. 40 ± 1. 69 0. 40 ± 0. 61 90 .8 0± 2 4. 85 0. 00 ± 0. 00 6. 40 ± 2. 31 0. 80 ± 0. 50 0. 80 ± 0. 50 24 .0 5 16 .6 0 t ita ga rh 75 7. 20 ± 40 .0 5 21 4. 60 ± 66 .9 9 13 2. 80 ± 29 .8 2 9. 80 ± 4. 81 1. 80 ± 1. 45 0. 80 ± 0. 93 15 6. 40 ±6 8. 61 0. 00 ± 0. 00 5. 40 ± 2. 90 0. 60 ± 0 .6 1 0. 60 ± 0. 61 28 .3 4 21 .4 5 m ad hy am gr am 56 4. 60 ±1 42 .0 5 14 9. 40 ± 53 .4 6 13 7. 20 ± 51 .7 4 7. 00 ± 2. 84 1. 80 ± 0. 93 3. 40 ± 1. 69 53 .6 0± 1 6. 32 0. 00 ± 0. 00 2. 80 ± 1. 22 0. 80 ± 0. 50 0. 80 ± 0. 50 26 .4 6 10 .1 3 d is til le d w at er 6 54 .0 0± 3 9. 87 32 2. 6 ± 64 .7 3 28 7. 40 ± 5 3. 67 29 .8 0± 1 3. 47 4. 00 ± 2. 08 1. 40 ± 2. 44 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 0± 0 0 49 .3 3 0. 00 a n o v a * ** ** ** * * ** a na ly si s of v ar ia nc e (a n o va ): si gn ifi ca nt e ffe ct s ar e in di ca te d by a s * = p < 0. 05 ( 5% ); ** = p < 0 .0 1 (1 % ) an d w hi le n on -s ig ni fic an t e ffe ct s ar e in di ca te d by n s. b ca d h lkji e f g figure 3. photomicrographs of cytological aberration in allium cepa l. (2n=16) root tip cells treated with different sewage water samples. (a) karyorrhexis (b) karyolysis (c) anaphage bridge and (d) fragmented, respectively in khardah water; e. karyorrhexis, f. karyolysis, g. anaphage bridge and h. fragmented, respectively in titagarh water; i. karyorrhexis, j. karyolysis, k. laggard, anaphase bridges and l. fragmented in madhyamgram water. 60 anup kumar sarkar, ranita saha, rupak halder tion with sewage wastewater to the food plants (iqbal et al. 2016; chary et al. 2008). trace elements accumulation in the food chain may harm different organism in the ecosystem along with humans. several studies have shown that presence of various metal in the industrial waste water can cause the various nature of chromosomal aberrations like lagging chromosome, fragmented chromosome, anaphase bridge and binucleated cells etc. in the meristematic root tip cells of allium cepa l. and plants with such abnormalities which may induce alterations in the genetic constitution not only the future progenies but correspondingly have triggered further complication in mankind when consumed as nourishment materials (sabeen et al. 2020). surveys on sludge samples from thirty-four cities in the usa have reports that there were no effects of chromosomal aberration a b c d e gf figure 4. photomicrographs of cytological aberration in zea mays l. (2n=20) root tip cells treated with different sewage water samples. a. karyorrhexis, b. fragmented, c. anaphage bridge, in khardah water; d. karyorrhexis and e. anaphage bridges in titagarh water; f. karyorrhexis, and g. anaphase bridge in madhayagram water. 61chromosomes damage by sewage water studies in the allium cepa l. and zea mays l. due to the treatment of effl uents (babish et al. 1983). conversely, there are also studies, revealing cytological eff ect of extracts from wastewater sludges collected from various american cities on the test of salmonella typhimurium sample (mumma et al. 1988; brown et al. 1991; blevins and brennan 1990). in 1998, white and rasmussen demonstrated that in the large areas of metropolis cities, wastewaters of diff erent municipalities are a multifaceted combination of effl uent resources from domestic and industrial sewage, containing a widespread series of heavy or light constituents from a source of diff erent varieties. siddiqui et al. 2011 had strongly recommended that seed germination of diff erent species such as brassica oleracea var. capitata, pennisetum glaucum and cucumis sativus are remarkable living beings for heav y metal toxicological monitoring of industrial effl uents and xad concentrated river water. furthermore, it was reported that significant quantities of diff erent types chromosomal abnormalities including fragmentation, bridges and stickiness were found by allium cepa test. cytogenetic eff ect of the carbon black factory industrial effl uents in allium sativum root meristem cells not only retarded germination percentage and radical growth but also induced chromosomal aberrations: karyolysis, fragmentation, laggards (ray and saha 1992). in the absence of telomeres, chromosomes turned out to be adhesive in nature which may join the end part of other fragmented chromosomes in the root tip of meristem cells of allium cepa in presence of alprazolam chemical compound (nefi c et al. 2013). th e presence of breaking fragments, laggards, chromosome bridges and stickiness with other abnormalities are viewed as mitotic irregularities are due to an-eugenic agents (zang and yang 1994; silveira et al. 2017; haq et al. 2017). grant (1982) told that chromosomes stickiness probably occurred due to degradation or de-polymerization of dna segment of the chromosome. it was also reported that the sticking of chromosomes resulted from dna compression and adhesiveness of inter-chromosome fi bers (schneiderman et al.1971). one of the abnormalities, which is stickiness and it shows high toxic substances are present along with irreversibility while acentric fragments that appear in anaphase stages are the result of chromatids or chromosome interruptions, representing interference with dna. bridges in anaphase stage are the outcome of the disruptions and joining of chromatids or chromosomes (turkoglu 2007). it is also described that anaphase bridges occur as an output of adhesiveness of chromosomes, unequal process of translocation or inversion in the segments of chromosome (gomurgen 2005). studies by nagajyoti et al. (2010) and fashola et al. (2016) indicates that among the heavy metal cadmium (cd) is known to be carcinogenic and mutagenic in biological system. as per the investigation reported by adhikari (2019) indicated that lead (pb) one of heavy metal act as a robust mutagenic mediator on lathyrus sativus. nickel with magnesium can be the cause for chromatin condensation of the cells (lee et al. 1995). whereas it also stated that the combination of nickel and chromium aff ected the cell division of mitotic spindle leading to chromosomal aberration in the root’s tips of allium cepa (anderson 1985). th e trace amount of few metals such as mn, fe, zn and cr combined together or individually has caused the observed cytogenotoxic eff ects and reported to induce aberrations in the larvae of newt (godet et al, 1993). it also reported that heavy metals induced the toxicity and mutagenicity on zea mays l. (vojtechova and leblova, 1991). th e mitotic index (mi) inhibition has been accredited to the eff ect of diff erent environmental substances on dna and synthesis of protein of the living organism (chauhan et al. 1998). nefi c et al. 2013 revealed that the occurrence of high concentration of heavyweight metals in the earth sample triggered the downward movement of the mitotic index of the meristematic root tips cells of allium cepa l. several heavy metals inhibit the cell division along with reduction of mi in the cortex of the meristematic root tips of zea mays l. (kozhevnikova 2009). in this study, the allium cepa l. and zea mays l. roots anaphase-telophase assay at diff erent stages of cells established that all the three wastewater samples had approximately same levels of toxicity. in the titagarh (22.7383° n, 88.3737° e) samples, the aberration frequency percentage was however higher in both species than figure 5. eff ect of sewage water and control samples (t1, t2, t3, andt4) on chromosomal aberration frequency % in allium cepa l. and zea mays l. 62 anup kumar sarkar, ranita saha, rupak halder the other two samples of wastewater. even the mitotic index percentage in all three wastewater location samples were half of the control sample (distilled water). conclusion the study indicates that the heav y metals present in the wastewater samples in khardah (t1); titagarh (t2); and madhyamgram (t3) in the district of north 24 parganas, west bengal, induced chromosomal aberrations: kar yorrhexis, kar yolysis, fragmented, laggard and anaphase bridge, reduced the mitotic index and morphological structure also such as germination % and root length inhibitions % in allium cepa l. and zea mays l. it may be concluded that presence of heav y metals such as ni, ld, mn, fe, cd leads to decrease cell reproduction and increase in the chromosome mutation frequency, posing a great potential threat to water ecosystem and human health as well. thus, the investigation advocates treatment of wastewater of three locations for decreasing contamination load before releasing for irrigation in the agricultural field or into the rivers. acknowledgement we are thankful to the honourable vice-chancellor of west bengal state university, berunanpukuria, malikapur, barasat, kolkata-700126, india, for providing the necessary facilities. references adhikari d. 2019. augmentation mitodepressive and cytogenotoxic effects of lead upon acute exposure on grass pea (lathyrus sativus l.) root tip cells. american journal of biological sciences. 1(1): 14-22. 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biology. 33:386-394. white p, rasmussen jb.1998. the genotoxic hazard of domestic wastes in surface waters. mutat res. 410:223-236. caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 83-92, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-116 citation: n.a. şuţan, a.n. matei, e. oprea, v. tecuceanu, l.d. tătaru, s.g. moga, d.ş. manolescu, c.m. topală (2020) chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts. caryologia 73(1): 83-92. doi: 10.13128/caryologia-116 received: january 9, 2019 accepted: february 22, 2020 published: may 8, 2020 copyright: © 2020 n.a. şuţan, a.n. matei, e. oprea, v. tecuceanu, l.d. tătaru, s.g. moga, d.ş. manolescu, c.m. topală. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 1 university of piteşti, faculty of sciences, physical education and informatics, department of natural sciences, 1 targu din vale str., 110040 pitesti, romania 2 university of bucharest, faculty of chemistry, department of organic chemistry, biochemistry and catalysis, blvd. regina elisabeta, no. 4-12, 030018, bucharest, romania 3 organic chemistry centre of the romanian academy “costin d. nenitescu”, 202b splaiul independentei, 78100 bucharest, romania *corresponding author. e-mail addresses: ancasutan@yahoo.com, mateiandreeanatalia@gmail., eliza_oprea2003@yahoo.com, vichi_tecu@yahoo.com, lavinia.tataru@upit.ro, sorin.g.moga@gmail.com, stefaniamanolescu@yahoo.com, carmen.topala@gmail.com. abstract. through time, in the traditional medicine ligularia genus has been used as a remedy to cure several diseases and affections. the paper represents an essential step in offering more information about the antioxidant activity, chemical composition and cytogenetic activity of l. sibirica (l.) cass. rhizomes and roots extracts. the antioxidant activity of the extracts has been achieved by analyzing the total phenolic content, total flavonoids, the organic chemical compound and trolox equivalent antioxidant capacity and their major polyphenolic constituents were quantified by liquid chromatography electrospray ionization-tandem mass spectrometry. the extracts were obtained by the supercritical fluid extraction (sfe-co2) technique, sfe-co2 extraction with co-solvent and absolute ethanol extraction. the best results for the antioxidant activity have been fulfilled through the last two techniques. high performance liquid cromatography (hplc) has been applied in order to identify and quantify selected phenolic acids and flavonoids in the ethanolic extracts of l. sibirica (l.) cass. the cytogenotoxic effects of the extracts completed the present study, with a furtherance of antiproliferative potential highlighted by the statmokinetic effect and an additional genoprotective effect. keywords. antioxidant activity, phenols, sfe extraction, hplc, cytogenotoxic effects, ligularia sibirica. abbreviation: tp total phenols, tf total flavonoids, dpph the organic chemical compound 2,2-diphenyl-1-picrylhydrazyl, teac trolox equivalent antioxidant capacity, ftir fourier transform infrared spectroscopy, hplc high-performance liquid chromatography, sfe supercritical fluid extraction, mir middle infrared region, atr attenuated total reflection, gae gallic acid equivalents, abts 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), mi mitotic index. 84 nicoleta anca şuţan et al. introduction the genus ligularia from the asteracae family has been extensively researched from phytochemical point of view especially in the last years, having a real significance in the traditional medicines as a remedy for bronchitis, asthma, tuberculosis, haemoptysis, soothing pain, rheumatoid arthritis, coughs, inflammations, jaundice, scarlet fever and hepatic diseases (pinglin et al. 2008; xie et al. 2010). ligularia genus has many biological activities such as antibacterial activity, antifeedant and insecticidal activities, antihepatotoxicity, antioxidant, antithrombotic and anticoagulant activity (yang et al. 2011). the pharmaceutical studies as well as the chemical studies on ligularia species demonstrate the specific presence of constituents such aseremophilane-type sesquiterpenes (wu et al. 2016). over the glacial and interglacial period, a large number of species migrated from the asian continent to europe, including the glacial relict ligularia sibirica (l.) cass. which is the target of the present study. l. sibirica (l.) cass., a perennial hemicryptophyte species, has a short and thick rhizome with long lateral fasciculated roots, leaves with long petioles, and inflorescence stem straight up to 200 cm. the blooming period starts from july till the end of august (pop, 1960; šmídová et al. 2011). an overall small number of studies have been published with regards to the chemical composition and cytogenotoxic effects of l. sibirica (l.) cass. scientific literature highlights the use of this species as a cure for treating phlegm and for reducing cough (liao et al. 1002; tori et al. 2008; yuan et al. 2013). it is worth to mention here that there is not a clear situation regarding the l. sibirica (l.) cass. populations in romania related to its distribution in the natura 2000 sites (brînzan et al. 2013), and the protection of the species through the bern convention, annex i strictly protected flora species (berne treaty no. 104), complemented by the iucn threat status, data deficient (bilz et al. 2011). due to the fact that the species of community interest is protected by the habitats directive through annexes iib and ivb, geo 57/2007 (law 49/2011): annexes 3, 4 a and mentioned in the carpathian list of endangered species in the category near threatened (mihăilescu et al. 2015), the material used in our study was collected after a complex survey regarding the quantitative and qualitative analysis of l. sibirica (l.) cass. population in each site, as follows: apa roşie peat bog, hărman marsh and zănoaga gorges. the necessary material for study was harvested in minimum amounts, in order to preserve the existing vegetal communities. in the present study, we analyzed the chemical composition, antioxidant activity and cytogenotoxic properties of ethanol extracts obtained from roots and rhizomes of l. sibirica (l.) cass. our study represents a first step in estimating the possible phytotherapeutic applications of l. sibirica (l.) cass., and to understand to what extent this species can be incorporated into farming systems as a medicinal herb. materials and methods plant material roots and rhizomes of l. sibirica (l.) cass were harvested in august 2017, from 3 distinct sites, both in terms of habitat, pedo-climatic conditions, but also with the same level of anthropogenic activity, in order to decide which of them hold a higher potential for further studies. the first sample was collected from zănoagei gorges (lat n45°28’, long e25°25’), which are part of bucegi natural park an interesting limestone mountain system, the studied area being characterized by a coolwet climate type, the average annual temperature being of 4.9°c where the rainfalls varies with the altitude, covered by rendzina soils (beldie, 1967). the second sample of l. sibirica (l.) cass. was collected from hărman marsh (lat n45°43’, long e25°39’), an eutrophic marsh with hydric and alluvial soils and caco3 deposits, located in the braşov depression, with an annual temperature of 7°c and low rainfall. the last sample was collected from apa roşie peat bog (lat n46°10’, long e26°15’), located in nemira mountains and characterized by a cool-wet climate type rich in precipitations, with an average annual temperature of 2–4°c, occupying hydric soils without caco3 deposits (brînzan et al. 2013). after weighing the plant material, the fresh rhizomes and roots were washed in tap water to remove the soil, rinsed well in distilled water, pat dried with paper towel, and then minced at room temperature. reagents and chemicals the reagents used: gallic acid monohydrate acs reagent ≥ 98% and folin ciocalteu’s phenol reagent of 2m concentration, caffeic, cinnamic, ferulic, rosmarinic and syringic, catechin, myricetin, naringenin, methanol, and ethanol were from sigma-aldrich (usa). chlorogenic acid and quercetin were from alfa aesar (germany) and fluka, respectively. ethanol (hplc degree) was obtained from merck co. (darmstadt, germany). the used water was double-distilled. the carbon dioxide (99.5% purity) 85chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts used in sfe extraction and the rest of the reagents used in the experiments were purchased from commercial sources. the weight of the samples was measured on an analytical balance of shimadzu corporation with a precision of 0.1 mg. extraction procedures ethanolic extract was prepared by mixing 5 g of each type of plant material, roots and rhizomes, in 50 ml absolute ethanol stored for 8 h at room temperature (22°c). using whatman filter paper no. 1, the extracts were filtered and the resulted filtrates were used to indicate their cytogenotoxic potential using allium cepa test. the sfe extracts were achieved through the specific equipment, a pilot unity called sft-110 supercritical fluid extractor (supercritical fluid technologies, inc.). the temperature of the restrictor valve was adjusted to 50° or 60°c for all extraction processes. the extract was collected in a 50 ml glass vial. the output of co2 in the system was of 6.0 g/min. the yields of extraction were around 2%. for the co-solvent extraction (sample 4), using 1% and 2% ethanol in relation to the co2 the experiment was performed in conditions of 50°c, 250 bar and flow of co2 at 6 g/min (erdogan et al. 2011). in this case the extraction yield was 2.5% from the raw material. five samples of extracts were prepared: hărman mash – 1, zănoagei gorges – 2 and apa roşie peat bog – 3 through sfe extraction with co2, then it was added a sfe extraction with co2 and etoh as cosolvent from apa roşie peat bog – 4 and the last one was the absolute ethanol extraction from apa roşie peat bog – 5. the extracts of every single assay were individualized by studying the antioxidant activity through: tp, tf, dpph and teac. the functional groups were analysed using ftir in the region 4000–400 cm-1. the ethanolic extract (sample 5) was used to evaluate cytogenotoxic activity. evaluation of chemical composition of the rhizome and roots extracts ft-mir the extracts of l. sibirica (l.) cass. were investigated for their chemical composition: extract with co2 (1, 2, 3), co2 and ethanol (4), ethanolic extract (5). for ftir spectroscopy, was used a jasco 6300 spectrometer. the ftir spectroscopy of each rhizomes and roots extract was recorded in the mir region. an atr accessory equipped with a diamond crystal (pike technologies) was used for sampling. samples were carried out at 100 scans with resolution of 4 cm-1 in the regions of 4000-400 cm-1. spectra manager ii software was used for processed the spectra data. total phenolic content in order to assess the tp content of the material extract, it has been used the folin-ciocalteu method (singleton and rossi, 1965). the tp content was brought out by mixing 0.5 ml of sample or standard (gallic acid) with 5 ml of folin ciocalteu reagent and 4 ml of 1m sodium carbonate. it was measured the absorbance at 746 nm (shimadzu uv-1800 spectrophotometer) and the calibration curve with standard solutions of gallic acid concentrations ranging from 0.05 to 1 mgl-1 was traced. equation of standard cur ve was y=0.0067x+0.0105 (r2=0.9960). the results were expressed as mg of gae in 100 g of the rhizomes and roots. extract constituents the identification and quantification of selected phenolic acids and flavonoids in the plant extracts was performed by high performance liquid chromatography (hplc) using a varian 310-ms lc/ms/ms triple quadrupole mass spectrometer (usa) fitted with an electrospray ionization interface (esi). a sample of each dry plant extract was dissolved in hplc-grade methanol. the mobile phase was double distilled water/methanol, fr = 20:80. a varian c18 column (150 mm 9 3.0 mm; 5 mm) was used at a flow-rate of 0.6 ml/min, in an isocratic elution. the injection volume was 20 ml and triplicate injections were used for each sample. the drying gas was air at a pressure of 131 kpa and 250 °c, and the nebulizing gas was nitrogen at 276 kpa. the capillary voltage was set at the potential -4500 v for negative ionization. the resulting deprotonated molecular ion was selected by the first quadrupole; in the second quadrupole, the deprotonated molecular ion was fragmented by collision with an inert gas (argon) at a pressure of 0.2 pa; the fragments were analyzed in the third quadrupole. prior to these experiments, the tuning of the mass spectrometer was performed using a polypropylene glycol standard for both positive and negative modes. the determinations have been carried out in triplicate. the results are expressed as μg of selected phenolic acid or flavonoid per 1 g of dry extract. determination of antioxidant activity 1. dpph antioxidant activity. in order to determine the radical scavenging activity of the ethanol 86 nicoleta anca şuţan et al. extract of l. sibirica (l.) cass. the brand-williams dpph assay has been used (brand-williams et al. 1995). dpph is stable organic nitrogen radical, with a purple colour solution, which reacts with the antioxidant compounds. the method it stands on the measurement of the reducing ability of antioxidants toward this radical. the ability can be evaluated by measuring the decrease of dpph absorbance at 517 nm. the percentage of the dpph remaining was calculated using the equation: i% = [(ablanck-asample)/ablanck] x100, where: ablank is the absorbance for the blank (ethanol dpph. ethanolic solution) and asample is the absorbance for the sample mixed with 0.04 mg/ml dpph solution. the results were expressed as ic50 (the extract concentration which inhibits the activity of dpph by 50%). in short, to 4 ml of extract at 1 ml of dpph (0.04 mg/ ml) ethanol solution was added. after incubating for 30 min in dark, the absorbance of the resulting solutions was measured at 517 nm using uv-vis jasco 730 spectrophotometer (kedare and singh 2011). 2. teac assay. a stock solution of abts•+ was obtained after reacting the abts chemical compound with potassium persulfate. then the mixture has been left at dark at room temperature for 12–16 hours before use. the abts•+ used solution was prepared by dilution of this solution with ethanol till the absorbance was around 0.70 (pellegrini et al 2003). the absorbance was measured at 734 nm. the calibration curve was made with trolox (with concentrations between 0.125 and 2 mm). the results were done in mmol of trolox per 100g rhizomes and root. equation of standard curve was y=45.432x+20.334 (r2=0.9896). evaluation of cytogenetic effects of l. sibirica ethanol extract the two samples extracted with ethanol (sample 4 and 5) showed the best antioxidant properties, taking into account a higher volume of extraction, the sample 5 was studied in order to evaluate the cytogenotoxic effects of l. sibirica (l.) cass. extracts. the cytogenotoxic effects were evaluated through the changes of the mitotic indices and through the frequency of the phases of mitosis (prophase, metaphase, anaphase, telophase), as well as through the chromosomal aberrations frequency and the nuclear anomalies induced in root tip cells of a. cepa l. the onion bulbs (a local variety), about 4 cm diameter, have been checked to fit in the phytosanitary standards. in order to expose root primordia, the outer scales have been gently removed and the bulbs were placed in 30 ml containers, with the discoid stem being in contact with distilled water. the allium test was performed through static exposure, initially at the action of distilled water for 24 or 48 hours as well as at various concentrations of l. sibirica (l.) cass. ethanol extracts for 24 or 48 hours. further the samples were defined by the extracts concentration, respectively 5%, 10%, and 15%, as well as by the incubation time of onion roots, 24 hours and 48 hours (l 5% 24h, l 5% 48h, l 10% 24h, l 10% 48h, l 15% 24h, l 15% 48h). for the cytogenetic analysis roots with a length of about 10 mm were used. the roots were fixed in a mixture of absolute ethanol: glacial acetic acid 3:1 overnight and then transferred to 70º ethanol for long-term preservation. for each experimental variant, a number of 5 roots were subjected to attenuated hydrolysis with hcl 1n for 15 minutes at 60°c. fixed and macerated roots were stained with aceto-orcein solution 1% at 60°c for 15 minutes. the root tips were cut on a glass slide, in a drop of 45% glacial acetic acid, and used to perform microscopic slides using the squash technique. the coverslips were glued with several layers of nail polish, and the resulted microscopic slides were analyzed on an olympus cx-31 microscope, at a 400x magnification. the microscopic analysis aimed at establishing the numbers of cells at different stages of mitosis, the frequency of chromosomal and nuclear aberrations related to about 3000 cells for each experimental sample. the mi was determined as the percentage ratio between the number of cells in mitosis and the total number of analysed cells (tedesco et al. 2012). depending on the total number of cells that carry out mitosis, it was determined the percentage of the cells mitosis stages. the frequency of chromosomal aberrations and nuclear abnormalities was calculated as the percentage between the number of damaged cells and total number of cells in the appropriate stage of the cell cycle and mitosis. the results were statistical analysed using the ibm spss statistics 20 program. significant differences among samples were determined using the analysis of variance (one-way anova), as well as the duncan multiple comparison test. the p≤0.05 values were considered statistically significant. the graphs and tables were elaborated based on average values ± the standard error (se) of more independent experiments. results and discussion evaluation of chemical composition of the rhizome and roots extracts by ft-mir the atr-mir spectra (4000-400 cm-1) of each extract was registered and the specific wavenumbers 87chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts and intensities were considered in order to present the ftir-atr spectra of alcoholic and co2 extracts (figure s1 in the supplementary material), as well as the ft-ir absorption bands for rhizomes and roots extracts (table s1 in the supplementary material). the vibrational assignments for extracts were compared with literature data (szymanska-chargot and zdunek 2013). the identification of the functional groups was based on the ftir peaks attributed to stretching and bending vibrations. eight areas were identified in the mir domain and the fingerprint region was localized between 900 and 1760 cm-1 (areas 1-6) (zavoi et al. 2011). figure 1 shows the spectral regions 1-7 for sample analysis. area 1 (< 1000 cm-1) corresponds to c-h bending vibrations from isoprenoids, area 2 (997-1140 cm-1) to stretching vibrations c-o of carbohydrates, with signals at 1005, 1009, 1040, 1080 and 1136 cm-1, while area 3 (1150-1270 cm-1) corresponds to stretching vibrations of carbonyl c-o or o-h bendings. the intense peaks at 1040 cm−1 and to nearly 1080 cm-1 are attributed to characteristic functional groups of polyphenols. c-o (amide) and c-c stretchings vibrations appear in the region 4 (13001450 cm-1) region 5 (1500-1600 cm-1) corresponds to aromatic group and n-h bending vibrations. domain 6 is a complex one (1600-1760 cm-1) and corresponds to bending vibrations n-h (amino acids), c=o stretchings (aldehydes and ketones, esters) as well to free fatty acids (1766 cm-1) and glycerides (1738 cm-1). area 7 (2800-2900 cm-1), corresponds to c-h stretching vibrations of ch3 and ch2 from lipids, lipid derivatives, c-h (aldehydes). region 8 (3350-3600 cm-1) is assigned to stretching vibrations of oh (from water, alcohols, phenols, carbohydrates). the spectra show relatively more bands in the region of 400-700 cm-1. the inorganic constituents can be observed in the region between 470-480 and 530-540 cm-1. the variation may be due to the differences in the extraction and purification methods. antioxidant activity and phenol content of plant extracts there are a few studies about compounds with antioxidant activity from the genus ligularia. according to them, l. fischeri (ledeb.) turcz. showed high total phenolic contents (215.8 ± 14.2 mg gallic acid equivalent/g) with low contents of total flavonoid (86.9 ± 3.8 mg rutin equivalent/g) (lee et al. 2013). liu (2010) has reported the isolation and structural elucidation of the furanoeremophilane-type sesquiterpenes and benzofuran derivatives (part of them with phenol groups) from l. veitchiana (hemsl.) greenm., and some results about its biologic activity (liu et al. 2010). l. macrophylla (ledeb.) dc. has been reported to contain at least two f lavonoids: 6-acetyl-8-methoxy-2,3-dimethylchromen4-one and 4-14 (2s)-3’-hydroxy-5’,7-dimethoxyflavanone in root and rhizome, while in l. duciformis’s root have been identified some derivatives of sinapyl alcohol and coniferyl alcohol, with known antioxidant activities (yang et al. 2011). from our knowledge, phenols, flavonoids or other compounds with antioxidant activity from l. sibirica (l.) cass. were not assessed. the table 1 presents the tp and tf content of l. sibirica (l.) cass. rhizome and root extracts and its teac. the highest concentrations of tp and tf were obtained for extracts of l. sibirica (l.) cass. prepared by the co-solvent method (etoh), as expected. our results have also shown that supercritical co2 extractions were the least efficient, predictable since phenols are polar compounds and thus are less extractible by co2. something more effective than this was the extraction method at cold temperatures with ethanol (method used for extraction of phenols from vegetal matrix (santos-buelga et al. 2012). the polyphenol constituents present in extract 5, as identified and quantified by lc–esi–ms/ms, are listed in table 2. based on these results, it was concluded that the antioxidant properties of the extract originate in figure 1. atr-mir spectra registered for sample 3 (sfe extraction with co2). table 1. tp, tf and teac assay for l. sibirica rhizome and root extracts. sample tp (mg gallic acid equivalent/100g root) tf (mg quercetol equivalent/100g root) teac assay (mmoli tolox equivalent/100g root) 1 2.13 n. d. a 0.03 2 3.08 n. d. 0.04 3 10.01 n. d. 0.09 4 494.26 5.25 1.42 5 49.25 n. d. 0.47 a not detected. 88 nicoleta anca şuţan et al. two main classes of compounds, namely flavonoids (catechin, myricetin, naringenin, quercetin) and, to a greater extent, phenolic acids (caffeic, chlorogenic, cinnamic, ferulic, gallic, rosmarinic, syringic acid). evaluation of cytogenetic effects of l. sibirica ethanol extracts starting with fiskejio (1988) the allium test was considerably used for the evaluation of cytotoxic and genotoxic effects, as well as for the genoprotective potential of various natural or synthetic chemical compounds. the allium test follows some endpoints, such as the mitotic index, chromosomal aberrations and the nuclear anomalies (bonciu et al. 2018). for each experimental sample, the results were compared with the control. figure 2 shows the mi variation in onion roots exposed for 24 or 48 hours at the action of l. sibirica (l.) cass. extracts of 5%, 10% and 25% concentration. statistical analyzis of the microscopic results revealed that ethanol extracts of l. sibirica (l.) cass. have determined a significant decrease in the frequency of cells in various phases of mitosis. the highest mi was determined for the control for which the calculated percentage was 7.64%. l. sibirica (l.) cass. extracts had a statistically important mitoinhibitory effect, when compared to the control, showing an indirect correlation with their concentration. the lowest mi (0.7%) was calculated after the roots incubation in the extract with the concentration of 5% for 48h. this decreased frequency of cells in the mitosis was followed at a statistically significant difference by that determined in the experimental sample, defined by the 5% concentration, respectively 24h. in the root tip cells incubated in l. sibirica (l.) cass. extracts, the highest mi with a 6.19% value was calculated for the l 15% 24h experimental sample. the overall interpretation of the microscopic results revealed that the variation of the mitotic index was independent of the exposure time. the decrease of the mi in a. cepa l. meristematic root cells demonstrates the presence of bioactive substances with antiproliferative potential. the effects of l. sibirica (l.) cass. extracts on the distribution of the mitotic division phases in the onion meristem cells are summarized in figure 3. the frequency of prophase and metaphase has significantly varied, in a concentration dependent manner. therefore l 5% 24h has induced the highest percentage of prophases, significantly different compared to the other tested concentrations. when compared to the control, the ethanol extracts of l. sibirica (l.) cass. have induced an increase and a significant increase of metaphases frequency. the table 2. the phenolic profile of the plant extract-5, as determined by lc–esi–ms/ms. class compound compound concentration (μg compounds/g dry extract) phenolic acids caffeic acid 69.778±1.0229 chlorogenic acid 189.114±1.7604 cinnamic acid 0.438±0.0177 ferulic acid 1.856±0.0764 gallic acid 7.420±0.1071 rosmarinic acid 25.359±0.1292 syringic acid 2.532±0.0955 flavonoids catechin 6.901±0.0124 myricetin 0.343±0.0158 naringenin 0.704±0.0283 quercetin 13.032±0.6992 figure 2. the influence of l. sibirica extracts on the mitotic index in root meristem cells of allium cepa l. (the data are the averages ± se of three repetitions; a, b, c, d, e, the interpretation of statistical significance and of significant differences through the duncan test, p <0.05). figure 3. the influence of l. sibirica extracts on the distribution of mitotic division phases in the radicular meristem cells of a. cepa l. (the data are ± se averages of three repetitions; a, b, c, d, e, f, g, h, i, j, k – the interpretation of statistical significance and of significant differences through the duncan test, p <0.05). 89chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts highest metaphases percentage was determined in l 10% 24h and l 10% 48h variants. the calculated percentage values for anaphase and telophase were not significantly different compared to the control. on the basis of these observations it can be appreciated that the mi decline is due to cells arresting in the metaphase at a 5% extracts concentration. the overall interpretation of the results also reveals a global slowdown of mitotic progression at a higher concentration of the extracts. the decrease of the mi may be reflecting a cytogenotoxic effect of l. sibirica (l.) cass. ethanol extracts and could be interpreted as cellular death (yuet ping et al. 2012). according to nieva-moreno et al. (2005) cited by stojković et al. (2013) the main mechanisms of cancer chemoprevention are anti-mutagenesis and anti-proliferation or mitotic anti-progression. an extensive review by yang et al. in 2011, as well as other recent studies (dong et al. 2015) shows that among the bioactive chemical constituents of ligularia species, the most common phytochemical types are sesquiterpenes, which have demonstrated a strong cytotoxic or inhibitory activity on some tumor cell lines. among the volatile compounds identified by gas chromatography with mass spectrometry detection in the l. sibirica (l.) cass. extracts obtained by microwave assisted hydrodistillation, there were sabinene, limonene and terpinolene (monoterpenoids), as well as alkaloids that were most likely tussilagine and isotussilagine. in figure 4. chromosomal aberrations and nuclear abnormalities identified in meristematic root cells of a. cepa l. (a) telophase bridge – l 5% 24h; (b) c-mitosis – l 5% 48h; (c) laggards – l 5% 24h; (d) micronucleus and nuclear buds l 5% 48h; (e) giant cell – l 10% 48h; (f ) vagrant – l 10% 24h; (g) anaphase bridges – l 15% 24h; (h) altered nuclear shape – l 10% 48h; (h) fragments – l 5% 24h. 90 nicoleta anca şuţan et al. our study, the statmokinetic effect may be attributed to these terpenoids. many of the isolated terpenoids from natural sources have chemopreventive effects (akihisa et al. 2003; yang et al. 2011). nuclear and chromosomal aberrations observed in the root tip meristem cells of a. cepa l. are shown in figure 4. their frequency in the various experimental samples is shown in supplementary material (table s2). the average percentage value of the nuclear and chromosomal aberrations calculated for the control it was only 0.35 ± 0.07, and the percentages 1.53 ± 0.12, 3.46 ± 0.55, 3.25 ± 0.94, 1.83 ± 0.22, 1.25 ± 0.42 and 1.26 ± 0.35 for l 5% 24h, l 5% 48h, l 10% 24h, l 10% 48h, l 15 % 24h, l 15% 48h. the significant higher frequency of the chromosomal and nuclear aberrations in l 5% 48h, l 10% 24h, l 10% 48h variants, may be attributed to the potentially lower antioxidant activity as a result of the dilution of the tested extracts. this antimutagenic activity may be attributed to the secondary metabolites or to synergistic action of the antioxidant compounds. besides that, the ft-mir analysis has revealed the existence of oh group characteristic for phenols, to which may be due the genoprotective activity. oxidative dna damage can contribute to single double strand breaks formation or to oxidation of the purines or pyrimidines bases, inducing a genomic instability and also the development of cancer (chobotokova 2009). the biologically active phenols are known for the antioxidant properties exerted by the absorption of the free radicals (hidalgo and almajano 2017; shahidi et al 2015) as well as for the wide spectrum of biological and physiological actions (durazzo 2017). mitoinhibitory effect of l. sibirica (l.) cass. extracts was supported by the c-mitosis high percentage, which indicates the spindle disturbance in metaphase (firbas and amon 2014; bonciu 2018). however, the nuclear aberrations identified in the meristematic root cells were much more varied and had a higher frequency compared to chromosomal aberrations. the nuclear abnormalities defined by the nuclear morphology alterations in the interphase, such as micronuclei, nuclear buds, altered nuclei shape may be an indicator of some processes such as cell death or tumorigenesis (pellegrini 2003; nefic et al. 2013). in our study, except for the l 10% 24h experimental sample, changes in the shape of the nucleus was observed with a very low frequency. nuclear envelope proteins ensure the nucleus rigidity to the distortion associated forces caused by the cytoplasmic microtubules. these changes may be the consequence of microtubule generated forces in the cytoplasm, if any of the membrane nuclear proteins is inactive (nefic 2013; king et al. 2008). other studies have shown that the inactivation of the associated proteins with the endoplasmic reticulum affects the nucleus form (higashio et al. 2000; matynia et al. 2002; webster et al. 2009). moreover, the chromosomal aberrations and nuclear abnormalities identified by microscopic analysis are indicative of both clastogenic and aneugenic effects and of genotoxic damage. in this context, for a deeper estimation of the potential of this species additional in vitro and in vivo studies are required, aiming the extraction conditions, extract concentration and exposure time. conclusion the paper represents an essential step in offering more information about the antioxidant activity, chemical composition and cytogenotoxic activity of ethanol extracts obtained from roots and rhizomes of l. sibirica (l.) cass. the best results for phenols and flavonoids extraction from roots and rhizomes of l. sibirica (l.) cass. were obtained in supercritical co2 extraction with cosolvent (ethanol) followed by ethanol extraction. the ethanol extracts of l. sibirica (l.) cass. have demonstrated a very strong mitoinhibitory effect on in vitro root meristem cells of a. cepa l. chromosomal aberrations and nuclear abnormalities identified by the microscopic analysis are key indicators of both clastogenic and aneugenic effects, and of genotoxic damage. supporting information the version of this article contains supplementary material: vibration assignments for the rhizome and roots of l. sibirica (l.) cass. extracts; 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fingerprint and extraction yield of medicinal herb phenolics with hepatoprotective potential, as determined by uv-vis and ft-mir spectroscopy. not bot horti agrobo. 39:82-89. doi: http://dx.doi.org/10.15835/nbha3926278 caryologia international journal of cytology, cytosystematics and cytogenetics volume 73, issue 1 2020 firenze university press karyotypic investigation concerning five bromus species from several populations in iran sara sadeghian, ahmad hatami, mehrnaz riasat high genetic diversity and presence of genetic structure characterise the endemics ruta corsica and ruta lamarmorae (rutaceae) marilena meloni1, caterina angela dettori2, andrea reid3, gianluigi bacchetta2,4,*, laetitia hugot5, elena conti1 cytogenetic effects of c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. and mathematical analysis cihangir alaca1, ali özdemir1, bahattın bozdağ2, canan özdemir2,* clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 74(3): 107-117, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1124 caryologia international journal of cytology, cytosystematics and cytogenetics citation: hasan genç, bekir yildirim, mikail açar, tolga çetin (2021) statistical evaluation of chromosomes of some lathyrus l. taxa from turkey. caryologia 74(3): 107-117. doi: 10.36253/caryologia-1124 received: november 01, 2020 accepted: march 29, 2021 published: december 21, 2021 copyright: © 2021 hasan genç, bekir yildirim, mikail açar, tolga çetin. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. statistical evaluation of chromosomes of some lathyrus l. taxa from turkey hasan genç1, bekir yildirim2,*, mikail açar3, tolga çetin4 1department of science education, faculty of education, burdur mehmet akif ersoy university, burdur, 15100, turkey 2department of plant and animal production, burdur food, agriculture and livestock vocational school of higher education, burdur mehmet akif ersoy university, burdur, 15100, turkey 3department of plant and animal production, tunceli vocational school of higher education, munzur university, tunceli, 62000, turkey 4science teacher, republic of turkey ministry of national education, seydikemer, muğla, 48850, turkey *corresponding author. e-mail: bytr33@yahoo.com abstract. in this study, a statistical analysis was performed on mitotic metaphase chromosomes of 26 lathyrus taxa, four of which are endemic. anova, correlation analysis, pca and cluster analysis were performed to determine the relationships between taxa based on chromosomal criteria. the morphological similarities of plant taxa and chromosomal statistics results may not be always parallel to each other. according to the findings obtained as a result of analysis, the following taxa, which are close to each other were determined: l. hirsutus l. odoratus, l. brachypterus var. haussknechtii l. phaselitanus, l. stenophyllus l. chloranthus, l. gorgoni var. gorgoni l. nissolia l. pratensis, l. tuberosus l. annuus. keywords: lathyrus, chromosome, endemic, statistical analysis, turkey. introduction lathyrus l., a genus belonging to fabaceae family consisting of more than 200 taxa is distributed almost all over the world (allkin et al. 1986). the main diversity centers of lathyrus are the mediterranean region, asia minor and north america, as well as temperate south america and east africa (klamt & schifino-wittmann 2000). in flora europaea, 54 species of lathyrus were reported from different areas of europe (tutin et al. 1968). lathyrus is represented by 79 taxa in turkey, 25 of which are endemic to turkey (davis 1970; davis et al. 1988; güner et al. 2000; genç & şahin 2008; genç 2009; genç & şahin 2011). some agriculturally important species of the genus lathyrus are grown for use as forage or human food (yamamoto et al. 1984; genç & şahin 2001). seeds of some lathyrus species are used in the preparation of regional human food in different countries in the world (kumar 1997; uncuer et al. 2016). 108 hasan genç et al. a lot of studies such as taxonomical, cytotaxonomical, morphological, anatomical, etc. have been carried out on lathyrus taxa which are so important in terms of agriculture. caryological studies were conducted on l. saxatilis (vent.) vis., l. vinealis boiss. & noe, l. inconspicuus l., l. setifolius l. (şahin & altan 1990) and l. rotundifolius willd. subsp. miniatus (bieb. ex stev.) davis, l. cassius boiss., l. cicera l., l. aphaca l. var. modestus p.h. davis (şahin 1993). anatomical, morphological and palynological features of l. inconspicuus, l. vinealis from orobastrum (taub.) boiss. section and l. sativus l., l. hirsutus l. from cicercula (medic.) gren. & godr. section were investigated qualitatively and quantitatively (mantar et al. 2002; 2003). there are also studies conducted on the cytotaxonomical properties of l. brachypterus čel. var. haussknechtii (širj.) davis, l. spathulatus čel., l. ochrus (l.) dc., l. odoratus l., l. belinensis n. maxted & d. j. goyder, l. clymenum l., l. phaselitanus hub-mor. & davis and the morphological characteristics of grass pea (l. sativus) (genç et al. 2009; grela et al. 2010). a numerical taxonomic study on 54 of 58 lathyrus taxa in flora of turkey was conducted (doğan et al. 1992). this study was carried out using the statistical evaluation of the findings of previous cytotaxonomical studies conducted by us (şahin et al. 1998; şahin et al. 2000; genç & şahin 2001; genç et al. 2009). in this study, by applying statistical analysis to the metaphase chromosome organization, it is aimed to see whether the taxa with the same chromosome morphology are similar taxonomically or not. material and methods material plant specimens and seeds belonging to lathyrus taxa were collected from natural habitats in turkey during 1995-2007. some plant specimens were stored at personal herbarium of genç, while other herbarium specimens were stored at the fuh (fırat university, elazığ, turkey) and gul (süleyman demirel university, isparta, turkey) herbariums. the eight sections of 28 investigated taxa according to morphological classification in davis 1970 and güner et al. 2000 are given in table 1. methods chromosome measurements determination of chromosome number and karyotype analyses of taxa were performed at mitotic metaphases. the seeds were germinated at room temperature in petri dishes covered with cotton. when the root tips reached 1 cm in length, they were cut off and pretreated with saturated paradichlorobenzene solution for 4 hours. at the end of the pretreatment process, root tips were washed and fixed with acetic acid-ethyl alcohol (1/3 v/v) for 24 hours. then, the root tips were washed again and stored in 70% ethyl alcohol at 2-4 °c (sharma & gupta 1982). after washed root tips had been hydrolyzed in 1 n hcl for 10-15 min at 60 °c. feulgen method was used in the dyeing process (elçi 1982; sharma & gupta 1982). squashed preparats were prepared using root tips. the karyotypes were discussed according to levan et al. (1964). chromosomes measurements of lathyrus taxa are given in table 2. data analyses for the analysis of karyotype characteristics, the following methods and formulas were used. the measurements were performed on haploid data sets. the following traits in each karyotype were measured: tlc (total length of chromosomes), mtlc (mean of total length of chromosomes), max (maximum length of chromosome), min (minimum length of chromosomes), mla (mean of long arms), msa (mean of short arms), mrv (mean of r value), mdv (mean of d value), mar (mean of arm ratio), mci (mean of chromosome index), mrlc (mean of relative length of chromosomes), drl (difference of range of relative length), tf% (total form percentage), s% (relative length of shortest chromosome), a1 (intrachromosomal asymmetry index), a2 (intertable 1. the sections of the investigated lathyrus taxa. section taxa platystylis l. brachypterus var. haussknechtii, l. digitatus (bieb.) fiori, l. spathulatus pratensis l. pratensis l., l. laxiflorus (desf.) o. kuntze subsp. laxiflorus lathyrus l. tuberosus l., l. belinensis, l. odoratus [l. odoratus is cultivated as an ornamental plant in turkey (davis 1970)] orobastrum l. sphaericus retz., l. inconspicuus, l. tauricola p. h. davis, l. setifolius cicercula l. annuus l., l. gorgoni parl. var. gorgoni, l. cicera, l. sativus, l. stenophyllus boiss. & heldr., l. phaselitanus, l. hirsutus, l. chloranthus boiss. clymenum l. clymenum, l. ochrus nissolia l. nissolia l. aphaca l. aphaca var. affinis (guss.) arc, l. aphaca var. pseudoaphaca (boiss.) davis, l. aphaca var. modestus 109statistical evaluation of chromosomes of some lathyrus l. taxa from turkey table 2. chromosomes measurements of lathyrus taxa (ch. no: chromosome no, c: total length of the chromosome, l: length of the long arm, s: length of the short arm, sat.: satellite). ch. no c l s sat. ch. no c l s sat. l. brachypterus var. haussknechtii l. digitatus 1 6.48 3.64 2.84 1 9.16 4.88 4.28 2 6.29 2.86 2.06 1.37 2 7.73 4.43 3.30 3 5.32 3.16 2.16 3 7.21 4.12 3.09 4 5.10 2.96 2.14 4 6.85 3.99 2.86 5 4.78 2.79 1.99 5 6.78 4.18 2.60 6 4.64 2.91 1.73 6 6.37 3.82 2.55 7 4.32 2.70 1.62 7 6.03 3.66 2.37 l. spathulatus l. pratensis 1 8.00 4.39 3.61 1 7.62 3.53 2.77 1.32 2 7.22 3.56 2.46 1.20 2 6.19 3.91 2.28 3 6.42 3.97 2.45 3 5.99 3.94 2.05 4 6.11 3.71 2.40 4 5.61 3.64 1.97 5 5.76 3.43 2.33 5 5.36 3.45 1.91 6 5.50 3.18 2. 32 6 4.99 3.18 1.81 7 5.09 2.95 2.14 7 4.65 2.77 1.88 l. laxiflorus subsp. laxiflorus l. tuberosus 1 9.27 5.33 3.94 1 8.10 3.76 2.86 1.48 2 8.48 4.93 2.49 1.06 2 6.51 4.15 2.36 3 8.15 5.24 2.91 3 6.27 4.18 2.09 4 7.83 5.30 2.53 4 5.95 3.76 2.19 5 7.43 4.89 2.54 5 5.77 3.77 2.00 6 7.04 4.78 2.26 6 5.53 3.47 2.06 7 6.32 3.54 2.78 7 5.01 3.11 1.90 l. belinensis l. sphaericus 1 6.56 3.70 2.86 1 6.92 3.83 3.09 2 5.88 3.47 2.41 2 6.31 3.64 2.67 3 5.20 3.18 2.02 3 5.84 3.49 2.35 4 4.95 3.11 1.84 4 5.51 3.49 2.02 5 4.81 3.06 1.75 5 5.27 3.22 2.05 6 4.64 2.76 1.88 6 5.08 3.18 1.90 7 4.48 2.75 1.73 7 4.68 2.74 1.91 l. inconspicuus l. tauricola 1 4.66 2.15 1.74 0.77 1 6.06 2.64 2.22 1.20 2 4.57 2.65 1.92 2 5.25 3.43 1.82 3 4.23 2.74 1.49 3 4.81 3.01 1.80 4 3.97 2.48 1.49 4 4.63 3.10 1.53 5 3.78 2.52 1.26 5 4.30 2.82 1.48 6 3.57 2.20 1.37 6 4.16 2.54 1.62 7 3.28 2.16 1.12 7 3.98 2.39 1.59 l. setifolius l. annuus 1 7.16 3.34 2.22 1.60 1 8.28 3.42 3.03 1.83 2 5.30 3.64 1.66 2 6.44 4.31 2.13 3 4.99 3.68 1.31 3 6.14 4.06 2.08 4 4.72 3.22 1.50 4 6.02 3.79 2.23 5 4.36 2.87 1.49 5 5.78 3.74 2.04 6 4.08 2.52 1.56 6 5.54 3.56 1.98 7 3.46 2.02 1.44 7 4.97 2.80 2.17 l. gorgoni var. gorgoni l. cicera 1 7.50 3.04 2.86 1.60 1 5.29 3.46 1.83 2 6.32 4.12 2.20 2 4.79 3.07 1.72 3 6.08 4.04 2.04 3 4.59 2.89 1.70 4 5.84 3.82 2.02 4 4.39 2.78 1.61 ch. no c l s sat. ch. no c l s sat. 5 5.68 3.68 2.00 5 4.18 2.23 1.55 6 5.43 3.45 1.94 6 3.81 2.23 1.58 7 4.77 2.74 2.03 7 3.30 2.04 1.26 l. sativus l. stenophyllus 1 6.21 3.74 2.47 1 7.16 4.21 2.95 2 5.85 2.49 1.96 1.40 2 6.31 4.15 2.16 3 5.53 3.34 2.19 3 5.90 3.75 2.15 4 5.36 3.44 1.92 4 5.38 3.46 2.12 5 5.11 3.31 1.80 5 5.25 3.24 2.01 6 4.87 3.29 1.58 6 4.90 2.86 2.04 7 4.47 2.71 1.76 7 4.65 2.64 2.01 l. phaselitanus l. hirsutus 1 6.41 3.64 2.77 1 8.36 4.78 3.58 2 5.43 2.24 1.90 1.29 2 7.08 4.93 2.15 3 5.33 3.29 2.04 3 6.53 4.52 2.01 4 5.16 3.26 1.90 4 6.30 4.15 2.15 5 4.85 2.85 2.00 5 5.93 4.01 1.92 6 4.73 2.83 1.90 6 5.48 3.55 1.93 7 4.36 2.65 1.71 7 5.07 2.89 2.18 l. chloranthus l. clymenum 1 7.35 4.12 3.23 1 7.22 3.68 2.13 1.41 2 6.56 3.67 2.89 2 6.62 4.72 1.90 3 5.94 3.77 2.17 3 5.76 4.06 1.70 4 5.65 3.54 2.11 4 5.36 3.18 2.18 5 5.34 3.46 1.88 5 4.13 2.93 1.20 6 4.99 3.07 1.92 6 3.40 2.03 1.37 7 4.70 2.90 1.80 7 2.83 1.80 1.03 l. ochrus l. nissolia 1 6.00 2.58 2.05 1.37 1 6.86 2.74 2.45 1.67 2 5.55 3.72 1.83 2 6.56 4.20 2.36 3 5.26 3.74 1.52 3 5.89 3.87 2.02 4 5.02 3.32 1.70 4 5.58 3.46 2.12 5 4.68 2.85 1.83 5 5.20 3.40 1.80 6 4.00 2.28 1.72 6 4.97 3.19 1.78 7 3.33 1.86 1.47 7 4.36 2.58 1.77 l. aphaca var. affinis l. aphaca var. pseudoaphaca 1 6.77 3.03 2.49 1.25 1 5.53 2.48 1.90 1.15 2 5.26 3.52 1.74 2 4.44 3.11 1.33 3 5.05 3.34 1.71 3 4.13 2.69 1.44 4 4.87 3.20 1.67 4 4.10 2.60 1.50 5 4.71 3.34 1.37 5 3.90 2.70 1.20 6 4.39 3.02 1.37 6 3.80 2.51 1.29 7 4.12 2.71 1.41 7 3.45 2.24 1.21 l. aphaca var. modestus l. odoratus 1 5.89 2.29 2.26 1.34 1 7.49 4.49 3.00 2 5.25 3.10 2.12 2 6.66 4.73 1.93 3 4.88 3.13 1.75 3 6.38 4.44 1.94 4 4.61 3.01 1.60 4 6.11 4.07 2.04 5 4.40 2.85 1.55 5 5.70 3.73 1.97 6 4.23 2.61 1.62 6 5.45 3.61 1.84 7 3.95 2.43 1.52 7 5.13 2.84 2.29 110 hasan genç et al. chromosomal asymmetry index), and a (degree of asymmetry). both arm ratios were assumed to be equally affected (adhikary 1974). all karyotype formulas were determined based on huziwara (1962) (tf%), levan et al. (1964) (r and d values), zarco (1986) (a1 and a2), watanabe (1999) (a), peruzzi and eroğlu (2013) (ci) as well. the abbreviations were taken from the rezeai et al. (2014) (rlc%, drl, s%). the formulas are as follows. formulas d value=length of the long arm of chromosome-length of the short arm of chromosome drl=(maximum relative length)(minimum relative length) (li = lengths of a long arm, si = lengths of a short arm, n = haploid chromosome number). (n = number of homologous chromosome pairs, bi = the average length of short arms in every homologous chromosome pair, bi = the average length of long arms in every homologous chromosome pair). (s = standard deviation of chromosome lengths, = mean of chromosome lengths). a data matrix was constructed according to 17 karyotype characteristics mentioned in table 3. the principal component analysis (pca) was used based on the data matrix (jolliffe 2002). the cluster analysis was made using gower (dis)similarity index for determining the relationships between chromosome properties of lathyrus taxa (romesburg 2004). in addition, the pearson correlation coefficient (r) analysis was performed to see strong and weak relationships between chromosome properties. at the same time, shapiro wilk normality test was performed. then, the one-way analysis of variance (anova) was performed to determine whether the difference between the data was statistically significant. all the analyses were carried out with paleontostatistics (past) (hammer et al. 2001). results statistical studies on the chromosome morphologies of 26 lathyrus taxa were conducted. images of the mitotic metaphase chromosomes of lathyrus taxa are given in figure 1. karyotype characteristics of lathyrus taxa are given in table 3. the chromosome properties of taxa are summarized in the stacked bar (figure 2). shapiro – wilk normality test and one way anova test results are given in figure 3 and table 4. according to the values obtained with the formulas using chromosome morphological properties of taxa, the data show a normal distribution (figure 3), and then the one-way anova test is statistically significant according to the p-value(p<0.05) (table 4). correlation analysis according to the correlation analysis, there are relations between the r-values of chromosome data according to the significance level less than p <0.05. especially a strong positive relationship between tlc, mtlc, max, min, mla, msa, and a strong negative relations between mrv, mdv and mar, mci and a1 and a values (figure 4). principal component analysis (pca) according to pca (table 5, figure 5), the first two components explained the majority of the variation according to chromosome data between the taxa. while the first two components explain 57.98 and 38% of the variance, respectively, these characters explained 96% of the total variation. the characters that affected the variation most were s%, tlc, drl and tf%. similarly, since some variables (such as a, a1) have lower values than calculations, the effects on variation in pca have been low. cluster analysis according to the upgma algorithm gower index cluster analysis results, the taxa are divided into 4 groups (figure 6). these groups are also divided into subgroups among themselves. especially l. hirsutus l. odoratus, l. brachypterus var. haussknechtii l. phaselitanus, l. stenophyllus, l. chloranthus, l. gorgoni var. gorgoni l. nissolia l. pratensis, l. tuberosus l. annuus taxa are closely related. 111statistical evaluation of chromosomes of some lathyrus l. taxa from turkey figure 1. mitotic metaphase chromosomes of lathyrus taxa (1. l. brachypterus var. haussknechtii, 2. l. digitatus, 3. l. spathulatus, 4. l. pratensis, 5. l. laxiflorus subsp. laxiflorus, 6. l. tuberosus, 7. l. belinensis, 8. l. sphaericus, 9. l. inconspicuus, 10. l. tauricola, 11. l. setifolius, 12. l. annuus, 13. l. gorgoni var. gorgoni, 14. l. cicera, 15. l. sativus, 16. l. stenophyllus, 17. l. phaselitanus, 18. l. hirsutus, 19. l. chloranthus, 20. l. clymenum, 21. l. ochrus, 22. l. nissolia, 23. l. aphaca var. affinis, 24. l. aphaca var. pseudoaphaca, 25. l. aphaca var. modestus, 26. l. odoratus). 112 hasan genç et al. ta bl e 3. k ar yo ty pe c ha ra ct er is tic s of l at hy ru s ta xa ( t lc : t ot al l en gh t of c hr om os om es , m t lc ( m ea n of t ot al l en gt h of c hr om os om es , m a x : m ax im um l en gt h of c hr om os om e, m in : m in im um l en gt h of c hr om os om e, m la : m ea n of l on g a rm s, m sa : m ea n of s ho rt a rm s, m rv : m ea n of r v al ue , m dv : m ea n of d v al ue , m a r : m ea n of a rm r at io , m c i: m ea n of c hr om os om e in de x, m r lc : m ea n of r el at iv e le ng th o f c hr om os om es , d r l: d iff er en ce o f r an ge o f r el at iv e le ng th , t f% : t ot al f or m p er ce nt ag e, s % : r el at iv e le ng th o f sh or te st c hr om os om e, a 1: in tr ac hr om os om al a sy m m et ry i nd ex , a 2: in te rc hr om os om al a sy m m et ry i nd ex ). la th yr us t ax a t lc m t lc m a x m in m la m sa m rv m dv m a r m c i m r lc d r l t f% s% a 1 a 2 a l. b ra ch yp te ru s va r. ha us sk ne ch tii 36 .9 3 5. 28 6. 48 4. 32 3. 00 2. 08 1. 47 0. 92 0. 69 0. 41 14 .2 8 5. 85 39 .3 7 66 .6 7 0. 90 0. 14 0. 18 4 l. d ig ita tu s 50 .1 3 7. 16 9. 16 6. 03 4. 15 3. 01 1. 41 1. 15 0. 72 0. 42 14 .2 8 6. 24 41 .9 9 65 .8 3 0. 90 0. 13 0. 16 6 l. s pa th ul at us 44 .1 0 6. 30 8. 00 5. 09 3. 60 2. 53 1. 43 1. 07 0. 70 0. 41 14 .2 8 6. 60 40 .1 6 63 .6 2 0. 90 0. 15 0. 17 6 l. p ra te ns is 40 .4 1 5. 77 7. 62 4. 65 3. 49 2. 09 1. 68 1. 39 0. 60 0. 37 14 .2 8 7. 35 36 .3 0 61 .0 2 0. 91 0. 16 0. 24 9 l. la xi flo ru s su bs p. la xi flo ru s 54 .5 2 7. 79 9. 27 6. 32 4. 86 2. 78 1. 79 2. 08 0. 58 0. 36 14 .2 8 3. 96 35 .6 7 68 .1 8 0. 92 0. 11 0. 27 2 l. tu be ro su s 43 .1 4 6. 16 8. 10 5. 01 3. 74 2. 21 1. 71 1. 53 0. 59 0. 37 14 .2 8 7. 16 35 .8 4 61 .8 5 0. 91 0. 15 0. 25 8 l. b el in en si s 36 .5 2 5. 22 6. 56 4. 48 3. 15 2. 07 1. 54 1. 08 0. 65 0. 39 14 .2 8 5. 70 39 .6 8 68 .2 9 0. 91 0. 13 0. 21 0 l. s ph ae ri cu s 39 .6 1 5. 66 6. 92 4. 68 3. 37 2. 28 1. 50 1. 08 0. 67 0. 40 14 .2 8 5. 65 40 .3 7 67 .6 3 0. 90 0. 12 0. 19 5 l. in co ns pi cu us 28 .0 6 4. 01 4. 66 3. 28 2. 41 1. 48 1. 66 0. 93 0. 62 0. 38 14 .2 8 4. 92 37 .0 3 70 .3 9 0. 91 0. 12 0. 24 1 l. ta ur ic ol a 33 .1 9 4. 74 6. 06 3. 98 2. 85 1. 72 1. 68 1. 08 0. 61 0. 38 14 .2 8 6. 27 36 .3 4 65 .6 8 0. 91 0. 14 0. 35 5 l. s et ifo liu s 34 .0 7 4. 87 7. 16 3. 46 3. 04 1. 60 1. 94 1. 44 0. 54 0. 35 14 .2 8 10 .8 6 32 .8 1 48 .3 2 0. 92 0. 22 0. 30 4 l. a nn uu s 43 .1 7 6. 17 8. 28 4. 97 3. 67 2. 24 1. 67 1. 43 0. 62 0. 38 14 .2 8 7. 67 36 .2 7 60 .0 2 0. 91 0. 16 0. 24 0 l. g or go ni v ar . g or go ni 41 .6 2 5. 95 7. 50 4. 77 3. 56 2. 15 1. 68 1. 40 0. 62 0. 38 14 .2 8 6. 56 36 .2 6 63 .6 0 0. 91 0. 13 0. 24 1 l. c ic er a 30 .3 5 4. 34 5. 29 3. 30 2. 67 1. 61 1. 65 1. 06 0. 61 0. 37 14 .2 8 6. 55 37 .0 7 62 .3 8 0. 91 0. 14 0. 24 2 l. s at iv us 37 .4 0 5. 34 6. 21 4. 47 3. 19 1. 95 1. 65 1. 23 0. 62 0. 38 14 .2 8 4. 65 36 .5 8 71 .9 8 0. 91 0. 10 0. 23 8 l. s te no ph yl lu s 39 .5 5 5. 65 7. 16 4. 65 3. 47 2. 20 1. 58 1. 27 0. 64 0. 39 14 .2 8 6. 35 39 .0 4 64 .9 4 0. 91 0. 14 0. 21 9 l. p ha se lit an us 36 .2 7 5. 18 6. 41 4. 36 2. 97 2. 03 1. 47 0. 93 0. 69 0. 41 14 .2 8 5. 65 39 .2 0 68 .0 2 0. 90 0. 12 0. 18 5 l. h ir su tu s 44 .7 5 6. 39 8. 36 5. 07 4. 12 2. 27 1. 86 1. 84 0. 56 0. 35 14 .2 8 7. 35 35 .5 7 60 .6 4 0. 92 0. 16 0. 28 9 l. c hl or an th us 40 .5 3 5. 79 7. 35 4. 70 3. 50 2. 28 1. 57 1. 22 0. 65 0. 39 14 .2 8 6. 54 39 .4 8 63 .9 4 0. 91 0. 15 0. 21 7 l. c ly m en um 35 .3 2 5. 05 7. 22 2. 83 3. 20 1. 64 1. 96 1. 55 0. 53 0. 34 14 .2 8 12 .4 3 32 .5 9 39 .2 0 0. 93 0. 30 0. 31 0 l. o ch ru s 33 .8 4 4. 83 6. 00 3. 33 2. 91 1. 73 1. 69 1. 17 0. 63 0. 38 14 .2 8 7. 89 35 .8 1 55 .5 0 0. 91 0. 18 0. 23 8 l. n is so lia 39 .4 2 5. 63 6. 86 4. 36 3. 35 2. 04 1. 65 1. 30 0. 62 0. 38 14 .2 8 6, 34 36 .2 8 63 .5 5 0. 91 0. 14 0. 23 7 l. a ph ac a va r. affi ni s 35 .1 7 5. 02 6. 77 4. 12 3. 17 1. 68 1. 95 1. 48 0. 53 0. 36 14 .2 8 7. 53 33 .4 4 60 .8 6 0. 92 0. 16 0. 31 1 l. a ph ac a va r. ps eu do ap ha ca 29 .3 5 4. 19 5. 53 3. 45 2. 62 1. 41 1. 90 1. 21 0. 54 0. 35 14 .2 8 7. 09 33 .6 3 62 .3 9 0. 92 0. 15 0. 30 0 l. a ph ac a va r. m od es tu s 33 .2 1 4. 74 5. 89 3. 95 2. 77 1. 77 1. 61 1. 00 0. 65 0. 39 14 .2 8 5. 84 37 .4 0 67 .0 6 0. 91 0. 13 0. 21 9 l. o do ra tu s 42 .9 2 6. 13 7. 49 5. 13 3. 99 2. 14 1. 90 1. 84 0. 55 0. 35 14 .2 8 5. 50 34 .9 7 68 .4 9 0. 92 0. 12 0. 29 7 113statistical evaluation of chromosomes of some lathyrus l. taxa from turkey discussion to best of our knowledge no statistical analysis of chromosomes belonging to such a number of taxa in the genus lathyrus is available in literature. in this study, 26 taxa belonging to 8 sections of genus lathyrus were investigated. among the investigated taxa, lathyrus brachypterus var. haussknechtii, l. belinensis, l. tauricola, l. phaselitanus are endemic to turkey. in some studies, the cluster analysis data can yield similar trees with the morphological classification of the taxa (açar & satıl 2019; dirmenci et al. 2019). figure 3. shapiro wilk normality test. figure 2. lathyrus taxa karyotype characteristics of stacked bar. 114 hasan genç et al. according to the data of doğan et al (1992), obtained in the study using forty morphological characters, the lathyrus genus was divided into two subgenus and nine sections. however, the results obtained do not show compatibility with davis (1970). the statistical results obtained in our study are also not consistent with davis (1970). this situation suggests that the statistical results obtained from taxa may not always be completely compatible with morphological features. however, in our study, the caryological data were not generally similar to the morphological classification of the taxa, but similarities and close relationships among some taxa were also similar to morphological data (figure 6). according to the pca scatter diagram, like the cluster analysis results, the sections were observed to be intertwined in the distribution formations of taxa (figure 5). cluster analysis made according to karyotype features successfully distinguished the taxa from each other. however, it was also found to be an inconsistency with morphological classification. according to the caryological examination, l. hirsutus l. odoratus, l. brachypterus var. haussknechtii l. phaselitanus, l. stenophyllus l. chloranthus, l. gorgoni var. gorgoni l. nissolia l. pratensis, l. tuberosus l. annuus taxa are closely related (figure 6). l. hirsutus and l. odoratus are morphologically similar, and have been observed to be close to each other as a result of caryological analysis. l. brachypterus var. haussknechtii and l. phaselitanus differ morphologically and are located in different sections; however, these taxa are similar according to caryological data we obtained. l. stenophyllus and l. chloranthus belonging to the same section are similar to each other according to caryological analysis. and  conversely, l. gorgoni var. gorgoni, l. nissolia and l. pratensis belonging to different sections are similar to each other according to the analysis of its metaphase chromosome morphology. similarly, the two species, l. tuberosus and l. annuus from different sections are similar to each other according to cluster analysis. table 4. one way anova test results. test for equal means sum of sqrs df mean square f p (same) between groups: 133605 16 8350.28 1464 0 within groups: 2423.77 425 5.70299 permutation p (n=99999) total: 136028 441 1e-05 omega2: 0.9815 table 5. principal component analysis of lathyrus taxa showing the eigen values of total variance. pc eigen value % variance 1 56.2106 57.978 2 36.8804 38.04 3 3.66766 3.783 figure 4. correlation analysis between karyotype characteristics. 115statistical evaluation of chromosomes of some lathyrus l. taxa from turkey in terms of similarities of the taxa, the presence of satellite and distribution was not found to be significant. this study revealed that the morphological similarities of plant taxa and chromosomal statistics results may not be always parallel to each other. figure 6. cluster analysis according to karyotype characteristics (same coloured taxa are located in the same section except l. odoratus. it is an ornamental plant). figure 5. pca scatter plot diagram (different colors refer to different sections and the lines with variables indicate the effect and direction of variation). 116 hasan genç et al. references 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(asteraceae) species that around the van lake in turkey pelin yilmaz sancar1,*, semsettin civelek1, murat kursat2 molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant jinxin cheng1,*, dingyu hu1, yaran liu2, zetian zhang1, majid khayatnezhad3 genetic variations and interspesific relationships in salvia (lamiaceae) using scot molecular markers songpo liu1, yuxuan wang1, yuwei song1,*, majid khayatnezhad2, amir abbas minaeifar3 development of female gametophyte in gladiolus italicus miller (iridaceae) ciler kartal1, nuran ekici2,*, almina kargacıoğlu1, hazal nurcan ağırman1 colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae) harshita dwivedi*, girjesh kumar statistical evaluation of chromosomes of some lathyrus l. taxa from turkey hasan genç1, bekir yildirim2,*, mikail açar3, tolga çetin4 use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater treatment and water of the kamniška bistrica river (slovenia) peter firbas1, tomaž amon2 the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bo shi1,*, majid khayatnezhad2, abdul shakoor3,4 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(2): 149-161, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1235 caryologia international journal of cytology, cytosystematics and cytogenetics citation: jing ma, wenyan fan, shujun jiang, xiling yang, wenshuai li, di zhou, amir abbas minaeifar (2021) molecular techniques in the assessment of genetic relationships between populations of consolida (ranunculaceae). caryologia 74(2): 149-161. doi: 10.36253/caryologia-1235 received: march 05, 2021 accepted: july 20, 2021 published: october 08, 2021 copyright: © 2021 jing ma, wenyan fan, shujun jiang, xiling yang, wenshuai li, di zhou, amir abbas minaeifar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. molecular techniques in the assessment of genetic relationships between populations of consolida (ranunculaceae) jing ma1, wenyan fan1,*, shujun jiang1, xiling yang1, wenshuai li1, di zhou1, amir abbas minaeifar2,* 1.agronomy college, heilongjiang bayi agricultural university, daqing, heilongjiang, 163319, china 2 department of biology, payame noor university, p.o. box19395-3697 tehran, iran corresponding author. e-mail: fwyjsj2021@126.com; aaminaeifar@pnu.ac.ir abstract. genetic diversity studies are essential to understand the conservation and management of plant resources in any environment. the genus consolida (dc.) gray (ranuculaceae) belongs to tribe delphinieae. it comprises approximately 52 species, including the members of the genus aconitella spach. no detailed random amplified polymorphic dna (rapd) studies were conducted to study consolida genetic diversity. therefore, we collected and analyzed 19 species from 12 provinces of regions. overall, one hundred and twenty-seven plant specimens were collected. we showed significant differences in quantitative morphological characters in plant species. unweighted pair group method with arithmetic mean and principal component analysis (pca) divided consolida species into two groups. all primers produced polymorphic amplicons though the extent of polymorphism varied with each primer. the primer opa06 was found to be most powerful and efficient as it generated a total of 24 bands of which 24 were polymorphic. the mantel test showed correlation (r = 0.34, p=0.0002) between genetic and geographical distances. we reported high genetic diversity, which clearly shows the consolida species can adapt to changing environments since high genetic diversity is linked to species adaptability. present results highlighted the utility of rapd markers and morphometry methods to investigate genetic diversity in consolida species. our aims were 1) to assess genetic diversity among consolida species 2) is there a correlation between species genetic and geographical distance? 3) genetic structure of populations and taxa. keywords: consolida, population structure, gene flow, network, genetic admixture. introduction genetic diversity is a vital feature that helps plant species survive in an ever-changing environment, and it sheds light on understanding the phylogenetic affinity among the species (erbano et al. 2015; ellegren and galtier 2016; turchetto et al. 2016 ). quite a significant number of genetic resources and materials programs of plant species have been carried out to preserve 150 jing ma et al. the plant species worldwide. scientific data indicate that genetic diversity plays a pivotal role in conservation programs (gomez et al. 2005; frankham 2005; cires et al. 2013). the genus consolida (dc.) gray (ranuculaceae) belongs to tribe delphinieae. it comprises approximately 52 species, including the members of the genus aconitella spach. iran is one of the richest countries for the genus in south-west asia, since it has 24 species (iranshahr et al., 1992). the genus consolida s.f. gray was considered as a separate genus based on one species (c. regalis) by gray (1821), who worked on british flora. but some researchers considered consolida as a section of delphinium (de candole 1824; boissier 1867; huth 1895; nevskii 1937). unlike the others based on annual life form, single spured petal, single follicle compared to 3 or 5 sessile follicles of delphinium recognized consolida as a separate genus (tutin et al. 1964; davis 1965; munz 1967; hayek 1970; iranshahr 1992; ertugrul et al. 2016; khalaj 2013). kemularia-nathades (1939) recognized a new genus aconitopsis from species of consolida based on peculiar formation of the petal, upper sepal, and spur. the name aconitopsis was later rejected by sojak (1969) and being replaced by aconitella because of nomenclature priority. some researchers have studied these genera taxonomically (soo 1922; munz 1967 ; davis 1965; iranshahr et al., 1992; constantinidis et al., 2001). consolida has about 40 species, of which 19 have been recorded from iran. aconitella with ca. 10 species (3 species in iran) and 31 species of delphinium (species in iran) are centred in irano-turanian and mediterranean phytogeographic regions (trifonova, 1990; hasanzadeh et al. 2017). consolida has been separated from delphinium by de candolle based on single spurred petals, one follicle and annual life cycle and has occurred in separate section. finally, it introduced as a separate genus by gray in 1821 (triffonova, 1990). based on phylogenetic studies of jabbour and renner (2011), aconitella is part of consolida, both being embedded in delphinium. the jabbour & renner (2011) results showed that consolida diverged from delphinium relatives in the early to middle miocene, a period of increasing aridity, caused primarily by decrease in sea level in the mediterranean (hayek 1970; iranshahr 1992; ertugrul et al. 2016) and desertification in asia (triffonova 1990). some biosystematic studies have carried out in various field such as chromosomal studies (trifonova 1990; koeva 1992; hong, 1986) chemical studies (aitzetmuller et al.1999), palynological studies (munz, 1967) and phylogenetic investigations by using dna sequence data (johansson 1995; ro et al.1997; jabbour and renner 2011; 2012; yosefzadeh et al. , 2012). in the recent molecular studies (jabbour and renner 2001; 2012) it was showed that consolida and aconitella form a clade embeded in delphinium and also aconitella is embedded within consolida. the jabbour and renner (2011) results showed that consolida diverged from delphinium relatives at least in the early of middle miocene. genetic diversity studies are usually tapped due to molecular markers. molecular markers are an excellent method to disentangle phylogenetic association between species and population. among molecular methods or markers, rapd (random amplified polymorphic dna) are sensitive to detect variability among individuals of species. rapd method is cost-effective and can work with limited sample quantities. in addition to this, rapd can amplify and target genomic regions with potential and several markers (esfandani-bozchaloyi et al. 2017a). taxonomical systematics studies were conducted in the past to identify the consolida species. according to the best of our knowledge, there is no existing rapd data on genetic diversity investigations in iran. we studied one hundred and twenty-seven samples. our aims were 1) to assess genetic diversity among consolida species 2) is there a correlation between species and geographical distance? 3) genetic structure of populations and taxa 4) are the consolida species able to exchange genes? materials and methods plant materials 19 consolida species were collected from different regions of iran (table 1). these species were studied via morphological and molecular methods. 127 plant samples (10-25 per plant species) were examined for morphometry purposes (figure 1). the random amplified polymorphic dna analysis method was limited to 110 samples. according to previous references, all the species were identified (iranshahr, 1992; ertugrul et al., 2016; khalaj, 2013). voucher specimens were deposited in herbarium of azad islamic university (haiu). morphometry we studied 18 qualitative and 7 quantitative morphological characters (table 2). data were transformed (mean= 0, variance = 1) prior to ordination . euclidean distance was implemented to cluster and ordinate plant species (podani 2000). 151molecular techniques in the assessment of genetic relationships between populations of consolida random amplified polymorphic dna we extracted dna from fresh leaves. leaves were dried. dna extraction was carried out according to the previous protocol (esfandani-bozchaloyi et al. 2019; niu et al., 2021; sun et al., 2021). dna quality was checked on an agarose gel to confirm the purity. we amplified the dna with the aid of rapd primers (operon technology, alameda, canada). these primers belonged to opa, opb, opc, opd sets. we selected those primers (10) which could show clear bands and polymorphism (table 3). overall, the polymerase chain reaction contained 25μl volume. this 25 volume had ten mm trishcl buffer, 500 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp; 0.2 μm of a single primer; 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). we observed the following cycles and conditions for the amplification. five minutes initial denaturation step was carried out at 94°c after this forty cycles of 1 minute at 94°c were observed. then 1-minute cycle was at 52-57°c followed by two minutes at 72°c. in the end, the final extension step was performed for seven to ten minutes at 72°c. we confirmed the amplification steps while observing amplified products on a gel. each band size was confirmed according to 100 base pair molecular ladder/standard (fermentas, germany). data analyses ordination methods such as multidimensional scaling and principal coordinate analysis were also performed (podani 2000). the morphological difference among species and population was assessed through analysis of variance (anova). pca analysis (podani 2000) was done to find the variation in plant population morphological traits. multivariate and all the necessary calculations were done in the past software, 2.17 (hammer et al. 2001). to assess genetic diversity, we encoded rapd bands as present and absent. numbers 1 and 0 were used to show the presence and absence of bands. it is essential to know the polymorphism infortable 1. location and herbarium accession numbers of the studied populations of consolida species collected by mehri in iran. no sp. locality latitude longitude altitude (m) sp1 c. tehranica (boiss.) rech.f. tehran: damavand tehran: rodehen golestan, ramian 38°52’37” 32°50’03” 35°50’03” 47°23’92” 51°52’08” 48°52’08” 1144 1066 1234 sp2 c. camptocarpa (fisch. &c.a.mey.) nevski khorassan: sarakhs, 14 km to mozduran 32°50’03” 51°24’28” 1990 sp3 c. lorestanica lranshahr, lorestan: 110 km khorram abad markazi:arak 29°20’07” 36°14’14” 51°52’08” 51°18’07” 1610 1807 sp4 c. leptocarpa nevski golestan: golestan national park, mirzabailoo 38°52’37” 47°23’92” 1144 sp5 c. persica (boiss.) grossh. fars: bamo national park fars: shiraz keramn: jiroft zanjan: abhar 33°57’12” 47°57’32” 2500 sp6 c. aucheri (boiss.) iranshahr khorassan: neyshabur 34°52’373 48°23’92” 2200 sp7 c. anthoroidea (boiss.) schrödinger east azerbaijan: kaleybar, cheshme ali akbar 38°52’373 47°23’92” 1144 sp8 c. hohenackeri (boiss.) grossh. arak: komayjan, pass of chehregan village, the margin road 35°50’03” 51°24’28” 1700 sp9 c. stocksiana nevski golestan: golestan national park, mirzabailoo 36°14’14” 51°18’07” 1807 sp10 c. rugulosa schrödinger esfahan: semirom to keikha 32°36’93” 51°27’90” 2500 sp11 c. ambigua (l.) ball & heywood tehran: between karaj and eshtehard 37°07’02” 49°44’32” 48 sp12 c. orientalis (gray) schrödinger azarbaijan: 20 km from jolfa to marand 28°57’22” 51°28’31” 430 sp13 c. regalis s.f. gray azarbaijan: tabriz 30°07’24” 53°59’06” 2178 sp14 c. oliveriana (dc.)schrod. kermanshah: 31 km to ghasre-shirin 28°57’22” 51°28’31” 288 sp15 c. flava (dc.)schrod khuzestan: do-gonbadan 34°46’10” 48°30’00” 1870 sp16 c. trigonelloides (boiss.) munz fars: bamo national park 35° 37’77” 46°20’25” 1888 sp17 c. oligantha (boiss.)schrod ardabil 33°47’60” 46°07’58” 1250 sp18 c. linorioides (boiss.) munz, esfahan: ghamishloo protected area 37°07’02” 49°44’32” 48 sp19 c. rugulosa f. paradoxa (bunge) iranshahr hamedan: khan abad 28°57’22” 51°28’31” 288 152 jing ma et al. mation content and marker index (mi) of primers because these parameters serve to observe polymorphic loci in genotypes (ismail et al. 2019). marker index was calculated according to the previous protocol (heikrujam et al. 2015). other parameters such as the number of polymorphic bands (npb) and effective multiplex ratio (emr) were assessed. gene diversity associated characteristics of plant samples were calculated. these characteristics include nei’s gene diversity (h), shannon information index (i), number of effective alleles (ne), and percentage of polymorphism (p% = number of polymorphic loci/number of total loci) (shen et al. 2017). unbiased expected heterozygosity (uhe), and heterozygosity were assessed in genalex 6.4 software (peakall and smouse 2006). neighbor-joining (nj) and networking were studied to fathom genetic distance plant populations (huson and bryant 2006; freeland et al. 2011). the comparison of genetic divergence or genetic distances, estimated by pairwise fst and related statistics, with geographical distances by mantel test is one of the most popular approaches to evaluate spatial processes driving population structure. the mantel test was performe as implemented in past. for this, nei genetic distance was determined for rapd data, while geographic distance of past was determined for geographical data. it is calculated based on the sum of the paired differences among both longitude as well as latitude coordinates of the studied populations (podani 2000). as we were interested in knowing the genetic structure and diversity, we also investigated the genetic difference between populations through amova (analysis of molecular variance) in genalex 6.4 (peakall and smouse 2006). gene flow (nm) which were calculated using popgene (version 1.31) program [yeh et al. 1999]. gene flow was estimated indirectly using the formula: nm = 0_25(1 _ fst)/fst. we also did structure analysis to detect an optimum number of groups. for this purpose, the evanno test was conducted (evanno et al. 2005). results morphometry significant anova results (p <0.01) showed differences in quantitative morphological characters in plant species. principal component results explained 80% variation. firs component of pca demonstrated 57% of the total variation. traits such as presence of petiole in caulin leaves, overtopping the bract from fruit, proportion of petal middle lobes to lateral lobes, presence of hair on the filament positively correlated with firs component (>0.7). the second and third components explained characters such as number of petal lobes, position of hair on filament, colour of anther, shape of follicle beak, shape of follicle. unweighted pair group method with arithmetic mean (upgma) and principal component analysis (pca) plots showed symmetrical results (figure 2). generally, plant specimens belonging to different species were separated from each other due to differences in morphology. our pca results also confirmed the application of morphological characters in separating and clustering the species in separate groups (figure 2). species identification and genetic diversity the primers, i.e., opd-05, could amplify plant (consolida ) dna (figure 3). 133 polymorphic bands were generated and amplified. amplified products ranged from 100 to 3000 bp. we recorded the highest polymorphic bands for opa-06. opd-08 had the lowest polymorphic bands. the average polymorphic bands ranged to 13.3 for each primer. the polymorphic information content (pic) had values in the range of 0.38 (opc-04) to 0.57 (opb-02). primers had 0.52 average polymorphic information content values. marker index (mi) values were 4.18 (opd-05) to 8.87 (opa-06), with an average of 6.87 per primer. effecfigure 1. map of distribtion of populations consolida species in iran; sp1= c. tehranica; sp2= c. camptocarpa; sp3= c. lorestanica; sp4= c. leptocarpa; sp5= c. persica; sp 6= c. aucheri; sp7= c. anthoroidea; sp8= c. hohenackeri; sp9= c. stocksiana; sp10: c. rugulosa; sp11: c. ambigua ; sp12= c. orientalis; sp13= c. regalis; sp14= c. oliveriana; sp15= c. flava; sp16= c. trigonelloides; sp17= c. oligantha; sp18= c. linorioides; sp19= c. rugulosa f. paradoxa. 153molecular techniques in the assessment of genetic relationships between populations of consolida tive multiplex ratio (emr) values are useful to distinguish genotypes. in our study, we reported 9.34 (opd08) to 16.55 (opa-05) emr values. emr values averaged 13.57 per primer (table 3). all the necessary genetic features calculated of 19 consolida species are shown (table 4). c. linorioides depicted unbiased expected heterozygosity (uhe) in the range of 0.15. c. orientalis showed a 0.34. uhe value heterozygosity had a mean value of 0.23 in overall consolida species. shannon information was high (0.32) in c. orientalis. c. linorioides showed the lowest value, 0.20. mean values for shannon information was 0.22. the observed number of alleles (na) ranged from 0.201 to 0.555 in c. regalis and c. oligantha. the effective number of alleles (ne) was in the range of 0.671.876 for c. flava and c. leptocarpa. analysis of molecular variance (amova) test highlighted genetic differences among consolida species (p = 0.01). amova revealed significant difference among the studied populations. it also revealed that, 46% of total genetic variability was due to within population diversity and 54% was due to among population genetic differentiation (figure 4). genetic similarity and dissimilarity assessed through genetic statistics (gst) showed significant differences i.e., (0.77, p = 0.001) and d_est values (0.256, p = 0.01). the neighbor-joining tree also revealed two major groups (figure 5). the neighbor-joining tree also repeated the same pattern as indicated in figures 2. in current work, molecular findings also coincided with the traditional taxonomical (morphology) approaches for consolida species. the neighbor-joining tree divided consolida species into two groups (figure 4). populations belonging to c. tehranica; c. camptocarpa; c. lorestanica; c. aucheri; c. rugulosa; c. orientalis and c. hohenackeri were in the first group. on the other hand, the second group consisted of two sub-groups. c. stocksiana; c. ambigua ; c. oliveriana; c. flava formed the first sub-group. c. trigonelloides; c. oligantha; c. linorioides; c. leptocarpa and c. persica formed the second sub-group. these groups table 2. characters used in this study from iran. character character states length of basal leaves 0: <55 mm 1: <55mm number of bracts 0: 0 1: 1 2: 2 broad of petal 0: 3-9 mm 1: 8-16 mm number of bracteole 0: variable 1: constant length of bracteole 0: ≤ 9mm 1: ≥12 mm length of spure 0: ≤ 25 mm 1: ≥ 25 mm shape of spure 0: curved 1: erect hair on lateral sepal 0: scattered 1: on the middle vein number of petal lobes 0: 5 1: 3 proportion of petal middle lobes to lateral lobes 0: equal 1: shorter 2: longer hair on the filament 0: absent 1: present hair on filament 0: wing 1: total of filament colour of anther 0: brown 2: yellow shape of follicle beak 0: erect 1: curved shape of follicle 0: falciform 1: erect hair on the follicle surface 0: absent 1: present shape of fruit stalk 0: antrorse 1: erect 2: decurved proportion of pedicle to flower 0: shorter 1: longer proportion of pedicle to fruit 0: shorter 1: longer presence of petiole in caulin leaves 0: present 1: absent presence of hair on the leaf surface 0: present 1: absent overtopping the bract from flower 0:yes 1: no overtopping the bract from fruit 0:yes 1: no position of bract 0: near the flower 1: far from the flower spure 0: present 1: absent 154 jing ma et al. and sub-groups were formed due to molecular differences among the individuals of consolida. gene flow (nm) was relatively low (0.54) in consolida species. genetic identity and phylogenetic distance in the consolida members are mentioned (table 5). c. camptocarpa and c. anthoroidea were genetically closely related (0.907) to each other. c. persica and c. rugulosa were dissimilar due to low (0.702) genetic similarity. mantel test after 5000 permutations produced significant correlation between genetic distance and geographical distance in these populations (r = 0.34, p = 0.0002). therefore, the populations that are geographically more distant have less amount of gene flow, and we have isolation by distance (ibd) in consolida species. the most popular approaches for estimating divergence include calculation of genetic distances and variance partitiontable 3. rapd primers and other parameters. note: tnb the number of total bands, npb: the number of polymorphic bands, ppb (%): the percentage of polymorphic bands, pi: polymorphism index, emr, effective multiplex ratio; mi, marker index; pic, polymorphism information content for each of cbdp primers. primer name primer sequence (5’-3’) tnb npb ppb pic pi emr mi opa-05 5’-aggggtcttg-3’ 15 15 100.00% 0.46 5.34 16.55 6.44 opa-06 5’-ggtccctgac-3’ 24 24 100.00% 0.57 5.88 14.56 8.87 opb-01 5’-gtttcgctcc-3’ 22 22 100.00% 0.55 6.23 12.23 6.47 opb-02 5’-tgatccctgg-3’ 15 14 91.74% 0.57 5.66 14.56 5.67 opc-04 5’-ccgcatctac-3’ 13 12 92.31% 0.38 3.21 15.60 5.55 opd-02 5’-ggacccaacc-3’ 14 13 97.74% 0.37 5.66 9.56 5.67 opd-03 5’-gtcgccgtca-3’ 13 12 92.31% 0.54 8.21 10.23 5.55 opd-05 5’ -tgagcggaca-3’ 12 12 100.00% 0.47 7.32 11.55 4.18 opd-08 5’-gtgtgcccca-3’ 11 9 80.89% 0.43 6.56 9.34 7.18 opd-11 5’-agcgccattg-3’ 10 10 100.00% 0.49 4.25 14.11 7.87 mean 14.5 13.3 96.22% 0.52 6.32 13.57 6.87 total 145 133 figure 2. pca plot of morphological characters revealing species delimitation in the consolida species; sp1= c. tehranica; sp2= c. camptocarpa; sp3= c. lorestanica; sp4= c. leptocarpa; sp5= c. persica; sp 6= c. aucheri; sp7= c. anthoroidea; sp8= c. hohenackeri; sp9= c. stocksiana; sp10: c. rugulosa; sp11: c. ambigua ; sp12= c. orientalis; sp13= c. regalis; sp14= c. oliveriana; sp15= c. flava; sp16= c. trigonelloides; sp17= c. oligantha; sp18= c. linorioides; sp19= c. rugulosa f. paradoxa. 155molecular techniques in the assessment of genetic relationships between populations of consolida ing among and within populations using wright’s fst and other related statistics, such as gst, ast, rst, θst and φst. for instance, the fst gives an estimate of the balance of genetic variability among and within populations, and is an unbiased estimator of divergence between pairs of populations under an island-model in which all populations diverged at the same time and are linked by approximately similar migration rates. however, migration rates usually vary proportionally with geographical distances, so that pairwise fst estimates between pairs of populations vary. evanno test performed on structure analysis produced the best number of k = 10 (figure.6). the structure plot has revealed the allele combination difference among the studied populations and the occurrence of genetic admixture among them. inspite of genetic stratification and isolation by distance observed in consolida species structure plot (figure 7) showed high degree of gene flow among the studied populations, although the studied populations contained some specific alleles. for example populations 8-14 and 2,19 (differently colored segments in figure.7), they shared some similar alleles too. for example, it showed genetic similarity between populations 3 and 4 (similarly colored), as well as 5, 6 and 15,16. the plants of population 1 had some alleles of population 10. similarly, population 5,6 had some alleles of population 14. nonetheless, we were able to construct a consensus tree that agreed with our molecular (rapd) and morphological findings (results not shown). the consolida populations showed divergence due to genetic and morphological characters. figure 3. gel electrophoresis image of dna fragments produced by opd-03 of consolida species. sp1= c. tehranica; sp2= c. camptocarpa; sp3= c. lorestanica; sp4= c. leptocarpa; sp5= c. persica; sp 6= c. aucheri; sp7= c. anthoroidea; sp8= c. hohenackeri; sp9= c. stocksiana; sp10: c. rugulosa; sp11: c. ambigua ; sp12= c. orientalis; sp13= c. regalis; sp14= c. oliveriana; sp15= c. flava; sp16= c. trigonelloides; sp17= c. oligantha; sp18= c. linorioides; sp19= c. rugulosa f. paradoxa. l = ladder 100 bp. arrows show polymorphic bands. table 4. genetic diversity parameters in the studied consolida species. sp n na ne i he uhe %p c. tehranica 12.000 0.287 1.233 0.271 0.184 0.192 51.91% c. camptocarpa 5.000 0.358 1.430 0.28 0.20 0.29 43.50% c. lorestanica 6.000 0.299 1.029 0.231 0.18 0.23 44.38% c. leptocarpa 5.000 0.462 1.876 0.288 0.29 0.28 62.05% c. persica 8.000 0.399 1.167 0.24 0.21 0.113 52.88% c. aucheri 5.000 0.336 1.034 0.23 0.25 0.19 51.83% c. anthoroidea 4.000 0.344 1.042 0.28 0.23 0.27 57.53% c. hohenackeri 5.000 0.455 1.234 0.277 0.24 0.22 55.05% c. stocksiana 3.000 0.255 1.021 0.25 0.18 0.22 42.15% c. rugulosa 3.000 0.288 1.024 0.23 0.35 0.30 64.30% c. ambigua 5.000 0.462 1.095 0.288 0.25 0.27 62.05% c. orientalis 8.000 0.399 1.167 0.322 0.398 0.344 65.77% c. regalis 8.000 0.201 1.00 0.23 0.17 0.17 42.23% c. oliveriana 5.000 0.341 1.058 0.24 0.27 0.20 53.75% c. flava 5.000 0.455 0.67 0.277 0.24 0.22 55.05% c. trigonelloides 8.000 0.499 1.067 0.24 0.13 0.24 49.26% c. oligantha 6.000 0.555 1.020 0.22 0.25 0.28 43.53% c. linorioides 10.000 0.431 1.088 0.20 0.12 0.15 41.53% c. rugulosa f. paradoxa 3.000 0.255 1.021 0.25 0.18 0.22 47.15% abbreviations: n = number of samples, na= number of different alleles; ne = number of effective alleles, i= shannon’s information index, he = genetic diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations. 156 jing ma et al. discussion the consolida is a relatively complex taxonomic group, and several morphological characters make it difficult to identify and classify consolida species (ertugrul et al., 2016). given the complexity, it is necessary to explore other methods that could complement the traditional taxonomical approach (erbano et al. 2015). advent and developments in molecular techniques have enabled plant taxonomists to utilize molecular protocols to study plant groups (erbano et al. 2015). consolida is an evolved genus with precise synapomorphies (reduction of carpels from three or more to one, complete loss of lateral petals, spur consisting of one petal) that are not found in any other species of delphinium and aconitum. most consolida species are adapted to the mediterranean type climate or more arid climate types of the irano-turanian zone (ertugrul et al., 2016). pronounced periods of drought in these areas have certainly favoured the exclusive annual life cycle of consolida. the biogeography of the genus indicates that turkey, in particular anatolia (c. 29 taxa) should be considered as the center of diversity, with further radiation of species into the irano-turanian area (c. 23 taxa), greece (c. 10 taxa) and countries around the mediterranean. consolida forms a coherent, monophyletic clade with delphinium and aconitum. some authors propose a direct evolution line of consolida from delphinium (tamura 1966). we examined genetic diversity in consolida by morphological and molecular methods. we mainly used rapd markers to investigate genetic diversity and genetic affinity in consolida. our clustering and ordination techniques showed similar patterns. morphometry results clearly showed the utilization or significance of morphological characters in consolida species. pca results also confirmed the application of morphological characters to separate consolida species. the present study also hightable 5. analysis of molecular variance (amova) of the studied species. source df ss ms est. var. % φpt among pops 20 1701.364 55.799 12.189 54% 54% within pops 120 354.443 1.905 4.55 46% total 150 2055.807 16.060 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance; φpt: proportion of the total genetic variance among individuals within an accession, (p < 0.001). figure 4. amova test of the studied populations. figure 5. neigbor-joingin tree produced while using rapd data. branch support values are given as bootstrap (bp) value above branches. 157molecular techniques in the assessment of genetic relationships between populations of consolida lighted that morphological characters such as bract exerting from fruit, presence of spore, shape of spore apex, the number of petal, the number of petal lobes, could delimit the consolida group. the consolida species highlighted morphological differences. we argue that such a dissimilarity was due to differences in quantitative and qualitative traits. present findings on morphological differences are in line with the previous studies (iranshahr, 1992; ertugrul et al., 2016; khalaj 2013). genetic structure and gene flow polymorphic information content (pic) values are useful to detect genetic diversity. the current study recorded average pic values of 0.52. this value is sufficient to study genetic diversity in the population (kempf et al. 2016). high genetic diversity among the consolida population was reported in the present study. the previous scientific data (kurata et al. 2019) supports our current high diversity results. genetic analysis conducted via analysis of molecular variance and structure showed genetic differences among the species. according to bru¨tting et al (2012) sampled 53 populations from 6 arable plant species throughout central germany. random amplified polymorphic dna analyses (rapd) were applied to calculate measures of genetic diversity at the population level and genetic differentiation. their results showed that genetic diversity was found to be lowest in bupleurum rotundifolium and anagallis foemina, and highest in consolida regalis and nigella arvensis. the highest levels of genetic differentiation were observed among populations of an. foemina and b. rotundifolium but within populations in all other species. ust values differed strongly ranging between 0.116 for c. regalis and 0.679 for an. foemina. patterns of genetic structure were related to the red list status for all the species studied except an. foemina, for which it should consequently be raised. them data confirm that even relatively recent threats are accompanied by detrimental genetic structure. figure 6. delta k plot of evanno’s test based on structure analysis. figure 7. structure plot of consolida species based on k = 10 of rapd data. 158 jing ma et al. genetic diversity and population size our data suggest that the 19 study species differed highly in their genetic diversity. populations of c. rugulosa; c. ambigua and c. orientalis showed the highest diversity, followed by c. leptocarpa and c. anthoroidea. lowest values were found in c. regalis and c. linorioides. it is widely accepted that the breeding system influences gene diversity dramatically ( mable and adam 2007). for example nybom and bartish (2004) extracted from literature that selfing taxa have a mean he of around 0.09. in contrast, plant species with a mixed or outcrossing breeding system show an he of around 0.22 to 0.26. for our study species, c. tehranica; c. camptocarpa; c. lorestanica; c. leptocarpa; c. anthoroidea; c. stocksiana; c. ambigua; c. orientalis and c. regalis tend to have a mixed breeding system and that c. oliveriana; c. flava; c. trigonelloides are more outcrossing species. this assumption is certainly true for c. regalis because it is not self pollinating (svensson and wigren 1986). as inflorescences of outcrossing taxa are generally larger than inflorescences of selfing species (hill et al. 1992), lower genetic diversity could be an indication of higher fragmentation, as fragmentation leads to limited gene flow (leimu et al. 2010). in fragmented populations pollinators struggle to reach the more distant populations and may even also decline in abundance (potts et al. 2010). however, the relationship is consistent with population genetic theory, predicting that genetic drift is particularly important in small populations (ellstrand and elam 1993) and population size is positively correlated to genetic variation (leimu et al. 2006). molecular markers (rapd) and morphometry analysis were useful to study genetic diversity and population structure in consolida species identification. all the species had distinct genetic differentiation. present results highlighted isolation and limited gene flow are the main deterministic factors that shape the consolida population. we also reported high genetic diversity, which clearly shows the consolida species can adapt to changing environments since high genetic diversity is linked to species adaptability. references aitzetmuller k,tsevegsuren n, werner f. 1999. seed oil fatty acid patterns of aconitumdelphiniumhelleborous complex (ranunculaceae). pl syst evol 213: 37-47. boissier e. 1841. voyage botanique dans le midi de l’espagne, livraison 16, vol. 2. paris: gide & cie. table 6. the matrix of nei genetic similarity (gs) estimates using scot molecular markers among 19 consolida species.sp1= c. tehranica; 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cytogenetics citation: somayeh saboori, masoud sheidai, zahra noormohammadi, seyed samih marashi, fahimeh koohdar (2021) genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates. caryologia 74(3): 151-168. doi: 10.36253/ caryologia-1089 received: september 20, 2020 accepted: september 09, 2021 published: december 21, 2021 copyright: © 2021 somayeh saboori, masoud sheidai, zahra noormohammadi, seyed samih marashi, fahimeh koohdar. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid zn: 0000-0003-3890-9001 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 1department of biology, science and research branch, islamic azad university, tehran, iran 2 faculty of life sciences and biotechnology, shahid beheshti university, tehran, iran 3date palm & tropical fruits research center, horticultural sciences research institute, agricultural research, education and extension organization (areeo), ahwaz, iran *corresponding authors. e-mail; marjannm@yahoo.com, z-nouri@srbiau.ac.ir abstract. date palm (phoenix dactylifera l.) is one of the oldest domesticated fruit trees. for future breeding program, knowledge on genetic structure of cultivars is necessary. therefore, the present study was performed with the following aims: 1to provide data on genetic diversity and genetic structure of 36 date palm cultivars, 2to provide data on the association between fruit characteristics and the genetic features of the cultivars. we used nine ssrs and est-ssr loci for our genetic investigation. the most of ssr loci obtained have a high gst value (0.70), and therefore have a good discrimination power for date palm cultivar differentiation task. k-means clustering grouped date palm cultivars either in two broad clusters, or in 16 smaller genetic groups. this was supported by delta k = 2 of the structure analysis. amova produced significant genetic difference among date palm cultivars (phipt = 0.70, p = 0.001). new genetic differentiation parameters estimated also produced significant difference among date palm cultivars (g’st (nei) = 0.673, p = 0.001; g’st (hed) = 0.738, p = 0.001). test of assignment revealed that some of the cultivars have 33-66% misassignment, probably due to genetic admixture. heatmaps of genetic versus morphological and agronomical characters in date palm cultivars differed from each other showing the cultivars morphological changes is not merely related to their genetic content. it points toward the potential role played either by environmental conditions or local selection practice. the new findings can be utilized in future conservation and breeding of date palms in the country. keywords: date palm, genetic variability, genetic structure, pst index, population assignment. introduction plant species of the family palmae/ or arecaceae are distributed mainly in tropical and subtropical areas, but a few species grow at higher latitudes in 152 somayeh saboori et al. the southern hemisphere. the main diversification centers of these taxa are the equatorial coast of africa, oceania, the brazilian coast, the amazon, indonesia and the antilles (moore & uhl, 1982). the palm trees greatly contribute to the economy of the people around the world. different sort of fruits, seeds, the ‘palmito’, honeys, ‘sagu’ (material with starch extracted from the centre of the trunks), different drinks from the sap or the fruits, and crystallized sugar from the sap, are only some of the palm tree products consumed by mankind (rivas et al. 2012). among date palm tree species, african oil palm (elaeis guineensis), the coconut tree (cocos nucifera), the date palm (phoenix dactylifera) and the betel nut palm (areca catechu), are considered as the main cultivated plant species. they are cultivated in about 14.585.811, 11.208.072, 1.264.611 and 834,878 hectares respectively (fao, 2010). the date palm (phoenix dactylifera l.) is one of oldest domesticated fruit trees, which its wild plants records date back to 5000-6000 bc in iran, egypt and pakistan (el hadrami & el hadrami, 2009). this important food plant produced about 7.048.089 tons of date only in algeria, saudi arabia, egypt, the arab emirates, iraq, iran, morocco, oman, pakistan and tunis (fao, 2010). successful future development of date palm industry and cultivation depends on proper evaluating, utilizing, and conserving date palm genetic resources, as well as efficient assessment of the present and potential future cultivars (jaradat, 2014). one of the main tasks in plant genetic resources investigation is evaluation of available genetic diversity. genetic diversity of date palm would be studied at different levels, including between cultivars, populations or individual clones, as well as between different geographical regions. genetic variability may be measured at the morphological, physiological, biochemical or molecular levels (jaradat, 2014). the degree and distribution amount of genetic diversity may vary among different oases and populations, due to historical, geographical, ecological and anthropogenic factors (jaradat, 2014). mankind can also influence the genetic diversity of date palms by his activities like cultivation practice, social behavior, artificial selection as well as spatiotemporal exchange and movement of germplasm (jaradat, 2014). date palm cultivars are reported to have a common genetic back-ground and therefore, proper differentiation of the cultivars and individual plant assignments in each cultivar is a difficult task and mistakes are inevitable in that. this may also be due to genetic admixture of the date palms (sharifi et. al. 2018, saboori et al. 2019, 2021 a,b, gros-balthazard et al. 2020). “in general, the question of individual assignment to population samples resulted in the development of different statistical methods distinguishing between resident individuals that are ‘’mis-assigned’’ (have a genotype that is most likely to occur in a population other than the one in which the individual was sampled) by error from real immigrant individuals (i.e., type i error, piry et al. 2004). “in assignment investigation, monte carlo resampling methods have been proposed to identify a statistical threshold beyond which individuals are likely to be excluded from a given reference population sample. the principle behind these resampling methods is to approximate the distribution of genotype likelihoods in a reference population sample and then compare the likelihood computed for the to-be-assigned individual to that distribution (piry et al. 2004)”. a combination of stable morphological characters and molecular markers may be used in date palm genetic diversity studies and discrimination among closely related date palm cultivars and clones (johnson et al. 2015). different molecular markers (neutral, multilocus and dna-sequence based markers) have been utilized in date palm genetic diversity investigations as well as cultivar phylogeny analyses (see for example, sharifi et al. 2018, saboori et al. 2019, saboori et al. 2020). among these molecular markers, the nuclear microsatellite markers (simple sequence repeat, ssrs) are known to be precise and accurate in genetic finger printing of date palm cultivars (ahmed et al., 2013, johnson et al. 2015, zehdi-azouzi. et al. 2015). moreover, zhao et al. (2013) developed several est-ssr (expressed sequence tagssr) gene based markers to investigate date palm (phoenix dactylifera l.) genetic finger printing. these genetic markers may provide a valuable genetic and genomic tool for further genetic research and varietal development in date palm, such as diversity study, qtl mapping, and molecular breeding. date palm comprises one of the most important horticultural crops of iran which is cultivates in several parts of the country but it is mainly in southern parts of iran (fig. 1). they have about 400 date palm cultivars, currently under cultivation. although domestic date palm identification started by 1960s in iran, it was basically relied on morphological features. however, recent genetic investigations utilize molecular approaches (hajia et al. 2015). the genetic investigations on iran date palms, are mainly focused on cultivar identification and evaluation, genetic diversity analyses and cultivars relationships, as well as male and female cultivars discrimination (see for example, hajian, 2007, marsafari and mehrabi, 2013, hassanzadeh khankahdani and bagheri, 2019. saboori et al. 153genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates 2020). however, with regard to 400 date palm cultivars and different geographical areas of their cultivation, we need a lot more detailed genetic studies in these cultivars. along with genetic diversity, significant difference in morphological and agronomic characters of date palm cultivars is important for breeding purpose. qst, is a quantitative genetic analog of wright’s fst (spitze 1993, prout and barker 1993). the fst gives provides a standardized measure of the genetic differentiation among presumed populations, while the qst provides the amount of genetic variance among populations relative to the total genetic variance. in fact, the average  qst  of a neutral additive quantitative trait is expected to be equal to the mean value of  fst  for neutral genetic loci.  the fst  can be readily measured on commonly available genetic markers, and  qst  can be measured by an appropriate breeding design in a common garden setting. therefore, qst  is an index of the effect of selection on the quantitative trait. if  qst  is higher than fst, it is taken as evidence of spatially divergent selection on the studied quantitative trait. if  qst  is much smaller than fst then this has been taken as evidence of spatially uniform stabilizing selection, which makes the trait diverge less than expected by chance. according to leinonen et al. (2006) and brommer (2011) “when qst estimates are not available, pst can be justified as a substitute.” according to brommer (2011) “divergence across populations of species that are less amenable for proper qst estimation may still be of considerable evolutionary or conservation interest’’ and it can be assessed by using pst. this in turn estimates the quantitative genetic differentiation (i.e., additive genetic variance) using quantitative trait measurements within populations (brommer, 2011). the pst index assesses the local adaptation through natural selection of wild populations and is an approximation of the quantitative genetic differentiation index (qst), obtained in common garden experiments (gentili et al. 2018). the relationship between the values of pst and fst can be used to estimate the relative importance of genetic processes and selection: (a) pst= fst indicates that divergence is compatible with a scenario of genetic drift; (b) pst > fst indicates directional selection (i.e., when one extreme phenotype (gentili et al. 2018). the quantification of population differentiation based on neutral genetic markers and quantitative traits can highlight the relative role of evolutionary processes such as natural selection, genetic drift and gene flow for patterns of local adaptation (brommer, 2011; leinonen et al., 2013). fixation index (fst) is widely used to estimate genetic differentiation with neutral loci (ssr, issr, aflp) by analyzing the variance in allele frequency (wright, 1965). in contrast, phenotypic differentiation index (pst) is an estimate of quantitative genetic differentiation (i.e., additive genetic variance) using quantitative trait measurements within populations (e.g., plant size, growth rate, etc.; brommer, 2011). material and methods plant materials and morphological features we used 36 cultivars including 122 trees were collected from ahwaz germplasm collection (omol-tomair station of date palm & tropical fruits research center, ahwaz, iran) and different date palm orchards located in hormozgan and kerman provinces, iran (saboori et al. 2019, saboori et al. 2020). the fruit characters were used based on saboori et al. 2020. they were including weight of fruit and seed, length and width of fruit, length, and width of the seed. est-ssr and ssr markers genomic dna of fresh leaves were extracted from date palm cultivars collected by modified ctab protocol (saboori et al. 2020). for genetic investigation we used three est-ssr and six ssr loci. two primers est-pdg3119-rubisco and est-dpg0633-laccase were selected (zhao et al., 2013), while est-gte primer was designed by primer3 and gene runner software. they were then checked for accuracy by blast algorithm. figure 1. the provinces that are under date palm cultivations in iran. numbers 113 are: hormozgan, kerma, fars, sistan & baluchestan, bushehr, khuzestan, south khorasan, isfahan, yazd, kermanshah, eilam, kohgiluie and boier-ahmad, and seman, respectively (hajian 2007, hajian et al. 2015). 154 somayeh saboori et al. six primers mpdcir078, mpdcir085, pdcuc3ssr2 , mpdcir090, mpdcir048 a nd mpdcir025 were selected for ssr marker (bodian et al, 2014). the sequences of primers of est-ssr and ssr markers are listed in table s1. pcr reaction for est-ssr and ssr loci were performed as following; a 25 µl volume containing 20 ng genomic dna and 5 u of taq dna polymerase (bioron, germany), 2x pcr buffer (50 mm kcl; 10 mm tris-hcl, ph;8), 1.5mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 µm of each primer. the pcr program for est-ssr and ssr markers were followed: the reactions for est-ssr were amplified in t100 thermal cycler (biorad, usa) using the following procedure, 5 min at 94 ºc, 35 cycles of 30 sec at 95 ºc, 30 sec at 50-60 ºc (est-pdg3119-rubisco 50 ºc, est-gte 52 ºc, est-dpg0633laccase 60 ºc) and 1 min and 30 sec at 72 ºc followed by 5 min at 72 ºc as final extension. the pcr program for ssr markers were performed as touch-up pcr; 94°c for 5 min, initial 10 cycles at 95°c for 30 sec, annealing step (mpdcir078 51°c, mpdcir085 47.5 °c, pdcuc3-ssr2 62 °c , mpdcir090 47.5 °c , mpdcir048 46.9 °c , mpdcir025 45 °c)for 1 min, 72°c for 1 min and 30 sec, followed by 40 cycles at 95°c for 30 sec, annealing step (mpdcir078 52°c, mpdcir085 49.9 °c, pdcuc3-ssr2 65 °c , mpdcir090 49.9 °c , mpdcir048 48.8 °c , mpdcir025 48 °c) for 1 min, 72°c for 1 min and 30 sec, a final cycle of 72 °c for 15 min. the pcr amplifications were separated on a 12% page (poly acrylamide gel electrophoresis) with a 100kb gene ruler (parstous, iran). data analyses genetic diversity analyses the ssr and est-ssr bands obtained were treated as binary characters (podani 2000) and used for further analyses. dca (dentrented correspondance analysis) was used to evaluate suitability of ssr and estssr bands obtained. discriminant power of the bands obtained was determined by popgene program. genetic diversity parameters in the date palm cultivars were estimated by genealex 4.2. a heat map was produced on these parameters by r package. genetic grouping of the cultivars in order to find the proper number of genetic groups within date palm studied, we followed two different statistical approaches. 1we used k-means clustering as performed in genodive program, which is based on likelihood method. 2delta k was obtained from struture analysis which is a bayesian-based method. details of these methods are according to sharifi et al. (2018). genodive provides two different statistics that can determine the number of clusters. these are pseudof-statistic; (the optimal clustering is the one with the highest value for the pseudo-f statistic), and the bayesian information criterion (bic, calculated using sum of squares and the optimal clustering is the one with the lowest value) (meirmans2020). both these criteria work well for clustering populations and individuals, especially when there is random mating within populations but bic has the benefit that it can be used to determine whether there actually is any population structure at all (meirmans 2020). based on the number of ks obtained we performed ward clustering as performed in past and structure analysis as implemented in structure program. the genetic differentiation of the studied cultivars was determined by amova as performed in genealex, as well asby gstnei and gst-hederick as performed in genodive. correlation between morphological characters studied was determined by pearson coefficient of correlation. in order to compare groups of the cultivars based on both molecular and morphological characters, heat maps were constructed by related commands in r package. population assignment was performed by two different methods: 1by discriminant analysis (da) as performed in spss program. in this analysis a summary table was produced which indicates relatedness of each case to its presumed population, and finally provide a percentage value for each population membership based on likelihood method. 2by using assignment test in genealex, which is also based on likelihood method and provides a total membership percentage for all data in question and also provide pairwise populations graph. phenotypic versus genetic differentiation pst index was used to estimate the role of local adaptation through natural selection in date palm populations, compared to that of genetic differentiation. for each population pair, pairwise pst values were calculated for each trait (and for an average pst), using the following formula: 155genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates in this formula, ð2b and ð2w are between-population and within population variance components for a trait, respectively; h2 expresses the heritability (the proportion of phenotypic variance that. is due to additive genetic effects); the scalar c expresses the proportion of the total variance that is presumed to be due to additive genetic variance across populations (broker,2011; leinonen et al., 2013). in the wild, the estimation of the additive genetic variance components is challenging as breeding design is impossible. therefore, qst is often approximated by pst (leinonen et al., 2006), which is directly calculated from the total phenotypic variance components with no distinction between the relative contribution of genetic and environmental variations. therefore, the phenotypic divergence between populations was estimated by the parameter pst as follows: in this formula, ð2b and ð2w are the respective phenotypic variances between and within populations, c is an estimate of the proportion of the total variance due to additive genetic effects across populations, and h2 is heritability, the proportion of phenotypic variance due to additive genetic effects (brommer, 2011). in present study pst was estimated by pstat of r package (da silva and da silva, 2018). results ssr and est-ssr analyses we obtained in total 40 ssr bands in 122 date palm trees studied. the lowest number of bands (13) occurred in cultivar “wardi” (male, no. 32), while the highest number of bands was observed in cultivars halili (no. 4), male (no. 11), khezrawi (no. 17), and barhi (no. 20). the cultivars investigated did not have private band. the suitability of ssr and est-ssr bands for date palm population genetic studies was determined by dca plot (fig. 2). the plot shows a well-scattered distribution of ssr loci, which indicated that these loci are from different regions of the genome and are not clustered to each other. such loci are useful in genetic diversity analyses of the populations. discriminating power of ssr and est-ssr bands versus migration (nm) is provided in table s2. the result shows that most of ssr loci obtained have a high gst value (0.70), and therefore have a good discrimination power for date palm cultivar differentiation task. this is also evidenced with the high mean gst value = 0.81 obtained. figure 2. dca plot of ssr and est-ssr bands/loci in date palm cultivars showing well-scattered distribution of loci obtained. 156 somayeh saboori et al. genetic diversity of date palm cultivars data with regard to genetic diversity parameters determined in 122 individual trees of 36 date palm cultivars are presented in table s3. the range of polymorphism percentage varied from 2.5 in cultivar kharook (no. 13), to 25 in. cultivar khadhrawi (no. 17). the mean value for polymorphism was 13.07%. usually, date palm cultivars show similar genetic contents, and therefore, about 13% genetic polymorphism is yet appreciable for further breeding studies if accompanied by some degree of morphological and agronomical desirable traits variation. heat-map constructed based on genetic diversity parameters (fig. 3), reveals that based on percentage of genetic polymorphism (p), nei’ gene diversity (he) and figure 3. heatmap of date palm cultivars based on genetic diversity parameters. abbreviations: na = no. of different alleles, ne = no. of effective alleles, i = shanon information. index, he = expected heterozygosity, uhe = unbiassed expected heterozygosity, and p% = polymorphism percentage. 157genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates shanon information index (i), date palms may be classified in 5 or 6 genetic groups. this classification is sharper by considering only genetic polymorphism parameter. grouping of the cultivars the nei genetic distance determined in the cultivars studied varied from 0.067 between cultivars 1 and 2, to 0.46 between cultivars estameran (no. 19) and mashtoom (no. 28). these low values of genetic distance, indicates a high degree of genetic alikeness in date palm cultivars cultivated in the country. for grouping of the cultivars based on ssr markers, we first performed k-means clustering by genodive program (table s4). the results indicated that these cultivars can be grouped either in two broad clusters according to calinski & harabasz’ pseudo-f: k = 2, or in 16 smaller genetic groups according to bayesian information criterion: k = 16. ward clustering of the date palm cultivars based on ssr and est-ssr data (fig. 4), also grouped the genotypes in two major clusters and about 16 sub-clusters which is in agreement with k-means clustering. ward dengrogram produced two main clusters or genetic groups in accord with k-means clustering result. the cultivars 1-13 comprise the first genetic group and form the first main cluster, while the other cultivars form the second major cluster or genetic group. in the first main cluster, the cultivars are distributed in three sub-clusters a-c. replicates of the cultivars 1-4 show a higher level of genetic similarity and are placed in a single sub-cluster, (a). replicates of the cultivars 9-13 comprise the second sub-cluster b, while replicates of the cultivar 5-9 form the sub-cluster c. replicates of the cultivar 4, were admixed in two sub-clusters a and c. few date palm plants of these cultivars also show some degree of admixture. since clustering is based on distance parameter only, we also tried structure analysis for genotype grouping, which is a bayesian-based method. for this, we first obtained k value by evanno method, which produced delta k = 2. this is in agreement with major growing obtained by k-means clustering. however, to obtain a better and more detailed picture on the cultivars genetic grouping, we carried out structure analysis based on k values 2-5 (fig. 5). the best genetic grouping obtained seems to be k =5. based on k =5, the cultivars 1-4 show genetic affinity and comprise the first genetic group. this is followed by the cultivars 5-13, then 14-2, 23-30, and finally the cultivars 31-36, form the fifth genetic group. all these five genetic groups show a low degree of genetic admixture with the other groups. figure 4. ward dendrogram of date palm cultivars based on ssr and est-ssr data. 158 somayeh saboori et al. genetic difference of the cultivars amova produced significant genetic difference among date palm cultivars (phipt = 0.70, p = 0.001). it also revealed that 70% of total genetic variability occurs due to among cultivar difference, while 31% occurs due to within population genetic variability. moreover, pairwise amova (table s5) produced significant genetic difference between the cultivars of the two main clusters as well as the cultivars of different sub-clusters in upgma dendrogram. new genetic differentiation parameters estimated produced significant difference among date palm cultivars (g’st(nei) = 0.673, p =0.001; g’st(hed) = 0.738, p = 0.001). these results indicate the presence of genetic figure 5. structure plot of date palms studied based on k = 2-5. 159genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates variability within date palm cultivar germplasm, which can be used in future breeding program. assignment of date palms assignment of individual date palm plants by discriminant analysis revealed that the cultivars 2, 9, 10, 13, 20, 21, 25, 29, 31 and. 32, have 33% mis-assignment, while cultivar 4 has 66% mis-assignment. genealex also revealed 67% self population assignment and 33% of other population assignment. in table s6, some parts of assignment result for 122 date palms have been given (only those samples inferred to be from other population are given). assignment is based on positive likelihood, and therefore the lower the value shows the correct assignment (inferred population). fst versus pst estimates details of morphological characters studied are given in fig. 6. anova produced significant difference (p <0.01), for these characters among the studied cultivars. most of these characters show significant correlation (p. <0.01) (see for example, fig. 7). heat-maps of the 45 date palm trees based on morphological versus genetic (ssrs), data are presented in fig. 8. comparison of the groupings obtained reveals difference in the clustering results. figure 6. anova of morphological characters studied in date palm cultivars. 160 somayeh saboori et al. moreover, the mantel test performed between the two clustering results did not produced a significant association between the two markers (r = 0.057, p = 0.16), supporting the heat maps. therefore, grouping and cultivar relationship illustrated by morphological characters studied do not accord with genetic relationship of the same date palm cultivars. fst versus pst analyses, revealed that in most of the studied morphological characters, the pst value greatly exceeds that of genetic fst value. for example, some of the pair-wise comparison between cultivar no. 3 and the others are provided in table s7. therefore, pst > fst indicates directional selection in quantitative fruit and seed characteristics has been occurred in the studied date palm cultivars. different factors may be responsible for these directional changes, like ecological and environmental conditions in which the cultivars grow, selection practiced by the breeders figure 7. representative pearson coefficient of correlation among morphological characters studied in date palm cultivars. 161genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates or locals, etc. in general, morphological difference along with genetic diversity present in the studied cultivars may contribute in future breeding of date palm. discussion genetic diversity present study revealed the presence of a low to moderate genetic diversity within date palm cultivars studied. this is in accord with the studies performed in iraq and tunesian date palms by jubrael et al. (2005) and zehdi et al. (2015), who suggested a common genetic basis among date palm genotypes despite the differences in fruit characters and tree morphology. low genetic diversity within date palm germplasm was revealed but both neutral molecular markers like, issrs and ssrs (see for example, sharifi et al. 2018, saboori et al. 2020), and sequence-based marker, like chloroplast dna (sharifi et al. 2018). cultivars genetic grouping the cultivars studied were placed in two major genetic groups by both k-means and bayesian-based delta k estimation more detailed analysis, revealed that they can be classified in 5 different genetic groups. such data may be used in future breeding program. cultivar grouping based on structure analysis were also utilized by the other researchers in date palms (see for example, sharifi et al. 2018). it is important in plants with almost common genetic background like date palms to classify them in different genetic classes. population assignment population assignment seems to be a prerequisite step in selecting plant individuals and breeding date palm, as these plants have a common genetic background and show overlapping genetic structure. this may also happen due to genetic admixture of the date palms (sharifi et. al. 2018, saboori et al. 2020). we obtained about 33% of incorrectly assigned date palms in respect to their presumed populations. this may be either due to improper plant sampling or identification within the germplasm, or due to gene flow and admixture among these cultivars. in any case, such cases should be considered in future breeding program. in a similar study concerned with genetic structure of tunesian date palms, zehdi et al. (2015) reported the presence of admixed cultivars too. they considered that the gene flows between eastern and western origins mostly from east to west following a human-mediated figure 8. heat maps of 45 date palm cultivars based on morphological and genetic (ssrs) data, showing different groupings of these cultivars. (abbreviations in morphological heat map are: sw = seed weight, swi = seed width, fw = fruit weigh, sl = seed. length, and. fl = fruit length). 162 somayeh saboori et al. diffusion of the species, is the reason for the formation of mixed genotypes. saboori et al. (2020), investigated the genetic structure of 13 date palm cultivars by scot molecular markers and reported some degree of genetic admixture among the cultivars. though, they did not study specifically assignment of the plants to their populations, by looking at the clustering result of their samples, it becomes evident that some of the plants a presumed cultivar has been placed intermixed with plants of another cultivar. however, sharifi et al (2018) investigated the gene flow and assignment in 16 date palm cultivars by using issr molecular markers and observed some degree of population admixture and few cases of incorrectly assigned date palms. in an elaborate and precisely studied report by grosbalthazard et al. (2020), they used a joint ethnographic study and genetic analysis of date palms to test whether named date palm types are true-to-type cultivars versus incorrectly assigned samples in desert nearby siwa (also known as “feral” in battesti in egypt). they recognized three categories of genotypes within their extensive collection namely, true-to-type cultivar samples, ethnovarieties and samples of local categories. therefore, there is a huge mistake in assigning date palms to their respective population or named cultivar. genetic versus phenotypic differential aljuhani (2016), studied the degree of dissimilarity and the impact of location on the genetic relationship between local cultivars in saudi arabia by using and twenty-four nuclear microsatellite loci. he reported a high level of genetic polymorphism in some of the loci, and could differentiate the studied cultivars by these markers. some of these cultivars were grouped according to their geographical area in which they were cultivated. we obtained a higher value for pst versus fst, almost in all date palm cultivars studied and for most of the fruit and seed characters. the pst is taken as index for morphological local adaptation through natural selection, but influenced by environment (brommer, 2011). if pst = fst, it indicates that divergence is due to genetic drift; and if pst > fst, it indicates the role of directional selection (i.e., when one extreme phenotype is favored over other ones) among populations; and finally, if pst< fst, it indicates that the same phenotypes are favored in different populations due to stabilizing selection. we may therefore, suggest that, due to some local environmental face or local practice of cultivation or selection, some adaptive changes have occurred in date palm cultivars in the country. qst–fst comparison has shown that trait divergence due to natural selection, as opposed to genetic drift have occurred in many taxa (leinonen et al. 2013). in present study, the mantel test did not produce significant association between the cultivar grouping and morphological grouping, in other words we did not see co-variation between genetic and morphological traits. however, in qst-fst investigation carried out by sˇurinová et al. (2018), in 11 populations of festuca rubra, they reported the existence of adaptive differentiation in phenotypic traits and their plasticity across the climatic gradient and observed statistically significant co-variation between markers and phenotypic traits, which is likely caused by isolation by adaptation. in a similar study, caré et al. (2018) investigated the high morphological differentiation in crown architecture in contrasts with low population genetic structure of german norway spruce stands by using pst-fst method and 11 nuclear ssr molecular markers. norway spruce trees have narrow crown phenotypes, whereas lowland trees have broader crowns. narrow crown phenotypes are likely the result of adaptation to heavy snow loads combined with high wind speeds. they observed a high differentiation of morphological traits (pst = 0.952–0.989) between the neighboring autochthonous and allochthonous stands of similar age contrasts with the very low neutral genetic differentiation (fst = 0.002–0.007; g”st = 0.002–0.030), suggesting that directional selection at adaptive gene loci was involved in phenotypic differentiation. it has been suggested that “the  qst–fst  method is still underused in ‘omics’ contexts, in which it may be useful for identifying evolutionary significance in large data sets in the absence of evolutionary models (leinonen et al. 2013)”. in conclusion we may sat that considering different molecular studies in date palm genotypes both around the world and in our country, and irrespective of molecular marker used (neutral versus sequence based markers), a low to moderate genetic diversity is present in limited number of cultivars investigated till now. we need to carry one further detailed population genetics analysis in much more number of accessions and cultivars to possibly broaden the genetic variability of date palm for future breeding. authors’ contributions z.n. and m.sh: conceptualization of the project; m.sh.: analyses of data; s.s and f.k data collection and lab work; s.m.: providing samples 163genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates acknowledgements we acknowledge science and research branch, islamic azad university for providing laboratory. we thank the iran national science foundation (insf), for partial financial support of this project (no.97010700). references ahmed j, al-jasass fm, siddiq m (2014) date fruit composition and nutrition. in: siddiq m, aleid s, kader a (eds) dates: postharvest science, processing technology and health benefit. wiley blackwell, chichester, uk, pp 261-283 aljuhani w s (2016) genetic diversity and the impact of geographical location on the relationships between phoenix dactylifera l. germplasms grown in saudi arabia. hereditary genet 5(3):1-11. https://doi. org/10.4172/2161-1041.1000172 bodian a, nachtigall m, frese l, elhoumaizi m a, hasnaoui a, ndir kn, sané d (2014) genetic diversity analysis of date palm (phoenix dactylifera l.) cultivars from morocco using ssr markers. j biodivers biopros dev 1(3): 2376-0214 brommer je (2011) whither pst? the approximation of qst by pst in evolutionary and conservation biology. j evol biol 24:1160-1168. https://doi.org/10.1111/ j.1420-9101.2011.02268.x caré o, müller, m, vornam, b, höltken, a, kahlert k, krutovsky k v, gailing, o, leinemann, l (2018) high morphological differentiation in crown architecture contrasts with low population genetic structure of german norway spruce stands. forests 9: 752. https://doi.org/10.3390/f9120752 da silva b, anne da silva a (2018) pstat: an r package to assess population differentiation in phenotypic traits by stéphane. the r journal 10(1): 447-454 el hadrami i, el hadrami a (2009) breeding date palm. in: jain sm, priyadarshan pm (eds) breeding plantation tree crops: tropical species, berlin, springer pp191-216 fao (2010) statistics division. <http://faostat.fao.org/ default.aspx>. online. accessed: dez. 01, 2010. gros-balthazard m, battesti v, ivorra s, paradis l, aberlenc f, zango o, zehdi s, moussouni s, abbas naqvi s, newton c, terral jf (2020) integration of ethno botany and population genetics uncovers the agrobiodiversity of date palms of siwa oasis (egypt) and their importance to the evolutionary history of the species. evol appl. https://doi.org/10.1111/eva.12930 gentili r, solari a, diekmann m, duprè c, monti gs, armiraglio s, assini s, sandra citterio s (2018) genetic differentiation, local adaptation and phenotypic plasticity in fragmented populations of a rare forest herb. peer j 6:e4929 https://doi.org/10.7717/ peerj.4929. hajia s, hamidi-esfahani z (2015) date palm status and perspective in iran. in: al-khayri j, jain s, johnson d. 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(2015) genetic structure of the date palm (phoenix dactylifera) in the old world reveals a strong differentiation between eastern and western populations. ann bot 116: 101– 112. https://doi.org/10.1093/aob/mcv068 wright s (1965) the interpretation of population structure by f-statistics with special regard to systems of mating. evol 19: 395-420. zhao y, williams r, prakash cs, he g (2012) identification and characterization of gene-based ssr markers in date palm (phoenix dactylifera l.). bmc plant biol 12(1): 237. https://doi.org/10.1186/1471-2229-12-237 table s1. est-ssr and ssr primer name, sequences and references. locus name estssr primer sequence ref est-pdg3119-rubisco-f catactgattattggcacacc (zhao et al. 2012) est-pdg3119-rubisco-r gtaccataccgtaccagttca est-dpg0633laccase -f agactggttaagttggtggag (zhao et al. 2012) est-dpg0633-laccase-r ctacaaaactgatgtggtggt est-gte-f gcttggccatctatgaaac -est-gte-r actctgagcatccatatcg -ssr primer sequence mpdcir025(ga)22-f gcacgagaaggcttatagt (bodian et al. 2014) mpdcir025(ga)22-r cccctcattaggattctac mpdcir048(ga)32-f cgagacctaccttcaacaaa (bodian et al. 2014) mpdcir048(ga)32-r ccaccaaccaaatcaaacac mpdcir078(ga)13-f tggatttccattgtgag (bodian et al. 2014) mpdcir078(ga)13-r cccgaagagacgctatt mpdcir085(ga)29-f gagagagggtggtgttatt (bodian et al. 2014) mpdcir085(ga)29-r ttcatccagaaccacagta mpdcir090(ga)26-f gcagtcagtccctcata (bodian et al. 2014) mpdcir090(ga)26-r tgcttgtagcccttcag pdcuc3-ssr2(ga)22-f acattgctcttttgccatgggct (bodian et al. 2014) pdcuc3-ssr2(ga)22-r cgagcaggtggggttcgggt 165genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates table s2. discrimination power (gst value), of ssr loci obtained. locus sample size ht hs gst nm locus1 122 0.0526 0.0385 0.2676 1.3687 locus2 122 0.3742 0.0354 0.9053 0.0523 locus3 122 0.4580 0.1177 0.7430 0.1730 locus4 122 0.2066 0.0438 0.7882 0.1343 locus5 122 0.3214 0.0792 0.7536 0.1635 locus6 122 0.2845 0.0083 0.9707 0.0151 locus7 122 0.1859 0.0333 0.8209 0.1091 locus8 122 0.1721 0.0136 0.9212 0.0428 locus9 122 0.2731 0.0625 0.7710 0.1485 locus10 122 0.0429 0.0302 0.2961 1.1888 locus11 122 0.4963 0.0490 0.9013 0.0548 locus12 122 0.1975 0.0000 1.0000 0.0000 locus13 122 0.0759 0.0136 0.8214 0.1227 locus14 122 0.1600 0.0469 0.7072 0.2071 locus15 122 0.2133 0.0906 0.5751 0.3694 locus16 122 0.4945 0.0678 0.8629 0.0794 locus17 122 0.3883 0.1199 0.6913 0.2233 locus18 122 0.4910 0.0604 0.8770 0.0702 locus19 122 0.2330 0.0271 0.8836 0.0659 locus20 122 0.0316 0.0136 0.5705 0.3765 locus21 122 0.4727 0.1042 0.7796 0.1413 locus22 122 0.2527 0.0552 0.7816 0.1397 locus23 122 0.0636 0.0083 0.8691 0.0753 locus24 122 0.4800 0.0521 0.8915 0.0609 locus25 122 0.2209 0.0250 0.8869 0.0637 locus26 122 0.4694 0.1230 0.7380 0.1775 locus27 122 0.2788 0.0354 0.8729 0.0728 locus28 122 0.0331 0.0219 0.3391 0.9744 locus29 122 0.3906 0.0604 0.8453 0.0915 locus30 122 0.4979 0.0469 0.9059 0.0519 locus31 122 0.4457 0.1334 0.7006 0.2136 locus32 122 0.1456 0.0271 0.8138 0.1144 locus33 122 0.0651 0.0438 0.3276 1.0262 locus34 122 0.4045 0.1593 0.6060 0.3250 locus35 122 0.0621 0.0271 0.5633 0.3876 locus36 122 0.3333 0.0219 0.9343 0.0351 locus37 122 0.2922 0.0521 0.8217 0.1225 locus38 122 0.3959 0.0438 0.8895 0.0621 locus39 122 0.4788 0.0761 0.8411 0.0945 locus40 122 0.4900 0.1020 0.7930 0.1340 mean 122 0.2804 0.0530 0.8109 0.1166 * nm = estimate of gene flow from gst or gcs. e.g., nm = 0.5(1 gst)/gst. abbreviations: hs = inbreeding due to sub-population, ht = hnbreeding in total population, gst = discrimination power, and nm = number of migration. 166 somayeh saboori et al. table s3. genetic diversity parameters in date palm cultivars. no cultuvar name na ne i he uhe p 1 mazafati 0.625 1.127 0.104 0.071 0.086 17.50 2 kalooteh 0.625 1.138 0.116 0.079 0.095 20.00 3 khalezohrei 0.650 1.138 0.116 0.079 0.095 20.00 4 holeileh 0.700 1.112 0.106 0.069 0.083 20.00 5 mordarsang 0.500 1.069 0.058 0.039 0.047 10.00 6 khazab 0.500 1.056 0.053 0.035 0.042 10.00 7 holoo 0.600 1.077 0.077 0.050 0.060 15.00 8 khenizi 0.600 1.117 0.092 0.064 0.077 15.00 9 negar 0.625 1.127 0.104 0.071 0.086 17.50 10 shahani 0.475 1.058 0.046 0.032 0.038 7.50 11 male isolate 0.675 1.101 0.094 0.062 0.074 17.50 12 alimehtari 0.525 1.069 0.058 0.039 0.047 10.00 13 kharook 0.450 1.024 0.017 0.012 0.015 2.50 14 gantar 0.550 1.082 0.063 0.044 0.053 10.00 15 zahidi 0.600 1.104 0.087 0.059 0.071 15.00 16 jowzi 0.575 1.104 0.087 0.059 0.071 15.00 17 khadhrawi 0.750 1.186 0.150 0.103 0.124 25.00 18 shekkar 0.475 1.032 0.036 0.022 0.027 7.50 19 istamaraan 0.525 1.045 0.041 0.027 0.033 7.50 20 barhi 0.650 1.104 0.087 0.059 0.071 15.00 21 hallawi 0.600 1.104 0.087 0.059 0.071 15.00 22 braim 0.500 1.069 0.058 0.039 0.047 10.00 23 dayri 0.525 1.080 0.070 0.047 0.056 12.50 24 beliani 0.650 1.101 0.094 0.062 0.074 17.50 25 owaidi 0.525 1.080 0.070 0.047 0.056 12.50 26 sowaidani 0.450 1.071 0.051 0.037 0.044 7.50 27 owaimri 0.525 1.056 0.053 0.035 0.042 10.00 28 mashtoom 0.600 1.104 0.087 0.059 0.071 15.00 29 fersee 0.600 1.114 0.099 0.067 0.080 17.50 30 sabzparak 0.475 1.080 0.070 0.047 0.056 12.50 31 ghannamisabz 0.500 1.053 0.060 0.037 0.045 12.50 32 wardi 0.400 1.071 0.051 0.037 0.044 7.50 33 ghannamisorkh 1 0.525 1.095 0.068 0.049 0.059 10.00 34 ghannamisorkh2 0.500 1.045 0.041 0.027 0.033 7.50 35 foreign male 1 0.525 1.106 0.080 0.056 0.068 12.50 36 foreign male 2 0.525 1.106 0.080 0.056 0.068 12.50 abbreviations: na = no. of different alleles, ne = no. of effective alleles, i = shanon information. index, he = expected heterozygosity, uhe = unbiassed expected heterozygosity, and p% = polymorphism percentage. 167genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates table s4. k-means clustering of date palm cultivars based on ssr and est-ssr data. ssd(t) ssd(ac) ssd(wc) r-squared pseudo-f bic 1376.672 202.482 1174.191 0.147 20.693 871.945 1376.672 319.18 1057.492 0.232 17.959 863.978 1376.672 411.173 965.499 0.299 16.751 857.679 1376.672 498.76 877.912 0.362 16.618 850.881 1376.672 566.45 810.222 0.411 16.22 845.896 1376.672 620.841 755.831 0.451 15.744 842.222 1376.672 660.327 716.346 0.48 15.012 840.48 1376.672 695.563 681.109 0.505 14.425 839.13 1376.672 724.476 652.196 0.526 13.824 838.642 1376.672 753.245 623.427 0.547 13.411 837.943 1376.672 780.048 596.625 0.567 13.074 837.385 1376.672 807.604 569.068 0.587 12.891 836.42 1376.672 832.456 544.217 0.605 12.708 835.777 1376.672 853.797 522.876 0.62 12.48 835.7 1376.672 874.463 502.209 0.635 12.305 835.584 1376.672 891.686 484.986 0.648 12.066 836.131 1376.672 909.553 467.12 0.661 11.912 836.356 1376.672 925.794 450.878 0.672 11.75 836.842 1376.672 940.632 436.04 0.683 11.581 837.564 * best clustering according to calinski & harabasz’ pseudo-f: k = 2 & best clustering according to bayesian information criterion: k = 16 abbreviations: ssd(t) = total sum of squares, ssd(ac) = among clusters sum of squares, and ssd(wc) = within clusters sum of squares. table s5. pair-wise amova showing significant genetic difference between the studied date palm cultivars (cultivar numbers are according to table s3). cultivar1 cultivar2 pvalue 3 21 0.001 4 8 0.001 4 16 0.001 5 10 0.001 5 35 0.001 6 7 0.001 6 11 0.001 6 27 0.001 6 29 0.001 6 1 0.001 7 12 0.001 8 22 0.001 8 30 0.001 11 23 0.001 14 15 0.001 15 36 0.001 21 23 0.001 18 24 0.001 20 24 0.001 20 36 0.092 21 36 0.001 24 26 0.001 25 34 0.001 27 31 0.001 table s6. assignment result of date palms (only samples inferred to be from other populations are given) based on positive likelihood. (cultivar numbers are according to table s3). home cultivar infered cultivar1 cultivar 1 2 3 4 5 6 7 1 3 4.432 4.15 3.516 6.055 10.076 12.431 14.59 12.748 1 2 6.034 4.099 4.789 5.549 10.356 13.829 15.386 14.793 2 3 4.592 4.533 3.789 4.453 8.18 10.812 12.845 13.6 2 1 3.373 4.724 3.947 5.708 13.331 12.732 10.891 9.901 2 3 3.704 3.413 3.227 4.453 10.414 14.352 14.289 14.969 3 2 3.579 2.798 3.617 4.152 8.812 12.13 12.067 12.873 3 2 3.771 2.798 3.77 4.328 11.59 14.051 11.988 13.094 4 2 5.057 4.439 5.537 5.554 8.796 10.431 10.368 9.997 8 7 8.698 9.4 9.588 6.106 7.683 7.414 4.429 5.641 9 5 8.379 9.044 9.093 10.565 7.239 7.271 11.271 9.776 9 10 8.328 7.597 8.093 7.935 10.96 12.829 12.908 11.316 10 11 10.93 9.09 10.19 8.759 11.106 11.13 14.085 13.316 11 10 9.93 8.898 9.792 9.537 12.437 12.829 14.607 12.89 11 12 11.555 11.032 10.588 10.236 11.692 11.829 10.243 8.043 11 10 12.708 12.713 13.588 10.333 10.68 11.607 15.306 13.316 15 16 11.437 14.442 14.257 13.379 16.294 17.574 15.431 15.288 15 16 9.592 10.965 11.081 9.981 13.118 14.352 15.908 13.714 168 somayeh saboori et al. table s7. fst versus pst values in cultivar no.3 with others. character pst fst pst fst pst fst pst fst fruit weight 0.053 0.16 0.43 0.16 0.36 0.16 0.053 0.16 fruit width 0.001 0.16 0.09 0.16 0.09 0.16 0.001 0.16 fruit length 0.99 0.16 0.46 0.16 0.01 0.16 0.99 0.16 seed weight 0.99 0.16 0.44 0.16 0.98 0.16 0.98 0.16 seed width 0.98 0.16 0.44 0.16 0.99 0.16 0.99 0.16 seed length 0.99 0.16 0.44 0.16 0.98 0.16 0.99 0.16 caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 3 2021 firenze university press chromomycin a3 banding and chromosomal mapping of 45s and 5s ribosomal rna genes in bottle gourd ahmet l. tek*, hümeyra yıldız, kamran khan, bilge ş. yıldırım development of a protocol for genetic transformation of malus spp federico martinelli1,*, anna perrone2, abhaya m. dandekar3 cytogenetic and cytological analysis of colombian cape gooseberry genetic material for breeding purposes viviana franco-florez, sara alejandra liberato guío, erika sánchez-betancourt, francy liliana garcía-arias, víctor manuel núñez zarantes* palynological analysis of genus geranium (geraniaceae) and its systematic implications using scanning electron microscopy jun wang1,2,*, qiang ye1, chu wang2, tong zhang2, xusheng shi2, majid khayatnezhad3, abdul shakoor4,5 influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats yuri a. kirillov1, maria a. kozlova1, lyudmila a. makartseva1, igor a. chernov2, evgeniya v. shtemplevskaya1, david a. areshidze1,* the morphological, karyological and phylogenetic analyses of three artemisia l. (asteraceae) species that around the van lake in turkey pelin yilmaz sancar1,*, semsettin civelek1, murat kursat2 molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant jinxin cheng1,*, dingyu hu1, yaran liu2, zetian zhang1, majid khayatnezhad3 genetic variations and interspesific relationships in salvia (lamiaceae) using scot molecular markers songpo liu1, yuxuan wang1, yuwei song1,*, majid khayatnezhad2, amir abbas minaeifar3 development of female gametophyte in gladiolus italicus miller (iridaceae) ciler kartal1, nuran ekici2,*, almina kargacıoğlu1, hazal nurcan ağırman1 colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae) harshita dwivedi*, girjesh kumar statistical evaluation of chromosomes of some lathyrus l. taxa from turkey hasan genç1, bekir yildirim2,*, mikail açar3, tolga çetin4 use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater treatment and water of the kamniška bistrica river (slovenia) peter firbas1, tomaž amon2 the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bo shi1,*, majid khayatnezhad2, abdul shakoor3,4 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(3): 21-30, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1081 caryologia international journal of cytology, cytosystematics and cytogenetics citation: viviana franco-florez, sara alejandra liberato guío, erika sánchez-betancourt, francy liliana garcía-arias, víctor manuel núñez zarantes (2021) cytogenetic and cytological analysis of colombian cape gooseberry genetic material for breeding purposes. caryologia 74(3): 21-30. doi: 10.36253/caryologia-1081 received: september 16, 2020 accepted: july 20, 2021 published: december 21, 2021 copyright: © 2021 viviana franco-florez, sara alejandra liberato guío, erika sánchez-betancourt, francy liliana garcía-arias, víctor manuel núñez zarantes. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid cvf-f: 0000-0002-1311-5419 salg: 0000-0003-3485-3004 es-b: 0000-0002-3024-3180 flg-a: 0000-0003-3112-9950 vmnz: 0000-0002-5087-9864 cytogenetic and cytological analysis of colombian cape gooseberry genetic material for breeding purposes viviana franco-florez, sara alejandra liberato guío, erika sánchez-betancourt, francy liliana garcía-arias, víctor manuel núñez zarantes* corporación colombiana de investigación agropecuaria – agrosavia. centro de investigación tibaitatá. km 14 vía mosquera bogotá, cundinamarca, colombia *corresponding author. e-mail: vnunez@agrosavia.co abstract. the cape gooseberry, physalis peruviana l., is a crop that is transitioning from a semi-wild rural food source to becoming an international export commodity fruit deserving of greater attention from the scientific community, producers, policy makers, and opinion makers. despite its importance, the crop has serious technological development challenges, mainly associated with the limited supply of genetically improved materials for producers and consumers. to bridge this gap, the present study determined the level of ploidy of 100 genotypes of cape gooseberry from a working collection by counting the number of chromosomes and chloroplasts, to include them in the breeding program. the number of chromosomes in dividing cells of root-tip meristems, as well as the number of chloroplasts per guard cell, from plants grown under in vitro and ex vitro conditions were determined. haploid with 24 chromosomes, doubled haploid, tetraploid with 48 chromosomes, aneuploid (44 and 49 chromosomes), and mixoploid genotypes with 36 to 86 chromosomes were found. the number of chloroplasts per guard cell ranged from 4-8, 6-16, 7-16 and 9-21 for the haploid, aneuploid, doubled haploid-tetraploid, and mixoploid genotypes, respectively. the results showed evidence of a high cytogenetic diversity in the evaluated genotypes. keywords: chloroplast number, chromosome number, mixoploidy, physalis peruviana, plant breeding. 1. introduction colombia is the world’s largest producer of cape gooseberry (physalis peruviana l), with a high-quality fruit desired for its aroma and flavor. during 2019, approximately 8,287 tons were mainly exported to the netherlands, united kingdom, united states, canada and belgium (agronet, 2019; analdex, 2019; procolombia, 2020). this makes cape gooseberry a crop with great competitive advantages for colombia, which being a tropical country can guarantee yearlong production to supply the international market (cotes et al., 2012). however, scientific, and technological progress is still lacking to 22 viviana franco-florez et al. position it as a stable and competitive crop in colombian agriculture. one way to address the agronomic and crop quality constraints is through the generation of genetically improved varieties for commercial production. the genetic pre-improvement of cape gooseberry in colombia – also known as uchuva in colombia, uvilla in ecuador, and aguaymanto in peru – is a relatively new activity since currently there are just two genetic improvement programs contributing to the solution of agronomic problems of the crop. one of these programs, at agrosavia, released the first two commercial varieties (núñez et al., 2016b, 2016a) and the other, at the university of nariño, is working toward the selection of genotypes for production under local conditions. however, the present commercial production process is mainly based on seeds of materials that producers select from their harvest. therefore, improvements in the breeding process to enhance availability of superior production material is an important endeavor. one essential prerequisite to breeding efforts is the elucidation of chromosome number in germplasm to be used as potential breeding parents. the ploidy level of the parents is a key factor that affects the efficiency of hybridization in the generation of new segregating populations, genetically stable in terms of chromosome number. in cape gooseberry, the ploidy variation has been supported at the cytogenetic level by several studies. vilmorin and simonet, (1928) determined a chromosomal complement of 2n = 48; yamamoto and sakai, (1932) described populations having 2n = 24 chromosomes; and bracamonte et  al., (1997) reported a chromosome complement of 2n = 16, in which they named p. peruviana as “capulí de la costa” – a term sometimes used in commercialized cape gooseberry in peru. among the most recent cytogenetic studies of p. peruviana was that of rodríguez and bueno, (2006), who recognized variation in the number of chromosomes associated with different ecotypes, finding plants with a chromosomal complement of 2n = 24, 2n = 32, and 2n = 48. according to lagos, (2006) the species presents three characteristic karyotypes: 2n = 24, 36 and 48; three rare ones with 32, 38 and 40 chromosomes; and cases of mixoploidy. bala and gupta, (2011) stickiness of chromosomes, multivalents and univalents, and unoriented bivalents during metaphase-i, non-synchronization in the separation of some bivalents, laggards, chromatin bridges and cytomixis at various meiotic stages besides aberrant microsporogenesis. in spite of all these abnormalities, distribution of chromosomes at anaphases found to be normal. microsporogenesis includes monads, dyads and tetrads with micronuclei besides normal tetrads, consequently reducing the pollen fertility (76% reported that p. peruviana has intraspecific polyploid cytotypes that include diploids, tetraploids, octoploids and hexaploids. recently, trevisani et al., (2018) determined that brazilian p. peruviana populations presented tetraploid cells 2n = 4x = 48; carbajal (2018) found in three peruvian ecotypes a ploidy level of 2n = 4x = 48 with chromosomal number variations in the same samples of analyzed cells in each of the studied ecotypes. on the other hand, the molecular genetic laboratory research group of agrosavia has advanced in the analysis of the ploidy level of cape gooseberry by flow cytometry, chromosome and chloroplast counting (franco, 2012; liberato et al., 2014; garcía-arias et al., 2018b). franco, (2012) and garcía-arias et al., (2018b) found chromosome numbers of 2n = 4x = 48 in the ecotypes colombia and kenya and chromosome variations induced by colchicine treatments; they additionally discovered that chromosome and chloroplast numbers were related to each other. liberato et al., (2014) determined chromosome numbers of 2n = 4x = 48 and 2n = 2x = 24 that correlated with the nuclear dna content estimated by flow cytometry. in addition, berdugo et al., (2015) carried out crosses between some genotypes analyzed by liberato et al., (2014) and found distortion in the segregation, possibly related to differences in the chromosome size or chromosome number of these materials. the cytogenetic variability, found in the collection of the germplasm bank maintained at agrosavia and in the working collections, highlights the importance of knowing the cytogenetic identity of each genotype before its inclusion in hybridization-based breeding programs. the ploidy knowledge of cape gooseberry genotypes is an essential factor in designing an appropriate breeding strategy (trevisani et al., 2018) for the genetic improvement of the crop. therefore, the objective of the present study was to determine the ploidy level of one hundred genotypes of cape gooseberry using conventional chromosome and chloroplast counting techniques. these materials have been characterized by agronomic and quality attributes, including candidate genes associated with yield, size and fruit quality to develop superior genotypes (garcía-arias et al., 2018a), and further exploring their ploidy level may inform the level of gene flow, and lead to the release of commercial improved varieties or hybrids. 2. materials and methods 2.1 plant materials one hundred genotypes derived from the national germplasm bank, which is administered by the cor23cytogenetic and cytological analysis of colombian cape gooseberry genetic material for breeding purposes poración colombiana de investigación agropecuaria agrosavia (table s1), were used for the study. the cape gooseberry population included wild genotypes, commercial genotypes from different producing areas of colombia, ecotypes and germplasm obtained from in vitro anther culture. the plants maintained under in vitro culture conditions were sub-cultured in ms medium (murashige and skoog, 1962) modified with half of the nitrates (nh4no3 825 mg/l and 850 mg/l), under a temperature of 25 ± 2ºc, a light intensity of 2000 lux and a photoperiod of 16 light hours. 2.2 determination of chromosome number for chromosome counts, roots of in vitro plants were collected after 15 days of culture at 11:00 am, time of the day in which mitotic activity in the radical apices of p. peruviana is at its peak, as previously determined by liberato et al., 2014. root tips of 2-3 cm in length were treated with 0.25% colchicine in a solution with 2% dmso for three hours at room temperature. after this treatment, the root samples were fixed for 12 hours in carnoy’s solution (96% ethanol and glacial acetic acid in a 3:1 ratio). subsequently, the roots were subjected to acid hydrolysis with 1n hcl for 25 minutes at room temperature. finally, they were transferred to distilled water and kept for one hour at 37 ° c. the root tips staining was done on a slide with two drops of 2% propionic orcein for 15 minutes. then, the tissue was crushed with a rubber bar. the cells were observed with the 40x and 100x lenses in an olympus microscope to count the chromosome number in a sample of 25 cells per genotype. 2.3 counting the number of chloroplasts the chloroplast counts were performed on 25 guard cells using the methodology proposed by orrillo and bonierbale, (2009) in potato. young leaves of each genotype were collected, and the epidermis was removed from the area close to the abaxial vasculature with sharp forceps. the sample was placed on a slide with two drops of iodine-potassium iodide (i-ki) solution in a 1:1 ratio in 70% alcohol. the preparation was observed under the microscope at 40x and 100x magnifications to determine the number of chloroplasts per stomatal guard cell. 2.4 data analysis to establish differential groups in relation to cytogenetic and cytological variability, a cluster analysis was performed using the ward method (semi-partial r2 = 0.10), complemented with a pearson correlation test (α = 0.05), using sas® (statistical analysis system, cary, north carolina) version 9.3. based on these analyzes, predictions were made on the possible results that could be found when carrying out intraspecific crosses between the genotypes studied. 3. results 3.1 chromosome counting when counting the number of chromosomes of genotypes from the work collection, the basic chromosome number x = 12 was predominant in the genotypes evaluated. we found that 85 of the 100 genotypes evaluated presented 4x = 48 chromosomes (table s1 and figure 1d, 1e -1f). of these, 66 genotypes from commercial and wild populations were tetraploid, and 19 doubled haploid genotypes derived from anther culture. seven haploid genotypes from anther culture showed n = 2x = 24 chromosomes (table s1 and figure 1a-1b). two aneuploids were observed: the genotype 09u012-5 with 44 chromosomes and the genotype 09u261-2 with 49 chromosomes (table s1 and figure 1c). mixoploidy was also present in six genotypes (table s1 and figure 1g-1i) related to five plants from anther culture and the genotype 09u136-3 from a working collection. the mixoploid genotypes presented different chromosomal complements in the same plant, with counts ranging from 36 to 86 chromosomes (36, 38, 40, 44, 48, 49, 52, 54, 57, 58, 60-74, 76-78, 80, 82, and 86 chromosomes). these results indicate that the population of one hundred cape gooseberry genotypes studied has a high cytogenetic diversity represented by tetraploids, aneuploids, mixoploids, haploids and doubled haploids. 3.2 chloroplast counts the number of chloroplasts per guard cell of haploid genotypes derived from anther culture ranged from 4 to 8, the aneuploid genotypes presented between 6 to 16 chloroplasts, while tetraploids and doubled haploid genotypes presented between 7 to 16 chloroplasts. additionally, the mixoploid genotypes ranged from 9 to 21 chloroplasts per guard cell. table s1 and figure 2 show the relationship of the chromosome and chloroplast counts of the analyzed genotypes. 3.3 cluster analysis according to the cluster analysis (figure 3 and table 1) the studied genotypes formed four groups. the first 24 viviana franco-florez et al. group was made up of seven haploid genotypes with an average of 5.69 chloroplasts. the second cluster grouped 35 genotypes corresponding to doubled haploids and tetraploids that presented a chloroplasts average of 11.04. the third group consisted of six mixoploid genotypes mostly derived from anther culture with a chloroplast average of 13.63. the fourth group was integrated by 52 genotypes that included doubled haploids, tetraploids and aneuploids with a chloroplast average of 9.82. pearson correlation coefficient between the number of chromosomes and number of chloroplasts per guard cell was of r = 0.89 (p < 0.0001). 4. discussion 4.1 diversity of chromosome counts a population of one hundred genotypes of cape gooseberry from commercial, wild, working collection and anther derived plants were analyzed in this study. a wide range of cytogenetic variations was observed from the four sources of genotype samples. specifically, the counts of 48 chromosomes from tetraploid genotypes found in this study coincide with that reported by vilmorin and simonet, (1928); menzel, (1951); gupta and figure 1. karyotypes representative of p. peruviana genotypes. a. and b. n = 2x = 24 genotypes 12u398 and 09u292-7. c. 49 chromosomes genotypes 09u261-2. d., e., and f. 2n = 4x = 48 genotypes 09u134-3, 09u048-1 y 09u120-3. g., h. and i. mixoploids 38, 48 y 71 chromosomes, genotypes 09u136-3, 09u296-2 and 14u422. scale of 10µm. all the photos were taken at 100x except for image 1h, which was taken at 40x. 25cytogenetic and cytological analysis of colombian cape gooseberry genetic material for breeding purposes roy, (1985); moriconi et al., (1990), and ganapathi et al., (1991). the results also agree with recently published studies that mention the 48-chromosome number as the most common event in p. peruviana l. genotypes from brazil (trevisani et al., 2018 ), peru (carbajal, 2018). the chromosome number 2n = 4x = 48 was also mentioned in the work of lagos, (2006), who reported for p. peruviana chromosome numbers of 24, 36 and 48 as the three characteristic karyotypic constitutions for the species. the results of this study also agrees with the 48 chromosome number of the kenyan ecotype reported by rodríguez and bueno (2006) and with the results of liberato et al., (2014) who determined 2n = 4x = 48 in several cultivated genetic material from colombia. seems that the 48 chromosome number es predominant in p. peruviana l. as shown by trevisani et al., (2018) who reported the chromosome number 2n =4x = 48 of four p. peruviana l. populations from brazil, colombia and peru, classifying them as polyploid with tetraploid cells, which agrees with the results of this study. among the genotypes analyzed there were several plants derived from anther culture with the genetic load of 48 chromosomes like their parents and defined as doubled haploid lines 2(n) = 4x = 48. reduction of chromosome number and double haploidization are events that occur through the process of androgenesis. this, may be due to spontaneous or induced chromosome duplication of the microspore under in vitro culture conditions, as reviewed by germanà, (2011). several studies have shown chromosome number related to diploidy level in several p. peruviana l. populations from different countries. rodríguez and bueno (2006), lagos (2006) and liberato et al., (2014) determined chromosomal constitutions of 2n = 2x = 24 in wild genetic materials from colombia. while azeez et al., (2019) and azeez and faluyi (2019) in two different studies found that p. peruviana l. has 2n = 2x = 24 chromosome constitution as compared to three different nigerian physalis species. in this study several genotypes derived from anther culture showed a reduction by half in the chromosomal load to the gametic number of 24, the same as the gametic chromosome number of the species. these anther culture derived genotypes are considered as haploid lines with n = 2x = 24 ploidy. these results were also observed by escobar et al., (2009) in a study of anther culture with different cultivars of mexican husk tomato (p. ixocarpa brot.). in the present study the results also show other chromosome numbers in several genotypes related to mixoploidy and aneuploidy, quite different from those tetraploids, diploids, doubled haploids and haploids genotypes analyzed. the mixoploid and aneuploid nuclei observed in several sample plants were not only observed in genfigure. 2. number of chromosomes and chloroplasts per guard cell from some representative genotypes of p. peruviana. a. somatic metaphase cell with 24 chromosomes and stomata with five chloroplasts. b. cell with 48 chromosomes and stomata with nine and ten chloroplasts in the guard cells. c. cell with 49 chromosomes and stomate with 12 chloroplasts in the left guard cell. d. cell with 69 chromosomes and stomate with 17 chloroplasts in the upper guard cell. 26 viviana franco-florez et al. otypes from anther culture derived plants, but also in plants from working collections derived from germplasm bank main collection. our results although do not show the same chromosome number for mixoploidy and aneuploidy, agree with the several published reports in p. peruviana l. rodríguez and bueno (2006) found that colombia ecotype had 2n = 3n = 32 chromosome number and lagos, (2006) reported chromosome numbers of 36. the results suggests that in cape gooseberry not all the cells analyzed from the same plant sample have the equal chromosome number, as already shown by carbajal (2018) who reported that 30%, 40% and 50% of the analyzed cells showed aneuploidy, with chromosome number range from 44 to 80 quite different from the commonly observed chromosome number of 2n = 4x = 48. mixoploidy is a very promising source to produce haploid plants in short time from somatic tissue. haploid plants once their genome is duplicated double haploid plants can be generated and used in genetic studies and crop improvement. in plants derived from anther culture or isolated microspore that arise through the process of androgenesis, besides expecting haploids and doubled haploids genotypes, for the case of p. peruviana, aneuploidy and mixoploidy are also generated, as was observed in our results. zagorska et al., (2004), mentioned that the variation in the chromosome number in gametes or gametophytic tissue plays an important role for gametoclonal variation during in vitro androgenesis and can result in haploid, doubled haploid, tetraploid, aneuploid and mixoploid plants. the variation in chromosomal contents of the plants derived from anther culture might be related to aberrant cell division in the immature pollen grains due to the stress generated by the in vitro culture conditions. this phenomenon was observed by sánchez, (2014) and garcía-arias et al., (2018b) in cape gooseberry anther culture-derived plants and other species such as solanum lycopersicum (arcobelli et al., 2014) and humulus lupulus (koutoulis et al., 2005). however, plants that come from natural populations as is the case of the genotype 09u136-3 of this study was mixoploid despite being from natural populations; suggesting that mixoploidy occurs naturally in cape gooseberry because of the continuous evolving process of the specie that is still under domestication. to this respect, trevisani et al., (2018) states that physalis peruviana possibly has not fixed its chromosomal structure yet. the aneuploid genotype 09u012-5 with 44 chromosomes analyzed in this study agrees in chromosome number with the ecotype peru reported by franco, (2012), while the genotype 09u261-2 with 49 chromosomes corresponds to a new report for p. peruviana. these chromosomal loads do not have a direct relationship with the basic number x = 12 reported for the genus physalis (menzel, 1951; gupta and roy, 1985; ganapathi et al., 1991; lagos, 2006, trevisani et al., 2018 and carbaja, 2018). therefore, these genotypes could arise from figure 3. dendrogram of grouping by number of chloroplasts in stomatal cells and chromosome number of 100 genotypes of p. peruviana. 27cytogenetic and cytological analysis of colombian cape gooseberry genetic material for breeding purposes the restructuring chromosomal set that involves the gain or loss of a chromosome (aneuploidy), structural rearrangement of chromosomes resulting in the increase or decrease in chromosome number, or hybridization between polyploids with different chromosome numbers (poggio and naranjo, 2004). 4.2 chloroplast counting as a proxy for ploidy level determination the number of chloroplasts found in the haploid genotypes from this investigation agrees with results reported by garcía-arias et al., (2018b) in p. peruviana haploid plants obtained from anther culture as well. in that report, the authors specified that those plants had 4-7 chloroplasts per guard cell. in an independent study, franco, (2012) working with three ecotypes reported ranges of 7-12 chloroplasts per guard cell for peru, and 8 to 13 chloroplasts for the colombia and kenya, the same as the results shown in the present study. additionally, the pearson correlation coefficient presented in this study is higher than the one reported by garcía-arias et al., (2018b), who showed a correlation of r = 0.61, probably due to differences in the plant material analyzed and the sample size. in both research works a relationship between chloroplast number and chromosome number, that suggest that the chloroplast number is influenced by de chromosomal content of a genotype in a proportional manner, as shown by rodríguez and bueno, (2006). therefore, the chromosomal variation, can affect the phenotypic characteristics of a genotype, justifying a direct proportional relationship between the level of ploidy and the size of different organs of the plant. recently garcía-arias et al., (2018b) reported that the increase of the number of chromosomes of haploid sterile cape gooseberry plants, is directly associated with the recovery of pollen fertility, variation in morphology of the leaf, flower bud, flower and normal fruit set. the results of the cluster analysis showed four groups based on chromosome and chloroplast number, differentiating the haploid, doubled haploid, tetraploid and mixoploid genotypes (table 1). a mixture of doubled haploid and tetraploid genotypes in the cluster two, formed two subgroups due to the amplitude of the range in the number of chloroplasts. the third group was composed of mixoploid genotypes and the fourth group was composed of tetraploid and aneuploid genotypes. these results suggest that ploidies can be estimated primarily by counting the number of chloroplasts in guard cells, but not in an exact way, since the aneuploid, doubled haploid and tetraploid genotypes formed a single group due to overlapping chloroplast count totals. 4.3 crossability strategies the ploidy level is very important for plant breeding and crop improvement strategies (udall and wendel, 2006; ochatt, 2011). this highly relates with natural divergence among subpopulations and the scale of local adaptation, as well as to the capability of gene flow and crossability in plants for successful interspecific and intraspecific hybridization. the observed chromosomal variations of 44, 48 and 49 can occur without noticeable changes in the phenotype (poggio and naranjo, 2004), while the cases of mixoploidy generate evident phenotypic changes. in p. peruviana, franco, (2012) and garcía-arias et al., (2018b) found an amorphous development in flowers and fruits, changes in the floral and vegetative structure, and an increase in the size of the fruit associated with mixoploidy events. table 1. cluster analysis. conformation of groups by chromosome number. group chromosomal number totalhaploid n = 2x =24 doubled haploid 2(n) = 4x =48 tetraploid 2n = 4x =48 aneuploid 44 49 mixoploid 36-86 1 fr eq ue nc y* 7 0 0 0 0 0 7 2 0 8 27 0 0 0 35 3 0 0 0 0 0 6 6 4 0 11 39 1 1 0 52 total 7 19 66 1 1 6 100 chloroplast average 5,69 10,40 10,27 9,34 13,63 chloroplast range 4-8 7-16 7-16 6-16 9-21 * number of genotypes that present determined chromosomal number. 28 viviana franco-florez et al. in crosses between genotypes with chromosomal dissimilarities, in terms of shape, size and number, there may be no fertilization or abortions due to irregularity in meiosis, a situation reported by menzel, (1951) in other physalis species. serrato-cruz et al., (2000) and laguado, (2007) unexpected ploidy levels, genomic instability, or odd chromosome numbers in hybrid progenies between parents with different ploidy level, which could lead to events of unreduced gametes, apomixis, and chimeras, leading to infertility and low levels of productivity. the genotypes which chromosomal complement have broad similarity can be successfully crossed since a normal meiotic division would occur, as menzel, (1951), ortiz et al., (1998) and serrato-cruz et al., (2000) pointed out. consequently, we could carry out intraspecific crosses among genotypes with the same chromosomal numbers, expecting to obtain viable and stable progeny. a precedent example of the success of crossing between genotypes with the same ploidy is the work of berdugo et al., (2015). in their study the authors made intraspecific crosses among accessions of p. peruviana with the same chromosome number, finding 100% viability in the progeny. in general, chloroplast counting could be useful as a quick method to assess the ploidy when the number of genotypes to be analyzed is large. 5. conclusions in this study, there was a close relationship between chromosome and chloroplasts number per guard cell, which was confirmed through the cluster analysis. it is clear, that the chloroplast count is an approximate and quick methodology to be used as a preliminary way for determination of the ploidy level, when dealing with many genotypes. as far we know, these are the first study that analyzed one hundred genotypes of p. peruviana l. from different sources for breeding purposes. in summary, this work contributes to the cytogenetic and cytological knowledge of cape gooseberry and is a useful tool in the selection of crossing parents in breeding programs. author contributions vmnz and eps-b conceived the study. vf-f, salg realized the experimental work. vf-f, sal-g and flg-a performed statistical analysis and prepared the first manuscript, vmnz refined the final version. all authors read and approved the final version of the manuscript. acknowledgments we thank the corporación colombiana de investigación agropecuaria agrosavia and minciencias for their financial support. funding this work was supported by the corporación colombiana de investigación agropecuaria – agrosavia and minciencias in the project “nuevos cultivares de uchuva con características genéticas, fisiológicas y fitosanitarias con énfasis en resistencia a fusarium oxysporum f. sp. physali y calidad de fruto”. references agronet (2019). reportes estadísticos. available at: http:// www.agronet.gov.co/estadistica/paginas/default.aspx. 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(2004). induced androgenesis in tomato (lycopersicon esculentum mill.). iii. characterization of the regenerants. plant cell rep. 22, 449–456. doi:10.1007/s00299003-0720-8. caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 3 2021 firenze university press chromomycin a3 banding and chromosomal mapping of 45s and 5s ribosomal rna genes in bottle gourd ahmet l. tek*, hümeyra yıldız, kamran khan, bilge ş. yıldırım development of a protocol for genetic transformation of malus spp federico martinelli1,*, anna perrone2, abhaya m. dandekar3 cytogenetic and cytological analysis of colombian cape gooseberry genetic material for breeding purposes viviana franco-florez, sara alejandra liberato guío, erika sánchez-betancourt, francy liliana garcía-arias, víctor manuel núñez zarantes* palynological analysis of genus geranium (geraniaceae) and its systematic implications using scanning electron microscopy jun wang1,2,*, qiang ye1, chu wang2, tong zhang2, xusheng shi2, majid khayatnezhad3, abdul shakoor4,5 influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats yuri a. kirillov1, maria a. kozlova1, lyudmila a. makartseva1, igor a. chernov2, evgeniya v. shtemplevskaya1, david a. areshidze1,* the morphological, karyological and phylogenetic analyses of three artemisia l. (asteraceae) species that around the van lake in turkey pelin yilmaz sancar1,*, semsettin civelek1, murat kursat2 molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant jinxin cheng1,*, dingyu hu1, yaran liu2, zetian zhang1, majid khayatnezhad3 genetic variations and interspesific relationships in salvia (lamiaceae) using scot molecular markers songpo liu1, yuxuan wang1, yuwei song1,*, majid khayatnezhad2, amir abbas minaeifar3 development of female gametophyte in gladiolus italicus miller (iridaceae) ciler kartal1, nuran ekici2,*, almina kargacıoğlu1, hazal nurcan ağırman1 colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae) harshita dwivedi*, girjesh kumar statistical evaluation of chromosomes of some lathyrus l. taxa from turkey hasan genç1, bekir yildirim2,*, mikail açar3, tolga çetin4 use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater treatment and water of the kamniška bistrica river (slovenia) peter firbas1, tomaž amon2 the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bo shi1,*, majid khayatnezhad2, abdul shakoor3,4 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(2): 131-139, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1056 caryologia international journal of cytology, cytosystematics and cytogenetics citation: xiao cheng, xiaoling hong, majid khayatnezhad, fazal ullah (2021) genetic diversity and comparative study of genomic dna extraction protocols in tamarix l. species. caryologia 74(2): 131-139. doi: 10.36253/caryologia-1056 received: august 18, 2020 accepted: july 22, 2021 published: october 08, 2021 copyright: © 2021 xiao cheng, xiaoling hong, majid khayatnezhad, fazal ullah. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. genetic diversity and comparative study of genomic dna extraction protocols in tamarix l. species xiao cheng1,*, xiaoling hong1, majid khayatnezhad2, fazal ullah3,4 1 jiangxi university of applied sciences, nanchang, jiangxi , 330100, china 2 department of environmental sciences and engineering, ardabil branch, islamic azad university, ardabil, iran 3 cas key laboratory of mountain ecological restoration and bioresource utilization and ecological restoration, biodiversity conservation key laboratory of sichuan province, chengdu institute of biology, chinese academy of science, p.o box 416, chengdu, sichuan 610041, china 4 university of chinese academy of science, beijing 100049, china *corresponding author. e-mail: chengxiao20212021@163.com abstract. the genus tamarix consists of about 54 species that mainly grow in saline areas of deserts and semi-deserts. this genus is chemically characterized by the presence of tannins, flavonoids, anthocyanins and essential oils which interfere with the extraction of pure genomic dna. thus it is necessary to optimize extraction protocols to minimize the influence of these compounds to the lowest level. the present study compares the efficiency of five different approaches to extract total genomic dna in tamarix species, showing significant differences in the extracted dna contents and quality,by using kit (dnp tm kit), ctab dna extraction method by murray and thompson, sahu et al., nalini et al. and bi et al., for the extraction of dna from tamarix species. our results showed significant differences in dna contents between these five methods. the quantity and quality of extracted genomic dna were checked by the spectrophotometer, nano-drop and and agarose gel electrophoresis analysis. finally, a pcr-based method was also applied to verify the amplification efficiency for two molecular markers (its and issr).. in the present study, the genetic diversity of 96 tamarix individuals species and 8 populations were studied using 10 issr markerswhile for nrdna its 8 species samples were used. the method of nalini et al., provided best results (207 ng/μl) in terms of quantity and quality ofdna. our results proposed that this method could be effective for plants with the same polysaccharides, proteins and polyphenols components. the advantage of this method is simple and fast as it does not involve time consuming steps such as incubation at higher temperatures, and also do not requires expensive chemicals such as proteinase k, liquid nitrogen. ,. the success of this method in obtaining high-quality genomic dna has been demonstrated in the tamarix species group and the reliability of this method has been discussed. keywords: dna yield, extraction protocols, tamarix, issr, secondary metabolites. 132 xiao cheng et al. introduction tamaricaceae is relatively a small family of 4 genera and 120 species (trease and evans, 2002). the genus tamarix l. (tamarisk, salt cedar) contain about 54 species that mainly distributed in saline areas of deserts and semi-deserts in europe, concentrated mainly in the mediterranean region and eastern europe (gaskin, 2003). they are typically adapted to arid climate with an efficient and deep root system (baum, 1978). thirty-five species of tamarix occur in iran reported by schiman-czeika (1964). these species have been used in plantation to prevent deforestation in iran. the species of tamarix are distributed in 21 provinces of iran. some species of the genus tamarix are used as ornamental plants (baum 1967; gaskin and schaal, 2002). tamarix species are frequently planted as windbreaks or grown for the stabilization and afforestation of sand dunes (gaskin and schaal 2003, gaskin and kazmer 2019, mayonde et al., 2019). tamarix are also famous for medicinal purposes such as the galls and bark are used as astringent. some species of the genus tamarix are utilized, as tonic, diuretic, stimulant, and stomachic action. they are also used as diaphoretic, diuretic,hepatotonic and to treat liver disorders, relieve headache, ease prolonged or difficulty during labor. some tamarix species are melliferous and are used as a sugar substitute (sharma and parmar 1998; abouzid et al. 2008; orfali et al 2009; bakr et al 2013; orabi et al., 2016). plastid dna (cpdna) and nuclear dna (ndna), can together be used to discourse different ecological queries. whereas the nuclear dna covers both unique single copy and repetitive regions (multiple copies), the chloroplast genome contains of coding segments such as ribosomal noncoding tandemly repeated units or rna genes (le roux and wieczorek, 2008). the its regions between the nuclear ribosomal dna (rdna) genes are commonly used for detecting changeability among species (sun et al., 1994). additionally, it is also a widely used molecular marker for rebuilding angiosperm phylogenies at different taxonomic levels as they always provide the correct level of difference at species level for well-resolved phylogenetic reconstruction (baldwin et al., 1995). the trns–trng primers are used to infer phylogenetic comparisons. moreover, chloroplast introns and intergenic spacer regions show the highest levels of intraspecific polymorphism since they are a lesser amount of inhibited through selection to preserve gene function (hamilton, 1999). the extraction and purification of high-quality dna is a critical step for genomic analysis especially from the plant materials with high accumulation of interfering substances including polysaccharides, proteins, and dna polymerase inhibitors such as tannins, alkaloids, and polyphenols. the presence of these compounds affects the quality and quantity of isolated dna, and therefore, renders the sample non-amplifiable (zamboni et al. 2008). pure and rapid dna extraction is a prerequisite for most advanced techniques such as genetic mapping, fingerprinting, marker-assisted selection, and for evaluating authenticity of exported cereal varieties. general problems in the isolation and purification of high molecular weight dna from medicinal and aromatic plant species include: (1) degradation of dna due to endonucleases, consolation of highly viscous polysaccharides, and (2) inhibitor compounds like polyphenols and other secondary metabolites which directly or indirectly interfere with the enzymatic reactions (weising et al. 1995; jenderek et al., 1997; zamboni et al. 2008; sahu et al. 2012). the presence of polyphenols, as oxidizing agents present in many plant species, can reduce the production of the purified extracted dna (loomis 1974; porebski et al., 1997). several methods to isolate dna from plant tissues are available; however, these methods produce either small amounts or dna of inconsistent quality. some of the dna extraction methods are modified versions of cetyltrimethyl ammonium bromide (ctab) extraction and differ in time and cost (doyle and doyle 1990; reichandt and rogers, 1994). doyle and doyle method (1990) are applied to extract dna in fruit trees (jenderek et al., 1997). the extraction technique of lodhi et al. (1994) has been utilized for the grape, apple, apricot, peach, cherry and snapdragon. sarkhosh et al. (2006) used the bi et al. (1996) method for some iranian pomegranate (punica granatum l.) genotypes. murray and thompson (1980) method were used for dna extraction in cabbage, olive, rose (csaikl et al., 1998) and sweet cherry (khadivi-khub et al., 2008). saghai-maroof et al. (1984) method was used for dna extraction in mangroves and salt marsh species (sahu et al. 2012). talebi baddaf et al. (2003) introduced murray and thompson (1980) method as the most appropriate method to achieve high-quality dna extraction from pomegranate leaves. because plants contain high amounts of many different substances, it is unlikely that just one nucleic acid isolation method suitable for all plants can ever exist (loomis, 1974). a perfect method is the one that is fast, simple, and reliable dna extraction method, which does not require long incubations, multiple dna precipitations, or commercial reagents, and could meet the pcr, sequencing, and next-generation library preparation requirements. therefore, the aim of this study was to compare quality 133genetic diversity and comparative study of genomic dna extraction protocols in tamarix l. species and quantity of five different dna extraction methods to isolate high-quality dna from leaf tissues of different tamarix species. in this study, we showed the results of tests from several dna extraction protocols that were made to overcome the problems that mainly arise from polysaccharide contamination. issr and its amplification was also performed to evaluate the suitability of the dna extraction methods for pcr-based techniques. as far as, we know, this’s the first report on dna extraction from tamarix leave at species level from iran, and we expect that the suggested protocol can be an incentive to perform further studies in order to investigate the genetic diversity among the plants with same chemical components as tamarix species. materials and method plant samples for dna isolation in this study leaves of 8 tamarix species were collected from different habitats in iran (table 1). one gram of young and mature leaf was collected and then frozen in liquid nitrogen and stored at -70 °c until extraction. for molecular studies, we used different number of plant individuals, as they were required. for example, in issr analysis, we used 96 individual samples of 8 species, while for nrdna its 8 individual of 8 species were used for the extraction of dna. dna extraction methods one gram of the frozen leaf samples of tamarix were grind into fine powder using pre-cooled mortar and pestle, and then homogenized with five different dna extraction methods based on randomized complete block design (rcbd) with five replicates. the five extraction methods were 1) murry and thompson (1980); 2) kit (dnp tm kit) 3) sahu et al. (2012),4) bi et al. (1996) 5) nalini et al. (2003) methods. after dna extraction and sedimentation, resulted pellet was rinsed with ethanol 75% and dissolved in 200 μl double distilled sterile water at 4 °c overnight and stored at -70 °c until next treatments. the chemicals used for the isolation of dna viz. tris, edta were obtained from sigma and sodium chloride, urea, sds, isopropanol, sodium acetate, chloroform, isoamlyalcohol, phenol, dntps, enzyme taq dna polymerase, 10x-assay buffer for taq dna polymerase, magnesium chloride and agarose. concentration, purity and quality of extracted dna the quantity (concentration and extraction efficiency) and quality (purity and intactness) of the dna obtained at the ratio of 1:49 (20 μl of dna stock solution + 980 μl of double distilled sterile water) were assessed using spectrophotometer at 260 and 280 nm, and the a260/a280 ratio was used to assess contamination with proteins through employing the spectrophotometry (hitachi u-2001 uv/vis), nano-droptm (thermo scientific) described by brodmann (2008) and wilmington (2008), agarose gel electrophoresis, pcr methods and molecular markers (its and issr). this spectrophotometric analysis was performed in triplicate on the samples of extracted dna using spectrophotometer. to verify dna integrity, 5 μl dna from 7 sample were subjected to gel electrophoresis at 0.8% (w/v) agarose gel, stained with ethidium bromide, and a constant voltage of 120  v for 90  min. the dna bands were visualized, and the images were acquired using gel doc xr+ imaging system (bio-rad laboratories inc., germany). issr amplifications the quality of extracted dna was examined at 0.8% agarose gel. in total, 10 issr primers; (agc) 5gt, (ca) table 1. tamarix species and populations, their localities and voucher numbers. r taxa locality alt (m) latitude longitude voucher no 1 tamarix arceuthoides bge. ardabil, khalkhal-asalem road 1500 37°57’36” 48°61’03” iauh1011 2 t. ramosissima ledeb gilan, damash 1700 36°75’54” 49°81’07” iauh1012 3 t. chinensis lour. fars, shahr miyan 2700 30°84’40” 52°06’76” iauh1013 4 t. szowitsiana bge. mazandaran,chalus, visar 1400 36°65’011” 51°31’051” iauh1014 5 t. meyeri boiss. gilan, damash 1700 36°75’54” 49°81’07” iauh1015 6 t. androssowii litw. golestan forest 700 37°47’50” 47°23’36.2” iauh1016 7 t. mascatensis bge. mazandaran, noshahr, kheyrud kenar forest 400 36°38’05” 51°29’05” iauh1017 8 t. aucheriana (decne. ex walp.) b.r. baum. ardabil,meshkin shahr, hatam forest 2700 38°18’77.1” 56°41’60” iauh1018 134 xiao cheng et al. 7gt, (agc) 5gg, ubc 810, (ca) 7at, (ga) 9c, ubc 807, ubc 823, (ga) 9t and (gt) 7ca commercialized by ubc (the university of british columbia) were used (see table 2). the final volume of 12 μl was tested in pcr reaction (2.5 μl pcr reaction buffer 10x, 0.875 μl mgcl2 50 mm, 0.5 μl dntps 10 mm, 1.0 μl primer 10 μm, 0.2 μl taq dna polymerase 5 unit/μl, 2.0 μl template dna (5 ng/μl). the amplification, reactions were performed in techne thermocycler (germany) with the following program: 5min initial denaturation step 94°c, followed by 38 cyclesfor 1 min at 95°c; 1 min at 50-55°c and 1 min at 72°c. the reaction was completed through a final extension step of 5-10 min at 72°c. the amplification products were observed at 1% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated using a 100 bp molecular size ladder (fermentas, germany). itssequences the its region was amplified using pcr with following primer pairs its-4 and its-5 (white et al. 1990). the final volume of 12 μl was tested in pcr reaction (2.5 μl pcr reaction buffer 10x, 0.875 μl mgcl2 50 mm, 0.5 μl dntps 10 mm, 1.0 μl primer 10 μm, 0.2 μl taq dna polymerase 5 unit/μl, 2.0 μl template dna (5 ng/μl). the amplification, reactions were performed in techne thermocycler (germany) with the following program: 5min initial denaturation step 94°c, followed by 38 cycles of 1 min at 94°c; 40 sec, at 55°c and 1 min at 72°c. the reaction was completed by a final extension step of 5-10 min at 72°c. the amplification products were observed at 1% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated using a 100 bp molecular size ladder (fermentas, germany). the its regions were amplified using primers reported as universal primers by white et al. (1990) and taberlet et al. (1991), respectively, for flowering plants (see table 2). results comparison of different dna extraction methods on agarose gel electrophoresis the quality of 8 extracted dna sample was verified spectrophotometrically using a nanodrop instrument and agarose gel electrophoresis. dna purity and yield were compared between these five extracted dna methods. plant genomic dna extraction of murry and thompson (1980); kit (dnp tm kit), sahu et al. (2012), bi et al. (1996) (fig. 1b: 1-4), did not give best results for tamarix species due to the presence of polysaccharides and proteins in the pellet and showed brown or yellow dna precipitate that presents the gdna gel image. the presence of phenolic compounds caused a brownish pellet (fig. 1b). the results confirmed that extracted dna by nalini et al. (2003) method from leaves showed better quality in comparison with the other extraction methods (fig.1a). due to the elimination of polysaccharides or protein contaminations dna has been extracted with high quality. we believe that this method will be efficient for molecular studies of many other arotable 2. primer sequences used in this study. region primer sequences (5’-3’) tm ref. tabc cgaaatcggtagacgctacg 56 taberlet et al. (1991). tabf atttgaactggtgacacgag 56 taberlet et al. (1991). its4 tcctccgcttattgatatgc 57 white et al. (1990). its5 gga agt aaa agtcgt aac aag g 57 white et al. (1990). ubs807 agagagagagagagagt 54 ubs set no. 9 ubs810 gagagagagagagagat 54 ubs set no. 9 ubc 823 tctctctctctctctcc 56 ubs set no. 9 (agc) 5gt agc agc agc agc agc gt 56 ubs set no. 9 (ca) 7gt cacacacacacacagt 56 ubs set no. 9 (agc) 5gg agc agc agc agc agc gg 56 ubs set no. 9 (ca) 7at cacacacacacacaat 56 ubs set no. 9 (ga) 9c gagagagagagagagagac 56 ubs set no. 9 (ga) 9t gagagagagagagagagat 55 ubs set no. 9 (gt) 7ca gtgtgtgtgtgtgtca 55 ubs set no. 9 135genetic diversity and comparative study of genomic dna extraction protocols in tamarix l. species matic and herbal plants. in this method high level of β-mercaptoethanol successfully removed the polyphenols of the leaf tissue which may be responsible forinhibition of the dna amplification during pcr reactions (suman et al. 1999). it was evident that high concentration of β-mercaptoethanol resulted in the high-quality of dna. using of nacl concentrations higher than 0.5 m, along with ctab, was previously recorded to be efficient in removing polysaccharides during dna extraction (moreira and oliveira 2011, paterson et al. 1993). it was also efficient in the present study with 0.5m of nacl concentration. polysaccharides and secondary metabolites of tamarix species were bounded by pvp and it is in concordance with previous studies (couch and fritz 1990, chaudhry et al. 1999, zhang and stewart 2000). more replications for using chloroform: isoamyl alcohol resulted in better removing of proteins in tamarix species. sahu et al. (2012) used of sodium acetate and isopropanol only in step (xv), but we used one more time of this material in order to have the better precipitation of dna and removing most of the secondary metabolites and polysaccharides from the dna. the presence of higher quantities of polyphenols and polysaccharides in mature leaves are proved by porebski et al. (1997), which makes it very difficult to isolate dna of good quality. so, we used fresh and young leaves to overcome this problem. clear banding patterns were observed in the issr study by nalini et al. (2003) method (fig. 2a). it possess better quality in comparison with the other extraction methods as well as murry and thompson (1980); kit (dnp tm kit), sahu et al. (2012), bi et al. (1996) (fig.2 b, 1-4). pcr tests findings of its are given in (figs. 3. a, b) which showed that extracted dna by the method of nalini et al. (2003) method (figs. 3a) from leaf samples brings an acceptable quality for pcr, and as the most appropriate method in aspect of quality of dna extractfigure 1. electrophoretic pattern of dna extracted by the five different methods from tamarix leaves. note. the electrophoresis was performed in 0.8% (w/v) agarose gel. the extraction methods were: a) nalini et al. (2003) (1tamarix arceuthoides 2t. ramosissima ,3t. chinensis, 4t. szowitsiana, 5t. meyeri, 6t. androssowii, 7t. mascatensis and 8t. aucheriana); b) 1murry and thompson (1980); 2 kit (dnp tm kit), 3sahu et al. (2012), 4bi et al. (1996); l) 100 bp dna ladder. figure 2. amplification of dna from tamarix leaf using five different extraction methods by issr amplification. note. fig. 2. a) nalini et al. (2003); fig. 2. b) 1murry and thompson (1980); 2kit (dnp tm kit), 3sahu et al. (2012), 4bi et al. (1996); l) 100 bp dna ladder. 136 xiao cheng et al. ed from young leaves of tamarix. the pcr-amplified dna fragments of its for 8 samples showed a clean single band product, when examined on an agarose gel (fig. 3a). the pcr products were of about 600 bp. uv spectrophotometer and nanodrop™ 1000 spectrophotometer analysis in spectrophotometer procedure, absorption of double-stranded dna in wavelength of 260 nm was 50 μg/μl. in fact, the ratio of absorption amount resulted in 260 nm to 280 nm was ranged from 1.7 to 2.12. it shows the most absorption was done by nucleic acids and therefore extracted dna was well-qualified and its purity was acceptable. if the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. the results showed that the dna yield and dna purity obtained from one gram of the fresh leaf tissue in different methods using uv spectrophotometer was statistically significant (p ≤ 0.01). a higher dna yield was obtained with the method of nalini et al. (2003) (333±58.1 ng/μl fresh weight), while the lowest was obtained with method of sahu et al. (2012) (120±64.4 ng/μl fresh weight) (table 3). therefore, the results confirmed that extracted dna by nalini et al. (2003) method from leaves of tamarix possess better qualitative and quantitative results as compared to other methods. dna sample was measured with a uv spectrophotometer for the ratio of od260/od280 using te buffer. the ratio of od260/od280 was determined to assess the purity and concentration of dna sample. dna concentration was calculated according to the equation of wilmington et al. (2008). dna concentration (ng/μl) = od260 × a (dilution factor) × 50 absorbance measurements made on a spectrophotometer, including any thermo scientific nanodrop spectrophotometer, will include the absorbance of all molecules in the sample that absorb at the wavelength of interest. the ratio of absorbance at 260 nm and 280 nm was used to assess the purity of dna and rna. a ratio of ~1.8 was generally accepted as “pure” for dna; a ratio of ~2.0 was generally accepted as “pure” for rna. if the ratio appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. some researchers encounter a consistent 260/280 ratio change, when switching from a standard cuvette spectrophotometer to a nanodrop spectrophotometer. the three main explanations for this observation were listed below: small changes in the ph of the solution will cause the 260/280 to vary*. acidic solutions will under-represent the 260/280 ratio by 0.2-0.3, while a basic solution will over-represent the ratio through 0.20.3. if comparing results obtained using a nanodrop spectrophotometer to results obtained using other spectrophotometers, it is important to ensure that the ph of an undiluted sample measured on our instruments was at the same ph and ionic strength as the diluted sample measured on the conventional spectrophotometer. the nanodrop absorbance was useful for detection of contaminants such as protein, salts, and polysaccharides, which can inhibit and interfere in dna sequencing. the nanodrop 1000 sspectrophotometer has the capability to measure highly concentrated samples without dilution. the ratio of 260 and 280 nm absorbance figure 3. agarose gel (1.5%) showing the pcr amplified its of the plant materials used in the present study. note. fig. a) nalini et al. (2003) (1tamarix arceuthoides, 2t. ramosissima, 3t. chinensis, 4t. szowitsiana, 5t. meyeri, 6t. androssowii, 7t. mascatensis and 8 t. aucheriana); fig. b) 1murry and thompson (1980); 2kit (dnp tm kit), 3sahu et al. (2012), 4bi et al. (1996); l) 100 bp dna ladder. 137genetic diversity and comparative study of genomic dna extraction protocols in tamarix l. species was used to assess the purity of dna and rna. this ratio was between 1.7 and 1.9, and this range was generally accepted as “pure” for dna (table 3). discussion the quality and quantity of dna required depends on the extraction method and plant group. dna isolated from plants often contains certain compounds that inhibit pcr amplification reactions (reichandt and rogers, 1994). in this method sodium chloride and β-mercaptoethanol were added in the extraction buffer to take care of the polysaccharides and the polyphenols in the leaf tissue which were the compounds that contribute to the inhibition of the dna amplification during pcr reactions. hence there were no additional steps needed for the removal of these compounds (khadivikhub et al., 2008]. the presence of the enzyme rnase a in the dna solution does not hamper the amplification. hence repurification of the dna is not needed (csaikl et al., 1998). our results showed that the dna isolation protocol could be successfully applied to a broad range of plant species. sarkhosh et al. (2006) in a study on genetic diversity of pomegranate cultivars of iran, using random amplified polymorphic dna (rapd) using four different genomic dna extraction procedures; murray and thompson (1980), j. j. doyle and j. l. doyle (1990), ziegenhagen et al. (1993) and jenderek et al., (1997) introduced murray and thompson’s method as the most appropriate and successful method in terms of quality of dna extraction from young leaves of pomegranate. jenderek et al. (1997) have found the method of j. j. doyle and j. l. doyle as the best quality resulting method for dna extraction form marshmallow, but its quantity was too low. saha et al. (2016) in a study on genetic stability of morus alba l. variety and nadha et al. (2011) on genetic diversity of guadua angustifolia kunth, using rapd and issr marker introduced murray and thompson (1980), and j. j. doyle and j. l. doyle (1990) methods as appropriate dna extraction procedures, respectively. bhatia et al. (2011) in a study on the genetic fidelity of gerbera jamesonii bolus using dna-based markers were used murry and thompson (1980). pcr tests finding showed that the extracted dna by bi et al. (1996) method from leaf samples brings an acceptable quality forth for pcr, and the candescence of amplified dna bands, in this study, five dna extraction methods were compared to isolate high-quality dna that can be efficiently amplified using pcr techniques. murry and thompson (1980); kit (dnp tm kit), sahu et al. (2012), bi et al. (1996) resulted in brown or yellow dna precipitate that could not be reliably amplified through pcr. therefore, we used the method of nalini et al. (2003) that produced good quality dna., the dna extracted by this method is successful in many land plants including; mangroves and salt marsh plants containing elevated concentrations of polysaccharide and polyphenolic compounds (nalini et al. 2003). nalini et al. (2003) method are helpful to provide a pure dna with high efficiency in tamarix species. advantages of the present method for studying medicinal plants with secondary metabolites are as follows: 1) omission of liquid nitrogen, 2) decrease of toxic effects, hazardous, expensive of some component as phenol in other methods, 3) lower amount of dried or fresh plant material, without any conservation specific condition. although this method has many advantages but its time-consuming. the dna extracted using this protocol can be used for whole-genome sequencing, advanced sequencing technologies, and bioinformatics tools. acknowledgements this project was supported by faculty life sciences and biotechnology, islamic azad university, ardebil, iran. table 3. comparison of means for efficiency of three different dna extraction methods in leaf samples of leaves tamarix using duncan’s multiple range test (p ≤ 0.01). methods spectrophotometer nano-drop dna yield (ng/μl) dna purity (ng/μl) dna yield (ng/μl) dna purity (ng/μl) nalini et al. (2003) 333±58.1 2.12±0.15 590.4±86.5 1.94±0.15 kit (dnp tm kit) 178±33.8 1.8±0.18 767.5±11.8 1.80±0.09 murray and thompson (1980) 292±34.4 1.7±0.19 534±76.4 1.78±0.07 sahu et al. (2012) 120±64.4 2.01±0.18 575±55.2 1.82±0.09 bi et al. 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doi: org/10.3832/ifor0465-0010122 zhang, j., stewart, j.m. 2000. “economical and rapid method for extracting cotton genomic dna,” – j. cotton. sci. 4: 193–201. caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 1 2021 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 75(1): 65-76, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1310 caryologia international journal of cytology, cytosystematics and cytogenetics citation: tinglu liu, shuangshuan zhang, yonghe hao, xiao liang, mohsen farshadfar (2022) genome survey of pistachio (pistacia vera l.) accessions revealed by start codon targeted (scot) markers. caryologia 75(1): 65-76. doi: 10.36253/caryologia-1310 received: may 10, 2021 accepted: august 24, 2021 published: july 6, 2022 copyright: © 2022 tinglu liu, shuangshuan zhang, yonghe hao, xiao liang, mohsen farshadfar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. genome survey of pistachio (pistacia vera l.) accessions revealed by start codon targeted (scot) markers tinglu liu1, shuangshuan zhang1,*, yonghe hao1, xiao liang2, mohsen farshadfar3 1 ordos agriculture and animal husbandry technology popularizing center, ordos, inner mongolia 017000 2 ordos city, this paper flag farming technology promotion center, ordos, inner mongolia 017000 3 department of agriculture, payame noor university (pnu), tehran, iran *corresponding author. e-mail: zhang123123301@163.com abstract. pistachio (pistacia vera l.) is the only cultivated and commercially important species in the genus pistacia, consisting of a deciduous, dioeciously and wind-pollinated at least 11 tree species. pistacia vera is native to north afghanistan, northeast iran, and central asian republics. to investigate the genetic diversity of pistachio (pistacia vera), we genotyped 30 cultivars of this species using 10 start codon targeted (scot) markers. the scot markers generated 9-25 alleles (155 in total) with an average of 16 per locus. the highest value of percentage polymorphism (61.99%) was observed in ghafori rafsanjan (cultivars no.27) which shows high value for gene diversity (0.42) and shanon, information index (0.39). genotype shahpasand (pust ghermez) (no.10) has the lowest value for percentage of polymorphism (20%) and the lowest value for shanon, information index (0.15), and he (0.010). genetic similarity values obtained from dice’s coefficient ranged from 0.66 (between akbari (pust ghermez) and badami dishkalaghi) to 0.88 (between populations menghar kalaghi and kaleghochi (pust ghermez). the main objectives of this study were to assess the genetic diversity and genetic relationship of pistachio cultivars in iran. these results could benefit irainian pistachio germplasm collection, conservation and future breeding. keywords: population structure, gene flow, network, genetic admixture, pistachio (pistacia vera l.). introduction genetic variability description specifies differences among individuals or populations of the same species and serves as a very good tool for plant breeding and conservation programmes (minn et al. 2015). different types of dna markers have been applied in evaluation of genetic diversity of different plants, considering also the effects of the plant growing environment and developmental stage (hopla et al. 2021; fikirie et al. 2020; gondal et al. 66 tinglu liu et al. 2021). the existing genetic variability of the individual species within and among the populations is connected to this species ability to mirror the shortand long-term specific regimes of their living habitats. the analysis of the distribution of the genetic variability patterns specific for landscape and ecological parameters is valuable for identification of the taxa most vulnerable to the anthropogenic impacts (brandvain et al., 2014). the genus pistacia is a member of the anacardiaceae family, which comprises 11 or more species (zohary 1952). pistacia vera l., is a diploid (2n=30) member of the anacardiaceae family (zohary 1952; whitehouse 1957). pistacia vera is native to north afghanistan, northeast iran, and central asian republics (browiez 1988; kafkas 2006). among the nut tree crops, pistachio tree ranks sixth in world production behind almond, walnut, cashew, hazelnut and chestnut (mehlenbacher 2003). iran is the main world producer with more than 400,000 tons followed by turkey, usa and syria (faostat 2004). the main cultivars grown in iran are ohady, kaleh ghochi, ahmad aghai, badami zarand, rezaii and pust piazi (esmailpour 2001). iran is the center of origin for four important pistacia species: p. vera, p. khinjuk stocks, p. eurycarpa yalt. (p. atlantica subsp. kurdica zoh.), and p. atlantica dsef. (karimi et al. 2009). three essential wild pistacia species, including p. vera, p. khinjuk, and p. atlantica grow in iran. although wild p. vera has spread to a territory of around 75,000 ha, in focal asia, which envelopes turk menistan, afghanistan, and northeast iran, where p. vera develops in the sarakhs region, covering around 17,500 ha (behboodi 2003). numerous studies have addressed genetic variability in pistacia that were based on evaluation of morphological, physiological, and biochemical characteristics (zohary 1952; barone et al. 1993; dollo 1993; tayefeh aliakbarkhany et al. 2013). among them, rapd (williams et al. 1990) has been the most commonly used method in pistachio cultivars characterization (hormaza et al. 1994, 1998; kafkas et al. 2002; katsiotis et al. 2003; golan-gpldhirsh et al. 2004; mirzaei et al. 2005). aflp and ssr techniques have been also used in pistachio to study genetic relationship among pistacia species and cultivars (golan goldhirsh et al. 2004; katsiotis et al. 2003; ibrahim basha et al. 2007; ahmad et al. 2003; ahmad et al. 2005; ahmadi afzadi et al. 2007). although previous studies have partially characterized pistachio diversity in iran, they did not conduct a full analysis regarding discrimination of wild pistacia and its potential breeding and implication of its conservation. induction of diversity in pistacia species are based on morphological characteristics which usually can be achieved by budding or grafting selected scions onto seedling rootstocks of the same species or other pistacia species. pistacia species have a high genetic diversity due to their dioecious character, pollination mechanism. because of these factors high selectivity in rootstocks breeding is required, and therefore knowledge of the genetic relationships among pistacia species would be very useful in pistachio rootstock breeding. with the progress in plant molecular biology, numerous molecular marker techniques have been developed and used widely in evaluating genetic diversity, population structure and phylogenetic relationships. in recent years, advances in genomic tools provide a wide range of new marker techniques such as, functional and genetargeted markers as well as develop many novel dnabased marker systems (collard and mackill 2009). start codon targeted (scot) polymorphism is one of the novel, simple and reliable gene-targeted marker systems. this molecular marker offers a simple dna-based marker alternative and reproducible technique which is based on the short conserved region in the plant genes surrounding the atg (collard and mackill 2009) translation start codon. this technique involves a polymerase chain reaction (pcr) based dna marker with many advantages such as low-cost, high polymorphism and extensive genetic information (collard and mackill 2009; wu et al. 2013; luo et al. 2011). the scot system has been successfully used to assess genetic diversity, carry out structure analysis, identify cultivars, map quantitative trait loci (qtl), as well as perform dna fingerprinting and diagnosis in different species (elshibli and korpelainen 2008; rhouma et al. 2009). the present study is the first attempt to use scot markers to assess the level of genetic diversity of irainian pistachio cultivars which were collected from the wild populations. the main objectives of this study were to assess the genetic diversity and genetic relationship of pistachio cultivars in iran. these results could benefit irainian pistachio germplasm collection, conservation and future breeding. materials and methods plant materials thirty specimens belonging to three geographical populations of pistacia vera were collected from different localities that were placed between three provinces semnan, damghan, khorasan, mashhad and kerman, rafsanjan. details of geographical populations are given in table 1, fig. 1. different references were used for the correct identification of species pistacia vera (zohary 1952; barone et al. 1993; dollo 1993). vouchers were deposited 67genome survey of pistachio (pistacia vera l.) accessions revealed by start codon targeted (scot) markers at the herbarium of islamic azad university, science and research branch, tehran, iran (iauh). dna extraction and scot-pcr amplification fresh leaves were used randomly from four to eleven plants in each of the studied populations. these were dried by silica gel powder. ctab activated charcoal protocol was used to extract genomic dna (esfandanibozchaloyi et al. 2019). the quality of extracted dna was examined by running on 0.8% agarose gel. a total of 25 scot primers developed by collard and mackill (2009), 10 primers with clear, enlarged, and rich polymorphism bands were chosen (table 2). pcr reactions were carried in a 25μl volume containing 10 mm trishcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). the thermal program was carried out with an initial denaturation for 1 min at 94°c, followed by 40 cycles in three segments: 35 s at 95°c, 40s at 55°c and 55s at 72°c. final extension was performed at 72°c for 5 min. the amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated by using a 100 bp molecular size ladder (fermentas, germany). data analyses morphological studies in total nineteen morphological (nineteen quantitative) characters were studied. four to twelve samples table 1. list of pistachio cultivars examined for genetic relatedness using scot marker system in this study.by majid khayatnezhad. no genotypes locality latitude longitude 1 sarakhs khorasan, mashhad 36.321247 59.532639 2 ebrahimi khorasan, mashhad 36.321247 59.532639 3 karimi khorasan, mashhad 36.321247 59.532639 4 aliabadi khorasan, mashhad 36.321247 59.532639 5 kaleghochi (pust sefid) semnan, damghan 36°9'52.6824' 54°21'27.52 6 shahpasand (pust sefid) semnan, damghan 36°9'52.6824' 54°21'27.52 7 akbari (pust ghermez) semnan, damghan 36°9'52.6824' 54°21'27.52 8 khanjari damghan semnan, damghan 36°9'52.6824' 54°21'27.52 9 kaleghochi (pust ghermez) semnan, damghan 36°9'52.6824' 54°21'27.52 10 shahpasand (pust ghermez) semnan, damghan 36°9'52.6824' 54°21'27.52 11 fakhri semnan, damghan 36°9'52.6824' 54°21'27.52 12 akbari (pust sefid) semnan, damghan 36°9'52.6824' 54°21'27.52 13 abbas-ali semnan, damghan 36°9'52.6824' 54°21'27.52 14 ahmad agaei semnan, damghan 36°9'52.6824' 54°21'27.52 15 menghar kalaghi semnan, damghan 36°9'52.6824' 54°21'27.52 16 pust khormaei kerman, rafsanjan 30.3548893 56.002705 17 ghazvini kerman, rafsanjan 30.3548893 56.002705 18 fandoghi kerman, rafsanjan 30.3548893 56.002705 19 javad aghaei kerman, rafsanjan 30.3548893 56.002705 20 badami dishkalaghi kerman, rafsanjan 30.3548893 56.002705 21 vahedi 30.3548893 56.002705 22 behesht abadi kerman, rafsanjan 30.3548893 56.002705 23 hasan zadeh kerman, rafsanjan 30.3548893 56.002705 24 gholamrezaei kerman, rafsanjan 30.3548893 56.002705 25 ohadi kerman, rafsanjan 30.3548893 56.002705 26 saiffodini kerman, rafsanjan 30.3548893 56.002705 27 ghafori rafsanjan kerman, rafsanjan 30.3548893 56.002705 28 ravare kerman, rafsanjan 30.3548893 56.002705 29 italiaei kerman, rafsanjan 30.3548893 56.002705 30 shasti kerman, rafsanjan 30.3548893 56.002705 68 tinglu liu et al. from each population were randomly studied for morphological analyses (appendix 1). morphological characters were first standardized (mean = 0, variance = 1) and used to establish euclidean distance among pairs of taxa (podani 2000). for grouping of the plant specimens, the upgma (unweighted paired group using average) and ward (minimum spherical characters) as well as ordination methods of mds (multidimensional scaling) were used (podani 2000). past version 2.17 (hammer et al. 2012) was used for multivariate statistical analyses of morphological data. molecular analyses excel 2013 was used to calculate the total number of bands (tnb), the number of polymorphic bands (npb), and the percentage of polymorphic bands (ppb). the polymorphism information content (pic) of scot primers was determined using powermarker v3.25. binary characters (presence = 1, absence = 0) were used to encode scot bands and used for further analyses. parameter like nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism (p% = number of polymorphic loci/number of total loci) were determined (weising et al. 2005; freeland et al. 2011). shannon’s index was calculated by the formula: h’ = -σpiln pi. rp is defined per primer as: rp = ∑ ib, were “ib” is the band informativeness, that takes the values of 1-(2x [0.5-p]), being “p” the proportion of each genotype containing the band. the percentage of polymorphic loci, the mean loci by accession and by population, uhe, h’ and pca were calculated by genalex 6.4 software (peakall and smouse 2006) nei’s genetic distance among populations was used for neighbor joining (nj) clustering and neighbor-net networking (freeland et al. 2011; huson and bryant 2006). the comparison of genetic divergence or genetic distances, estimated by pairwise fst and related statistics, with geographical distances by mantel test is one of the most popular approaches to evaluate spatial processes driving population structure. the mantel test was performed as implemented in past ver. 2.17 (hammer et al. 2012). for this, nei genetic distance was determined for scot data, while geographic distance of past was determined for geographical data. it is calculated based on the sum of the paired differences among both longitude as well as latitude coordinates of the studied populations. the mantel test, as originally formulated in 1967, is given by where gij and dij are, respectively, the genetic and geographic distances between populations i and j, considering n populations. because zm is given by the sum of products of distances its value depends on how many populations are studied, as well as the magnitude of their distances. amova (analysis of molecular variance) test (with 1000 permutations) as implemented in genalex 6.4 (peakall and smouse 2006), and nei,s gst analysis as implemented in genodive ver.2 (2013) (meirmans and van tienderen 2004) were used to show genetic difference of the populations. moreover, populations, genetic differentiation was studied by g’st est = standardized measure of genetic differentiation (hedrick 2005), and d_est = jost measure of differentiation (jost 2008). to assess the population structure of the pistachio genotypes, a heuristic method based on bayesian clustering algorithms were utilized. the clustering method based on the bayesian-model implemented in the software program structure (pritchard et al. 2000; falush et al. 2007) was used on the same data set to better detect population substructures. this clustering method is based on an algorithm that assigns genotypes to homogeneous groups, given a number of clusters (k) and assuming hardy-weinberg and linkage equilibrium within clusters, the software estimates allele frequencies in each cluster and population memberships for every individual (pritchard et al. 2000). the number of potential subpopulations varied from two to ten, and their contribution to the genotypes of the accessions was calculated based on 50,000 iteration burn-ins and 100,000 iteration sampling periods. the most probable number figure 1. map of iran shows the collection sites and provinces where of pistacia vera species were obtained for this study. 69genome survey of pistachio (pistacia vera l.) accessions revealed by start codon targeted (scot) markers (k) of subpopulations was identified following evanno et al. (2005). in k-means clustering, two summary statistics, pseudo-f, and bayesian information criterion (bic), provide the best fit for k (meirmans 2012). gene flow (nm) which were calculated using popgene (version 1.31) program (yeh et al. 1999). gene flow was estimated indirectly using the formula: nm = 0.25(1 fst)/ fst. in order to test for a correlation between pair-wise genetic distances (fst) and geographical distances (in km) between populations, a mantel test was performed using tools for population genetic analysis (tfpga; miller 1997) (computing 999 permutations). this approach considers equal amount of gene flow among all populations. results scot polymorphisms twenty-five scot primers were tested with four of pistacia vera cultivars as dna templates; all primers produced amplification products, and only primers showing clear and reproducible band patterns were selected for further analysis. the size of the amplified fragments ranged from 100 to 2500 bp (fig. 2). ten primers were then chosen for the genotypes identification and phylogenetic analysis. as shown in table 2, all 10 primers used for scot analysis. a total of 155 fragments were obtained, and 143 of the fragments were polymorphic. the number of polymorphic fragments for each scot primer ranged from 8 (st3) to 25 (st14), with an average of 12. the percentage of polymorphic fragments was from 84.57% to 100.00%, with an average of 94.55% polymorphism. polymorphism information content (pic) values were 0.22 to 0.59, with an average of 0.41. the number of different alleles was 0.43 at the species (table 3). these results indicated that a high level of polymorphism could be detected among pistacia vera cultivars using scot markers. populations, genetic diversity genetic diversity parameters determined in three geographical populations of pistacia vera are presented in table 3. the percentage of polymorphic loci (p) and nei’s gene diversity (h) were important parameters for measuring the level of genetic diversity. in table 3, the genetic diversity parameters of the 30 pistacia vera cultivars are shown. the highest value of percentage polymorphism (61.99%) was observed in ghafori rafsanjan (cultivars no.27) which shows high value for gene diversity (0.42) and shanon, information index (0.39). genotype shahpasand (pust ghermez) (no.10) has the lowest value for percentage of polymorphism (20%) and the lowest value for shanon, information index (0.15), and he (0.010). population genetic differentiation amova (phipt = 0.29, p = 0.010), revealed significant difference among the studied genotypes (table 4, fig. 3). it also revealed that, 23% of total genetic variability was due to within genotypes diversity and 55% was due to among genotypes genetic differentiation. moreover, pair-wise amova revealed significant genetic difference almost among all the studied genotypes. these results indicate that of pistachio genotypes are genetically differentiated and we can use such genetic difference in future breeding programs of this figure 2. electrophoresis gel of pistacia vera species from dna fragments produced by scot-11 molecular markers, (population numbers are according to table 1). 70 tinglu liu et al. table 2. scot primers used for this study and the extent of polymorphism. tnp: total number of bands; npb: number of polymorphic bands; ppb: percentage of polymorphic bands; pic: polymorphism information content. primer name primer sequence (5’-3’) tnb npb ppb pic scot-1 caacaatggctaccacca 13 13 100.00% 0.55 scot-3 caacaatggctaccaccg 9 8 86.99% 0.43 scot-6 caacaatggctaccacgc 19 19 100.00% 0.34 scot-11 aagcaatggctaccacca 17 16 94.33% 0.47 scot-14 acgacatggcgaccacgc 25 25 100.00% 0.35 scot-15 acgacatggcgaccgcga 14 12 94.74% 0.59 scot-16 ccatggctaccaccggcc 15 12 92.31% 0.49 scot-17 catggctaccaccggccc 10 10 100.00% 0.22 scot-18 accatggctaccaccgcg 12 10 84.57% 0.50 scot-19 gcaacaatggctaccacc 24 24 100.00% 0.37 mean 16 12 94.55% 0.41 total 155 143 table 3. genetic diversity parameters in the studied populations of pistachio cultivars (n = number of samples, na = number of different alleles, ne = number of effective alleles, i= shannon’s information index, he = genetic diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations). code genotypes n na ne i he uhe %p sarakhs 5.000 0.555 1.020 0.22 0.25 0.28 43.53% ebrahimi 8.000 0.431 1.088 0.20 0.22 0.25 49.53% karimi 8.000 0.255 1.021 0.25 0.28 0.22 37.15% aliabadi 5.000 0.261 1.024 0.292 0.23 0.23 53.15% kaleghochi (pust sefid) 5.000 0.886 1.183 0.184 0.116 0.122 24.29% shahpasand (pust sefid) 8.000 0.686 1.157 0.30 0.11 0.22 39.43% akbari (pust ghermez) 4.000 0.344 1.042 0.28 0.23 0.20 33.53% khanjari damghan 5.000 0.455 1.077 0.277 0.24 0.22 53.05% kaleghochi (pust ghermez) 3.000 0.255 1.021 0.15 0.18 0.19 48.45% shahpasand (pust ghermez) 3.000 0.643 1.173 0.154 0.010 0.010 20.00% fakhri 8.000 0.431 1.088 0.20 0.22 0.25 49.53% akbari (pust sefid) 9.000 0.255 1.021 0.25 0.28 0.22 37.15% abbas-ali 6.000 0.261 1.024 0.292 0.23 0.23 40.15% ahmad agaei 10.000 0.287 1.253 0.266 0.254 0.28 50.99% menghar kalaghi 5.000 0.358 1.430 0.28 0.20 0.29 23.50% pust khormaei 6.000 0.299 1.029 0.231 0.28 0.23 24.38% ghazvini 5.000 0.462 1.095 0.288 0.29 0.22 22.05% fandoghi 8.000 0.399 1.167 0.24 0.21 0.213 32.88% javad aghaei 5.000 0.336 1.034 0.23 0.25 0.29 41.83% badami dishkalaghi 4.000 0.344 1.042 0.28 0.23 0.20 57.53% vahedi 5.000 0.455 1.077 0.277 0.24 0.22 55.05% behesht abadi 3.000 0.255 1.021 0.15 0.18 0.19 38.45% hasan zadeh 3.000 0.643 1.173 0.154 0.102 0.109 30.00% gholamrezaei 8.000 0.431 1.088 0.20 0.32 0.25 41.53% ohadi 9.000 0.255 1.021 0.25 0.28 0.22 27.15% saiffodini 6.000 0.261 1.024 0.292 0.23 0.23 43.15% ghafori rafsanjan 10.000 0.287 1.253 0.396 0.424 0.44 61.99% ravare 3.000 0.567 1.062 0.24 0.224 0.213 34.73% italiaei 3.000 0.499 1.067 0.24 0.281 0.24 49.26% shasti 9.000 0.352 1.083 0.23 0.22 0.24 45.05% 71genome survey of pistachio (pistacia vera l.) accessions revealed by start codon targeted (scot) markers valuable plant species. the results of this study showed that there is a relatively low level of genetic diversity in the studied samples which are expected in view of the dioecius and outbreeding nature of the cultivated pistachio cultivars and high level of heterozygosity due to the cross-pollinating nature of the plant established during the evolution and domestication processes which have been conserved by the propagation of clones through vegetative reproduction. the pairwise comparisons of ‘nei genetic identity’ among the studied populations of pistacia vera (table not included) have shown a higher a genetic similarity (0.887) between populations menghar kalaghi (province semnan) and kaleghochi (pust ghermez) (province semnan), while the lowest genetic similarity value (0.667) occurs between akbari (pust ghermez) (province semnan) and badami dishkalaghi (province kerman). populations, genetic affinity nj tree and neighbor-net network produced similar results therefore only nj tree is presented and discussed (fig. 4). this result show that molecular characters studied can delimit pistacia vera genotypes in two different major clusters or groups. in general, two major clusters were formed in nj tree (fig. 3), four genotypes of cultivars sarakhs, ebrahimi, karimi and aliabadi formed a single cluster, and these genotypes were all from khorasan, mashhad province. cluster ii contained two sub-clusters, and most of individuals kaleghochi (pust sefid); shahpasand (pust sefid); akbari (pust ghermez); khanjari damghan and kaleghochi (pust ghermez), shahpasand (pust ghermez); fakhri; akbari (pust sefid); abbas-ali and ahmad agaei (semnan province) formed cluster ii. there were 26 individuals in this cluster. besides, principal coordinate analysis (pcoa) was performed to visualize the association among the genotypes in more detail. the pcoa results showed that the first three principal coordinates account for 64.88% of the total variation (not shown). based on the results of pcoa analysis, cultivars sarakhs, ebrahimi, karimi and aliabadi genotype showed the highest dissimilarity with other genotypes. additionally, the results from bayesian clustering analysis using structure software (fig. 4) confirmed the groupings we observed in nj and pcoa clusterings. table 4. analysis of molecular variance (amova) of the studied species. source df ss ms est. var % among regions 12 39.211 23.648 0.266 19% among pops 15 96.822 18.802 0.114 55% among indiv 57 64.553 21.130 0.283 20% within indiv 71 15.500 0.284 0.204 8% total 141 215.007 1.678 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance. figure 3. amova test of the studied populations. figure 4. nj tree of populations in pistacia vera based on scot molecular markers. 72 tinglu liu et al. th e present study indicated that a higher genetic diversity was found in the older genotypes. th is fact confi rms our speculation that pistachio cultivations have increasingly led to the reduction of their genetic variation due to deployment of improved cultivars and to the availability of private or public graft ed seedling nurseries for pistachio, as well as the changing livelihood conditions. recently, the method of pistachio cultivation is changing leading towards an increased reduction of crop diversity deployed on farm. in the past, pistachio diversity was maintained high in the fi eld through a number of cultivation practices, s. a. use of male varieties derived from seed, use of wild pistacia species to boost pollination and hence the fruit setting, use of natural populations of wild pistacia (p. atlantica) as a rootstock due to their wellknown resistance to stony and calcareous soils. th is is in agreement with amova and genetic diversity parameters presented before. mantel test aft er 5000 permutations produced signifi cant correlation between genetic distance and geographical distance in these populations (r = 0.87, p = 0.001). th erefore, the populations that are geographically more distant have less amount of gene fl ow, and we have isolation by distance (ibd) in pistacia vera genotypes. th e most popular approaches for estimating divergence include calculation of genetic distances and variance partitioning among and within populations using wright’s fst and other related statistics, such as gst, ast, rst, θst and φst. for instance, the fst gives an estimate of the balance of genetic variability among and within populations, and is an unbiased estimator of divergence between pairs of populations under an island-model in which all populations diverged at the same time and are linked by approximately similar migration rates. however, migration rates usually vary proportionally with geographical distances, so that pairwise fst estimates between pairs of populations vary. th erefore, the populations that are geographically more distant have less amount of gene fl ow, and we have isolation by distance (ibd) in pistacia vera genotypes. populations genetic structure th e number of genetic groups was determined by two methods of 1—k-means clustering which is based on the maximum likelihood approach, and 2—evanno test which is based on structure analysis and is a bayesian approach based method. k-means clustering based on pseudo-f and bic (bayesian information criterion) recognized 3 and 5 genetic groups, respectively. th is is in agreement with amova result, showing signifi cant genetic diff erence among date populations of pistacia vera genotypes. evan test based on delta k (fig. 5) identifi ed the optimum number of genetic groups 3. we performed structure analysis based on k = 3, to identify the genetic groups (fig. 6). in the plot of k = 3, the cultivars sarakhs, ebrahimi, karimi and aliabadi (red colored) are placed in the fi rst genetic group, while the populations of kaleghochi (pust sefi d); shahpasand (pust sefi d); akbari (pust ghermez); khanjari damghan and kaleghochi (pust ghermez), shahpasand (pust ghermez); fakhri; akbari (pust sefi d); abbas-ali and ahmad agaei (semnan province) (blue colored) formed the second genetic group and fi nally the populations of kerman province (green colored) formed the third genetic group. th ese diff erent genetic groups may be used in future breeding and hybridization programs of iranian date pistacia vera genotypes. th e mean nm = 0.65 was obtained for all scot loci, which indicates low amount of gene fl ow among the populations and supports genetic stratifi cation as indicated by k-means and structure analyses. th is result is in agree with grouping we obtained with pca plot, as these populations were placed close to each other. as evidenced by structure plot based on admixture model, these shared alleles comprise very limited part of the genomes in these populations and all these results are in agreement in showing high degree of genetic stratifi cation within of pistacia vera genotypes. morphometric analyses in present study we used 30 plant accessions (six to fourteen samples from each populations) belonging to four diff erent populations. in order to determine the most figure 5. delta k plot of evanno’s test based on structure analysis. 73genome survey of pistachio (pistacia vera l.) accessions revealed by start codon targeted (scot) markers variable characters among the taxa studied, pca analysis has been performed (fig. 6). it revealed that the first three factors comprised over 73% of the total variation. in the first pca axis with 40% of total variation, such characters as length of leaves; width of leaves; length of petioles; length of the terminal leaf; width of the terminal leaf; length of inflorescence have shown the highest correlation (> 0.7), fruit length; fruit width; fruit thickness; number of fruit per inflorescence; kernel infestation were characters influencing pca axis 2 and 3, respectively. different clustering and ordination methods produced similar results therefore, pca plot of morphological characters are presented here (fig. 6). the result showed morphological difference/ divergence among most of the studied populations. this morphological difference was due to quantitative characters only. discussion the coupling of ecological and genetic data will provide the most suitable background for preserving the ability of the biota to respond the rapid environmental changes ((sawadogo et al. 2021; paul et al. 2021)). the literature reports the following basic factors influencing the distribution of genetic variation: habitat specify, plant-insect interactions, connectivity and disturbance, dispersal ability, species lifespan, reproductive rates fig. 6. top: structure plot of pistacia vera populations based on k = 3, numbers are according to table 1.bottom: pca plot of pistacia vera populations based on morphological characters. numbers are according to table 1. 74 tinglu liu et al. and existing genetic diversity (esfandani–bozchaloyi, et al. 2018a, 2018b, 2018c, 2018d). genetic diversity when analysed by neutral markers does not correspond to the adaptive ability of plant populations, but these types of markers are very useful for the interpretation of the past landscapes, refugia and gene flow (wankiti et al. 2021; lucena et al. 2021). that is, why the selected genes or markers of active parts of plant genomes are used to interprete the plant genome response to the changes to the local climate and environment. molecular-based population genetic data are very useful for determining the ecological and habitat events in the past and for detection of patterns of the recent genetic divergence. this can be achieved using different types dna markers. scot markers are novel molecular markers that target the translation initiation site and preferentially bind to genes that are actively transcribed. these primers have been shown to exhibit relatively high levels of polymorphism (collard and mackill 2009). it was more informative than irap and issr for the assessment of diversity of plants (collard and mackill 2009). pistachio has important socio-economic and ecological impacts in the arid and semi-arid agricultural regions of iran (kafkas et al. 2006). in addition, iran hosts a wide genetic diversity of pistacia spp. and more than 300 pistachio genotypes have been collected across the country. iran therefore possesses valuable germplasm for pistachio improvement and conservation programs. assessing genetic diversity and relationships among cultivars of iranian pistachio, using discriminative and robust markers, is therefore important (mirzaei et al. 2005). in the present work, 30 p. vera cultivars were characterized with 10 scot markers. the results confirm the efficiency of microsatellite markers for fingerprinting purposes. our results demonstrated that the polymorphism information content (pic) ranged from 0.22 to 0.59 with an average value of 0.41, while the percentage of polymorphism (p%) ranged from 0.20 to 0.61 with an average value of 0.42 and also the expected heterozygosity (he) varied from 0.011 to 0.42 with an average of 0.20.these values were higher than those reported by arabnejad et al. (2008), who detected an average of 3.69 alleles per primer pairs and an average pic of 0.46 detected in 20 commercial cultivars of iranian pistachio; and also higher than those reported by baghizadeh et al. (2010) (an average of 2.75 alleles per primer pairs and an average of 0.44 for detected in 31 iranian pistachio cultivars) and by ahmad et al. (2005) (an average of 3.30 alleles per locus in 17 pistachio cultivars). kolahi-zonoozi (2014) assessed genetic diversity of 45 commercially iranian cultivars using 12 nssr markers and detected that pic varied from 0.19–0.56 with an average of 0.33 and the mean of ho and he were 0.49 and 0.35, respectively. mirzaei et al. (2005) reported 80.00%polymorphism among 22 iranian pistachio cultivars and wild pistachio species. in a study reported by golangoldhirsh et al. (2004) in assessing polymorphisms among 28 mediterranean pistachio accessions, 27 selected primers produced 259 total bands (an average of 9.59). some cultivars in different locations have the same name and some morphological identity, while molecular results showed differences between them. for instance, badami-zarand cultivar was differentiated from badamikaj and badami-zoodras. also, ghazvini-zodras showed differences with ghazvini. these differentiations can be due to the intrinsic nature of nssrs, since it is very unlikely that the microsatellites amplified correspond to the mutated dna region when they have been randomly isolated from the whole genome. the results from this study showed that the studied cultivars had high genetic variation due to the species’ dioeciously and cross-pollination nature (ahmad et al. 2005). conclusion: this study was aimed at evaluating the genetic diversity of iranian pistachio in order to aid the conservation of its germplasm. the obtained information about the genetic variation between and within different populations will prepare the ground for the formulation of appropriate conservation strategies. the present analysis revealed that iranian-cultivated pistachio germplasm is highly variable, presumably due to specific local genetic backgrounds, breeding pressure and/or limited interchange of genetic material. the unique nature of the iranian pistachio germplasm revealed by our results, supports the case for the implementation of more intense characterization, conservation and breeding strategies. also, the scot markers used were useful for determination of genetic diversity among pistachio cultivars in iran. acknowledgment the authors thank anonymous reviewers for valuable comments on an earlier draft. references arabnejad h, baharm, taj a. 2008. genetic diversity of pistacia khinjuk stocks by ssrmarkers. agri sci tech. 12:207–217. 75genome survey of pistachio (pistacia vera l.) accessions revealed by start codon 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(2nd ed.), boca raton, fl., usa: crc press, pp. 472. yeh francis, c., r.c. yang, b.j. boyle timothy, z.h. ye, x. mao judy, 1999. popgene version 1.32, the user-friendly shareware for population genetic analysis, molecular biology and biotechnology centre, university of alberta, canada, wu jm, li yr, yang lt, fang fx, song hz, tang hq, wang m, weng ml 2013. cdna-scot: a novel rapid method for analysis of gene differential expression in sugarcane and other plants. ajcs 7:659–664. zohary m 1952. a monographic study of the genus pistacia. palest j bot jerus ser 5:187–228. caryologia. international journal of cytology, cytosystematics and cytogenetics 73(4): 65-75, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-1029 caryologia international journal of cytology, cytosystematics and cytogenetics citation: m. mert, b. betül (2020) investigation of the cytotoxic and genotoxic effects of the euphorbia rigida bieb. extract. caryologia 73(4): 65-75. doi: 10.13128/caryologia-1029 received: july 23, 2020 accepted: january 01, 2021 published: may 19, 2021 copyright: © 2020 m. mert, b. betül. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. investigation of the cytotoxic and genotoxic effects of the euphorbia rigida bieb. extract1 metin mert2,*, bürün betül3 1 this study is a part of a phd thesis 2 mugla sıtkı koçman university, faculty of milas veterinary, department of biochemistry, 48200 muğla, turkey 3 mugla sıtkı koçman university, faculty of science, department of biology, 48170 muğla, turkey *corresponding authore-mail: mertmetin@mu.edu.tr abstract. the present study was conducted to evaluate and compare the cytotoxic and genotoxic effects of the aqueous extracts of euphorbia rigida bieb. which is a natural pesticide. the comparison was done using the allium test to the chemical pesticides elandor® and goldplan®. according to allium test results, it had negative impacts on mitosis and showed cytotoxic and genotoxic effects on the existing cells. the lowest level of mi (2.95 %) was observed in the 200 ppm treatment of e. rigida extract. number of the aberrant cells were 88.1, 84.1 and 82.5 in the treatments with elandor®, 50 ppm, e. rigida extract and goldplan® respectively. the highest cytological anomalies were chromosome stickiness, irregular metaphase and anaphase, pole deviations and c-mitosis. according to the present results of this research, we can suggest that the extract obtained from e. rigida plant with water up to 50 ppm can be used as an alternative to chemicals used as biopesticide. antibacterial, antifungal and antiviral properties of euphorbia extracts are well known, so it could mean that they can be used as additives or a disinfectant for inanimate surfaces in the pharmaceutics industry. no matter what purposes the extract of this plant is used, great care should be taken while using it because it can cause damage on cells and chromosomes. hence, through more detailed and comprehensive studies about its capacity for medical and biopesticide purposes should be investigated. keywords: allium test, biopesticide, cell aberrations, cytotoxic, genotoxic and euphorbia rigida bieb. introduction extracts and essential oils of some naturally grown plants show antibacterial and antifungal activities and these activities are used as a biopesticide in agricultural control and nutrient preservation (baytop 1991). in recent years, researchers have been focusing on studies to obtain compounds harmless to human health and the environment that can be used instead of chemical pesticides against plant diseases and pests, which are of great economic value. although pesticides have a fast and strong effect in the con66 metin mert, bürün betül trol of pests, they cause environmental pollution and accumulate over time in all living things through the food chain, creating toxic hazard (güler and çobanoğlu 1997). most of the euphorbia species are rich in phenolic compounds, aromatic esters, diterpenoids, tetracyclic and pentacyclic triterpenoids, essential oils, resins, resinoids and many bioactive compounds (wu et al. 2009; kumar et al. 2010; ekeke and ndukwu 2014; ghareeb et al. 2018; ghosh et al. 2019). diterpenoids of euphorbia have biological activities such as antitumor, cytotoxic, anti-viral and anti-inf lammatory, but f lavonoids and tannins are known for their antitumor, hepatoprotective and antioxidant activities (wu et. al. 2009; ghareeb et al. 2018; ghosh et al. 2019). the plants of euphorbia species are used for the treatment of hypertension, destruction of wart cures, skin diseases, gonorrhoea, migraine and intestinal parasites (kırbağ et al. 2013; ghareeb et al. 2018). e. hirta possesses antibacterial, anthelmintic, antiasthmatic, sedative, antispasmodic, antifertility, antifungal, and antimalarial properties (kumar et al. 2010; özbilgin and çitoğlu-saltan 2012). compounds isolated from e. paralias showed moderate antiviral activity against hiv-1 replication. seven triterpenoids isolated from e. antiquorum and steroids isolated from e. chamaesyce also have strong inhibitory activity against epstein-barr virus early antigen (ebv-ea) activation (shi et al. 2008). e. paralias, e. maschallian and e. myrsinites species have different diterpene species that have been identified as antiviral compounds. some of the other euphorbiaceae species, e.g. e. pekinensis, e. peplus, phyllanthus nanus and p. amarus are effective against virus infections (gyurıs et al. 2009). the aqueous and 50% meoh extracts of e. hirta shows direct antiviral effects on hiv-1, hiv-2 and siv (mac251) reverse transcriptase (rt) activity which were determined in mt4 cells in-vitro (gyurıs et al. 2009; alam et al. 2016). the antibacterial effect of e. hirta (linn.) comes from tannins, alkaloids and flavonoids contained in the ethanol extract (ogueke et al. 2007). e. orientalis l. was found containing bioactive compounds that has essential antibacterial and antioxidant activities (avcı et al. 2013). the aqueous extract of e. hirta also inhibits aflatoxin contamination in rice, wheat, maize, and mustard crops (kumar et al. 2010). e. platypyllos l. extracts showed significant cytotoxic effect and dna damaging effects in mcf-7 cells (aslantürk and aşkın-çelik 2013). the active ingredients of 17-acetoxyjolkinolide b and 13-hexadecanoyloxy-12-deoxyphorbol were obtained from the roots of e. fischeriana. cytotoxic effects of these compounds against ramos b cells were already determined (wang et al. 2006; özbilgin and çitoğlusaltan 2012). dafnan and tigliane diterpenoids isolated from latex of e. poisonii showed selective and potent cytotoxic effects on human kidney carcinoma (a-498) cell lines (wang et al. 2006; shi et al. 2008). ingenol 3-angelate that is euphorbia diterpenes, which was approved by the fda in 2012 and the ema in 2013 for the treatment of actinic keratosis, a precancerous skin condition. e. retusa extract was effective for the prevention of ccl4-induced hepatic damage in rats (ghareeb et al. 2018). extracts of e. hirta have been found to show anticancer activity (kumar et al. 2010). the latex and plant extracts derived from the roots of the e. rigida plant did not cause gene change in bacteria in two sensitive ames test strains, such as ta 98 and ta 100, while in the comet experiment, they showed mutagenic effects in human lymphocytes (başaran et al. 1996). fumigant effect of e. aleppica extract has been demonstrated with an average of 88% mortality against grain storage pests, sitophilus garanarius and s. oryzae (şahin et al. 2006). the strong molluscoidal activity of water extracts and partially purified latex of e. pulcherima and e. hirta plants is known (shi et al. 2008). isolated compounds from e. paralias had strong molluscicidal activity on biomphalaria alexandrina (ehrenberg) and antifeedant effects on third‐instar larvae of spodoptera littoralis (boisd) (abdelgalil 2002). the petroleum ether fraction of e. hirta is indicated as an herbicidal biopesticide with a larvicidal effect in anopheles stephensi known as the malaria vectors (huang et al. 2012). methanolic extract of aerial parts of e. hirta was effective against p. falciparum parasites, polyphenolic extract of e. hirta inhibited the growth of entamoeba histolytica (kumar et al. 2010) and aqueous leaf extracts (1:10 w/v) of e. hirta had a maximum killing efficacy (45%) against the zonabris pustulata thunb. (oudhia 2000). e. fischeriana has been used as an anthelmintic and insecticide in china (lee et al. 1991). the aqueous extract of e. hirta reduced the egg counts of intestinal parasites in nigerian dogs’ feces as a potential anthelmintic and antiparasiticide agent (huang et al. 2012). the ethanol extracts from the leaves and flowers of e. cyparissias l., had the highest larvacidal activity against the codling moth (cydia pomonella l.) with strong acaricidal activity on the treated population of two-spotted spider mite (tetranychus urticae koch.) on the third day post treatment (velcheva 2001). extracts of e. hirta and the root exudate exhibit nematicidal activity against juveniles of meloidogyne incognita (kumar et al. 2010). e. myrsinites extracts against root-knot nematodes (nematoda: meloidogyne spp.) in greenhouse tomato cultivation were found to be significantly more effective than synthetic pesticide cyromazine (c6h10n6) (civelek 67investigation of the cytotoxic and genotoxic effects of the euphorbia rigida extract and weintraub 2004). most plant extracts have been used as topical antiseptics, or have been reported to have antimicrobial properties (avcı et al. 2013). most of the euphorbia species and other plant extracts, which are used for some medicinal and agricultural purposes, show potentially toxic, mutagenic, carcinogenic and teratogenic effects. for this reason, it is necessary to test the potential harmful effects of plant extracts to be used in both medical (antibacterial, antifungal, antiviral, antitumor, antioxidant, antiseptic, anthelmintic etc.,) and agricultural purposes (biopesticide, insecticidal, acaricidal, antiparasitic, larvicidal, molluscoidal, antimalarial, antifeedant etc.,). in the current study, the cytotoxic and genotoxic effects of e. rigida (bieb.) being used for medical and biopesticide purposes were investigated. the objective of this study was to evaluate and compare the cytotoxic and genotoxic effect of the extract of e. rigida, a natural pesticide, with the chemical pesticides, elandor® and goldplan®. materials and methods collection of the plants and their extraction the aerial parts of euphorbia rigida (bieb.) in the flowering period were collected from sıtkı koçman üniversity campus (gps:37°09’40,1”n 28°22’34”e) in muğla at turkey. the taxanomic identification of plant materials was confirmed and deposited in the herbarium by voucher specimen dr. olcay ceylan at the department of biology at muğla sıtkı koçman university (herbarium number: o0388). the samples were air-dried at room temperature and protected from direct sunlight. seventyfive (75 g) grams of dried and powdered aerial parts of e. rigida were extracted with 2.5 l boiling water for 60 min. decoction (aqueous phase) was filtered with a 2.5 µm filter paper (whatman no. 42) to remove suspended particles and the extract was kept at least 3 days at -20°c and later lyophilized to obtained crude (6.72 g) extract which was stored at -20°c. allium test method the united nations environment program (unep) and the us environmental protection agency (usepa) have standardized the use of plants as bioindicators in the determination of toxicity (sivas and gökbayrak 2011; girasun et al. 2019). unep and the international chemical safety program (ipcs) certified the allium cepa (onion) root tip test in 1991 as a highly effective biotester for imaging mutagenic effects (oney-birol and gündüz 2019). the onion genotoxicity test provides for easy screening of chemicals or toxic agents with genotoxic, cytotoxic, physiologic, clastogenic and aneugenic effects, especially to plants. the a. cepa assay is an efficient test for chemical screening and in situ monitoring for genotoxicity of environmental contaminants (sivas and gökbayrak 2011; pandey et al. 2014; girasun et al. 2019; adhikari 2019). the test has been used widely to study genotoxicity of many pesticides revealing that these compounds can induce chromosomal aberrations in root meristems of a. cepa. the most important advantage of this test is that it is a low budget method, which besides being fast and easy to handle, it also yields reliable results (fiskesjö 1985, rank 2003; çelik and aslantürk 2006; eren et al. 2017; karaismailoğlu 2016; adhikari 2019; liman et al. 2020). allium test results show a good correlation with the other eukaryotic and prokaryotic test results (bonciu et al. 2018; pirdal and liman 2019; oney-birol and gündüz 2019; adhikari 2019). the effects of the extract on cells and chromosomes were investigated through allium test (fiskesjö 1981). for the allium test, a. cepa were purchased from the market. 0, 50, 100, 200 and 400 ppm (v/v) solutions of e. rigida extract were prepared with distilled water and also 200 ppm elandor® (imidacloprid) and 400 ppm goldplan® (acetamiprid) solution (v/v) (recommended doses) was prepared. elandor® and goldplan® are commercially available pesticides, which are contact and systemic insecticide for the control of hemiptera, especially aphids, thysanoptera and lepidoptera (öncüer 2004). all onions were grown in distilled water for first 48 hours. then a. cepa bulbs were placed in beakers including 0 (control= distilled water), 50, 100, 200 and 400 ppm (v/v) solutions of e. rigida, 200 ppm elandor® and 400 ppm goldplan® (v/v) solution (for the treatment 24 h). at the end of 24 h (after total 72 h), the root number and length of a. cepa were determined. also the least ten root tips from each treatment were fixed in carnoy solution (absolute alcohol: chloroform: glacial acetic acid, 6:3:1). thereafter, roots tips were applied to cold hydrolysis and the meristem tissue cells were painted with feulgen, then squashed and examined under the light microscope (nikon ufx-2a) (elçi 1994). from these squashed root tips, ten random areas were selected for the observation at 10x40 microscopic magnification (approximately 2000 cells were counted in an area), minimum 20.000 mitotic cells were counted from each of the slides. on each slide, abnormalities of chromosome stickiness, binucleate cells, micronuclei, c-mitosis, lagging chromosome, fragmentation in the metaphase, bridges and pole deviations in anaphase, irregular metaphase or anaphase etc. were detected. moreover, the 68 metin mert, bürün betül mitotic index (mi) was calculated for each treatment as a number of dividing cells/100 cells (metin and bürün 2008). statistical analysis statistical analyses were performed using the spss 14.0 software package program, data were evaluated with one-way anova and lsd (least significant difference) tests. results and discussion the effects of the extract of e. rigida, a natural pesticide were compared with elandor® and goldplan®, chemical pesticides. aqueous extracts of e. rigida plants (50, 100, 200 and 400 ppm), 200 ppm elandor® and 400 ppm goldplan® were observed to cause the occurrence of aberrant cells and considerably decrease cell division depending on the increase in extract concentration. the number and length of root according to treatments the roots of all onions left for rooting in distilled water for 48 hours were healthy and well developed, and the differences were observed in the average root length and the total number of roots due to the onion’s own characteristics. in the 24 hours following the first 48 hours, the development of the roots of onions exposed to different doses of e. rigida extracts and chemical pesticides slowed down, the number of roots did not change and the root lengths were in different statistical groups (p<0.05) (table 1). changes in root length of the treatments were compared to the control at the inhibition percent. growth in onion root apical meristems of the treatment samples was 45% less than that of the control. accordingly, the extracts and chemicals applied most likely contain toxic substances with a sublethal effect (p<0.05). inhibition of root growth could be not only related to apical meristematic activity but also cell elongation during differentiation or enzyme activation that promote the elongation and loosening of the cell wall in the differentiation process (eren et al. 2017; pirdal and liman 2019). the lowest root length (highest inhibition) in 24-hour treatment of extract and chemica ls was observed in 400 ppm e. rigida treatment (p<0.05) (table 1). the root elongation % inhibition of elandor® and goldplan® treatments remained below the 50 ppm dose of the extract (p<0.05). root elongation inhibition increased with concentration increase of plant extract. this means that the plant extract prevents mitosis by showing toxic effects on the meristematic cells of a. cepa. heavy metal induced toxicity and mutagenicity on various plant species have been already reported. primary toxic effect of pb in higher plants had been the inhibition of root growth possibly due to the inhibition of cell division in the root tip region. the reduction in root lengthening is strongly correlated to the mitotic index of the root tips of lathyrus sativus. the reductions in the number of mitotic cells in root tips of seeds exposed to pb could be due to its mechanism of action on cell cycle progression (adhikari 2019). effect on the mitotic index (mi) of treatments mitotic index (mi) is a cytogenetic parameter that helps to measure the proliferation (m phase) of mitotic cells (oney-birol and gündüz 2019). after applications, the highest level of mi (15.70 %) was observed in the elandor® treatment, and this was followed by the goldplan®, 50 ppm, e. rigida extract and control. the lowest level of mi (2.95 %) was observed in the 200 ppm treatment of e. rigida extract (p<0.05) (table 2). a. cepa meristem cells are not affected by the level of toxicity of these chemical pesticide solutions. in contrast, some chemicals involved in elandor® and goldplan® or the lowest plant extracts (e. rigida 50 ppm) may promote the cells into mitosis. however, by increasing the treatment doses of plant extracts, the toxic impact prevents cell division, and, as a result mi decreases (p<0.05) (table 2). the decline in mi value shows interference in the cell cycle (oloyede et al. 2009). the reduction in number of the dividing cells in the roots shows the cytotoxic effects of the substances that are found in the plant aqueous extracts. mi, number of aberrant cells and its percent were high in the control and a. cepa meristematic tissue cells which were exposed to e. rigida 50 ppm, elandor and goldplan solutions. this indicates that at the end of 24-hour exposure, treatment doses have enough influence on stopping the mitosis except the e. rigida 200 ppm (p<0.05). on the other hand, in e. rigida 100 ppm and e. rigida 400 ppm treatments, the defence systems preventing mitosis become active and this results in the decrease of mi. the high mi exposed to elandor, goldplan and 50 ppm e. rigida extract indicates that the damage on living cells from these extracts can be recoverable and tolerable. significant reduction in mi may be due to disturbed cell cycle such as blockage of g1 phase and suppressing dna synthesis or inhibition of dna synthesis at the s phase or blocking of g2 phase preventing the cell from entering in mitosis or mitotic phase changes (pandey et al. 2014; karaismailoğlu 2017a; 69investigation of the cytotoxic and genotoxic effects of the euphorbia rigida extract liman et al. 2020). the causes of the decrease in the mi can be physiological response of cells that have entered the mitotic cycle and are not protected against extract components; partial inhibition of energy, protein, rna and dna synthesis in treatment groups and inhibition or postponement of the mitotic spindle formation in treated groups due to the high percentage of prophase in some concentrations (karaismailoğlu 2016). formation aberrant cells according to treatments increase in the frequency of c-mitosis cells, multipolar anaphases, sticky and diffuse chromosomes together with the decrease in the mitotic index are defined as cytotoxic, and other nuclear abnormalities as genotoxic (kanev et al. 2017). chromosome abnormalities occur as a result of damage at the dna level and are considered to be highly reliable analyzes for the evaluation of genotoxicity. the abnormal cells in mitosis were observed at different levels in all treatment doses of extract and in the pesticides treatments. the highest number of aberrant cells was observed in elandor® treatment with 88.1 aberrant cells, followed by the 50 ppm e. rigida extract with 84.1 and goldplan® with 82.5 aberrant cells (table 3). total abnormal cell formation was the highest in applications where mi was high. total abnormal cell counts were also observed to be the highest in these treatments as there was no toxic effect at the lowest dose of extract (e. rigida 50 ppm) and chemical pesticides. when the table 1. mean root length (mm) and number of allium cepa before and after the treatment with the different doses of e. rigida extract and the chemical pesticides. first 48 h (before the treatment) after the treatments (after 72 hours) at the end of 48 h root length (mean) (mm)± sd treatments and doses (ppm) at the end of 72 h root length (mean) (mm)± sd increase in root length (mm ±sd) increase in percent of root length (%) root number ± sd% growing % i̇nhibition 19.00 ± 5.25 d control (0) 22.00 ± 5.31 e 3.00 100 0 71 f 14.60 ± 4.70 c e. rigida (50) 16.10 ± 4.64 c 1.50 50 50 30 a 19.34 ± 6.08 d e. rigida (100) 20.23 ± 6.12 de 0.89 29.6 70.4 47 e 9.48 ± 2.51 a e. rigida (200) 10.33 ± 2.43 a 0.85 28.3 71.7 33 c 12.40 ± 4.33 b e. rigida (400) 13.00 ± 4.32 b 0.60 20 80 37 d 10.73 ± 2.58 ab goldplan (400) 12.76 ± 3.14 b 2.03 67.6 32.4 30 a 17.62 ± 4.96 d elandor (200) 19.34 ± 5.02 d 1.72 57.3 42.7 32 b variability around the mean was represented as ± sd (standart deviation). data having the same letter in a column were not significantly differed by lsd’s multipli comparison test (p<0.05). table 2. the number of normal, total normal and total aberrant dividing cells and percentage of total aberrant dividing cells in mitotic phases and mitotic index (mi %) for the chemical pesticides and the different treatment doses of e. rigida extract. treatments and doses (ppm) prophase ± sd metaphase ± sd anaphase ± sd telophase ± sd the number of total normally dividing cells ± sd total aberrant cell number ± sd % aberrant cells ± sd total dividing cell number ± sd % mi (mean ±sd) control (0) 81 ± 11.97 c 61 ± 10.08 c 28.2 ± 6.95 c 42.9 ± 11.60 c 213.1 60.7 22.16 273.8 13.69 d e. rigida (50) 82 ± 18.13 c 57.8 ± 18.89 c 30.3 ± 9.26 cd 31.7 ± 7.70 b 201.8 84.1 29.41 285.9 14.29 d e. rigida (100) 59 ± 11.97 b 41.3 ± 10.39 b 17.9 ± 7.74 b 23.8 ± 9.02 b 142 48.5 25.45 190.5 9.52 c e. rigida (200) 18.2 ± 6.12 a 9.6 ± 3.83 a 6.3 ± 2.54 a 7.3 ± 2.35 a 41.4 17.6 29.83 59 2.95 a e. rigida (400) 56.1 ± 8.99 b 39.9 ± 10.99 b 18 ± 4.26 b 24.4 ± 6.61 b 138.4 46.5 25.14 184.9 9.24 b goldplan (400) 90.3 ± 14.29 c 55.2 ± 20.38 c 36.7 ± 11.47 d 46.3 ± 12.32 c 228.5 82.5 26.52 311 15.55 e elandor (200) 88.5 ± 8.51 c 57.3 ± 18.01 c 37 ± 8.89 d 43.2 ± 11.69 c 226 88.1 28.04 314.1 15.70 e variability around the mean was represented as ± sd (standart deviation). data having the same letter in a column were not significantly differed by lsd’s multipli comparison test (p<0.05). 70 metin mert, bürün betül dividing cells were suddenly exposed to treatments after 48 hours, these cells, which were not affected by the lowest dose of extract and chemical pesticides at a toxic level, completed their division with abnormalities. on the other hand, in the cells exposed to high doses (100, 200 and 400 ppm) of plant extract, toxic effects and mitodepressive effects were observed, mi decreased and fewer abnormal cells were recorded. the highest anomalies were chromosome stickiness (figure 1a), irregular metaphase (figure 1b), irregular anaphase (figure 1c), pole deviations in the anaphase (figure 1d), c-mitosis and aneuploidy (hipoploidy) (figure 1e) (table 3). other anomalies observed in this study were fragmentation in the metaphase (figure 1f ), lagging chromosome (figure 1g), bridges in anaphase (figure 1h), binucleate cells (figure 1i), micronuclei (figure 1j). in addition to these anomalies, granulation in the prophase nucleus (figure 1k), irregular prophase (figure 1m), split in the interphase nucleus (figure 1n), increases in the number of nucleolus in the nucleus (figure 1o), nucleus erosion and granulation in the interphase nucleus (figure 1p), nucleus vacuolization in the prophase (figure 1q), multipolar anaphase with polyploidy (figure 1r), polyploidy with c-mitosis and fragments (s) were observed (p<0.05). micronucleus (mn) occurs as a result of clastogenic and aneugenic effects (andrade-vieira et al. 2012; adhikari 2019; rosculete et al. 2020). micronucleus analysis has an important role in assessment of the genotoxic and cytotoxic impacts of chemicals or pesticides (karaismailoğlu 2015; 2017b). disturbed ana-telophase and chromosome laggards may result from deformation of the spindle structure or degraded microtubules and remaining acentric chromosome fragments (türkoğlu 2007; andradevieira et al. 2012; pirdal and liman 2019; rosculete et al. 2020). laggard chromosomes are considered indicators of spindle poisoning (rank 2003). the induction of spindle disturbances in the cell of a. cepa by extracts may lead to aneuploidy and lagging chromosome(s) or micronucleus formation at the next stage of cell division. the lagging chromosome(s) may be lost or form nuclear membrane around itself thereby forming micronucleus (grant 1978). the lagging chromosome(s) usually arises from irregular separation of chromosomes at anaphase thereby making some chromosomes to reach the poles before the other (grant 1978; pandey et al. 2014; adhikari 2019; rosculete et al. 2020). c-mitosis, binucleate cells, and increases in the number of nucleolus in the nucleus were also observed. ta bl e 3. t yp e an d pe rc en ta ge o f m ito tic a bn or m al iti es o bs er ve d in th e tr ea tm en ts w ith p es tic id es a nd d iff er en t d os es o f e . r ig id a ex tr ac t. d os es ( pp m ) st ic ki ne ss ± sd . fr ag m en t ± sd ir re gu la r m et ap ha se ± sd c -m ito s ± sd la gg ar d c hr om os om e ± sd ir re gu la r a na ph as e ± sd po le d ev ia tio n ± sd br id ge ± sd bi nu cl eu s ± sd m ic ro nu cl eu s ± sd o th er a no m al ie s ± sd to ta l a be rr an t c el l n um be r ± sd c on tr ol ( 0) 23 .4 ± 6 .9 4 bc d 0. 2 ± 0. 63 a 12 .5 ± 5 .0 8 bc 1. 4 ± 1. 34 a 0. 8 ± 0. 91 a b 12 .3 ± 5 .2 7 b 6 ± 0. 81 d 0. 6 ± 0. 84 a b 1. 3 ± 1. 05 a b 0. 4 ± 0. 69 a 0 ± 0 a 60 .7 e. r ig id a 50 31 .2 ± 1 3. 72 d 0. 7 ± 0. 82 a 19 ± 1 1. 46 c d 10 .6 ± 1 1. 85 b 2. 3 ± 2. 35 c 12 .7 ± 4 .8 5 b 2. 9 ± 1. 96 b 0. 3 ± 0. 48 a b 3 ± 1. 76 b c 1. 3 ± 0. 82 b 0. 1 ± 0. 31 a 84 .1 e. r ig id a 10 0 18 .4 ± 7 .8 0 bc 0. 4 ± 0. 69 a 9. 5 ± 4. 06 a b 3. 6 ± 4. 37 a 2 ± 2. 10 b c 6. 8 ± 4. 61 a b 2. 7 ± 2. 66 b 0. 2 ± 0. 42 a 2. 7 ± 2 ab c 2. 2 ± 1. 54 c 0 ± 0 a 48 .5 e. r ig id a 20 0 5. 4 ± 2. 22 a 0. 4 ± 0. 51 a 4. 6 ± 1. 77 a 0. 6 ± 0. 69 a 0. 8 ±0 .7 6 ab 3. 2 ± 1. 03 a 0. 8 ± 0. 78 a 0. 3 ± 0. 48 a b 1. 2 ± 0. 78 a 0. 3 ± 0. 48 a 0 ± 0 a 17 .6 e. r ig id a 40 0 16 .2 ± 8 .1 3 b 0. 3 ± 0. 48 a 9. 5 ± 4. 30 a b 1 ± 0. 69 a 0. 4 ± 0. 51 a 10 .4 ± 6 .7 1 b 3. 6 ± 2. 11 b c 0. 5 ± 0. 52 a b 3. 5 ± 2. 54 c 1 ± 0. 66 a b 0. 1 ± 0. 31 a 46 .5 g ol dp la n 40 0 25 .1 ± 9 .7 6 cd 0. 6 ± 0. 84 a 22 .3 ± 9 .4 2 d 3. 1 ± 2. 55 a 1. 2 ± 0. 91 a bc 21 .5 ± 8 .8 0 c 5 ± 1. 82 c d 1. 2 ± 1. 93 b 1. 9 ± 1. 1 ab c 0. 4 ± 0. 51 a 0. 2 ± 0. 42 a 82 .5 el an do r 20 0 27 .5 ± 7 .3 6 d 1. 6 ± 2. 01 b 23 .3 ± 9 .4 0 d 1. 8 ± 0. 91 a 1 ± 0. 66 a bc 23 ± 7 .9 3 c 5. 2 ± 2. 82 c d 1 ± 0. 66 a b 3. 2 ± 2. 2 c 0. 5 ± 0. 70 a 0 ± 0 a 88 .1 v ar ia bi lit y ar ou nd t he m ea n w as r ep re se nt ed a s ± sd ( st an da rt d ev ia tio n) . d at a ha vi ng t he s am e le tt er in a c ol um n w er e no t si gn ifi ca nt ly d iff er ed b y ls d ’s m ul tip li co m pa ri so n te st (p <0 .0 5) . 71investigation of the cytotoxic and genotoxic eff ects of the euphorbia rigida extract a b c d e f g h i j k m n o p q r s figure 1. (a) stickiness in metaphase (100 ppm e. rigida) (b) irreguler metaphase (100 ppm) (c) irreguler anaphase, (100 ppm) (d) pole deviation (goldplan) (e) c-mitosis and aneploidy (100 ppm) (f ) fragments (400 ppm) (g) laggard chromosome (50 ppm) (h) bridge in anaphase, (goldplan) (i) binucleus (200 ppm) (j) micronucleus (50 ppm) (k) granulation of nucleus (50 ppm) (m) irreguler prophase (100 ppm) (n) nucleus deformation (goldplan) (o) increase in the number of nucleolus in the nucleus (goldplan) (p) nucleus erosion in interphase (200 ppm) (q) vacualation of nucleus in interphase (200 ppm) (r) multipolar anaphase (elandor) (s) polyploidy with cmitosis and fragments (50 ppm) (10x40). 72 metin mert, bürün betül it is thought that the formation of binucleate cells may result from incomplete cell division or wall fusion as a result of halting protein synthesis (sivas and gökbayrak 2011). c-mitosis constitutes with stopping spindle form (badr 1983). mitotic toxicity affects spindle mechanism therewith c-mitosis is observed (yüzbaşıoğlu 2003; grisolia et al. 2004; türkoğlu 2007; sivas and gökbayrak 2011; andrade-vieira et al. 2012; pandey et al. 2014). the cytotoxic effects of urea, fungicides (afugan, carbendazim)(yüzbaşıoğlu 2003; bonciu et al. 2018), synthetic plant growth regulators (2,4-dichlorophenoxyacetic acid)(özkul et al. 2016), pesticides, herbicides, insecticides (pentachlorophenol, maleic hydrazide, dichlorvos and glyphos)(grant 1978; kaymak 2005; fındıklı and türkoğlu 2010), heav y metal (chromium=k2cr2o7, pb(no3)2)(güler et al. 2018; girasun et al. 2019) and some plant extracts containing bioactive compounds (triterpenoids, tannins etc.)(başaran et al. 1996; shi et al. 2008) were observed from the occurrence of fragments on dna doubled spindles (çavuşoğlu 2019). fragmentation might have arisen due to stickiness of chromosomes and consequent failure of separation of chromatids to poles. in addition to this, dna double strand breaks induced by reactive oxygen species can lead to chromosome fragments (adhikari 2019; liman et al. 2020). it is claimed that the stickiness, bridges and fragments which are scored as indicators of clastogenicity in chromosomes are induced by chemicals regarded as clastogenic agents (p<0.05) (rank 2003; kaymak 2005; sivas and gökbayrak 2011; andrade-vieira et al. 2012; adhikari 2019). these alterations can form an irreversible and genotoxic influence (fiskesjö and levan 1993). stickiness aberration forms as a result of chromatid irregularity (badr 1983). stickiness in the chromosomes is an indication that chemical substance has a high toxicity, and may cause the death of cells by inducing unrecoverable damages (fiskesjö 1985; türkoğlu 2007; andrade-vieira et al. 2012; pandey et al. 2014). chromosome bridges may be due to the chromosomal stickiness and subsequent failure of free anaphase separation or may be attributed to unequal translocation or inversion of chromosome segments (gömürgen 2005; türkoğlu 2007; sivas and gökbayrak 2011). in this study, the highest stickiness in the chromosomes was seen in e. rigida 50 ppm, elandor and goldplan (p<0.05). nucleus deformation increases depending on the increase in extract concentration, which indicates that the cells are affected cytotoxically and genotoxically, dna synthesis is pressured. vacuolisations were observed in e. rigida 200 ppm extract treatment, then this indicated that the chemical pesticides are more destructive and comprehensive mutagens, and those high concentrations of e. rigida 200 ppm extract give the same results. the anomalies occurred in all doses of e. rigida extract, but the highest anomalies was usually observed in e. rigida 50 and 100 ppm. elandor and goldplan are routinely used against control of hemiptera, especially aphids, thysanoptera and lepidoptera (öncüer 2004). when these pesticides were compared to extract of e. rigida, it was seen that even the high doses of e. rigida extract are more mutagenic (p<0.05). in this study, 200 and 400 ppm e. rigida extracts were found to be more cytotoxic and genotoxic than 200 ppm goldplan or 400 ppm elandor (p<0.05). it is obvious from the results of the present study and based on the literature that the production of atp is suppressed in the cells of a. cepa meristem tissue exposed to increased doses of e. rigida extract and chemical pesticides for 24-hour. also, the metabolic activities in the cell slow down or stop. at the same time, depending on the clastogenic and aneugenic effects of plant extract and chemical pesticides, cells that tend to enter mitosis are eliminated. thus, mitosis was suppressed in tissues and cells exposed to high doses and less abnormal cell formation were observed. e. paralias, e. antiquorum and e. chamaesyce showed moderate antiviral activity (shi et al. 2008). e. paralias, e. maschallian and e. myrsinites species have antiviral compounds. e. pekinensis, e. peplus, phyllanthus nanus and p. amarus are effective against virus infections and e.hirta shows direct antiviral effects on hiv1, hiv-2 and siv (mac251) reverse transcriptase (rt) activity (gyurıs et al. 2009; alam et al. 2016). e. rigida plant did not cause gene change in bacteria such as ta 98 and ta 100, they showed mutagenic effects in human lymphocytes (başaran et al. 1996). e. rigida extract may also have strong effects on viruses’ capsid, reverse transcriptase (rt) and dna/rna structures. although e. rigida extract is a natural and organic product, it is clear that it is more toxic, cytotoxic and genotoxic. therefore, the possibility of using e. rigida extract as an antiseptic for sterilization or disinfection of large and inanimate surfaces can be explored (avcı et al. 2013). according to the data obtained from cytotoxic and genotoxic tests in this study, the aqueous extract up to 50 ppm of e. rigida is seen to be promising for using as biopesticide purposes as an alternative to the chemicals we use in our experiments. however, studies on the evaluation of systematic toxicity and safety of euphorbia species are very few. in the studies that have been conducted, only the organs that are targeted and side effects are emphasized (huang et al. 2012). although e. rigida extract is a natural and organic product, it is clear that it can be dangerous because it is more toxic, cytotoxic and 73investigation of the cytotoxic and genotoxic effects of the euphorbia rigida extract genotoxic than goldplan and elandor used as a chemical pesticide in agricultural control (p<0.05). in order to reach more information and certain conclusions on this subject, however, further research should be performed with different test systems. acknowledgements we thank scientific research projects unit (09/21 bap) of muğla sıtkı koçman university for their support in this study. references abdelgalil sam, el-aswad af, nakatani m. 2002. molluscicidal and anti-feedant activities of diterpenes from euphorbia paralias l. pest manage. sci. 58: 479482. adhikari 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cytogenetics citation: asim iqbal bazaz, irfan ahmad, tasaduq h. shah, nafhat-ularab (2022) karyomorphometric analysis of fresh water fish species of india, with special reference to cold water fishes of kashmir himalayas. a mini review. caryologia 75(1): 109-121. doi: 10.36253/caryologia-1362 received: july 14, 2021 accepted: march 28, 2022 published: july 6, 2022 copyright: © 2022 asim iqbal bazaz, irfan ahmad, tasaduq h. shah, nafhat-ul-arab. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyomorphometric analysis of fresh water fish species of india, with special reference to cold water fishes of kashmir himalayas. a mini review asim iqbal bazaz1, irfan ahmad2,*, tasaduq h. shah1, nafhat-ularab3 1 division of fisheries resource management, faculty of fisheries, skuast – kashmir 2 division of fish genetics and biotechnology, faculty of fisheries, skuast – kashmir 3division of aquatic environmental management, faculty of fisheries, skuast – kashmir *corresponding author. e-mail: ahmadirfan@skuastkashmir.ac.in abstract. cytogenetics is the diagnostic study of chromosomal structure and properties, as well as cell division, using a variety of methods, one of which is “karyotyping.” it refers to a method of photographing a stained preparation in which the chromosomes are organised in a uniform pattern. the advent of modern techniques such as “karyotyping” has made it feasible to visualize  undetected chromosomal abnormalities such as short chromosome segments and chromosome translocations. because such techniques enabled  each pair of chromosomes to be identified separately, they have further  aided our understanding  of the chromosomal basis of a certain  significant genetic diseases. every organism has its own unique karyotype, which is defined by its number and shape. karyotypic variation, on the other hand, occurs in different individuals of the same species, as well as between different species. monitoring cytogenetic data of economically significant fishes as well as threatened fishes can hold importance of the succeeding generations.  this review article highlights the variation in the chromosomal number & classification, methods of chromosome preparation and karyotypic analysis of various fish species of india with a special reference to fishes of kashmir himalayas. keywords: fish chromosomes, fish karyology, freshwater fish, kashmir, himalaya. introduction the study of chromosome number, morphology and size at the metaphase stage is the basis of cytogenetics, which involves karyotype analysis. a species’ chromosomes can be arranged in size order. the karyotype is the full set of chromosomes grouped according to their number shao et al. (2010). karyotyping is the method of pairing and ordering all of an organism’s chromosomes, resulting in a genome-wide snapshot of the chromosomes of an individual. karyotypes are produced using standardised stain110 asim iqbal bazaz et al. ing procedures that expose each chromosome’s specific structural features. changes in chromosome number associated with aneuploid conditions may be revealed through karyotypes. more subtle structural shifts, such as chromosomal deletions, duplications, translocations, or inversions, can be identified by carefully studying karyotypes. indeed, karyotypes are increasingly being used to diagnose birth defects, genetic disorders, and even cancers o’connor (2008). dna, which is packed into chromosomes, provides the blueprint for the development and maintenance of an organism. chromosomes are the elements that differentiate one species from another and allow genetic information to be passed down from generation to generation. chromosomes are the vehicles that allow a species to replicate and sustain itself ciccone et al. (2005) and de ravel et al. (2006). methods of chromosome preparation the study of fish cytogenetics started as early as the last decade of the nineteenth century, when some idea of chromosomes was made possible by the studies of retziat (1989) on agnathan myxine glutinosa, using histologically cut gonadal material. it was later understood that chromosome preparation could be obtained from all the tissues in which mitosis occurs. since the 1960s, several methods have been used to study the chromosome of fish. those employed involves  colchicine injections and squashes of the testes or haematopoietic tissues ohno et al. (1965), corneal and conjunctival epithelium drewry (1964), gill epithelium chen and ebling (1968), embryonic material simon and dollar (1963), in vitro tissue growth roberts (1967), blood leukocytes in culture ojima et al. (1970) and epithelium scale denton and howell (1969). improved techniques for the preparation of fish chromosomes were developed after the 1970s nagpure et al. (2001).tissue cultures roberts (1964), squashing technique of the testis, and other karyotypic techniques have accompanied the advancement in cytogenetical studies of teleostean fishes. roberts (1964); ohno et al. (1965), embryonic tissues or haematopoetic materials simon (1963); yamada (1967), smearing technique from gill epithelium mcphail and jones (1966); stewart and levin (1968), solid tissues like kidney ojima et al. (1972); arai (1973); ueno and ojima (1977), from regenerating fin tissue cattin and ferreira (1989); volker et al. (2005) and air drying techniques eicher (1966); bertollo et al. (1978); thode et al. (1988), together with colchicine treatment yamazaki (1971). dropping method was used in most previous studies to distribute cells from different tissues for chromosome preparation. for spreading and flattening metaphase chromosomes, the squash technique is the oldest process denton (1973). the air drying process, on the other hand, is the most commonly used method for preparing animal chromosomes evans et al. (1964). according to chourrout and happe (1986), modern chromosome preparation techniques using air drying after colchicine injection in young fish resulted in sufficient metaphase spreads mcphail and jones, (1966); kligerman and bloom (1977). however, these methods yielded some results, it was discovered that a large number of cells were lost during the cell dropping. furthermore, dropping the cells precisely on the preheated slides requires a high degree of technical ability. some researchers have also attempted to prepare chromosome spreads by lowering cells from a certain height onto frozen slides ojima et al. (1964); ida et al. (1978). for chromosome preparations, researchers also tried incorporation of  hot steam and metal plates with a temperature gradient across their surface henegariu et al. (2001). squash method squashes have been the most widely used methods of providing karyotyping material. although an ancient  technique, it is still very effective because preparations are possible from small pieces of tissue that can be separated without causing serious injury to the animal. for example, chromosome slides can be made from epithelium of  scale, gill  epithelium,  marginal barbels and fins, and corneal tissue. one can even greatly improve mitotic activity in these tissues by triggering local injury and allowing it to heal. squashing is typically performed in some stain-fixatives, such as aceto-carmine or aceto-orcein, after pre-treatment with hypotonic solution. in place of potassium chloride, sodium citrate, tap water or distilled water or even a 1.0 per cent tissue culture medium may be used. the ideal treatment time is between 20 and 30 minutes. a  good metaphase spread from the kidneys and gills of labeo rohita and cirrhinus mrigala following the squash method was obtained by khuda-bukhsh and chakraborty (1994). cell culture method in terms of chromosome preparation, cell culture yielded promising results. successful attempts have been made by amemiya et al. (1984). however, chen and ebling (1975) contended that this method requires the killing of fish before tissues can be excised. blood lymphocyte culture helps to overcome many of the limitations mentioned above, as fish do not need to be killed, 111karyomorphometric analysis of fresh water fish species of india so repeated samples can be taken if necessary and the number of mitoses is increased due to the need to stimulate mitogen lymphocytes blaxhall (1983). the author also identified the use of phytohaemagglutinin-purified blood lymphocyte culture for karyotyping of salmo trutta l. and cyprinus carpio l. (pha-p). staining of chromosomes to distinguish the colourless chromosomes from the similarly colourless cytoplasm, staining is needed. for chromosome visualisation, the slides are stained with the required solution. most commonly, the  giemsa stain is used to stain slides. the use of filtered acetoricine as a stain was recorded by mc phail and jones (1966). arcement and rachlin (1976) experimented with various stains, including giemsa (normal or buffered), aceto-orcein, and aceto-cannine, and found that giemsa standard provided the best results. however, the majority of the workers proposed diluting giemsa with phosphate buffer. chromosome banding techniques the chromosomal band is defined as a segment of chromosomes, which can be differentiated from adjacent segments by being either lighter or darker depending on the staining technique involved. chromatin is the substance that makes up chromosomes, and there are two types: euchromatin, which stains softly, and heterochromatin, which stains darkly. the arrangement and organisation of chromosomes can be better understood with chromosome banding. the unambiguous identification of chromosomes in the karyotype, as well as the study of heteromorphism between and within organisms, is two of the most important applications of the banding technique. banding techniques may also be used to identify chromosome rearrangements that have taken place during the course of evolution. the different banding techniques used today for the cytogenetic study are c-, g-, r-, q-, and nor-banding. among these, norand c-banding are commonly used in fish hartley and horne (1985). g-banding was also tried, but only with little success blaxhall (1983).the approach to slide preparation is vital, as incorrect spreading strategies can result in the chromosomes being washed out during the staining process. after slide  preparations, various staining techniques such as classic staining (e.g., aceto-orcein, haematoxylin, giemsa, wright and leishman stains) or banding techniques are employed to stain chromosomes for various purposes e.g., q-banding, g-banding, r-banding, c-banding and high resolution banding  calado et al. (2013); moore and best (2001); wang et al. (2010). concentrated staining solutions and/or over incubation result in a dark background filling the space between chromatids, whereas diluted staining solutions and/or a short incubation period produce chromosomal spreads that are indistinguishable. use of colchicine the most common microtubular poison is colchicine. colchicine inhibits spindle microtubules and disperses metaphase chromosomes in the cytoplasm before nuclear envelope breakdown (neb) in metaphase cells caperta et al. (2006), whereas in d. rerio both colchicine concentration and incubation period were significantly influenced by larval age, and in c. gariepinus only colchicine incubation period was significantly influenced by larval age. it has previously been demonstrated that microtubule polymers are sensitive to physical and chemical parameters tilney and porter (1967); weisenberg (1972). therefore, ageand species-dependent cellular parameters may influence the sensitivity of cells towards depolymerizing effects of colchicine. a spindle poison (e.g., colchicine) is used to arrest the cells at their metaphase stage in conventional chromosome preparation processes kligerman and bloom (1977). in order to achieve clear and identifiable metaphase chromosome spreads, it is essential to select the appropriate concentration and incubation time for the poison rieder and palazzo (1992). while the cells cannot be stopped at metaphase stage with insufficient concentration and/or time spindle poisons exposure, extremely high concentrations or excessively long durations of exposure might lead to chromosome condensation rieder and palazzo (1992); wood et al. (2001). cells or larvae must be incubated in a hypotonic solution following mitotic spindle inhibition to swell the nuclei and disperse the chromosomes on slides moore and best (2001). using an appropriate hypotonic solution is the other critical element that has been emphasized in the current study. potassium chloride (kcl 0.075 m) is one of the most commonly used hypotonic solutions in chromosomal preparation protocols. analogously, the efficacy of distilled water as a hypotonic treatment has been shown in some other protocols. when distilled water was used instead of kcl, the amount of clear metaphase chromosome spreads in c. gariepinus increased significantly. the use of kcl resulted in a lot of cell burst and chromosomal loss. changes in the hypotonic solution, on the other hand, had no impact on the amount of metaphase chromosome spreads in d. rerio. karami et al. (2015) also 112 asim iqbal bazaz et al. reported that the type of hypotonic solution used can be changed depending on the fish species and/or larval age in order to obtain a desired number of consistent chromosome spreads. besides these aforementioned  elements, the researchers attempted to alter other essential aspects of chromosomal preparation protocols in their research. their preliminary research also showed that the larvae should be killed before being incubated in colchicine solution, as incubating live larvae in the solution did not result in chromosome spread. furthermore, the yolk sac should be extracted prior to incubation in colchicine to obtain direct chromosome spread hussain and mcandrew (1994); pradeep et al. (2011), because the yolk’s high lipophilicity can limit the penetration of colchicine or hypotonic solution into the cells hussain and mcandrew (1994); pradeep et al. (2011). hypotonic treatment hypotonic treatment is an important and crucial factor in improving the chromosome spreads. this treatment helps in removal of lipid and denatures proteins. hypotonic treatment allows the swelling of the cell, which facilitates cell disruption and the dispersion of chromosomes when the cell contents are spread on slides. ida et al. (1978) reported that the use of potassium chloride showed the best chromosome spreads as compared to other two hypotonic solutions of sodium citrate and distilled water. chourrout and happe (1986) reported that the chromosome spreading was insufficient at 0.56% kcl for hypotonic treatment at a lower temperature in the rainbow trout. however, the same concentration of kcl showed slightly better results when the experiments were performed at ambient temperature. according to the same author trisodium citrate as hypotonic treatment gave significant improvement in chromosome spreading. pradeep et al. (2011) used 50% acetic acid during the chopping of tissues, but simple distilled water, produced better suspensions. in the modified technique, different durations of staining along with different concentrations of giemsa stain were also tried. a concentration of 5% giemsa stain for 20 minutes of treatment as described by bayat and woznicki (2006) was not very effective. moreover, counting of the chromosomes was found difficult at a concentration of 20% as suggested by don and avtalion (1986). changing timing and concentrations of giemsa stain significantly affected the visibility and brightness of the spreads on the slides. a concentration of 10% giemsa stain prepared in 0.01 m phosphate buffer of ph 7 for 20 minutes, as described by hussain and mcandrew (1994) was very effective in obtaining clear images. the majority of genetic defects are caused by chromosomal abnormalities. cytogenetics is the diagnostic study of chromosome structure and properties, as well as cell division, using a number of techniques, one of which is “karyotyping.” it refers to a method of photographing a stained preparation in which the chromosomes are organised in a uniform pattern. the advancement of newer techniques such as “karyotyping” has made it possible to see previously undetected chromosomal abnormalities such as small chromosome segments and chromosome translocations, veerabhadrappa (2016). colchicine injections and squashes of the testes or haematopoietic tissues are among the techniques used roberts (1964); ohno et al. (1965), corneal and conjunctival epithelium sick et al. (1962); drewry (1964), gill epithelium mcphail and jones (1966); chen and ebling (1968), embryological material simon (1963); simon and dollar (1963), sectioning of testes nogusa (1960), growth of various tissues in vitro roberts (1964); (1966); (1967), blood leukocytes in culture heckman and brubaker (1970); ojima et al. (1970), scale epithelium denton and howell (1969). a good quality review of some of these methods was made by roberts (1967). in several classes of plants and animals, karyological characteristics have proved to be a useful tool in taxonomic and evolutionary studies. fish cytology has been used by few ichthyologists because the chromosomes are tiny and the available techniques have often yielded distorted counts and limited morphological information. the use of squash preparations of gill arch epithelial cells in karyological methods produced satisfactory results. the gill arch technique defined by mcphail and jones (1966) was used with modifications that improved the performance lieppman and hubbs (1969). in aquaculture, the study of karyotype is also significant because of the use of chromosome manipulation techniques such as induction of polyploidy, gynogenesis, androgenesis, and inter or intra-specific hybridization wu et al. (1986); diter et al. (1993). karyological studies can help resolve a number of evolutionary and genetic questions about animals macgregor (1993), and chromosomal analysis can help determine changes that transformed an ancestral karyotype as it transformed into new lines winkler et al. (2004). chromosomal analysis is also important for genetic regulation, taxonomy, and evolutionary studies macgregor and varly (1993); fister et al. (1999); suleyman et al. (2004) and is widely use in various investigations pisano et al. (2007). when comparing karyotypes among related fishes, chromosome number, arm number, and dna volume can be exemplified. when one is viewed alone, it may 113karyomorphometric analysis of fresh water fish species of india lead to erroneous conclusions. centromeric fusion can minimise chromosome number without affecting chromatin content fundamentally. similarly, unequal reciprocal translocations may change arm numbers but have little impact on chromatin booke (1968). polyploidy can result in substantial changes, suggesting greater phylogenetic effects than previously thought ohno et al. (1967). in india, the analysis of fish chromosomes began in the 1960s, and of the approximately 2000 species of inland and marine fish studied for karyological information, over 200 species are native to the region, including both freshwater and marine species. das and barat (1995) for instance, schizothorax richardsonii sharma et al. (1992); lakara et al. (1997); barat et al. (1997), schizothorichthys prograstus rishi et al. (1983), s. kumaonensis, lakara et al. (1997); rishi et al. (1998), catla catla and mystus vittatus john et al. (1992), labeo john et al. (1993), tor khudree and tor mussullah kushwaha et al. (2001), heteropneustes fossilis kushwaha et al. (2002), labeo rohita nagpure (1997), clarias gariepinus nagpure et al.(2000), labeo rohita, catla catla and cirrhinus mrigala nagpure et al. (2001). labeo dussumieri, horabagrus brachysoma and puntius filamentosus nagpure et al. (2004), horabagrus nigricollaris, puntius denisonii and puntius sarana subnasutus nagpure et al. (2004). though their taxonomy has been studied by several workers in the past heckel (1838); mcclelland (1839); silas (1960); talwar and jhingran (1991); kullander et al. (1999), there is still a lot of uncertainty about the exact number of species occurring in different aquatic ecosystems of the valley. this is complicated even further by the fact that hybrids of some of these species have been reported heckel (1838) and hora (1936). as a result, despite its value as a food fishery, this species complex has not been studied for its nutritional and biochemical components, nor has it been commercially cultured. ganai et al. (2011) studied five recognized species of schizothorax viz., schizothorax niger, s. esocinus, s. curvifrons, s. plagiostomus and s. labiatus for various karyological features. somatic complement of schizothorax niger showed a diploid number of 98 chromosome pairs, including 12 metacentric pairs, 16 sub-metacentric pairs, 11 sub-telocentric pairs, and 10 telocentric pairs. the diploid complement of schizothorax esocinus was 98, with 15 metacentric chromosome pairs, 11 sub-metacentric pairs, 5 sub-telocentric pairs, and 18 telocentric pairs. the diploid complement of schizothorax labiatus was 98, with 12 metacentric pairs, 10 sub-metacentric pairs, 1 sub-telocentric pair, and 26 telocentric pairs. the somatic complement of schizothorax plagiostomus was 96, with 12 metacentric pairs, 9 sub-metacentric pairs, and 27 telocentric pairs. schizothorax curvifrons had a diploid chromosomal complement of 94 chromosomes: 13 metacentric pairs, 10 submetacentric pairs, 10 subtelocentric pairs, and 14 telocentric pairs. s. niger, s. esocinus, and s. labiatus, three of the five species examined, had a diploid number of 98 and a fundamental arm number of fn of 154, 150, and 142, respectively. intra-chromosomal changes involving pericentric and paracentric inversion, as well as centromeric shifts, could explain the difference in the fundamental arm number without a change in the 2n rishi et al. (1998). the karyotypes of the two species indicate that in s. esocinus, there was simultaneous fusion of telocentric and fission of metacentric chromosomes, resulting in the karyotype of s. niger. this is due to the fact that s. niger has more biarmed chromosomes than s. esocinus, and a karyotype of biarmed chromosomes is generally considered to reflect a derived condition ohno et al. (1968); ohno (1970); denton (1973); gold (1979). the karyotype of s. labiatus tends to be characterized by the same forms of chromosomal rearrangements. except for s. plagiostomus, the chromosomes of all five schizothorax species were divided into four groups: metacentric, submetacentric, subtelocentric, and telocentric, according to levan et al. 1964.  the overall similarity in chromosome number and morphology suggested that schizothorax species are closely related in that they have not been separated as evolving organisms long enough for random chromosome changes to have occurred and become set, and that a particular karyotype will be selected implies an adaptive advantage for that specific configuration. for chromosome differences observed in fundulus chen (1971) and rivulines, this hypothesis has been proposed scheel (1972). cyprinid karyotypes have had systematic implications joswiak et al. (1980) since comparative karyology has been a useful method in fish systematic studies arai (1982); buth et al. (1991) because chromosome number and morphology indicate changes that altered an ancestral karyotype as it developed into new lines winkler et al. (2004) and are useful for addressing a range of genetic, genetological, and evolutionary genetic and cyto-taxonomic questions about animals kirpichnikov (1981); mcgregor (1993). table 1. nomenclature for designating chromosome type levan et al. (1964). centromeric position arm ratio chromosome type symbol median 1.00-1.70 metacentric m sub-median 1.71-3.00 sub-metacentric sm sub-terminal 3.01-7.00 sub-telocentric st terminal >7.01 acrocentric a 114 asim iqbal bazaz et al. variation in the chromosomal number & classification ganai and yousuf (2011) observed diploid number per metaphasic plate ranged from 47 to 50. a modal diploid number of 2n = 50 constituted 72.5% (22 m+16 sm+12 t) and 2n = 48 constituted 20% of the counted metaphase plates. other diploid numbers other than 2n = 50 are usually the result of losses or additions during the karyotype preparation, including splashing due to their downfall from various heights from nearby cells, as reported in other studies (suleyman et al. (2004); esmaeli and piraver (2006); nasri et al. (2010). ganai and yousuf (2011) obtained proper metaphasic plate chromosomal indicators including eleven metacentric, eight sub-metacentric and six telocentric pairs respectively and fundamental number as fn = 88. comparison with already worked out species of p. conchonius in jammu and other parts of the country sharma and agarwal (1981); tripathi and sharma (1987) reveals that it is a new cytotype, inhabiting dal lake, kashmir. the most commonly occuring diploid number in family cyprinidae is 50, considered to be the modal number in case of this family manna (1984); rishi (1989). according to the studies performed by various workers on puntius species of india tripathi and sharma (1987), it seems that 2n table 2. chromosome classification of various schizothorax species, worked out in kashmir valley (m = metacentric; sm = sub-metacentric; st = sub-telocentric; t = telocentric; nf = fundamental arm number). s. no. name of the species 2n m sm st t nf value author and year 1 schizothorax niger 98 24 32 22 20 154 ganaie et al. (2011) 2 schizothorax esocinus 98 30 22 10 36 150 ganaie et al. (2011) 3 schizothorax labiatus 98 24 20 2 52 142 ganaie et al. (2011) 4 schizothorax plagiostomus 96 24 18 54 138 ganaie et al. (2011) 5 schizothorax curvifrons 94 26 20 20 28 140 ganaie et al. (2011) table 3. karyotypic analysis of various fresh water fish species. species family diploid (2n) chromosome formula (2n) authors 1. oncorhynchus mykiss salmonidae 56-65 24 m +20 sm + 16 t vasave et al. (2016) 2. cyprinus carpio cyprinidae 97 24 m +24 sm + 52 t khuda-bukhsh and barat (1987) 3. ctenopharyngodon idella cyprinidae 48 14m+20sm+8st+6t manna (1983) 4. botia birdi botiidae 98 14 m+18 sm+ 4st + 62 t khuda-bukhsh and nayak (1982) 5. tor tor cyprinidae 100 24 m+ 24sm+ 6 st + 46 a khuda-bukhsh (1980) 6. schizothorax curvifrons cyprinidae 94 26m+20sm+20st+28t ganai et al. (2014) 7. schizothorax niger cyprinidae 98 22 m +26 sm + 8 st + 42t khuda-bukhsh and nayak (1982) 8. tor putitora cyprinidae 100 10 m+24 sm+ 14st + 52 t khuda-bukhsh (1980) 9. schizothorax esocinus cyprinidae 98 30m+22sm+10st+36t ganai et al. (2014) 10. schizothorax plagiostomus cyprinidae 96 24m+18sm+54t ganai et al. (2014) 11. cirrhinus mrigala cyprinidae 50 12 m+ 18sm + l0st + l0 t zhang and reddy (1991) 12. crossocheilus dipiocheilus cyprinidae 48 12m+36a manna (1983) 13. carassius carassius cyprinidae 98/ 100 24 m +26 sm + 12st + 36a 20 m +36 sm + 44 (st + a) singh (1983) spoz et al. (2014) 14. carassius auratus cyprinidae 96 12 m +36 sm + 48 a rishi (l981) 15. garra gotyla cyprinidae 50 14 m +10 sm + l0 st + 16 t khuda-bukhsh (1984) 16. hypophthalmichthys molitrix cyprinidae 48 20 m +12 sm + 6st+ l0t manna and khuda-bukhsh (1977) 17. puntius conchonius cyprinidae 48 50 10 m +20 sm + 10st + 8 t 16 m +24 sm + 2 st + 8 t barat (1985); khuda-bukhsh et al. (1986) 18. nemacheilus moreh nemacheilidae 50 24 m + 22 sm + 4 t chanda (1989) 19. puntius ticto cyprinidae 50 14m+ 18sm + 14st + 4t bano et al. (2015) 20. schizothorax richarsonii cyprinidae 96 18 m +16sm +12st+ 50t vasave et al. (2016) 115karyomorphometric analysis of fresh water fish species of india = 50 in the genus puntius, as in many other cyprinids. despite the similarity of the diploid number in species of puntius, there are differences in their karyotype formulae. nayyar (1964) reported the presence of all acrocentric chromosomes in p. conchonius. barman (2003) also confirms the presence of both biarmed and acrocentric chromosomes. the primitive teleost karyotype is thought to have consisted of 46 to 48 acrocentrics, nayyar (1966); ohno et al. (1968); ohno (1970); fitzsimons (1972); legrande (1975). karyotypes with biarmed chromosomes are generally regarded to represent a derived condition ohno et al. (1968); ohno (1970); denton (1973); gold (1979). ganai et al. (2011) reported both the species of schizothorax analysed cytologically, revealed a high number of chromosomes ranging from 94 to 98. all the schizothorax species studied karyologically till date s. richardsonii gray and s. kumaonensis menon, lakara et al. (1997); s. zarudnyi, nikolskii, kalbassi et al. (2008); s. plagiostomus and s. esocinus ganai et al. (2011) show a high chromosome number ranging from 96 to 98. species with high numbers are considered to have resulted through polyploidy from ancestral 2n= 48 or 50 rishi et al. (1998). such genomic enlargements have been hypothesised as key factors that enable or even drive diversification in various vertebrate groups holland et al. (1994); meyer and malaga-trillo (1999); navarro and barton (2003a, b); ohno (1970). variation in the karyotypic configuration of s. niger (24m + 32sm + 22st + 20t and fn=154) and s. curvifrons (26m+20sm+20st+28t) and fn=140 can easily be explained by centric fusion and fission events. both centric fission and fusion probably provide important mechanisms to explain the diverse range of chromosome numbers observed in many mammalian and non-mam-malian animal taxa todd (1970); imai et al. (1986), kolnicki (2000). decrease in 2n and fn in s. curvifrons may be attributed to robertsonian arrangements and pericentric inversion choudhury et al. (1982); ganai et al. (2011) also reported despite overlap in the general morphological features, the two species of schizothorax investigated are genetically different and hence definite species as the chromosomal differentiation in animal species usually precedes strong morphological differentiation howell and villa (1976). most morphologic features of fishes have been shown to have the potential of being modified by the environmental conditions svardson (1965); fowler (1970). therefore, a morphologically based classification should be tested by the features not likely to be environmentally false and chromosome structure is best suited for this purpose as it reflects genetic divergence and is least affected by environmental distortion, campos (1972). barat et al. (2012) reported the majority (85%) of cells had metaphase complements containing 2n = 50 chromosomes, though a few metaphases had a range of 46 to 52 chromosomes. the karyotypic formula was detected as 2n = 12m (metacentric) + 14sm (sub-metacentric) + 10st (subtelocentric) + 14t (telocentric) with a fundamental arm number (nf) of 80. however, most of the members of the family cobitidae had a diploid chromosome number (2n) of 50, with just a few species – botia birdi, b. macroracantha and b. dario – with a diploid chromosome number of 90–98 khuda-bukhsh et al. (1986). therefore, the modal chromosome number in this family could be ascertained as 50 barat et al. (2012). advantages & applications • karyological studies have made a substantial contribution to various fields in fisheries like systematics, evolution, mutagenesis, aquaculture, phylogenetic relationship and hybridization kligermann and bloom (1977). • chromosomal analysis is important for fish breeding from the viewpoint of genetic control kirpichnikov (1981). • besides, karyological studies also generate information about genetic diversity in natural fish population, which is imperative in the conservation and stock management kligermann and bloom (1977). • karyological studies have provided basic information on the number, size and morphology of chromosomes that is important to undertake chromosome manipulations in fish khan et al. (2000). • since 1960s, karyological studies in teleost fish have made noteworthy contributions to increasing knowledge in the fields of genetics, taxonomy and environmental toxicology cucchi and baruffaldi (1990). • karyotyping helps in analyzing the entire genome. it can visualize individual cells and individual chromosomes. • many cy togenetic techniques are useful in fish breeding and culture practices such as: • ploidy determination rishi and haobam (1984) • hybrid identification manna (1989) • sex determination manna (1989) • genotoxicity study of the pollutants rishi (1989). • further cytogenetic characterization of threatened species is useful in drawing programmes for conservation and stock management john et al. (1994). 116 asim iqbal bazaz et al. conclusion karyological studies have provided basic information on the number, size and morphology of chromosomes that is important to undertake chromosome manipulations in fish. the development of newer techniques such as “karyotyping” has made it possible to visualize undetected chromosomal anomalies such as small portions of chromosomes and translocations of tiny parts of chromosomes to one another. because such procedures also enabled each pair of chromosomes to be distinguished individually, it has helped to further our understanding of chromosomal basis of certain important genetic disorders. chromosomal analysis is important for fish breeding from the viewpoint of genetic control. indigenous species of kashmir (schizothorax sps.) analysed cytologically, revealed a high number of chromosomes ranging from 94 (schizothorax curvifrons) to 98 (schizothorax niger). the nf value of schizothorax species of kashmir valley ranged from 138 (schizothorax 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of cytology, cytosystematics and cytogenetics 74(2): 11-19, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1013 caryologia international journal of cytology, cytosystematics and cytogenetics citation: dilek akyil (2021) mutagenic and cytotoxic activity of insecticide napoleon 4ec in allium cepa and ames test. caryologia 74(2): 11-19. doi: 10.36253/ caryologia-1013 received: july 06, 2020 accepted: july 20, 2021 published: october 08, 2021 copyright: © 2021 dilek akyil. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. mutagenic and cytotoxic activity of insecticide napoleon 4ec in allium cepa and ames test dilek akyil department of molecular biology and genetic, faculty of arts and sciences, afyon kocatepe university, afyonkarahisar/turkey e-mail: dilekakyil9@gmail.com abstract. the objective of this study was to explore the mutagenic and cytotoxic effects of napoleon 4ec pesticide used in turkey to control insect pest by using two standard assays. the allium cepa test was used for determined the cytotoxic effects of this pesticide. for this test, onion seeds were exposed to napoleon 4ec (100, 200, and 400 ppm) for 24, 48, and 72 hours. for each test group root tip cells were stained with feulgen and five slides were prepared for each concentration and counted microscopically. the concentrations napoleon 4ec was compared with the value for the negative control using dunnet-t test, 2 sided. the results indicated that mitotic index was clearly decreased with increasing the concentration of napoleon 4ec in each treatment group as compared to the controls. the percentage of mitotic phases has been markedly impacted. five different doses of the pesticide (50, 100, 200, 400, 800 μg/plate) were examined with ames test using salmonella typhimurium strains ta98 and ta100 with and without s9 metabolic activation for mutagenic activity. ames test results showed a dose dependent effect, but not twice the negative control for s. typhimurium ta98 and ta100, with or without s9 mix except 800 μg/plate doses. in 800 μg/plate doses, colony numbers are two-fold increase according to colony number of control group. so, this places the this compound as a weak mutagen according to the parameters. keywords: allium test, ames test, cytotoxicity, mutagenicity, napoleon 4ec. introduction the identification of chemicals capable of inducing mutations has become an important procedure in safety estimation and such substances can potentially induce to fertility problems in future generations. mutagenic materials are also capable of inducing cancer, and this problem has been increased of the importance the mutagenicity testing systems (kumar et al. 2013). earlier studies have showed that some pesticides are clastogenic and mutagenic in different biological test systems (siroki et al. 2001; celik 2003; stivaktakis et al. 2010; moulas et al. 2013; akyıl et al. 2014; akyıl and konuk 2015; özkara et al. 2015a). organophosphorus pesticides (ops) are one of the most important groups of pesticides which have been broadly used in industry, hygiene and agriculture (bello-ramı́ rez et al. 2000; ballesteros and par12 dilek akyil rado 2004; wu et al. 2007). napoleon 4ec is also an ops which has an important role to control crops. ops are strong inhibitors of cholinesterase enzymes and they were improved to replace organohalide pesticides in the late 1950’s due to relatively easier to degrade via microbial or environmental processes (obare et al. 2010). on the other hand, these substances or their derivatives can accumulate in the living organisms and induce mutagenicity, teratogenicity, immunotoxicity and carcinogenicity. short-term test methods have been used for many years from past to present to identify the genotoxic activities of pesticides (miao et al. 2017; özkara 2017; khallef et al. 2018; özkara 2019; akyıl 2019). the shortterm test systems can detect different types of genetic dna damage: (i) gene or point mutations; (ii) primary dna damage and repair; (iii) chromosomal alterations. one of these test systems is the ames test which is used to evaluate the mutagenic activity of chemicals; it is a short-term bacterial reverse mutation assay (mortelmans and zeiger 2000; liman et al. 2010; arriaga-alba et al. 2013; escobar et al. 2013). the other test system is the allium test which is one of the well-known and reliable test systems to determine the toxicity in the laboratories (konuk et al. 2007; liman et al. 2010; özkara et al. 2015; bonciu et al. 2018). to analyze the effects of different substances, higher plants (vicia faba, tradescantia paludosa, pisum sativum, hordeum vulgare, crepis capillaris and allium cepa, etc.) have proven to be useful when used as bioindicators (enan 2009; siddiqui and alrumman 2020). however, allium cepa has as an advantage due to its large chromosomes, easily observed with a light microscope; also in relation to the features that may reveal an effect even at relatively low level of interaction of the tested substance with the genetic material, besides its long history of use as cytotoxicological test. as it is an in vivo test, the data can be used for assessment of genotoxicity on plants and even for eukaryotes in general, including humans (bonciu et al. 2018). the use of insecticides has become increasingly widespread throughout the world so additional studies are necessary to determinate the potential toxic risk of insecticides on non-target organisms through bacterial mutational/ames test and allium test, a plant assay (yaduvanshi et al. 2012). to our knowledge, there is no study on the cytotoxicity and mutagenicity of napoleon 4ec except in the present paper. the aim of this experiment was to evaluate both the mutagenic and cytotoxic effects of different doses of napoleon 4ec by the bacterial reverse mutation assay in s. typhimurium ta98 and ta100 strains with or without s9 mix and allium cepa test, respectively. materials and methods chemicals and test strains the lt-2 ta98 and ta100 histidine demanding auxotrophs of s. typhimurium were kindly obtained from prof. b.n. ames (university of california, berkeley). these strains were incubated for 16 h in liquid nutrient broth and kept at -80°c. their genetic markers and other properties, such as the numbers of spontaneous revertants and responses to positive controls, were controlled as described by maron and ames (1983). the test substance napoleon 4ec was purchased from a local market in afyonkarahisar/turkey and dissolved in sterille distilled water. allium cepa onion bulbs, 25–30 mm diameter, were obtained from a local market without any treatments. the other chemicals were obtained from merck and riedel. ames plate incorporation test the mutagenicity of the napoleon 4ec was determined using the standard plate incorporation assay. salmonella typhimurium strains ta98 and ta100 were used with or without s9 mix in this test (maron and ames 1983). the tester strains were tested for the presence of the strain-specific markers as described by maron and ames (1983). before the experiment, determination of the cytotoxic doses of the substance to be used during the experiment is carried out as a preliminary step. cytotoxic doses of napoleon 4ec (10.000, 1.000, 100, 10, 1 and 0.1 µg/plate) were determined by the method of dean et al. (1985). the strains were selected according to the strategies of mortelmans and zeiger (2000). the stock solutions of the test materials were dissolved in sterile distilled water and stored at 4°c. the s. typhimurium strains were incubated in nutrient broth at 37°c for 16h with shaking. a specific positive control was always used to test the experimental defect, if any, for each tester strain. positive controls were 4-nitro-o-phenylenediamine (npd) for ta98 and sodium azide (sa) for ta100, applied without metabolic activation, and 2-aminofluorene (af) for ta98 and 2-aminoanthracene (2aa) for ta100 used with metabolic activation. determination of cytotoxic doses for the test of cytotoxic doses were prepared by adding 0.1 ml of the test suspension for each concentration and 0.1 ml bacterial suspension of ta100 from an over13mutagenic and cytotoxic activity of insecticide napoleon 4ec in allium cepa and ames test night culture to 2 ml top agar which kept in 45°c water bath. the mixture was shaken for 3 s with a vortex mixer, and added into the nutrient agar. all test plates were incubated for 24 h at 37°c and then the revertant colonies were counted for each plate and determinated toxic and non-toxic doses which used in the experiment. for the test of without s9 mix were prepared by adding 0.1 ml of the test suspension for each concentration, 0.1 ml bacterial suspension from an overnight culture, and 0.5 ml phosphate buffer to 2 ml top agar which kept in 45°c water bath. the mixture was shaken for 3 s with a vortex mixer, and added into the minimal agar. for the test plates with s9 mix were prepared by adding 0.5 ml of s9 mix instead of the phosphate buffer. all test plates were incubated for 72 h at 37°c and then the revertant colonies were counted for each plate. samples were evaluated on triplicate plates in two independent parallel experiments and all results of the experiment were analysed by the statistical analysis. allium test the root inhibition test procedure was performed as described by fiskesjo (1985). preliminary experiments were conducted to determine the concentrations of each pesticide to be used in the actual cytotoxicity experiments. the pesticides were dissolved in distilled water. outer scales of the bulbs and the dry bottom plates were removed without damage the root primordia. the onions were germinated in freshly distilled water for the first 24 h and then exposed for 96 h to the different doses of napoleon 4ec (25, 50, 100, 200 and 400 ppm, respectively). the roots from each group including control group were cut off on the fifth day and length of each root was measured in order to determine the ec50 values. ec50 value was determined as the concentration which retards the growth of root 50% less when compared with the control group. root growth inhibition test (ec50 determination) the onions were grown in freshly made distilled water for 24 h and then exposed for four days to the control group and other concentrations of extracts. in order to determine efficient concentration (ec50) values, ten roots from each onion were cut off at the end of the treatment period, and the root’s length was measured. the concentration that decreased root growth about 50% when compared to the negative control group (distilled water), was accepted as ec50 value. to determine the possible toxic effects on roots, ec50/2, ec50 and ec50×2 concentrations of root were used in allium mitotic index test. the ec50 value was approximately 200 ppm for napoleon 4ec. in order to show possible concentration-dependent effects of this pesticide, the root tips were treated with 100 ppm (ec50/2), 200 ppm (ec50) and 400 ppm (ec50x2) concentrations of napoleon 4ec and all application groups were performed 24, 48 and 72 h treatment periods. after treatment, the roots were washed in distilled water and fixed in 3:1 ethanol:glacial acetic acid for 24 h and then roots were taken in 70% alcohol and stored +4°c. feulgen was used for staining root tip cells. slides were randomly coded and scored blindly. for mitotic index (mi), the different stages of mitosis were counted in a total of 5000–6000 cells (1000 cells/slide) per concentration, and expressed as a percentage. statistical analysis root length and mi datas were given as percentages. the levels of difference in treatment groups were analyzed statistically by spss 15.0 version for windows. dunnett-t test (2 sided) was used on both the allium and ames tests in the analyses. results ames test results was carried out for mutagenicity determination of the tested material. for this test histidine mutant strains of s. typhymurium, ta98 and ta100 were used, and control group colony numbers were compared with the test material. the concentrations which caused two-fold increase in the colony number of control group were accepted as mutagenic ones. a compound is considered a weak mutagen if it produces a reproducible, dose-related increase in the number of revertant colonies in one or more strains but the number of revertants is not double the background number of colonies (mortelmans and zeiger 2000). it was found that only 1000 μg/plate concentration was cytotoxic against s. typhymurium strains among six tested concentrations for cytotoxicity tests (1000, 800, 400, 200, 100, 50 and 25 μg/plate). so this toxic concentration was not applied in ames test. in ames test positive controls were 4-nitro-o-phenylenediamine (npd) for ta98 and sodium azide (sa) for ta100, used without metabolic activation, and 2-aminofluorene (af) for ta98 and 2-aminoanthracene (2aa) for ta100 used with metabolic activation, while distilled water was used as a negative control group. most of the results, whether 14 dilek akyil increasing or decreasing relative to the negative control group, were not statistically significant at p<0.05 (dunnett-t test, 2 sided) except for in the 800 μg/plate doses of the napoleon 4ec in the ta98 without s9 mix. ames test results showed a dose dependent effect, but not twice the negative control for s. typhimurium ta98 and ta100, with or without s9 mix except 800 μg/ plate doses. in 800 μg/plate doses, colony numbers are two-fold increase according to colony number of control group. so, this places the this compound as a weak mutagen according to the parameters. when s9 was added, revertant colony numbers in ta98 and ta100 became stronger and ames test data’s is summarized in table 1. table 2 are summarized in allium root growth test results. the effective concentration (ec50) was determined as 200 ppm. table 3 gives the effect of napoleon 4ec on mi and mitotic phase in the root meristematic cells of a. cepa treated for 24, 48 and 72 h. at all concentrations treated in the incubations of root diminished mi compared to negative control at each exposure time. the highest values were showed from 24 h examination of 100 ppm, and the lowest one in 72 h application of 400 ppm concentrations of napoleon 4ec. the reduced of mi indicates statistically significant results (p < 0.05) all concentrations and all treatment time. all doses of napoleon 4ec applied in the experiment caused changes in the percentage of particular phases’ distribution in comparison to the control. discussion while the use of pesticides are planned to eliminate pests and develop the quality and quantity of yield in agriculture, there is concern about their use because some have cytotoxic/mutagenic and carcinogenic effects and harm non-target organisms (asita and mokobo 2013). earlier studies have reported that some pesticides have mutagenic and clastogenic activities in several biological test systems (yaduvanshi et al. 2012; topcu et al. 2013; asita and mokobo 2013; özkara et al. 2015b; karaismailoğlu 2016; khallef et al. 2017). the bacterial reverse mutation test uses amino-acid requiring strains of s. typhimurium to identify point mutations, which include substitution, deletion or addition of one or a few dna base pairs. the cause of many human genetic diseases are originated point mutations and their occurrence in oncogenes and tumor suppressor genes of somatic cells are caused in tumor formation table 1. the mutagenicity assay results of napoleon 4ec for s. tyhimurium ta98 and ta100 strains. test substance concentration (µg/ plate) no of his+ revertants/plate, mean±sd ta98 ta100 s9 + s9 s9 + s9 napoleon 4ec 800 70.20±4.14* 83.24±6.25 110.54±6.04 197.54±11.58 400 52.10±3.21 83.40±3.46 102.21±4.54 168.21±6.52 200 45.21±3.56 76.45±4.32 90.54±6.87 154.31±10.25 100 40.90±3.68 65.20±6.25 91.21±9.62 142.30±11.54 50 31.62±1.83 46.70±5.54 83.02±7.04 138.12±12.26 neg. control 100 32.70±4.68 45.21±2.57 81.21±11.24 126.32±5.83 sa 10 2824.56±68.31* 2aa 5 2468.24±70.15* 2af 200 988.50±17.57* npd 200 1445.60±22.23* *mean statistically significant at p<0.05 (dunnett t-test), sa:sodium azide, npd: 4-nitro-o-phenylendiamine, 2af: 2-aminofluorene, 2aa: 2-aminoanthracene, sd: standard deviation, negative control: distilled water. table 2. allium root growth inhibition test. test substance concentrations (ppm) mean of root length±sd negative control 3.86±0.41 positive control 1.12±0.14* napoleon 4ec 25 3.41±0.26* 50 2.20±0.14* 100 1.94±0.31* 200 1.62±0.21* 400 1.27±0.14* *significantly different from negative control (p<0.05 dunnet-t test, 2-sided) sd: standart deviation. 15mutagenic and cytotoxic activity of insecticide napoleon 4ec in allium cepa and ames test in experimental animals and humans (malev 2012). the salmonella/microsome assay is broadly used for evaluating the mutagenicity of chemicals including pesticides (yaduvanshi et al. 2012 ). in the present studies, ames plate incorporation assay with the different concentrations of napoleon 4ec showed a mutagenic response only ta98 tester strain. in order to characterize the possible mechanism of mutagenicity, the important bacterial strains, sensitive to different mutational events due to their specific geno-types, were used. there are much reports on the mutagenic effects of ops determined with the ames test (aufderheide and gressmann 2007; coral et al. 2009; wu et al. 2012; akyıl and konuk 2014). however, no study has yet reported on the in vitro mutagenicity of napoleon 4ec using the ames assay. s. typhimurium ta98 strain is defined by the -1 frameshift deletion hisd3052, which effects the reading frame of a close by repetitive –c–g– bases and can be reverted by frameshift mutagens. ta100 contains the marker hisg46, which causes from a base-pair substitution of a leucine (gag/ctc) by a proline (ggg/ccc): this mutation is reverted by mutagens causing base substitutions at g-c sequences (di sotto et al. 2008). in this context these bacterial properties, our results can be said that udimo 75 wg mutagenicity in ta98 strain is caused by frameshift mutations and that of ta100 strain is due to base change (di sotto et al. 2008). all concentrations of this pesticide were weak mutagenic in the ta98 and ta100 strains, with or without the s9 fraction except 800 μg/plate doses of the napoleon 4ec in the ta98 without s9 mix. in 800 μg/plate doses, colony numbers are two-fold increase according to colony number of control group. exposure to the pesticide induced g–c base pair mutations (maron and ames, 1983) causing a frameshift reversion of the histidine-dependent tester strain (ta98) to the wild type (his+). however, the addition of s9 mixture resulted in a reduction of the mutagenic effect of napoleon 4ec but not significant according to negative control. due to biotransformation, a compound that is active biologically can be changed to an inactive metabolite. similarly an inactive compound can be changed to an active metabolite (paolini and forti 1997). in other words, the presence of an eukaryote enzyme in s9 fraction resulted in eliminate of the mutagenic activity of the tested substance. therefore, it is vital to use the s9 fraction in the ames test. yaduvanshi et al. (2012) reported that negative results for chlorpyrifos-an organophosphate pesticide with all three tester strains (ta97, ta98, ta102) of salmonella used in the presence or absence of metabolic activation. however, chlorpyrifos was toxic in ta98 tester strain at the dose of 5000 μg/plate in absence of metabolic activation while reduction in toxicity was seen on addition of s9 mixture. in another study, five different concentrations of the chlorthiophos were tested by ames test using salmonella typhimurium strains ta97, ta98, ta100, and ta102, with and without s9 metabolic activation. no concentrations of chlorthiophos showed mutagenic activity on the ta97, ta100, and table 3. the effects of napoleon 4ec on mi and mitotic phases in the root cells of a. cepa. concentration (ppm ) treatment time counted cell number mitotic index ± sd mitotic phases (%) ± sd prophase metaphase anaphase telophase negative control 24 hour 5102 87.40±9.68 83.21±10.66 1.86±0.23 1.01±0.45 0.88±0.75 positive control 4802 43.36±4.76* 38.27±4.26* 0.62±0.17* 0.55±0.54 0.50±0.28 100 4685 41.45±4.06* 41.10±2.71* 1.02±0.27* 0.71±0.22 1.05±0.46 200 5002 38.21±4.25* 38.44±4.02* 0.98±0.24* 1.00±0.53 1.27±0.24 400 5012 39.26±2.85* 36.11±2.45* 0.87±0.14* 0.45±0.12 0.62±0.47 negative control 48 hour 5045 78.34±4.54 75.21±6.47 1.70±0.12 1.34±0.42 1.26±0.18 positive control 5065 38.22±3.12* 36.85±3.54* 0.60±0.19* 0.64±0.31* 0.72±0.31 100 5145 31.54±3.65* 37.62±3.53* 0.79±0.49* 0.76±0.12* 1.21±0.24 200 5214 28.42±3.06* 32.54±2.08* 0.62±0.14* 0.60±0.20* 1.02±0.23 400 5162 23.40±1.48* 30.54±2.78* 0.45±0.23* 0.51±0.11* 0.92±0.41 negative control 72 hour 5252 65.45±2.23 62.25±3.14 1.54±0.41 1.50±0.24 0.94±0.25 positive control 5265 31.12±3.16* 32.02±3.69* 0.42±0.24* 0.86±0.24* 0.85±0.23 100 5189 28.17±5.44* 30.27±3.58* 0.65±0.19* 0.66±0.54* 0.52±0.14 200 4989 25.01±2.14* 24.24±2.45* 0.53±0.14* 0.62±0.27* 0.54±0.47 400 4980 18.17±2.25* 17.45±2.07* 0.38±0.47* 0.49±0.14* 0.61±0.51 * significantly different from negative control (p< 0.05 dunnet-t test. 2-sided) sd: standart deviation. 16 dilek akyil ta102 strains, with and without s9 fraction, but were all mutagenic to the ta98 strain without s9 (akyıl and konuk 2014). many researchers who have studied ames test systems with ops also reported mutagenic or nonmutagenic result (aiub et al. 2002; kumar et al. 2013; arroyo et al. 2015; akyıl et al. 2017). chemicals which tested with different test methods can be genotoxic or not genotoxic depending on a number of factors such as chemical structure, biological activity, the positions of the binding location and having rings in the structure (kutlu et al. 2011). furthermore, it might be related to differences in test conditions, such as exposure time, concentrations of substances, the dispersal of the materials in the cell and physico-chemical characteristics of the chemicals (ema et al. 2012; kaur et al. 2014). therefore, it could be explained why some studies find an increase of genetic damage while in others result as negative. it is well known that plants are direct recipients of toxins and the allium cepa assay is one of the plant assay method used broadly to study the genotoxicity of pesticides (fernandes et al. 2007). also, allium cepa showed a good correlation with the results from other established test systems using eukaryotic as well prokaryotic cells (yıldız et al. 2009). mitotic index is a parameter that allows to estimate the frequency of cellular division (marcano et al. 2004) and the reduction of mitotic activities has been used frequently to determine the cytotoxicity (linnainmaa et al. 1978). many investigators have reported the change mitotic index following the treatment of test organisms with pesticides (panda and sahu 1985; amer and farah 1974). in this experiment, mitotic index mostly decreased with increase napoleon 4ec concentrations at each treatment times in comparison with control groups (p<0.05). when the phase frequencies are compared with control in different treatment groups, significant outcomes were obtained statistically (p<0.05). the percentages of the mitotic phases were clearly influenced in totally almost all applications (p<0.05). chlorpyrifos, the active ingredient in napoleon 4ec, was shown to a dose-dependent increase in dna damage in the liver and brain of rats using the single cell gel electrophoresis (or comet) assay (mehta et al. 2008). chlorpyrifos is known to generate oxidative stress, induce lipid peroxidation, and cause depletion of reduced glutathione (gsh), increase in oxidized glutathione (gssg), and decrease in the ratio of gsh/gssg in rat erythrocytes and tissues (gultekin et al. 2001; verma and srivastava, 2003). chlorpyrifos exposure causes inhibition of antioxidant enzyme activities and increase in the levels of hydrogen peroxide (h2o2) in rat brain and liver (gultekin et al. 2001; verma and srivastava 2003). additionally, chlorpyrifos was not mutagenic in the ames salmonella mutagenicity assay and mammalian cell cultures (cho/hgprt assay), cytogenetic abnormalities in mammalian cells both in vitro (rat lymphocyte chromosomal aberration test, rlcat) and in vivo (mouse bone marrow micronucleus test) and induction of dna damage and repair in rat hepatocytes in vitro (gollapudi et al. 1995). dursban 4 (chlorpyrifos-ethyl) was decreased mitotic index and induced chromosome aberrations in the root meristem cells of allium cepa (topcu et al. 2013). in the present study with allium cepa root tip meristem cells, the three doses of napoleon 4ec tested induced cytotoxicity thus corroborating the findings of these studies but not the in vitro studies with s. typhimurium cited above. there are some possible mechanisms for chemically decreased mi in plant cells. the significant decline in the mitotic index could be due to the inhibition of the dna synthesis or the blocking of the g1 suppressing the dna synthesis or effecting the test compound at the g2 phase of the cell cycle (sudhakar et al. 2001; majewska et al. 2003). when a pesticide penetrates the cells and reaches a critical dose, it could be an active form, causing lesions during several following cellular cycles (marcano et al. 2004). the decrease of the mitotic index in our study can be related to this. in this study, all the concentrations of napoleon 4ec caused the changes in the percentage of the particular phases’ distribution when compared to the control group. pesticides accumulate in the cell due to this substance not being able to emerge out of the cell easily after once penetrating the cell and it may be highly toxic in the cell (antunes-madeira and madeira 1979). the safety evaluation of a fragrance material includes a broad range of toxicological information, both for the substance itself and for structurally related chemicals belonging to the same chemical group (bickers et al. 2003). among toxicological information, genotoxicity is a systemic consideration, as it can be related to carcinogenicity (di sotto et al. 2008). normally, to evaluate a potential genotoxic risk due to a chemical exposition, in vitro assays for detecting point mutations (ames test) and extended treatment (e.g., micronucleus assay, allium test, single cell gel electrophoresis assay or comet assay) are used in the first instance (emea 2008; di sotto et al. 2013). if the results of these studies are positive, in vivo studies, for example a mammalian cytogenetic study, are performed (efsa 2014). in conclusion, napoleon 4ec was determined to be cytotoxic due to reducing of mi in allium test and weak mutagenic in ames test. for this reason, further inves17mutagenic and cytotoxic activity of insecticide napoleon 4ec in allium cepa and ames test tigations are needed to determine the toxicity of this compound using other in vivo and in vitro biological test systems. a single test system is not enough to determinate a compound whether it is toxic or non-toxic. in this study we performed two different test methods. 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dicrotophos, an organophosphorus pesticide, assessed with different assays in vitro. environmental toxicology. 27: 307–315. yıldız m, cigerci ih, konuk m, fidan af, terzi h. 2009. determination of genotoxic effects of copper sulphate and cobalt chloride in allium cepa root cells by chromosome aberration and comet assays. chemosphere. 75: 934-938. caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 1 2021 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 74(4): 59-68, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1337 caryologia international journal of cytology, cytosystematics and cytogenetics citation: lu feng, fariba noedoost (2021) genetic diversity and relationships among glaucium (papaveraceae) species by issr markers: a high value medicinal plant. caryologia 74(4): 59-68. doi: 10.36253/caryologia-1337 received: june 20, 2021 accepted: november 18, 2021 published: march 08, 2022 copyright: © 2021 lu feng, fariba noedoost. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. genetic diversity and relationships among glaucium (papaveraceae) species by issr markers: a high value medicinal plant lu feng1,*, fariba noedoost2 1 college of medicine, veterinary & life sciences, university of glasgow, glasgow g12 8qq, scotland, uk 2 department of biology, faculty of science, behbahan khatam alanbia university of technology, khuzestan, iran *corresponding author. e-mail: fatemeh.fat1990@gmail.com; fl337551127@outlook.com; abstract. glaucium mill. (horned poppy), belonging to the family papaveraceae, is represented by a total of 25 species worldwide, and especially distributed throughout western, northern and eastern asia, europe, northern africa, and australia. as a country, iran harbors relatively more species of the genus glaucium (11-13 species) and hence, this country is considered as the hot spot of the genus. as a result, we conducted a molecular analysis of the data for this genus due to the relevance of these species of plants. we employed 75 plants from seven species and seven provinces that were randomly picked for this investigation. five primers were used to amplify genomic dna, yielding 78 bands, 73 of which were polymorphic (97.78%). issr primers have a great capability to recognise polymorphic loci among glaucium species, as evidenced by the high average pic and mi values obtained. the genetic similarity of seven samples was calculated to be between 0.77 and 0.92. glaucium corniculatum var. corniculatum and glaucium elegans var. elegans showed the lowest similarity, while glaucium oxylobum and glaucium grandiflorum had the highest similarity, according to inter-simple sequence repeats (issr) markers analysis. the following are the study’s goals: 1) is it possible to identify glaucium species using issr markers? 2) in iran, how are these taxa genetically structured? 3) what is the inter-species relationship? according to this study, issr markers can be utilized to distinguish species. keywords: iran, species identification, population structure, glaucium species, issr markers. introduction having a better understanding of any biological investigations requires determining the exact boundaries of a species. as a result, in the context of biology, species delimitation is a topic that receives a lot of attention (collard & mackill 2009, wu et al. 2013; esfandani-bozchaloyi et al. 2018a, 2018b, 2018c, 2018d; pandey et al. 2008). additionally, the research of intra-specific 60 lu feng, fariba noedoost levels of genetic diversity and the examination of genetic sequence of wild populations are essential for the development of effective conservation methods (fujita et al., 2012; hendrixson et al., 2013; mckay et al., 2013). chelidoniodeae ernest, eschscholzioideae ernest, papaveroideae ernest, and platystemonoideae ernest were the four subfamilies of the papaveraceae s. str (ernest 1962 kadereit 1993). later on, kadereit et al. (1994) included the subfamily of platystemonoideae in papaveroideae as well. glaucium is a genus belonging to papaveraceae subfam. chelidonoideae ernest that contains about 23 species (kadereit 1993). fedde (1909) listed 20 species, ten varieties, and one subvariety, but boissier (1867) only approved 12 species. mory (1979) divided the genus into two segments based on fruit dehiscence, morphological and structural characteristics of leaves, stems, seeds, and pollen grains: g. sect. acropetal mory, with four species having acropetal dehiscence, and g. sect. glaucium, with 18 species having basipetal dehiscence. the genus can be discovered in both dry and wet environments throughout europe’s atlantic coasts and the canary islands, as well as mongolia’s altai (mory 1979). (kadereit 1993). in iran, it was represented by 11 (cullen 1966) to 13 (mobayen 1985; gran and sharifnia 2008) species, of these, five are endemics: g. calycinum boiss., g. contortuplicatum boiss., g. elegantissimum mobayen, g. mathiolifolium mobayen and g. golestanicum gran & sharifnia. several taxonomic investigations have demonstrated that seed and trichome micromorphology can be used to classify and delimitate taxa at all taxonomic levels and even across plant families (barthlott 1981, krak and mraz 2008, salmaki et al. 2009, satil et al. 2011, salimi moghadam et al. 2015, tavakkoli and assadi 2016, arabi et al. 2017). gran and sharifnia also researched the seed ornaments of 14 glaucium taxa in iran (2008). light microscopy (lm) and scanning electron microscopy (sem) were used to examine the seeds and trichomes of 15 glaucium taxa found in iran (tavakkoli and assadi, 2019). the seeds are semicircular to reniform in shape, however reniform and extended reniform seeds have been seen in g. oxylobum and g. elegans, respectively. the sculpturing of the testa surface are verrucate–rugulate (most frequent type), verrucate–granulate, verrucate–perforate, verrucate–lineolate, rugulate– granulate, rugulate and ocellate. their findings reveal that seed and ovary trichome micromorphological traits give helpful and critical information for separating species and taxa within species, as well as a diagnostic key for the taxa. glaucium taxa were researched in terms of their morphological, palynological, and phylogenetical characteristics, according to fatma mungan kiliç et al (2019). several of these characteristics differ between taxa, particularly in micromorphology and the establishment of clades in phylogenetic trees based on matk and its3-6 dna sequence data, according to their findings. the genus glaucium of turkey was separated into subsections glabrousae and pubescentae based on the results of dna investigations and morphological data (stem trichomes). for researching genetic diversity, molecular markers are a useful tool. random amplified polymorphic dna (rapd) and inter simple sequence repeats (issr) markers are among the most commonly utilized advanced genetic markers for diversification assessments (pharmawati et al. 2004). the rapd method is quick and easy to use, and it doesn’t need any clear insight of sequences. using a single primer of any nucleotide sequence, the approach detects nucleotide sequence polymorphism (moreno et al., 1998). a single 16-18 bp. long primer consists of a repeating sequence attached at the 3’ or 5’ end of 2-4 arbitrary nucleotides is used to amplify dna for issr markers. the method is faster, easier, less expensive, and more repeatable than rapd (esfandani-bozchaloyi et al. 2017a, 2017b, 2017c, 2017d; collard & mackill 2009, wu et al. 2013). the current study used new gene-targeted molecular markers, namely issr markers, to assess the genetic diversity and relationships among different glaucium species. because this is the first research of issr markers in the glaucium genus, we conducted a molecular analysis on 75 collected specimens from seven glaucium species. we attempt to respond to the following questions: 1) does the researched species have infraspecific and interspecific genetic diversity? 2) is there a link between genetic distance and geographical distance among these species? 3) how do populations and taxa differ genetically? 4) does the glaucium genus exchange genes with other glaucium species in iran? materials and methods plant materials during the months of july to august 2016, 75 individuals representing seven geographical populations of glaucium species were sampled in the iranian provinces of lorestan, guilan, mazandaran, esfahan, golestan, hamadan, and kohgiluyeh, as well as boyerahmad (table 1). 75 plant accessions (eight to thirteen samples from each population) were collected from seven distinct pop61genetic diversity and relationships among glaucium (papaveraceae) species by issr markers ulations of different eco-geographic features and stored in -20 until used for issr analysis. table 1 and fig. 1 provide more information on the geographical distribution of accessions. morphological studies morphometry was performed on eight to thirteen samples from each species. a total of 36 morphological features (13 qualitative, 23 quantitative) were investigated. the data was normalized (mean=0, variance=1) and used to calculate euclidean distance for clustering and ordination analysis (podani 2000). corolla form, bract shape, calyx shape, calyx length, calyx width, calyx apex, calyx margins, bract length, corolla length, corolla width, corolla apex, leaf length and width, leaf apex, leaf margins, leaf shape, leaf gland, and bract margins are among the morphological features analyzed. dna extraction and issr assay young leaves were utilized at random from one to twelve plants in each of the populations studied. silica gel powder was used to dry them. to extract genomic dna, the ctab activated charcoal procedure was applied (esfandani-bozchaloyi et al. 2019). the purity of the extracted dna was tested using an 8% agarose gel. 22 primers from the ubc (university of british columbia) series were evaluated for dna amplification for the issr study. based on band reproducibility, ten primers were chosen for issr study of genetic diversity (table 2). pcr reactions were carried in a 25μl volume containing 10 mm tris-hcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). the reactions and amplifications were carried out in a techne thermocycler (germany) using the following program: initial denaturation at 94°c for 5 minutes, then 40 cycles of 1 minute at 94°c, 1 minute at 52-57°c, and 2 minutes at 72°c. a last extension step of 7-10 minutes at 72°c brought the reaction to a close. running the amplification results through a 1% agarose gel and staining with ethidium bromide revealed the amplification products. using a 100-bp molecular size ladder, the fragment size was determined (fermentas, germany). data analyses morphological investigations first, morphologica l characters were norma lized (mean = 0, variance = 1) and utilized to calculate euclidean distance between taxonomic pairs (podani 2000). the upgma (unweighted paired group using average) ordination methods were utilized to group the plant specimens (podani 2000). anova (analysis of variance) was used to show morphological differences between table 1. voucher details of glaucium species in this study from iran. no sp. locality latitude longitude altitude (m) sp1 g. corniculatum var. corniculatum (l.) curtis kohgiluyeh and boyer-ahmad 38°52’37” 47°23’92” 1144 sp2 g. elegans var. elegans fisch. & c.a.mey. mazandaran, haraz road, emam zad-e-hashem 32°50’03” 51°24’28” 1990 sp3 g. oxylobum var. oxylobum boiss. & buhse guilan, sangar, road sid 29°20’07” 51°52’08” 1610 sp4 g. flavum var. serpieri (heldr.) halácsy esfahan:, ghameshlou, sanjab 38°52’373 47°23’92” 1144 sp5 g. fimbrilligerum boiss. lorestan, oshtorankuh, above tihun village 33°57’12” 47°57’32” 2500 sp6 g. contortuplicatum var. cantortuplicatum boiss. golestan, gorgan 34°52’373 48°23’92” 2200 sp7 g. grandiflorum boiss. & a.huet hamedan, nahavand 38°52’373 47°23’92” 1144 figure. 1. map of iran shows the collection sites and provinces where glaucium species were obtained for this study. 62 lu feng, fariba noedoost groups, while a biplot of pca (principal components analysis) was employed to determine the most variable morphological features among the populations investigated (podani 2000). for multivariate statistical analysis of morphological data, hammer et al. (2012) employed past version 2.17 (hammer et al. 2012). molecular analyses the issr bands were coded as binary characters (presence = 1, absence = 0) and utilized to analyze genetic diversity. to quantify the capacity of each primer to distinguish polymorphic loci among the genotypes, two measures, polymorphism information content (pic) and marker index (mi), were utilized to assess its discriminatory ability (powell et al. 1996). mi = pic× emr is the formula for calculating mi for each primer, where emr is the product of the number of polymorphic loci per primer (n) and the fraction of polymorphic fragments (β) (heikrujam et al. 2015). for each primer, the number of polymorphic bands (npb) and effective multiplex ratio (emr) were measured. the number of effective alleles, nei’s gene diversity (h), shannon information index (i), and percentage of polymorphism (p percent = number of polymorphic loci/number of total loci) were all calculated (weising et al, 2005, freeland et al. 2011). shannon’s index was calculated by the formula: h’ = -σpiln pi. rp is defined per primer as: rp = ∑ ib, were “ib” is the band informativeness, that takes the values of 1-(2x [0.5-p]), being “p” the proportion of each genotype containing the band. genalex 6.4 software was used to calculate the percentage of polymorphic loci, the mean loci by accession and population, uhe, h’, and pca (peakall & smouse 2006). neighbor joining (nj) clustering and neighbornet networking were based on nei’s genetic distance between populations (freeland et al. 2011, huson & bryant 2006). the mantel test was used to see if there was a link between the analyzed populations’ geographical and genetic distances (podani 2000). past ver. 2.17 (hammer et al. 2012) and darwin ver. 5 (2012) software were used to conduct these searches. for demonstrating genetic differences between the populations, the amova (analysis of molecular variance) test (with 1000 permutations) was utilized, which was performed in genalex 6.4 (peakall & smouse 2006). the genetic organization of populations was investigated using the bayesian-based model structure analysis (pritchard et al. 2000) and genodive ver. 2’s maximum likelihood-based k-means clustering approach (2013). data were evaluated as dominating markers for structure analysis (falush et al. 2007). by using δk value, the evanno test was run on the structure output to identify the right number of k. (evanno et al., 2005). two summary statistics, pseudo-f and bayesian information criterion (bic), give the best fit for k in k-means clustering (meirmans, 2012). gene flow was calculated by (i) using popgene ver. 1.32 (1997) to calculate nm, an estimate of gene flow from gst, as follows: nm = 0.5(1 gst)/gst. this method takes into account the same amount of gene flow in all populations (yeh et al. 1999). results species identification and inter-relationship. morphometry in quantitative morphological features, anova revealed significant differences (p<0.01) among the samples analyzed. pca analysis was used to discover the most changeable characteristics among the taxa investigated. the first three factors accounted for more than 75% of the overall variation. characters like corolla form, calyx shape, calyx length, bract length, and leaf shape had the largest correlation (>0.7) in the first pca axis, with 33 percent of total variation, whereas leaf apex, corolla length, leaf length, and leaf width influenced pca axis 2 and 3 accordingly. because the findings of several clustering and ordination approaches were similar, a pca plot of morphological features is shown here (fig. 2). plant samples from different species were put together and generated various groups in general. this finding indicates that the examined species were divided into various groups based on both quantitative and qualitative morphological characteristics. we found no transitional forms in the specimens that we looked at. species identification and genetic diversity to examine genetic links among glaucium species, five issr primers were tested; all of the primers yielded replicable polymorphic bands in all seven glaucium species. figure 3 depicts the issr amplification produced by the issr-2, issr-4 primer. seven glaucium species yielded a total of 73 amplified polymorphism bands. the amplified fragments had different size from 100 to 3000 bp. issr-3 had the most polymorphic bands (22), whereas issr-2 had the fewest (only 7), with an average of 14 polymorphic bands per primer. the average pic of the 5 issr primers was 0.22, ranging from 0.14 (issr-3) to 0.29 (issr-5). the primers’ mi ranged from 2.85 (issr2) to 5.47 (issr-5), with an average of 3.7 per primer. 63genetic diversity and relationships among glaucium (papaveraceae) species by issr markers issr primers had an emr ranging from 2.56 (issr4) to 6.23 (issr-5), with an average of 4.6 per primer (table 2). the primers with the highest emr values were thought to be more useful in separating the genotypes. for all 7 glaucium species amplified with issr primers, the genetic parameters were computed (table 3). unbiased predicted heterozygosity (h) ranged from 0.10 to 0.30 (glaucium corniculatum var. corniculatum), with a mean of 0.21. with a mean of 0.26, shannon’s information index (i) showed a similar pattern, with the maximum value of 0.38 in glaucium corniculatum var. corniculatum and the lowest value of 0.15 in glaucium contortuplicatum var. cantortuplicatum. glaucium oxylobum var. oxylobum has a number of alleles (na) ranging from 0.261 to 0.667. the effective number of alleles (ne) ranged from 1.011 (glaucium contortuplicatum var. cantortuplicatum) to 1.495 (glaucium elegans var. elegans). the amova test revealed a substantial genetic difference (p = 0.001) between the species investigated. it was discovered that 55 percent of overall variance occurred between species and 45 percent occurred within species (table 4). furthermore, significant nei’s gst (0.88, p = 0.001) and d est (0.389, p = 0.001) values revealed genetic difference between these species. in comparison to within-species genetic diversity, these findings demonstrated a larger distribution of genetic variety among glaucium species. because the findings of other clustering and ordination approaches were similar, nj clustering is reported here (figure 4). in general, two main clusters appeared in the nj tree (figure 4). populations of glaucium fimbrilligerum, g. contortuplicatum, and g. oxylobum were put in the first major cluster, separated from the other species by a great distance. two sub-clusters made up the second major cluster. the first sub-cluster consisted of glaucium corniculatum var. corniculatum and g. grandiflorum plants, whereas figure 2. pca plots of morphological characters revealing species delimitation in the glaucium species. figure 3. electrophoresis gel of studied ecotypes from dna fragments produced by issr-2, issr-4; 1, 8= g. corniculatum var. corniculatum; 2, 9= g. elegans var. elegans; 3, 10= g. oxylobum; 4, 11= g. flavum var. serpieri; 5, 12=; g. fimbrilligerum; 6, 13= g. contortuplicatum; 7, 14= g. grandiflorum 64 lu feng, fariba noedoost the second sub-cluster consisted of g. flavum var. serpieri and g. elegans var. elegans plants. in general, issr data aligns well with morphological data in terms of species relationships. this is in line with the amova and genetic diversity factors discussed previously. the species are genetically distinct. these findings show that issr molecular markers can be utilized to classif y glaucium species. the nm analysis by popgene software also produced mean nm= 0.768, that is considered very low value of gene flow among the studied species. isolation by distance (ibd) occurred among the glaucium species tested, as the mantel test with 5000 permutations revealed a substantial correlation (r = 0.87, p=0.0002) between genetic distance and geographical distance. the genetic identity of nei and the genetic distance between the species examined (table not included). glaucium corniculatum var. corniculatum and g. elegans var. elegans had the highest degree of genetic similarity (0.92), according to the findings. between g. oxylobum and g. grandiflorum, there was the least genetic resemblance (0.77). the low nm value (0.768) indicates minimal gene flow or ancestrally shared alleles between the species investigated, as well as considerable genetic divergence between and within glaucium species. the δk =6 was obtained by structure analysis and the evanno test. the organization plot (figure 5) revealed further details regarding the genetic structure of the species investigated, as well as common ancestral alleles and/or gene flow between glaucium species. due to shared common alleles, this plot demonstrated genetic affinity between g. corniculatum var. corniculatum and g. grandiflorum (similarly colored, no. 1, 7) and g. fimbrilligerum and g. contorttable 2. issr primers used for this study and the extent of polymorphism. primer name primer sequence (5’-3’) tnb npb ppb pic pi emr mi issr-1 dbdacacacacacacaca 14 14 100.00% 0.22 2.66 3.55 4.45 issr-2 ggatggatggatggat 8 7 84.99% 0.25 4.91 4.43 2.85 issr-3 gacagacagacagaca 22 22 100.00% 0.14 5.34 5.55 5.44 issr-4 agagagagagagagagyt 13 13 100.00% 0.27 2.88 2.56 3.85 issr-5 acacacacacacacacc 12 12 100.00% 0.29 1.23 6.23 5.47 mean 16 14 97.78% 0.22 3.5 4.6 3.7 total 78 73 note: tnb the number of total bands, npb: the number of polymorphic bands, ppb (%): the percentage of polymorphic bands, pi: polymorphism index, emr, effective multiplex ratio; mi, marker index; pic, polymorphism information content for each of caat boxderived polymorphism (cbdp) primers. table 3. genetic diversity parameters in the studied glaucium species. sp n na ne i he uhe %p g. corniculatum var. corniculatum (l.) curtis 13.000 0.358 1.380 0.384 0.30 0.31 66.50% g. elegans var elegans fisch. & c.a.mey. 8.000 0.299 1.495 0.231 0.18 0.23 44.38% g. oxylobum var. oxylobum boiss. & buhse 13.000 0.667 1.062 0.24 0.224 0.213 44.73% g. flavum var. serpieri (heldr.) halácsy 8.000 0.499 1.067 0.19 0.181 0.14 49.26% g. fimbrilligerum boiss. 9.000 0.261 1.034 0.172 0.13 0.13 33.15% g. contortuplicatum var. cantortuplicatum boiss. 11.000 0.545 1.011 0.15 0.10 0.10 23.53% g. grandiflorum boiss. & a.huet 13.000 0.352 1.083 0.23 0.22 0.14 45.05% abbreviations: n = number of samples, na= number of different alleles; ne = number of effective alleles; i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations. table 4. analysis of molecular variance (amova) of the studied species. source df ss ms est. var. % φpt among pops 48 1201.364 22.789 17.154 55% 55% within pops 50 104.443 1.805 1.888 45% total 98 1355.807 19.060 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance; φpt: proportion of the total genetic variance among individuals within an accession, (p < 0.001). 65genetic diversity and relationships among glaucium (papaveraceae) species by issr markers uplicatum (sp no. 5,6). this aligns with the nj dendrogram that was previously displayed. the allele compositions of the other species are different. discussion in the biology of long-term evolution of a taxon or population, genetic diversity plays a significant role. the foundation for a taxon’s existence, expansion, and evolution. to recognize the taxonomy, origin, and evolution of a taxon, it is necessary to investigate its genetic diversity. furthermore, such research will provide a theoretical foundation for the conservation, development, use, and breeding of germplasm resources (lubbers et al., 1991; ma, et al., 2021; peng, et al 2021; jia, et al., 2020; karasakal, et al., 2020a; 2020b). the recent study discovered fascinating information about genetic divergence, genetic differentiation, and physical differences in iran’s north and west. the degree of genetic diversity inside a species is intimately linked to its breeding technique; the higher the percentage of open pollination/cross breeding, the higher the level of genetic divergence in the clade under investigation (meusel et al., 1965). a primer’s pic and mi features aid in establishing its efficacy in genetic diversity analysis. according to sivaprakash et al. (2004), the level of polymorphism may be more directly related to an indicator technique’s ability to address level of genetic diversity. pic values of zero to 0.25 indicate very low genetic variation among genotypes, 0.25 to 0.50 indicate a mid-level of genetic diversity, and 0.50 indicate a high level of genetic diversity (tams et al., 2005; si et al., 2020; sun et al., 2021). the pic values of the issr primers in this study ranged from 0.14 to 0.29, with a mean value of 0.22, indicating that issr primers had a mid-level ability to determine genetic diversity among glaucium species. in the glaucium taxon, all five primer pairs demonstrated good polymorphism. for the species under investigation, a total of 78 alleles were discovered. the total number of polymorphic bands per primer ranged from 8 to 22, and the average allele number in loci was 16. in most studies, population size is limited to several vegetative accession (meusel et al., 1965; uotila, 1996). this population may have experienced genetic drift, as evidenced by the high degree of fis and minimal genetic diversity. the isolation of the population and absence the gene flow led to fragmentation of the glaucium populations. between genetic diversity parameters and population size were showing positive correlations that confirmed various studies (leimu et al. 2006). the positive association across genetic diversity and size of population figure 4. upgma tree of issr data revealing species delimitation in the glaucium species. figure 5. structure plot of glaucium species based on issr data. 1= g. corniculatum var. corniculatum; 2= g. elegans var. elegans; 3= g. oxylobum; 4= g. flavum var. serpieri; 5=; g. fimbrilligerum; 6= g. contortuplicatum; 7= g. grandiflorum. 66 lu feng, fariba noedoost can be explained in two ways (leimu et al., 2006). 1a positive connection may indicate the existence of an extinction vortex, in which a decrease in population size reduces genetic variety, resulting in inbreeding depression. plant fitness separates populations depending on habitat quality changes, which is the second cause (vergeer et al., 2003). low genetic variety, according to booy et al. (2000), can impair plant fitness and limit a population’s capabilities to react to changes in environmental conditions by selection and adaptation. within populations, 45 percent of genetic variety was achieved, while 55 percent of genetic variance was gained among the assessed groups. the reproductive system in plant species is important member of the primary elements controlling the distribution of genetic variation (duminil, 2007). couvet (booy et al., 2000) found that one migrant per generation is insufficient to maintain the long-term existence of small populations, but also that the numbers of immigrants is governed by phenotypic traits and population genetics (vergeer et al., 2003). despite the fact that the genetic variations across the three groups were identical, they were statistically meaningful. for the lack of distinctions across isolated groups, there are two explanations. the initial hypothesis proposed that genetic variety within and between populations demonstrates gene flow patterns, resulting in population fragmentation (dostálek et al., 2010). according to the second hypothesis, populations that are geographically close are more clearly related through gene transfer than species that are divided by a great distance. the morphological, palynological, and phylogenetic parameters of ten glaucium taxa were investigated (fatma mungan kiliç et al., 2019). although several of the morphological attributes of the taxa surveyed were matched with those listed in cullen’s flora of turkey (cullen, 1965), certain properties were revealed to be different. in particular, the results of mory’s (1979) study were compared to those acquired by our methods. in this assessment, the morphological and palynological characteristics were determined to be the most equivalent. gran and sharifnia (2008) identified g. haussknechtii as homologous with g. grandiflorum depending on 28 qualitative and 37 quantitative features in a micromacromorphological examination of 18 glaucium taxa. according to fatma mungan kiliç et al (2019) the glaucium taxa were divided into two groups with respect to stem hairs. taxa with pubescence stems were g. corniculatum subsp. corniculatum and g. corniculatum subsp. refractum, g. grandiflorum var. grandiflorum, g. grandiflorum var. torquatum, g. grandiflorum var. haussknechtii and g. secmenii, while the taxa with hairless stems were g. flavum, g. leiocarpum, g. acutidentatum and g. cappadocicum. the results of phylogenetic analyses showed that the glaucium taxa were grouped into two main clades in the ml trees based on the matk and its3-6 dna sequences, which is in compatible with the hairness of their stems, petal color and testa outline of the seeds. the taxa included in these two sub-clades were also compatible with ovary tubercle. finally, the findings of this study revealed that primers obtained from issr were more successful than other molecular markers in determining the genetic diversity of the glaucium genus. in addition, the dendrogram and pca clearly distinguished glaucium species, demonstrating that the issr approach is more effective in identifying glaucium species. acknowledgment the authors express their gratitude to anonymous reviewers who provided helpful feedback on an earlier edition. references arabi, z. et al. 2017. seed micromorphology and its systematic significance in tribe alsineae (caryophyllaceae). – flora 234: 41–59. barthlott, w. 1981. epidermal and seed surface characters of plants: systematic applicability and some evolutionary aspects. – nord. j. bot. 1: 345–355 booy g, hendriks rjj, smulders mjm, van groenendael jm, vosman b. 2000. genetic diversity and the survival of populations. plant biol. 2: 379–395. collard bcy. mackill dj. 2009. start codon targeted (scot) polymorphism: a simple novel dna marker technique for generating gene-targeted markers in plants. plant mol biol rep 27:86–93. cullen, j., 1966: glaucium. in: rechinger, k. h. 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(caprifoliaceae), using scot molecular markers fengzhen chen1, dongmei li2,* , mohsen farshadfar3 the new chromosomal data and karyotypic variations in genus salvia l. (lamiaceae): dysploidy, polyploidy and symmetrical karyotypes halil erhan eroğlu1,*, esra martin2, ahmet kahraman3, elif gezer aslan4 cytogenetic survey of eight ant species from the amazon rainforest luísa antônia campos barros1, gisele amaro teixeira2, paulo castro ferreira1, rodrigo batista lod1, linda inês silveira3, frédéric petitclerc4, jérôme orivel4, hilton jeferson alves cardoso de aguiar1,5,* molecular phylogeny and morphometric analyses in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f. neda atazadeh1,*, masoud sheidai1, farideh attar2, fahimeh koohdar1 a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) melika tabasi1, masoud sheidai1,*, fahimeh koohdar1, darab hassani2 genetic diversity and relationships among glaucium (papaveraceae) species by issr markers: a high value medicinal plant lu feng1,*, fariba noedoost2 morphometric analysis and genetic diversity in rindera (boraginaceae-cynoglosseae) using sequence related amplified polymorphism xixi yao1, haodong liu2,*, maede shahiri tabarestani3 biosystematics, fingerprinting and dna barcoding study of the genus lallemantia based on scot and remap markers fahimeh koohdar*, neda aram, masoud sheidai karyotype analysis in 21 plant families from the qinghai–tibetan plateau and its evolutionary implications ning zhou1,2, ai-gen fu3, guang-yan wang1,2,*, yong-ping yang1,2,* some molecular cytogenetic markers and classical chromosomal features of spilopelia chinensis (scopoli, 1786) and tachybaptus ruficollis (pallas, 1764) in thailand isara patawang1,*, sarawut kaewsri2, sitthisak jantarat3, praween supanuam4, sarun jumrusthanasan2, alongklod tanomtong5 centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates) simona ceraulo, vanessa milioto, francesca dumas* repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil marina souza cunha1,2,*,#, silvana melo1,3,#, filipe schitini salgado1,2, cidimar estevam assis1, jorge abdala dergam1,* karyomorphology of some crocus l. taxa from uşak province in turkey aykut yilmaz*, yudum yeltekin variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l. egizia falistocco1,*,+, gianpiero marconi1,+, lorenzo raggi1, daniele rosellini1, marilena ceccarelli2, emidio albertini1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(3): 169-175, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1120 caryologia international journal of cytology, cytosystematics and cytogenetics citation: cynthia aparecida valiati barreto, marco antônio peixoto, késsia leite de souza, natália martins travenzoli, renato neves feio, jorge abdala dergam (2021) further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae). caryologia 74(3): 169-175. doi: 10.36253/caryologia-1120 received: october 21, 2020 accepted: july 22, 2021 published: december 21, 2021 copyright: © 2021 cynthia aparecida valiati barreto, marco antônio peixoto, késsia leite de souza, natália martins travenzoli, renato neves feio, jorge abdala dergam. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid map: 0000-0003-0564-7068 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 1laboratório de sistemática molecular – beagle, av. ph holfs, s/n, departamento de biologia animal, universidade federal de viçosa (ufv), cep 36570-900, viçosa, minas gerais, brazil 2laboratório de biometria – labio, av. ph holfs, s/n, departamento de biologia geral, universidade federal de viçosa (ufv), cep 36570-900, viçosa, minas gerais, brazil 3museu de zoologia joão moojen – mzufv, vila gianetti, n° 32, departamento de biologia animal, universidade federal de viçosa (ufv), cep 36570-900, viçosa, minas gerais, brazil *corresponding author. e-mail: cynthiavaliatibarreto@gmail.com abstract. the genus enyalius is composed of 11 described species inhabiting forest areas in amazônia, cerrado and atlantic forest biomes. currently, eight species with high levels of chromosome variation have been karyotyped. the study aims to characterize the karyotype of enyalius boulengeri, with classical and molecular techniques, and improve knowledge about the karyotype evolution of the lizard genus enyalius. the species has 2n = 36 chromosomes (8m + 4sm + 24mc), fn = 24; nors and 18s rdna were subtelomeric and located on chromosome pair 2. repetitive dna probes (cat)10 accumulated on centromeric and terminal regions of some macrochromosomes. (ga)15 probe showed conspicuous accumulation on the pericentromeric region of chromosome pairs 1 and 6. repetitive fish patterns obtained with (gc)15 probe marked the pericentromeric region of the first chromosome pair. all probes showed accumulation in the microchromosomes. the chromosomal formula found in e. boulengeri has been considered the ancestral karyotype for pleurodont iguania. the genus enyalius is characterized by two distinctive chromosomal groups; one with highly conserved karyotypes, whereas the other is karyotypically diverse. our molecular cytogenetics data are promising and will increase knowledge about the genus enyalius chromosome evolution. keywords: ag-nor banding; cytogenetics; fish; lizards; rdna 18s; repetitive dna probes. 170 cynthia aparecida valiati barreto et al. introduction cytogenetic studies on lizards of pleurodont iguania infraorder suggest that there are two distinct trends on chromosome evolution in this taxon: some genera present chromosome variability, such as supernumerary chromosomes, sexual elements and large differences in chromosomal number and size (e. g. liolaemus, norops, and sceloporus); on the other hand, many families show a conserved karyotype (gorman & atkins 1967; pinnasenn et al. 1987; pellegrino et al. 1999; bertolotto et al. 2002). based on these results, the karyotype with 2n = 36 chromosomes and distinction between macrochromosomes (m) and microchromosomes (mc) (12m + 24mc) has been proposed as the ancestral karyotype for iguania (paull et al. 1976). however, chromosome banding reveals that these conservative karyotypes present some polymorphisms, such as different positions of nucleolar organizing region (nor) in some chromosome pairs, varying patterns of heterochromatin and different mc morphology (bertolotto et al. 1996; kasahara et al. 2004). the advent of techniques banding heterochromatin regions in dna is promising for the advance of the comprehension of the genome structure and evolution (martins et al., 2011). microsatellite regions apparently accumulate on regions with low levels of replication, such as telomeric and centromeric ones, and are easily detected by fluorescence in situ hybridization (fish) techniques, as indicated in plants, anurans and fishes (soares-scott et al. 2005; peixoto et al. 2015; cunha et al. 2016). although cytogenetic studies using molecular tools are still scarce on reptiles, they allow to understand relations between populations or/and species, and identify sexual elements at karyotypic components of species (martins et al. 2011). the genus enyalius (wied, 1821) is composed of 11 described species inhabiting forest areas in amazônia, cerrado and atlantic forest biomes (rodrigues et al. 2014; costa & bérnils 2018). moreover, cryptic species are indicated to occur along the atlantic forest (rodrigues et al. 2014). currently, eight species of the genus have been karyotyped (bertolotto, 2006; rodrigues et al., 2014), showing a certain degree of karyotypic variation. some phylogenetic related species (clade a sensu rodrigues et al. 2014) are proposed as bearers of the ancestral karyotype of iguania. on the other hand, related species present variation in chromosome number and size and supernumerary elements (clade b sensu rodrigues et al. 2014). however, two characters are highly conserved in enyalus: the nucleolar organizing region is located on the chromosome pair number 2 and heterochromatic regions occur in the centromeric position, on m and on almost all mc (bertolotto et al. 2002). phylogenetic relationships within this family are still unresolved, and studies employing banding techniques associated to molecular cytogenetics should reveal undetected synapomorphies. enyalius is a widely distributed genus and a potential model for biogeographical analyses and chromosome evolution in squamata. the study aims to characterize the karyotype of enyalius boulengeri, with classical and molecular techniques and improve knowledge about the karyotype evolution of this genus. material and methods specimens collection seven specimens of enyalius boulengeri were analyzed: four specimens (two females mzufv 13581359and two males – mzufv 1353, 1362) from the apa bom jesus, divino (20°35’52.85”s; 42°14’25.89”w) and three specimens (one female – mzufv 1356 – and two males – mzufv 1354-1355) from the estação de pesquisa, treinamento e educação ambiental (eptea), mata do paraíso, viçosa (20°48’0.40”s; 42°51’47.80”w), both in minas gerais state, brazil. proceedings were carried out according to the animal welfare commission of the universidade federal de viçosa and the current brazilian laws (concea 1153/95). all vouchers were deposited in the herpetological collection of the museu de zoologia joão moojen, at the universidade federal de viçosa (mzufv), viçosa municipality, in minas gerais state, brazil. conventional staining and molecular cytogenetic analyses the specimens were fed 24 hours before being sacrificed. each specimen was injected intraperitoneally with 0.1% solution of colchicine (0.1 ml per 10 g of body weight) 6 hours before euthanasia (carried out intraperitoneally with hypnol solution 0.01 ml. mg-1) to induce local anesthesia and pentobarbital (60 mg.kg-1 – lethal dose). mitotic chromosomes were obtained from gut epithelial cells, according to schmid (1978) and stained using conventional protocols (5 % giemsa diluted in sorensen buffer). the best metaphases were photographed in digital olympus bx53 light microscope with a dp73 olympus camera, using the cellsens dimensions® software system. chromosome pairing and measurements were performed using image pro plus® (ipp version 4.5) to determine the modal value (2n) and the fundamental number (fn) for the species. homologs were paired and grouped according to the centromere position, in decreasing size order, and classified in meta171further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri centrics (m), submetacentrics (sm), subtelocentrics (st) and telocentrics (t) (green & sessions 1991). active nors in the preceding interphase were identified using silver nitrate precipitation (ag-nors) (howell & black 1980), whereas the heterochromatinrich regions were detected using c-banding protocol (sumner 1972). the fish technique was performed according to pinkel et al. (1986). the 18s rdna probe was obtained from e. boulengeri, via polymerase chain reaction (pcr) with the following primers: 18sf (5’-ccg ctt tgg tga ctc ttg at-3’) and 18sr (5’-ccg agg acc tca cta aac ca-3’) (gross et al. 2010). the 18s probe was labeled by nick-translation with digoxigenina 11–dutp, and the signal detection and amplification were performed using anti-digoxigeninrhodamine (roche applied science). the dna repetitive probes were thynilated with cy3 at the 5’ position (sigma-aldrich), using the following repetitive dna probes: (a)30, (c)30, (ca)15, (ga)15, (gc)15, (ta)15, (cat)10, (caa)10, (cag)10, (gag)10, (cgg)10 , and the protocols followed cioffi et al. (2011). fish images were captured in a bx53 olympus microscope with a xm10 camera. all procedures were carried out in the laboratório de sistemática molecular beagle, at the universidade federal de viçosa, viçosa municipality, minas gerais state, brazil. cytogenetic tree in order to comprehend the relationship between the phylogenetic hypothesis and cytogenetic data of the genus enyalius, we overlapped our results on rodrigues et al. (2014) hypothesis. the cytogenetic data were derived from the available data on literature (gorman et al. 1967; pellegrino et al. 1999; bertolotto et al. 2002; bertolotto 2006; rodrigues et al. 2006; rodrigues et al. 2014). the species tree was reconstructed in tnt 1.6 (goloboff et al. 2008), and some rearrangements were made on figtree software v1.3.1 (rambaut 2009) and illustrator v. cs3. results a karyotype with diploid number equal to 2n = 36 chromosomes comprised of 12 macrochromosomes (m) and 24 microchromosomes (mc) (12m + 24mc) characterized the e. boulengeri populations (figure 1). the m pairs 1, 3, 4, and 5 are metacentrics, and the pairs 2 and 6 are submetacentrics in all metaphases. the karyotype formula was 8m + 4sm + 24mc, with fundamental number (fn) equal to 24. a secondary constriction was observed in the distal end of the long arm of chromosome pair 2 (figure 1). heteromorphic sex chromosomes or supernumerary elements were not detected in any of these specimens. the nors were detected at the subtelomeric region of the long arm from both homologues on chromosome pair 2. nors location corresponded to the conspicuous secondary constriction observed with giemsa stain, and to the location of the 18s rdna probe (figure 1b). the c-banding results did not highlight heterochromatin regions from any chromosome pair. this result is probable related to the technique used in this study, once the presence of heterochromatin are reported to the other species of the genus (bertolotto, 2006). repetitive dna probes (ga)15 showed conspicuous accumulation on the pericentromeric region of chromosome pairs 1 and 6 and several mc (figure 2a), whereas (gc)15 probe marked the pericentromeric region of the first chromosome pair and a few mc (figure 2b). repetitive fish patterns obtained with (cat)10 accumulated on the centromeric and terminal regions of some macrochromosomes and several microchromosomes (figure 2c). the repetitive dna probes (a)30, (c)30, (ca)15, (ta)15, (caa)10, (cag)10, (cgg)10, and (gag)10 did not label any chromosome pair. the two clades of enyalius (rodrigues et al. 2014) diverged on their cytogenetic patterns (figure 3). the clade a (composed of five species with cytogenetic data available for three of them) presents the same chromosome formula (8m + 4sm + 24mc), and one species with cytogenetically differentiated sex chromosomes (enyalius perditus 2). on the other hand, clade b (composed of seven species, six of them with cytogenetic data available), comprises species with high levels of caryological instability. the species possess different formulae (i. e. e. pictus: 8m + 4sm + 24mc and e. bibronii: 8m + 2sm + 2t + 24mc), as well as b chromosomes (i. e. e. bilineatus: 8m + 4sm+ 24mc + 1b/2b), sex chromosomes (e. bilinetus and e. leechii), besides some unusual telocentric chromosomes (i. e. e. catenatus 1: 6m + 2sm + 6t + 24mc and e. erythroceneus: 24t + 24mc). discussion enyalius boulengeri showed a 2n = 36 (12m + 24mc) karyotype that is ubiquitous among species of the genus and pleurodont iguania (gorman et al. 1967; pellegrino et al. 1999; bertolotto et al. 2002; bertolotto 2006; rodrigues et al. 2006; rodrigues et al. 2013). furthermore, it was observed a similarity between the nucleolar organizing region (nor) and the 18s rdna labeling at the distal end of the long arm of both 172 cynthia aparecida valiati barreto et al. homologues on chromosome pair 2. this same pattern (similarity between nor and 18s rdna probes and labeling in chromosome pair 2) is reported for all other species of the genus enyalius and from leiosauridae family (bertolotto et al. 2002; bertolotto 2006; rodrigues et al. 2006). here, the first enyalius species was labelled with repetitive dna probes, providing additional karyological data. this result will contributes to disentangling the phylogenetic relationships within the genus when similar studies are available to other species of the genus. the fundamental number of 24 is also shared with the other species from clade a of enyalius (e. perditus and e. iheringii). the invariable number of mc (24 mc) in all species of the genus seems to be the rule in squamata. although clades a and b (rodrigues et al. 2014) differ on macrochromosome constitution, mc are the same on all species of enyalius. thus, mc seem to be constituted by dna sequences that represent a conserved part of the karyotypes of enyalius. patterns of repetitive dna probes within this genus will be informative to test this hypothesis. two techniques corroborate that nors are located on the subterminal region of the long arm of the second chromosome pair in e. boulengeri, with eight species of the genus presenting this same pattern (bertolotto et al. 2002; bertolotto 2006; rodrigues et al. 2006). the conservation of chromosomal position of nors also suggests the stability of this chromosome segment in leiosauridae family (bertolotto et al. 2002). the pattern of nor banding should representing a phylogenetic sign for close related species. in addition, another pattern grabbing attention was the location of ag-nor and fish 18s rdna probe in the same chromosome region, which has been reported for several species from different families of clade iguania (i. e. agamidae (patawang et al. 2015), leiosauridae (bertolotto et al. 2002), liolaemidae (bertolotto et al. 1996), polychrotidae (bertolotto et al. 2001), and tropiduridae (kasahara et al. 1987). figure 1. giemsa-stained karyotypes of enyalius boulengeri. insets present chromosome pairs with ag-nor (above) and 18s rdna (below) identified on chromosome pair number 2. scale bars indicate 5 μm. figure 2. mitotic chromosomes of enyalius boulengeri labeled with the repetitive dna probes: a: (ga)15; b: (gc)15; c: (cat)10. scale bar indicated 10 μm. 173further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri e. boulengeri showed distribution of microsatellite repeats on pericentromeric and centromeric regions of the macrochromosome and many microchromossomes. no uneven accumulation of repetitive dna probes was observed in the chromosome pairs, which corroborates the hypothesis this species have no system of sexual chromosomes. studies using fish to evaluate the genome distribution of microsatellite repeats on sex chromosomes was realized by porkorna, 2011, this study showed certain microsatellite sequences are extensively accumulated over the whole length or parts of the w chromosome in eremias velox (pokorná et al. 2011). giovannotti et al, (2018) isolated the repetitive element imo-taqi satdna in several species of lacertidae and found this element to be very abundant in the constitutive heterochromatin of the w-sex chromosome of the four lacerta species investigated. on the other hand, repetitive probes also have evidenced chromosome stability among some species and populations of the scinax perpusillus group and ololygon tripui (peixoto et al. 2015; peixoto et al. 2016). our results highlighted that the chromosomal formula reported in e. boulengeri (8m + 4sm + 24mc) is shared with all species with described data in clade a. this pattern are possible result of the phylogenetic relation among the species that occurs in warmer climates in sout heastern a nd nor t heastern brazi l and once they belonged to the same clade inside the enyalius genus. this character has been considered an ancestral karyotype for pleurodont iguania, which includes the leiosauridae family (paull et al. 1976; bertolotto et al. 2002) and might also be present in the two remaining species with unknown karyotypes (e. brasiliensis and e. perditus 1). clade b members inhabit the cooler climates in southeastern and southern brazil (rodrigues et al. 2014), and presents a second distinct trend on chromosome evolution in iguania, showing considerable karyotype variability. this trend is exemplified by anolis, norops, ctenonotus also with relation to sex chromosomes (castiglia et al. 2013; giovannotti et al. 2017; lisachov et al. 2019; kichigin et al. 2019). for instance, some species present supernumerary chromosomes, different chromosomal formulae, besides some unusual telocentric chromosomes. in e. erythroceneus (24t + 24mc), the number of telocentric chromosomes seems to derive from f ission events of chromosomes, assuming that the ancestral karyotype is 8m + 4sm. the karyotypes of two species in clade b may also indicate fission events: enyalius catenatus and e. bibroni are evidenced by the presence of telomeric elements (6m + 2sm + 6t + 24mc and 8m + 2sm + 2t + 24mc, respectively). figure 3. compiled information about cytogenetic data in the genus enyalius and some related species on a condensed phylogenetic hypothesis resulting from the analyses of rodrigues et al. (2014). 174 cynthia aparecida valiati barreto et al. conclusion our results support classical cytogenetics (diploid number, fn and nor number and location) as an efficient tool to characterize lizard species in a family level, which corroborates the position of e. boulengeri within its genus. repetitive dna probes complement this conservative pattern and, if apply to other species of the genus, may allow to detect synapomorphies so as to improve knowledge about chromosome evolution and phylogenetic relationship within this genus. acknowledgements the authors are grateful to the anonymous reviewers that read the first draft of this manuscript. we also thank priscilla hote for the collection of some individuals. funding this work is a contribution to the project “biogeografia e conservação da anurofauna no complexo serrano da mantiqueira, sudeste do brasil” supported by conselho nacional de desenvolvimento científico e tecnológico (project #068437-2014/06). the authors acknowledge the 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available from: http://ivrtpm.cpac.embrapa. br/homepage/capitulos/cap_9.pdf sumner at. 1972. a simple technique for demonstrating centromeric heterochromatin. exp cell res. 75:304– 306. caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 3 2021 firenze university press chromomycin a3 banding and chromosomal mapping of 45s and 5s ribosomal rna genes in bottle gourd ahmet l. tek*, hümeyra yıldız, kamran khan, bilge ş. yıldırım development of a protocol for genetic transformation of malus spp federico martinelli1,*, anna perrone2, abhaya m. dandekar3 cytogenetic and cytological analysis of colombian cape gooseberry genetic material for breeding purposes viviana franco-florez, sara alejandra liberato guío, erika sánchez-betancourt, francy liliana garcía-arias, víctor manuel núñez zarantes* palynological analysis of genus geranium (geraniaceae) and its systematic implications using scanning electron microscopy jun wang1,2,*, qiang ye1, chu wang2, tong zhang2, xusheng shi2, majid khayatnezhad3, abdul shakoor4,5 influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats yuri a. kirillov1, maria a. kozlova1, lyudmila a. makartseva1, igor a. chernov2, evgeniya v. shtemplevskaya1, david a. areshidze1,* the morphological, karyological and phylogenetic analyses of three artemisia l. (asteraceae) species that around the van lake in turkey pelin yilmaz sancar1,*, semsettin civelek1, murat kursat2 molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant jinxin cheng1,*, dingyu hu1, yaran liu2, zetian zhang1, majid khayatnezhad3 genetic variations and interspesific relationships in salvia (lamiaceae) using scot molecular markers songpo liu1, yuxuan wang1, yuwei song1,*, majid khayatnezhad2, amir abbas minaeifar3 development of female gametophyte in gladiolus italicus miller (iridaceae) ciler kartal1, nuran ekici2,*, almina kargacıoğlu1, hazal nurcan ağırman1 colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae) harshita dwivedi*, girjesh kumar statistical evaluation of chromosomes of some lathyrus l. taxa from turkey hasan genç1, bekir yildirim2,*, mikail açar3, tolga çetin4 use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater treatment and water of the kamniška bistrica river (slovenia) peter firbas1, tomaž amon2 the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bo shi1,*, majid khayatnezhad2, abdul shakoor3,4 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(1): 41-53, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1264 caryologia international journal of cytology, cytosystematics and cytogenetics citation: anahita shariat, fatemeh sefidkon (2022) enhanced morphologic traits and medicinal constituents of octaploids in satureja mutica, a highyielding medicinal savory. caryologia 75(1): 41-53. doi: 10.36253/caryologia-1264 received: march 26, 2021 accepted: march 21, 2022 published: july 6, 2022 copyright: © 2022 anahita shariat, fatemeh sefidkon. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. enhanced morphologic traits and medicinal constituents of octaploids in satureja mutica, a high-yielding medicinal savory anahita shariat1,*, fatemeh sefidkon2 1 biotechnology resesarch department, research institute of forests and rangelands, agricultural research, education and extension organization (areeo), tehran, islamic republic of iran 2 medicinal plants research division, research institute of forests and rangelands of iran, agricultural research, education and extension organization (areeo), tehran, islamic republic of iran *corresponding author. e-mail: shariat@rifr-ac.ir anahita shariat: conceptualization, methodology, investigation, writing reviewing & editing, supervision. fatemeh sefidkon: phytochemical analysis, reviewing & editing. abstract. satureja mutica is a tetraploid and perennial semi-bushy plant cultivated for different medicinal purposes. to induce polyploidy, two-leafed seedlings were exposed to different concentrations (0.00, 0.05, 0.1, and 0.2 % w/v) and durations (6, 12, and 24 h) of colchicine. the seedlings were then transferred to a culture medium for recovery and propagation. after clones were prepared from each seedling, octaploid clones were identified using flow cytometry. chromosome counting was also used to confirm flow cytometric results in tetraploid (2n = 4x = 60; 2c dna= 1.90 ± 0.01 pg) and octaploid (2n = 8x = 120; 2c dna= 3.82 ± 02 pg) plants. the highest polyploidy induction efficiency with 32% was related to 0.05 % colchicine and 12 h duration. the results showed that the phenotypic traits of anatomical (stomata size, leaf guard cell size), morphological (stem diameter, length, width, and leaf area), physiological (soluble sugars, phenols, and flavonoids), and phytochemical (essential oil yield, p-cymene, γ-terpinene, α-thujene, and α-pinene) properties significantly increased in octaploid plants, while the density of leaf stomata decreased compared to tetraploid plants. our results verified that octaploid induction in satureja mutica is an effective breeding method, remarkably increasing the quantitative and qualitative characteristics, which could be used as a new genetic resource in future breeding programs. keywords: octaploid plants, polyploidy, phytochemical properties, genome size. introduction plant evolution includes polyploid development, which has facilitated adaptation and speciation. researchers have since discovered that artificial polyploidy induction can be a useful method for the plant. cell size and nuclear volume are directly related to ploidy levels in plants (símová 42 anahita shariat, fatemeh sefidkon and herben, 2012; robinson et al., 2018), but organ size varies due to regulatory mechanisms and a decrease in cell number (tsukaya, 2008; czesnick and lenhard, 2015). in arabidopsis thaliana, doubling the ploidy level increased cell size by 70% (4x versus 2x and 8x versus 4x), while organ size increased by only 20% (robinson et al., 2018). organ size depends on cell size and cell number. the number of cells is also affected by the regulation of cell proliferation and differentiation, so an increase in ploidy may not coordinate with organ size increment (orr-weaver, 2015). also, ploidy does not affect nuclear size in all organisms and it is regulated by genetic factors (vuković et al., 2016). for example, in saccharomyces pombe, there is no difference between ploidy levels 2x to 32x in terms of nuclear volume and the cell area (neumann and nurse 2007). polyploidy induction is performed in plants with different purposes such as increasing the size of leaves, stems, inf lorescences, f lowers, fruits, and seeds (wei et al., 2011; huang et al., 2014; shariat et al., 2021). in medicinal plants, ploidy induction is performed to increase the amount of secondary compounds, for example, the amount of parthenolide in tetraploid tanacetum parthenium (majdi et al., 2010); alkamines and caffeic acid derivatives in tetraploid echinacea purpurea (xu et al., 2014); wedelolactone in tetraploid eclipta alba (salma et al., 2018). unexpectedly, the production of new secondary compounds or the reduction of active compounds due to polyploidy induction has also been reported, for example, loss of α-bergamotene and isocarveol geranial and a new component of β-bisabolene in tetraploid citrus limon (bhuvaneswari et al., 2020); lack of α-terpineol in tetraploid trachyspermum ammi l. (noori et al., 2017); loss of citral in triploid and lack of linalool in tetraploid lippia alba (julião et al., 2020), new compounds such as viridiflorol, α-terpineol and α-humulene in tetraploids tetradenia riparia (hannweg et al., 2016). species of savory are used in food and pharmaceutical industries due to their pleasant smell and medicinal properties such as antifungal, antibacterial, antiviral, antioxidant, and analgesic properties (shariat et al., 2018a). recognition of the therapeutic effects of s. mutica has led to increasing demand for fresh and dried leaves so that the amount of consumption is more than the amount of production. due to the limited level of cultivation and agricultural inputs, cultivating highyielding polyploid cultivars is a goal for a producer. previous research on the species mutica has focused on its chemical composition and antimicrobial properties. to the best of our knowledge, breeding and polyploidy induction of s. mutica has yet to be reported. among savory species, some species such as s. mutica and s. spicigera are naturally tetraploid and have larger bushes and more biomass than other satureja species. the amount of essential oil yield in s.mutica populations varies from 0.17% to 4.22% (w / w) (karimi et al., 2014). it should be noted that the population used in this study had an essential oil yield of 3.88% w/w. since s. mutica is tetraploid, polyploidy refers to upwards of tetraploidy. questions addressed in this study include: (1) is it possible to produce octaploid plants using colchicine? (2) are the induced octaploids flowering? (3) do octaploid plants exhibit superior morphological and physiological traits compared to tetraploids? (4) do the content of essential oil (v/w) and yield (%) differ between tetraploid and octaploid plants? the purpose of octaploid induction in this study was to increase the biomass, quantity, and quality of essential oil, along with an efficient protocol for inducing octaploidy in s. mutica. materials and methods plant material  the seeds were collected from mature plants of s. mutica in keshanak, north khorasan province, in the northeast of iran. in order to surface-sterilize the seeds, the following solutions were used: (1) thiram 0.2% (v / v) (10 min) as a fungicide, (2) sodium hydrochloride 5% (2 min), (3) 70% alcohol (20 s). after each step, the seeds were rinsed with sterile water and then placed on a moistened filter paper in a petri dish. petri dishes were sealed with parafilm to retain moisture. the seeds were incubated at temperatures 25 ± 1 with controlled photoperiods (16 h: 8 h, light: dark interval) under standard cool white fluorescent tubes (35 µmol s−1 m−2). in vitro polyploid induction after about eight days, the germinated seeds, which had reached the two-leaf stage, were transferred to erlenmeyer containing different concentrations of colchicine. a factorial experiment with two factors was conducted in a completely randomized design (crd) with three replications. the first factor was different concentrations of colchicine (0.00, 0.05, 0.10, and 0.20% w/v), and the second factor was the duration (6, 12, and 24 h). the dimethyl sulfoxide (dmso) solution was added to each treatment to increase the penetration of colchicine into plant tissue (manzoor et al. 2018). after that, the erlenmey43enhanced morphologic traits and medicinal constituents of octaploids in satureja mutica ers containing the seedling samples were placed on a shaker with a rotation speed of 80 rpm. each seedling was rinsed three times with sterile water after the treatment duration was over, then transferred to a small vial containing ½ murashige and skoog (ms) medium (murashige and skoog, 1962). the parameters of the growth chamber, including darkness, temperature, and light quality, were similar to those used for seed germination. a large number of seedlings were lost after one month, and the remaining seedlings were transferred to a suitable culture medium. the tissue culture medium used in the second month was a ½ms medium that four hormones were added [2-isopentenyl-adenine (2ip) (0.3 mg l-1), 1-phenyl-3-(1,2,3-thidiazol-5-yl) urea (tdz) (0.1 mg l-1), indole-3-butyric acid (iba) (0.05 mg l-1), 6-benzylaminopurine (bap) (0.5 mg l-1)]. after three regenerations (3 months), when the number of explants reached a sufficient number, the ploidy level was determined using f low cytometry, and octaploids were selected. then, selected plants were subcultured to produce clones. as the next step, two rooting hormones were added to the ms medium [iba (1 mg l-1), alpha-naphthalene acetic acid (naa) (0.1 mg l-1)] (shariat et al. 2016). after rooting proliferation (1 month), plantlets were transplanted in pots containing sterilized cocopeat and covered with polystyrene bags and then placed in a greenhouse at a temperature of 20 ± 1 °c with daylight intensity 8000– 12000 lux.  plastics were punctured after a week. this was done to reduce the humidity around the plant and make it more accustomed to the greenhouse environment. in the present study, to reduce mixoploidy results, petri dishes containing sprouted seeds were kept in the refrigerator at 4°c for 24 h and then placed in the incubator at 25°c for 4 h under standard cool white fluorescent tubes (35 µmol s−1 m−2). then colchicine treatments were applied immediately. this method leads to synchronization of meristematic cell division and as a result, the percentage of mixoploid production is greatly reduced. flow cytometry analysis  it is necessary to measure the ploidy level of the seedlings before they can be propagated in large numbers and transferred to the greenhouse. flow cytometry was used for this purpose. maize (zea mays ce-777, 2c dna = 5.43 pg) was also selected as the standard reference plant (doležel et al. 2007). 0.5 cm2 of maize leaf with 1 cm2 of savory leaves were placed in a petri dish and 1 ml of wpb extraction buffer was added (2007 et al. louirero). crushing was performed with a sharp razor. the resulting mixture was passed through a 30 μm nylon mesh filter made by partec. then 50 μl of rnase solution (1 mg ml-1, sigma-aldrich corporation, mo, usa) and 50 μl of pi dye (1 mg ml-1, pi, fluka) were added to the solution (shariat et al. 2018b) and then incubated for five min at room temperature and then injected into bd facscanto ii flow cytometer (b.d. biosciences, bedford, ma, usa). the results were analyzed in flomax 2.4e software and the value of cv value and mean g1 peak for each replication were calculated. then, the amount of 2c dna in each sample was calculated using the following formula: chromosome counting flow cytometry confirmed the presence of octaploidy, so octaploid explants were transferred to ms medium containing rooting hormones as described before (shariat et al. 2016). root samples were collected before transferring the explants to the greenhouse. the roots were immersed in the following solutions respectively: (1) 2 h in α-bromonphthalein pretreatment solution (1 in 10,000 ml), (2) 16 h in carnoy fixative solution (3 alcohols: 1 acetic acid), (3) roots then kept in 70% alcohol at 4°c(4) 12 min at 60°c in 1 m hcl hydrolysis solution. it should be noted that root tips were rinsed with distilled water after each step (5) 6 h at 60°c in 4% hematoxylin as staining dye. a needle was used to crush the root tip on the slide, and a drop of 45% acetic acid was dropped on it. slides were protected with lamellae and squashed (shariat et al. 2013). stomata photographs were captured using optical microscopy (nikon coolpix p90 digital camera interfaced to a bh2-rfca olympus microscope).  anatomical and morphological analysis anatomical properties including abaxial and adaxial stomata length, width, area, and density were measured using the nail varnish technique (smith et al. 1989). stomata photographs were captured using a digital camera (nikon coolpix p90 digital camera interfaced with a bh2-rfca olympus microscope). stem diameter, internode length, leaf length, width and area, floral leaf length, and width, corolla length, and width, calyx length, calyx lower, and upper teeth length, filament, and style length, peduncle length was measured with a caliper. 44 anahita shariat, fatemeh sefidkon measurement of physiological traits the method of anthron (irigoyen, 1992) was used to measure soluble sugars. proline and f lavonoids were measured by using protocols described by bates, 1973 and kamtekar et al., 2014 respectively. plant pigments were also measured using the acetone method (lichtenthaler and welburn 1983). folin ciocalteu phenol reagent was used to measure total phenol according to singleton and rossi’s method (singleton and rossi, 1965). essential oil content and composition the aerial parts of tetraploid and octaploid plants were harvested at the 50% flowering stage and air-dried in the shade (25°c). oil was produced from the ground aerial parts of the plant (100 g) by hydro-distillation for 3 h using a clevenger-type apparatus. the oil was dried over anhydrous calcium chloride and stored in a refrigerator at 4 °c before analysis. oil yield was calculated as a weight percentage (w/w) according to the equation (alizadeh et al., 2018): gas chromatography (gc) analysis was performed using a shimadzu gc9a gas chromatograph (japan). gas chromatography coupled with a mass spectrometer (gc-ms) analyses were also carried out on a varian 3400 gcms system (m.s. usa). both systems were equipped with a db-5 fused silica column (30 m × 0.25 mm i.d. film thickness 0.25 µm). in the present study, the protocol as described by sefidkon and jamzad was used (sefidkon and jamzad 2004). compounds were identified using various indicators such as time and inhibition index, mass spectra, and comparison of these spectra with standard compounds (adams 2007). data analysis: the effect of different concentrations of colchicine and durations on seedling traits (seedling vigor, survival rate, and polyploidy induction efficiency) were analysed in a completely randomized design with three replications. mean comparisons were performed by the least significant difference test (l.s.d.) at 1% and 5% levels of significance. octaploid induction efficiency was calculated using the following formula (tarkesh esfahani et al. 2020): to calculate the octaploid induction efficiency, the genome size of the regenerated explants was determined by using flow cytometry, and the percentage of octaploid induction was calculated. normalization of data was performed using kolmogorov-smirnov test. a significant comparison of anatomical, morphological, and physiological traits in tetraploid and octaploid plants was performed using the t-student test. analyzes were performed using excel 2016 software and ibm spss statistics 24. results polyploid induction two-leafed seedlings were affected by colchicine toxicity, which was accompanied by symptoms such as necrosis of seedlings, blackening of roots, deformation of leaves and stems, or growth stops. at the same time, several seedlings were growing normally. the analysis of variance demonstrated that increasing the concentration of colchicine decreased seedling survival rate, while there was no significant relationship (p > 0.05) between treatment duration and survival rate. the highest survival rate among colchicine treatments was related to the concentration of 0.05% colchicine. the interaction between colchicine concentrations and durations on survival was not significant (p > 0.05) (table 1). according to the anova analysis, different concentrations of colchicine, durations, and the interaction between them had a significant impact (p < 0.01) on seedling vigor and polyploidy induction efficiency. in other words, colchicine concentrations at various durations did not affect the two traits in the same manner.  at the concentration of 0.05% colchicine solutions, survival rate and seedling vigor were 64 ± 2.5% and 59.5 table 1. anova results (mean square) for the influence of colchicine concentration and duration on seedling vigor, survival rate, and octaploid induction efficiency in s. mutica. source of variation survival rate seedling vigour polyploid induction efficiency colchicine concentration (c) 6325.7** 14028.1** 1577.9** treatment duration (d) 3.5ns 40.8** 42.2** c*d interaction 20.4ns 25.1** 1609** error 23.1 5.2 1.4 ns: non significant, **: significant at 1% (p < 0.01). 45enhanced morphologic traits and medicinal constituents of octaploids in satureja mutica ± 2.4%, respectively, while at the concentration of 0.2% was 22 ± 3.8%, and 4.5 ± 1.4% respectively (figure 1). therefore, increasing the concentration of colchicine leads to a sharp decrease in seedling vigor. octaploony induction efficiency was the lowest in the presence of 0.2% colchicine treatment (15.3%) and was the highest in the presence of 0.05% colchicine treatment (32%). flow cytometric analysis the genome size of plants treated with colchicine was determined by flow cytometry. the results classified the explants into three groups: tetraploid, octaploid, and mixoploid. the position of the standard maize plant relative to the tetraploid and octaploid plants is critical (figure 2). the amount of 2c dna in tetraploid s. mutica plants was 1.90 ± 0.01 and in induced octaploids was 3.82± 0.02 (table 4). 15% of individuals were mixoploid, half of it was related to 0.05% colchicine treatment, and the rest was related to 0.1% and 0.2% colchicine treatments. in mixoploids, there are two types of cells with two ploidy levels. during plant growth, those cells with lower ploidy levels divide more rapidly, and after a while, the number of polyploid cells decreases or is eliminated. (touchell et al., 2020). therefore, due to the instability of mixoploids, they were excluded in this study and were not examined. it should be noted that only the percentage of octaploids obtained was used to calculate the efficiency of polyploidy induction. chromosome counting to confirm the flow cytometry results, chromosome counting was performed using the microscopic method. tissue culture seedlings (colchicine treated and control) were used for chromosome counting. the results of chromosomal counting showed that the number of chromosomes in control plants was 2n = 4x = 60 and in octaploid plants was 2n = 8x = 120 (table 2, figure 3). physiological traits octaploid induction had a significant effect (p < 0.01) on all measured physiological parameters including plant pigments (total chlorophyll and carotenoids), phenol content, flavonoids, and soluble sugars. comparisons between traits measured in 4x and 8x plants were performed using the t-student test (table 3). the amount of total phenol (according to the gallic acid standard) in the tetraploid plant increased from 20.34 ± 0.97 to 62.16 ± 1.90 µg g-1 dw in the octaploid (205% increase). the amount of flavonoids was also increased from 2.00 ± 0.04 to 6.58 ± 0.17 µg g-1 dw due to octaploid induction (229% increase). total chlorophyll and carotenoids increased by 28% and 32% in octaploid plants, respectively. the amount of soluble sugars also increased from 939 ± 18 in tetraploids to 2,820 ± 62 µg g-1 dw in octaploids (200% increase) anatomical and morphological properties octaploid induction had a significant effect (p < 0.05) on the enlargement of stomata and morphological traits. internode length was the only parameter that was not significant (p > 0.05) among the measured parameters. vegetative leaf length increased from 19.70 figure 1. effect of colchicine concentration and treatment duration on seedling vigor (a), survival rate (b), and polyploidy induction efficiency in s. mutica explants (mean ± standard error). 46 anahita shariat, fatemeh sefidkon ± 0.54 in tetraploids to 28.90 ± 0.60 mm in octaploids (46% increase), and leaf width increased from 3.96 ± 0.16 to 6.00 ± 0.16 mm. leaf area was also 123% larger in octaploids (p < 0.01) (table 3, figure 4). stem diameter increased from 0.97 ± 0.04 in tetraploids to 2.18 ± 0.05 in octaploids (124% increase) (figure 5). an average of 50 stomata data was used to compare anatomical traits at two ploidy levels. the length of abaxial and adaxial stomata in octaploid plants increased by 62% and 58%, respectively. the width of the abaxial and adaxial stomata also increased by 32 and 31%. the area of abaxial and adaxial stomata increased from 48.30 ± 2.74 and 69.24 ± 3.69 mm2 in tetraploids to 97.60 ± 4.63 and 145.73 ± 8.18 mm2 in octaploids, respectively. therefore, the area of the abaxial and adaxial were 102 and 110% bigger (figure 7). while the density of the abaxial and adaxial stomata decreased from 82.00 ± 1.26 and 68.25 ± 1.27 in tetraploids to 46.20 ± 1.36 and 37.40 ± 1.63 in octaploids. that means the number of stomata in the abaxial and adaxial stomata decreased by 44% and 46%, respectively (p <0.01) (table 3) there was a significant increase (p < 0.01) in all f lower characteristics (table 3, figure 6). floral leaf length and width, calyx length, corolla length, and width, filament, and style length increased by 124%, figure 2. flow cytometric histograms of nuclei isolated from in vitro-derived leaves of s. mutica tetraploid (a), octaploid (b), and mixoploid (c) plants. the right peaks (s) refer to the g1 of the standard maize reference plant (zea mays  ce-777, 2c dna = 5.43 pg), the left peaks (4x, 8x) refer to the g1 of tetraploid and octaploid plants, respectively. figure 3. chromosome numbers of s. mutica. tetraploid control plant (2n = 4x = 60) (a) octaploid plant (2n = 8x = 120) (b). bars = 5 µm. 47enhanced morphologic traits and medicinal constituents of octaploids in satureja mutica 80%, 15%, 53%, 42%, 63%, and 57% in the induced octaploids, respectively (table 3). phytochemical traits the essential oil yield in tetraploid and induced octaploid plants was 3.88 ± 0.07 and 5.68 ± 0.14%. as a result, octaploid induction increased the essential oil yield by 49%. identification of compounds in essential oils using gc and gc-ms devices led to the identification of 14 compounds, representing more than 99% of the oil (table 4; figure 8). table 4 shows the essential oil components of tetraploid and octaploid plants. the compounds are listed in order of their elution on the db-5 column. in octaploid table 2. mean genome size (2c dna) in the tested s. mutica (n = 5). ploidy level 2n 2c dna value (pg) ± se 1c dnavalue (pg) holoploid genome size (1c dna, mbp) monoploid genome size (1cx dna,mbp) 4x 60 1.901± 0.01 0.95 929.10 464.55 8x 120 3.82± 0.02 1.91 1866.02 466.51 table 3. effects of induced polyploidy on physiological and anatomical characteristics of satureja mutica. reported values are mean ± s.e., p-values based on t-test for independent samples (n = 10). traits (unit) abbr. 4x 8x t(df=8) p-value phenol (µg g-1 dw) phenol 20.34 ± 0.97 62.16 ± 1.90 -19.61 < 0.001 flavonoid (µg g-1 dw) flavonoid 2.00 ± 0.04 6.58 ± 0.17 -26.82 < 0.001 soluble sugar (µg g-1 fw) ssugar 939 ± 18.0 2,820 ± 62.2 -29.04 < 0.001 oil yield (%) oily 3.88 ± 0.07 5.68 ± 0.14 -11.83 < 0.001 total chlorophyll (mg g-1 f.w.) chl 1.76 ± 0.03 2.27 ± 0.04 -9.70 < 0.001 carotenoid (mg g-1 f.w.) car 14.02 ± 0.26 18.58 ± 0.59 -7.01 < 0.001 stem leaf length (mm) sll 19.70 ± 0.54 28.90 ± 0.60 -11.41 < 0.001 stem leaf width (mm) slw 3.96 ± 0.16 6.00 ± 0.16 -8.89 < 0.001 leaf area (mm2) la 38.80 ± 3.25 86.65 ± 2.45 -11.76 < 0.001 internode length (mm) inl 17.60 ± 1.44 21.00 ± 0.71 -2.13 0.07ns stem diameter (mm) sd 0.97 ± 0.04 2.18 ± 0.05 -19.14 < 0.001 adaxial stomata length (µm) adsl 13.42 ± 0.35 21.30 ± 0.87 -8.38 < 0.001 adaxial stomata width (µm) adsw 9.46 ± 0.42 12.44 ± 0.86 -3.10 < 0.05 adaxial stomata area (µm2) adsa 69.24 ± 3.69 145.73 ± 8.18 -8.52 < 0.001 adaxial guard cell density adgcd 68.25 ± 1.27 37.40 ± 1.63 14.93 < 0.001 abaxial stomata length (µm) absl 11.20 ± 0.28 18.22 ± 0.73 -8.96 < 0.001 abaxial stomata width (µm) absw 7.94 ± 0.19 10.44 ± 0.28 -7.46 < 0.001 abaxial stomata area (µm2) absa 48.30 ± 2.74 97.60 ± 4.63 -9.16 < 0.001 abaxial guard cell density (no.) abgcd 82.00 ± 1.26 46.20 ± 1.36 19.30 < 0.001 floral leaf length (mm) fll 17.80 ± 0.80 40.00 ± 1.05 -16.83 < 0.001 floral leaf width (mm) flw 3.90 ± 0.09 7.02 ± 0.08 -26.00 < 0.001 calyx length (mm) cl 5.52 ± 0.17 6.36 ± 0.12 -4.10 < 0.01 calyx lower teeth length (mm) cltl 2.66 ± 0.05 3.68 ± 0.07 -11.40 < 0.001 calyx upper teeth length (mm) cutl 1.94 ± 0.04 2.79 ± 0.05 -13.12 < 0.001 peduncle length 1 (mm) pl1 1.82 ± 0.03 2.42 ± 0.03 -16.64 < 0.001 peduncle length 2 (mm) pl2 2.21 ± 0.03 3.05 ± 0.07 -10.98 < 0.001 corolla length (mm) col 44.60 ± 1.03 68.60 ± 1.03 -16.48 < 0.001 corolla width (mm) cow 11.80 ± 0.46 16.80 ± 0.25 -9.45 < 0.001 filament length (mm) fl 8.34 ± 0.20 13.62 ± 0.24 -17.22 < 0.001 style length (mm) stl 16.00 ± 0.29 25.16 ± 0.40 -18.41 < 0.001 48 anahita shariat, fatemeh sefidkon plants several compounds such as α-thujene, α –pinene, sabinene, α –terpinene, ρ-cymene, limonene, 1,8-cineol, γ-terpinene, methyl ether thymol increased but terpinolene, thymol, carvacrol, e-caryophyllene, germacrene d decreased (table 4). the major components were p-cymene (5.9, and 17.7%), γ-terpinene (10.7, and 14.9%), thymol (47.7, and 29.2%) carvacrol (24.8, and 22.5%), in tetraploid and octaploid plants, respectively. discussion the purpose of polyploidy induction in medicinal plants with economic value, including s. mutica, is to improve the quantity and quality of essential oils and increase plant yield. in this study, successful octaploid induction was performed using a specific concentration and duration of colchicine. colchicine is the most widely used chemical that is a mitotic spindle inhibitor and used for polyploidy induction (tsai et al., 2021). concentrations of 0.005–0.5% colchicine and durations of 6 h to 6 d are traditionally used for chromosomes duplication (ahmadi and ebrahimzadeh, 2020).  researchers have shown that colchicine tolerance thresholds are not the same in different plant species and colchicine concentrations do not have the same effects on polyploidy induction in different plants. for example, the highest tetraploid induction efficiency (33%) in papaver bracteatum lindl, was obtained at a concentration of 0.05% colchicine with a duration of 24 hours (tarkesh esfahani et al., 2020). the highest polyploidy induction efficiency in rhododendron fortunei lindl (36.6 %), sophora tonkinensis gapnep (23.3%), thymus persicus (26%) was obtained at a concentration of 0.1, 0.2, and 0.3% colchicine after 24, 30, and 12 h respectively (tavan et al., 2015; wei et al., 2018; figure 4. comparison of the morphology between tetraploid (a) and octaploid (b) s. mutica. bars = 5 mm. figure 5. comparison of the stem diameters between tetraploid (a) and octaploid (b) s. mutica, tetraploid plant. bars = 500µm. figure 6. comparison of the flower morphology between tetraploid (4x) and octaploid (8x) s. mutica. bars = 5 mm. 49enhanced morphologic traits and medicinal constituents of octaploids in satureja mutica figure 7. abaxial stomata in leaves of s. mutica; stomatal density in tetraploid (a) and octaploid plant (b) (bars = 50 µm); stomatal size in tetraploid (c) and octaploid plant (d) (bars = 5 µm). table 4. comparison between tetraploid and octaploid components of essential oils resulted by gc, and gc-ms. component ri tetraploid octaploid α-thujene 929 0.4 1.8 α -pinene 938 0.9 sabinene 977 0.8 1.0 α -terpinene 1032 1.6 2.8 ρ-cymene 1044 5.9 17.7 limonene 1046 0.2 0.5 1,8-cineol 1050 0.2 γ-terpinene 1075 10.7 14.9 terpinolene 1090 1.1 0.6 methyl ether thymol 1258 1.3 3.4 thymol 1315 47.7 29.2 carvacrol 1324 24.8 22.5 e-caryophyllene 1448 2.5 1.6 germacrene d 1522 1.9 1.1 figure 8. gc-ms chromatogram of tetraploid (a) and induced octaploid (b) of satureja mutica. 50 anahita shariat, fatemeh sefidkon mo et al., 2020). in the present study, the highest polyploidy induction efficiency (32%) was obtained by 0.05% colchicine after 6 h. prolonged exposure to colchicine decreased seedling vigor and polyploidy induction efficiency, but did not affect seedling viability. flow cytometry results showed that the genome size in s. mutica octaploid plants doubled. several reports indicate a direct relationship between genome size and ploidy level. for example, in annual ryegrass (lolium multif lorum lamarck), tetraploid induction doubled genome size from 6.13 ± 0.36 in diploids to 12.30 ± 0.83 pg in tetraploid (rios et al., 2015). another study on polyploidy induction of two species of acacia (acacia dealbata link. and acacia mangium willd.) showed that by increasing the ploidy level from diploid to triploid and tetraploid, the size of the genome increases by about 50%, respectively. thus, the genome size of tetraploids is almost twice as large as diploids (blakesley et al., 2002). genome size in d. rotundifolia and d. anglica with three levels of diploid, tetraploid, and octaploid were 2.73, 5.34, and 11.12 pg, respectively, indicating a direct relationship between ploidy level and genome size (rauf et al., 2021). however, there are reports that the size of the genome does not increase directly as the ploidy level increases. as an example, one study found that tetraploid species of rhododendron had 2c dna = 1.3-1.5 pg, but hexaploid species had 2cdna = 4.27 pg (kumar de et al. 2010). in the present study, one of the consequences of octaploid induction was a significant reduction (p < 0.01) in the number of stomata per unit area. stomatal density is not affected by external factors such as temperature and water content of tissues, so stomatal counting is a convenient and easy method that can be used to determine the ploidy level in a species (silva et al., 2000). according to several studies, including nianmaohuangqin (radix scutellariae viscidulae) (huang et al., 2014) and stevia (stevia rebaudiana bertoni) (zhang et al., 2018), stomatal size and density have been used as markers to differentiate polyploid seedlings and their control genotypes. however, stomatal size and density are not reliable factors in identifying chimer samples. as a result of polyploidy, plants are generally bigger and have thicker, darker leaves (huang et al., 2014).  according to a study of rose chromosome duplication, polyploid roses possess a longer stem, more living pollen, and more leaflets with a greater width-tolength ratio (kermani et al., 2003). in another study, polyploid lily plants also had larger leaves, roots, and stomata, but fewer stomata than diploid plants (fang et al., 2009). an increase in leaf size and leaf area leads to an increase in biomass and yield. in medicinal plants, where leaves, stems, and f lowers are all sources of active ingredients, biomass is an important characteristic. (hannweg et al., 2016) in the present study, calyx length, calyx lower and upper teeth length, floral leaf length, and width, filament, and style length, corolla length, and width were significantly longer (p < 0.01) in octaploid plants. the results are compatible with those reported by other researchers for the african violet (teixeira da silva et al., 2017) and the chamomile (majdi et al., 2010). even though the overall flower size in tetraploid plants increased significantly in african violets, the inflorescence length and the number of petal buds per inflorescence decreased significantly which differs from our finding in this regard. several reports confirm the increase in flower size and number per stem (tulay and unal, 2010), and some confirm the lengthening of the inflorescence stem (takamura and miyajima, 1996). in addition to morphological characteristics, physiological traits including the number of plant pigments, soluble sugars, phenols, and flavonoids were also affected by octaploid induction in this study. the purpose of polyploidy induction in medicinal plants is to increase the yield of active ingredients. for example, tetraploid induction in dendrobium hybrid increased the amount of shikunidine (grosso et al., 2018), in bletilla striata, superior phenolic and polysaccharide compounds (li et al., 2018), in stevia rebaudiana over accumulated stevioside (hegde et al., 2015), in scutellaria baicalensis higher baicalin (gao et al., 2002), in dendrobium officinale richer polysaccharides (song et al., 2016) and in salvia officinalis l. increased flavonoids, total phenol, and antioxidants such as polyphenol oxidase, catalase, and peroxidase (hassanzadeh et al., 2020). secondary compounds increase in polyploid plants as cells multiply, leaves thicken, and roots develop (hegde et al., 2015). increasing gene expression can also result from the duplication of chromosomes, which leads to an increase in secondary compounds (majdi et al., 2010). according to a study of arabidopsis thaliana, increasing ploidy levels reduced cellulose and lignin in the cell wall, increasing saccharification function (corneillie et al., 2019). tetraploid induction in thymus vulgaris l demonstrated that tetraploid and diploid plants contain similar levels of total terpenes but differ in the proportion of each terpene. thus, the terpene ratios for the five compounds were higher, indicating that polyploidy induction had altered the quality of the essential oil (navrátilová et al., 2021). there was an increase in fenchone content in tetradenia riparia tetraploids. furthermore, tetraploids contained several compounds (alpha-humulene, viridifloral, and alpha-terpinene) that were not present in diploids. 51enhanced morphologic traits and medicinal constituents of octaploids in satureja mutica conclusion the present study is the first report of octaploid induction using colchicine in s. mutica. as s. mutica is tetraploid, it was expected that octoploid would not 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10.13128/caryologia-112 citation: h.p. shambhavi, p. makwana, b. surendranath, k.m. ponnuvel, r.k. mishra, a.n.r. pradeep (2020) phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection. caryologia 73(1): 93-106. doi: 10.13128/caryologia-112 received: january 9, 2019 accepted: february 22, 2020 published: may 8, 2020 copyright: © 2020 h.p. shambhavi, p. makwana, b. surendranath, k.m. ponnuvel, r.k. mishra, a.n.r. pradeep. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* 1 proteomics division, seribiotech research laboratory, csb-kodathi campus, carmelaram p.o., bangalore-560035, karnataka, india 2 central sericultural research and training institute, berhampore, india 3 central tasar research and training institute, ranchi, india *corresponding author. e-mail: arpradeepnair@gmail.com abstract. microbial infections induced humoral and cellmediated immune events in hemocytes. after infection by the microsporidian nosema bombycis in the commercially important silkworm, bombyx mori, hemocytes exhibited deformed nucleus and degranulation of structural granules by exocytosis. granulocytes showed signs of phagocytosis included formation of microvilli, pseudopodia, engulfment of spores, phagosome formation and membrane porosity. association of membrane disintegration with infection – induced lipid peroxidation (lpo) was revealed by testing level of malondialdehyde, a byproduct of lpo. lpo activity enhanced significantly (p < 0.0002) throughout infection with peak activity in later stages of infection from day 11 accompanied by hemocyte plasma membrane disintegration. partial increase in lpo activity coupled with increased peroxidase activity recorded in early and mid stages of infection. in later stages, peroxidase activity decreased however lpo increased accompanied by phagocytosis events. in hemocytes, phagocytic events are initiated by activation of genes encoding recognition proteins, aggregation factors and immune associated proteins. ß-grp expression was down regulated after the infection whereas ctl-11 enhanced expression on day 10. humoral lectin enhanced expression on day 6 whereas apolipophorin showed 2.59 fold increase on day 10 after infection. gene encoding cytoskeletal protein, ßactin showed stable enhanced expression throughout infection showing positive correlation (r2 = 0.65) with age after infection. phagocytosisassociated gene eater from drosophila showed enhanced heterologous expression. altogether phagocytic events induced by microsporidian infection are accompanied by increased lpo, decreased peroxidase activity and modulated gene activity in hemocytes of b. mori. keywords. phagocytosis, lipid peroxidation, peroxidase activity, bombyx mori, microsporidian infection, hemocytes. 94 hungund p. shambhavi et al. introduction insects have developed functionally active immune system for survival in the widespread habitat. insect immune system comprises humoral and cellular responses as well as phenol oxidase cascade culminates in melanisation. innate immunity components included activation of different pathways such as toll, imd and jakstat pathways effecting in production of antimicrobial proteins (hoffmann 2003; govind 2008). cellmediated responses, facilitated instantaneously by hemocytes against pathogens (barillas-mury et al. 2000) involved nodulation, encapsulation and phagocytosis depending on size of the pathogen/ parasite (rosales 2011). phagocytic response of a cell is evolutionarily conserved from protozoan to mammals (faurschou and borregaard 2003; nazario-toole and wu 2017) which involved recognition, binding and ingestion of parasites (rosales 2011; kwon et al. 2014). phagocytic receptors evoke different signalling pathways such as draper activated draper/src/syk/ced-6 pathway (fullard et al. 2009) whereas tep (thioester containing protein) activated csd6 pathway (blandin et al. 2004). activation of signalling leads to cytoskeletal rearrangement, insertion of new membrane for pseudopodia and microvilli formation for parasitic engulfment (bajno et al. 2000). in drosophila, plasmatocytes and granulocytes act as major phagocytic cells against bacteria (castillo et al. 2006; lamprou et al. 2007) whereas in lepidopterans granulocytes are modified to become phagocytic cells (ling and yu 2006; rebeiro and brehelin 2006). though commercially important silkworm, bombyx mori (lepidoptera) is domesticated and reared under protected conditions, worms are exposed to pathogenic attack causing major losses to silk industry. in b. mori, infection by the obligate intracellular microsporidian parasite nosema bombycis causes devastating disease, pebrine. n. bombycis infects either through transovarian transmission or through secondary contamination by feeding contaminated mulberry leaves (hukuhara 2017). on reaching midgut, spores germinate and inject sporoplasm into midgut epithelial cells (franzen 2005) where it multiplies and spread to other host tissues including haemolymph and hemocytes. though pathogens spread through eggs to newly hatched larvae, symptoms or presence of spores could not be identified in initial stages of infection and spores could be identified only after six days of infection (ma et al. 2013). in b. mori, infection by n. bombycis suppressed host responses, inhibited melanization events and down regulated immune genes in midgut (pan et al. 2013; ma et al. 2013). however hemocytemediated cellular responses against microsporidian infection is not known. in this investigation, cellular, biochemical and molecular immune responses of hemocytes in b. mori were revealed showing induction of lpo activity and peroxidase activity in association with phagocytosis events against n. bombycis infection. materials and methods infection and sample collection b. mori larvae were reared on mulberry (morus sp) leaves under standard rearing conditions of 25 ± 2oc, 70% relative humidity and natural photoregime (13l : 11d). initially larvae were grown till third instar and separated after third moulting to fourth instar. day 0 fourth instar larvae were collected and exposed to experimental infection by feeding spores of n. bombycis (standard strain: nik-1s_mys) smeared on mulberry leaf with a dose of 1 x 106 spores / larva that induced 50% mortality within the lethal time lt50 (rao et al. 2004). non-infected b. mori larvae of the same age group were used as control and reared separately. in order to analyse hemocyte responses after n. bombycis infection, haemolymph samples were collected from control and infected larvae on day 0, 2, 6, 8, 9, 10, 11 and 12 after the infection. haemolymph was collected by piercing first abdominal proleg using a sterilized needle. haemolymph of pupae were collected from newly formed pupa (14 days after infection) by puncturing leg impressions on ventral side. the hemocytes were separated by centrifugation at 880 g for 10 min at 4oc. the hemocyte pellets were washed with anticoagulant solution (0.098m naoh, 0.186m nacl, 0.017m edta, 0.041m citric acid, ph 4.5 adjusted using nacl) twice and stored at -80oc for protein analysis. for total rna extraction, hemocyte pellets were stored in rna stabilization reagent rna later (qiagen). light microscopy and transmission electron microscopy (tem) hemocytes of infected and non-infected control larvae were observed under inverted tissue culture microscope (leica) and the hemocytes and spores were counted using hemocytometer. in order to carryout tem analysis, hemocyte samples were processed essentially as described earlier (pradeep et al. 2013). briefly, hemocytes were fixed in 3 % glutaraldehyde upto 24 h before fixation in 1% osmium tetroxide. the samples were dehydrated through alcohol series, brought to acetone and then stained with 2 % uranyl acetate. using an embedding kit 95phagocytosis and lipid peroxidation in hemocytes of b. mori induced by microsporidian infection (araldite embed812) hemocyte samples were embedded in araldite for 48h. ultrathin sections (100nm) were cut using ultramicrotome (leica –em uc6) and placed on a copper grid. the ultrathin sections were stained with uranyl acetate and lead citrate and observed under tem. ultrastructural variations in the hemocytes (n = 50 each) were observed at 60 kv in a tecnai g2 transmission electron microscope attached with megaview soft imaging system and photographed (at the tem facility in national institute for mental health and neuro sciences (nimhans), bangalore, india). lipid peroxidation in order to examine lipid peroxidation in the hemocytes of b. mori larva infected with n. bombycis, hemocyte samples were collected at different time points after infection along with sample from same aged noninfected control. the assay was performed using ezassaytm tbars lipid peroxidation estimation kit following manufacturer’s (himedia) protocol. membrane lipids are destructed by phagocytes to form lipid peroxides (mylonas and  kouretas 1999) which in turn breakdown to form a by-product malondialdehyde (mda) which is measured by absorbance. the lipid peroxidation was estimated as malondialdehyde (mda) equivalents (hodges et al. 1999) in a 96 well microtiter plate using absorbance reading at 532 nm in a microtiter plate reader (multiskan spectrum, thermo). from 110 µm mda, a standard curve was obtained by using a slope of standard curve (y = 0.00457+0.00416x) and mda concentration in the sample was calculated (yagi 1998; fatima et al. 2011). ascorbate peroxidase activity in order to test whether peroxidase activity varied in hemocytes after the infection, ascorbate peroxidase (prx) activity was determined as described earlier (nakano and asada 1981). hemocyte pellet was collected from haemolymph at different time points after the infection and then lyzed in insect cell lysis buffer and quantified the protein (lowry et al. 1951). briefly, prx assay was performed using 750 µl 100 mm phosphate buffer (ph7.0), 150 µl of 5.0 mm ascorbate and 300 µl 0.5 mm of h2o2. the reaction was carried out in 1.5 ml eppendorf tubes using 300 µl of hemocyte lysate sample containing 100 µg protein. the reaction mixture was transferred to 96 well plate. the rate of decrease in absorbance was read at 290 nm at every one minute interval for ten minutes duration. the enzymatic activity was calculated using a molar extinction coefficient of 2.8 mm-1 cm-1 and the result was expressed as micromoles per minute per milligram of protein (nakano and asada 1981). hydrogen peroxide activity in order to test variation in hydrogen peroxide (h2o2) level after infection, assay was performed using hemocyte extract in phosphate buffered saline (pbs) buffer (ph 7.1) as described earlier (velikova et al. 2000; pooja et al. 2017). the absorbance was measured at 390 nm in the microplate reader and h2o2 content was calculated based on a standard curve. total rna extraction and cdna synthesis total rna was extracted from hemocytes collected from control and n. bombycisinfected b. mori larva. genomic dna contamination was removed from total rna by incubation with rnase free – dnase i (takara). complementary dna (cdna) was synthesized from 1 µg total rna using oligo d(t) primer and m-mulv (moloney murine leukemia virus) reverse transcriptase using cdna synthesis kit as per manufacturer’s protocol (primescript; takara). rt-pcr was performed to analyse semiquantitative expression on 0, 2, 6, 8 and 10 days after infection using genespecific primers (table 1) which was validated by qpcr. quantitative pcr (qpcr) qpcr was performed using dynamo sybr green qpcr master mix (thermo finzyme) with 0.3x rox as passive reference dye, on agilent stratagene mx3005p qpcr system. a 25 μl reaction mixture contained 2.5 μl cdna template, one pmol each of forward and reverse primers and 12.5 μl sybr green qpcr master mix (1x) containing 4 mm mgcl2. the thermal program was set as 95°c for 15 min, followed by 40 cycles of 95°c for 30 seconds and at primerspecific annealing temperature (tm) for a minute (table 1).the pcr products were resolved by 1.5 % agarose gel electrophoresis and confirmed target-specific amplification. for housekeeping gene, ribosomal protein gene was used and fold change in expression relative to calibrator was calculated. cloning and sequencing from b. mori genome phagocytosisassociated genes tep-1 and eater are not reported. in order to con96 hungund p. shambhavi et al. ta bl e 1. k ey to th e ge ne s an d en co di ng p ro te in s as so ci at ed w ith p ha go cy to si s in du ce d in h em oc yt es o f b . m or i l ar va a fte r in fe ct io n w ith th e m ic ro sp or id ia n n . b om by ci s. g en e na m e n uc le ot id e a cc es si on n o. pr im er (l eft ( l) a nd r ig ht ( r )) tm (° c ) pr ot ei n a cc es si on n o. fu nc tio ns r ef er en ce t ep 1 a y 43 37 51 5’ g t g g c ta a g c c c a g t t t c a g 3’ l 5’a g t c a c t c g g a a g c a c t c g t 3’ r 59 .4 a b g -0 03 44 13 h em oc yt e m ed ia te d co m pl em en t l ik e pr ot ei n, pl as m od iu m b er gh ei b in di ng a nd m ed ia te s ki lli ng bl an di n et a l, 20 04 ea te r n m _1 43 27 6 5’a ta g c c g c t g c t g a t g a c t c 3’ 5’ t c t t c g a t c c g g c a a a a c 3’ 56 .5 q 9v b 78 tr an sm em br an e pr ot ei n, s ca ve ng er r ec ep to r, ba ct er ia l r ec og ni tio n an d ph ag oc yt os is . k oc ks e t a l, 20 05 ; c hu ng e t a l., 2 01 1 βg r p 2 n m _0 01 04 39 85 5’a a t g a c a c t g t t c g c g t t c c 3’ 5’ t c g c a c t c t c g t c t t t g t t g 3’ 57 .3 h 9j q 04 r ec og ni tio n of β 1, 3 g lu ca n fr om fu ng i a nd m ed ia te c el lu la r re sp on se s. k im e t a l, 20 00 x ia ng -j un r ao e t a l 2 01 4 βg r p 4 n m _0 01 16 61 42 5’a c c t t g t c g a a t c c a g a g g c 3’ 5’ c g g g t c ta t t g t t g a a g c c g 3’ 59 .4 q 9n l8 9 r ec og ni tio n of β 1, 3 g lu ca n fr om fu ng i a nd m ed ia te c el lu la r re sp on se s. k im e t a l, 20 00 x ia ng -j un r ao e t a l 2 01 4 c t l 1 1 b g ib m g a 00 66 23 5’ t c t g g t c g g t c g g c t g ta ta 3’ 5’ g a g c t g c t c c g c ta t g a a c t 3’ 59 .4 d 2x 2f 7 a ct s as o ps on in a nd in cr ea se p ha go cy to si s. pe nd la nd e t a l, 19 88 c t l 1 7 n m _0 01 13 08 99 5’a c g t c c t g c a ta c c g a a a a g 3’ 5’ g c c t c g t c ta a c g a t t c a g g 3’ 58 .3 q 06 fj 6 a ct s as o ps on in a nd in cr ea se p ha go cy to si s. pe nd la nd e t a l, 19 88 a po lip op hor in i /i i a b 64 06 23 5’ t g g c g g a ta a a t g c t c g t t g 3’ 5’ t c t t c t t c c g c g c a a a t c t g 3’ 57 .3 g 1u is 8 a ct a s pa tt er n re co gn iti on p ro te in a nd b in ds to fu ng al β 1, 3 g lu ca n, a id in p ha go cy to si s. w hi tt en e t a l. 20 04 ; b ar ab as a nd c yt ry ńs ka , 2 01 3 h um or al le ct in n p_ 00 11 04 81 7. 1 5’ g g c g g ta c a a c g t ta a g g a g 3’ 5’a a c g a g c a c c g a c a c a a g ta 3’ 58 .3 p9 80 92 a dh es iv e pr ot ei n an d re la te s to h em os ta si s or en ca ps ul at io n of fo re ig n su bs ta nc es fo r se lfde fe ns e. k ot an i e t a l. 19 95 be ta a ct in a 4 n m _0 01 12 62 55 5’a t c c t g c g t c t g g a c t ta g c 3’ 5’a a g a c t t c t c g a g g g a g c t g 3’ 59 .4 p8 41 83 c yt os ke le ta l p ro te in , h el ps in fo rm at io n of ce llu la r pr oj ec tio ns . m ay e t a l. 20 01 ; m ay a nd m ac he sk 20 01 ri bo so m al p ro te in n p_ 00 10 37 25 9. 1 5’ t g g a g c g c c t ta c a a a c t c t 3’ 5’ g c c a g a t t g c t t g g t t g a c t 3’ 57 .3 q 5u a n 9 h ou se ke ep in g ge ne lu e t a l 2 01 3 97phagocytosis and lipid peroxidation in hemocytes of b. mori induced by microsporidian infection firm heterologous expression of tep-1 and eater, mrna sequence of tep-1 of aedes aegypti (acc no.ay432915.1) and eater of drosophila melanogaster (acc. no. hm165182) were collected from ncbi database and primers were designed (primer 3 program) and used for qpcr (table 1). the amplicons were resolved on 1.2 % agarose gel containing the fluorescent dye ethidium bromide. the eater gene amplified at expected product size. in order to confirm the sequence identity eater amplicon from rtpcr was purified using a pcr purification kit (qiaquick, qiagen) and cloned into a vector pjet 1.2/blunt using clonejet pcr cloning kit (thermo scientific) as per manufacturer’s protocol. the cloned product was transformed into jm 109 competent cells. through colony pcr, plasmids were confirmed and the plasmid dna was isolated using qiaprep spin miniprep kit (qiagen). presence of the fragment was confirmed by pcr and sequenced using sanger method at a facility (applied biosystems) available at eurofins genomics india pvt. ltd., bangalore, india. the nucleotide sequence was used to perform blastn 2.8.1+ (ncbi) search against non-redundant database. the nucleotide sequence was then translated to protein sequence using expasy – translate tool (https:// web.expasy.org/translate/). the 3’5’ frame 2 of the translated sequence was used for analysis by blastp 2.8.1+ (ncbi) and searched against non-redundant genbank cds translations, pdb, swissprot, pir and prf. statistical analyses the data were presented as mean ± sd. significance of difference between means was evaluated by students’ ttest or single factor anova. correlation between variables was analysed by linear regression (y = a + bx). quantitative gene expression was performed using cdna synthesized from total rna of hemocytes of control and infected b. mori larva, relative to the calibrator using mx3500p real time pcr system (agilent). average threshold cycle (ct) value of transcript expression was calculated from triplicates using δδ ct method (livak and schmittagen 2001) and normalized by housekeeping gene encoding ribosomal protein. comparative ct values of the genes were standardized by ct values for the house keeping gene encoding ribosomal protein. ct values were standardized with average control value, providing δct value which is standardized to make the average control value ‘1’ (the δδ ct values) (gerardo et al. 2010). fold change in gene expression was calculated with reference to the calibrator, which in turn presented down regulated relative quantities as negative values. the data (mean ± sd) denoted is the gene expression induced by infection after eliminating the changes in control. results organismal variations day 0 fourth instar larvae were experimentally infected by feeding spores of n. bombycis smeared on mulberry leaf with ld50 dose of 1 x 106 spores / larva. after the infection, changes were not observed in larval behaviour and activity till day 2. larval death was recorded from four days after infection. moulting of infected larvae to fifth instar delayed by 24 h in comparison to control. infected larvae were smaller in size and showed reduced growth from day 8. infected larvae initiated cocoon spinning at 24 h after control larvae spun silk. cocoons of infected larvae were smaller and flimsy with lower silk content. infected larvae were black and showed melanization under cuticle. infected pupae were smaller in size and acutely melanized (fig 1 a-c). n. bombycis spores were absent in noninfected control larval haemolymph and tissue samples. in n. bombycisinfected larvae (n = 30 each), spores were not found till day 6. on day 6, spore count was 0.1 x 105 / ml haemolymph. on day 9 the count was 0.95 x 105 spores/ ml showing significant (p< 0.001) increase in comparison to day 6. spore count then enhanced significantly (p < 0.001) to 4.4 x 105 spores / ml on day 10 and to 5.6 x 105 spores / ml on day 11 after infection showing the increase in a sigmoid fashion. within 11 days, average of 60% larval mortality was recorded after the infection. cellular variations in hemocytes total hemocyte count of control and n. bombycisinfected larvae did not vary significantly on day 0 of infection, however significant (p < 0.005; anova) decrease observed in later stages (8 to 11 days) of infection. in control, total hemocyte count on day 0 fourth instar larvae was 14.4 x 105 cells/ml, which did not vary significantly (p < 0.1), on day 0 after infection. the count increased to 16.55 x 105 cells/ ml on day 6 however hemocyte count significantly (p< 0.002) increased to 17.95 x 105 cells/ml after infection. in control, hemocyte count significantly (p < 0.001) increased on day 8 to 37.05 x 105 cells/ ml whereas after infection it decreased to 32.35 x 105 cells/ ml. on day 9 the count was 49.15 x 105 cells / ml in control and 45.05 x 105 cells / ml after infection. in control on day 10 and 11, mean hemocyte count was 50.4 x 105 cells/ ml in comparison to 42.1x 105 cells/ ml after infection. under light microscope, four types of hemocytes viz., granulocytes, plasmatocytes, spherulocytes and oenocytes were observed in addition to the precursor 98 hungund p. shambhavi et al. prohemocytes. in control, hemocytes appeared intact with clear cytoplasm and less granules whereas after infection, cy toplasm becomes granulated on day 6. many cells ruptured and degranulated from day 8 after infection (fig.1 d e). in order to examine subcellular variations induced by n. bombycis infection, infected and control hemocytes were examined under tem on day 6, 8 and 11 after infection. in hemocytes of control day 6 larvae, cytoplasm was clear. rer and mitochondria with well developed cristae distributed in cytoplasm (fig. 2 a c). in plasmatocytes, nuclei were round or ovoid and in granulocytes, smooth or branched. chromatin was uniformly spread in nucleoplasm. granulocytes showed presence of few granules and plasmatocytes with few electron dense particles (edp). plasma membrane was smooth and with pinocytic vesicles showing active membrane transport. on day 6 after infection, several packs of structured granules appeared in granulocytes. largesized vacuoles occupied major cytoplasmic area. mitochondria increased in number. many granulocytes featured highly irregular branched nucleus with condensed chromatin. plasma membrane showed few cellular projections (fig. 3 a). on day 8 after infection, granulocytes showed plasma membrane with cytoplasmic extensions, lysosomes and phagosomes with engulfed spores (fig. 3 b). more granulocytes were with pseudopodia and microvilli (fig. 3 c). phagosomes enclosing spores were observed and were associated with lysosomes (fig. 3 b). on day 11 after infection, hemocytes showed porous plasma membrane that lost integrity. in few cells, cell membrane completely degenerated. degranulation by exocytosis observed in close vicinity of spores (fig. 4 a-b). many phagocytic granulocytes were observed with cy toplasmic extensions, developed pseudopodia, whirled sporoplasm, engulfed mature spores and ghost spores (fig. 3 d f). rough endoplasmic reticulum (rer) and several mitochondria were observed in figure 1. organismal effects of microsporidian infection on b. mori larva: in comparison to control larva (a), infected larvae showed retarded growth and both larvae and pupae melanised (b – c). control larval hemocytes illustrate clear cytoplasm and less granules (d) whereas infected larval hemocytes were with granulated cytoplasm and few showed degranulation (e). figure 2. transmission electron microscopy (tem) of hemocytes of control fifth instar larvae of b. mori (a) showed smooth plasma membrane, clear cytoplasm and cells with few granules and electron dense particles (edp), nucleus (n) with oval or branched nuclear membrane and evenly distributed chromatin, several mitochondria (m) with developed cristae (b) and rough endoplasmic reticulum (rer) (c). figure 3. tem observations on hemocytes of fifth instar larvae of b. mori showing subcellular variations after infection with n. bombycis: granulocytes turn phagocytic on day 6 (a-b) showing highly deformed nucleus with condensed chromatin (cc), cellular projections (cp) and few vacuoles (v) in cytoplasm. on day 10 well differentiated pseudopodia (c; black arrow heads), multivesicular body (mv) and phagosomes (p) in association with lysosomes (l) were found. on day 11, hemocytes (d f) with dense cytoplasmic contents, invaginated nucleus (ni) with condensed chromatin, packets of structured granules (sg), engulfed mature spores (s; white arrows), ghost spores (gs) and whirled sporoplasm (ws) in vacuoles found. cytoplasm was with rer, mitochondria (m), and vacuoles with cellular remnants (e). 99phagocytosis and lipid peroxidation in hemocytes of b. mori induced by microsporidian infection dense cytoplasm. nucleus demorphed, highly intended and showed deep invagination (fig. 3d). number of lysosomes increased and located adjacent to spores or fused with vacuoles formed phagosomes. lipid peroxidation in order to examine involvement of lipid peroxidation (lpo) in inducing membrane permeability, lpo was assayed by measuring malondialdehyde (mda) which is a by-product of lipid peroxidation. after n. bombycis infection, mda levels significantly (p < 0.012; anova) increased from day 2 to day 14 with significantly (p < 0.000004) larger increase from day 11 indicating increased lpo activity after infection (fig. 5 a). the increase in lpo activity showed positive correlation with increase in age after infection (r2 = 0.58) as well as number of spores increased exponentially (y = 0.0003e0.5572x; r² = 0.80) with increasing age. in order to verify relation between infection level and lpo increase, correlation analysis was performed which showed significant linear correlation (r2= 0.65) between increase in spore number and lpo activity. ascorbate peroxidase activity in order to examine change in activity of ascorbate peroxidase (prx) with increase in infection, prx activity was measured in hemocytes using ascorbic acid as substrate. prx activity significantly (p < 0.025; anova) enhanced from day 2 to 10 after infection with peak activity on day 10 (fig. 5 b) whereas control hemocytes showed prx activity at basic level and increased activity on day 12, during spinning duration. relation between increase in lpo activity (mda level) with changes in prx activity in infected hemocytes was analyzed by correlationregression which showed positive linear correlation though with low correlation coefficient value (r² = 0.294) during initial ten days of infection. notably, from day 11 after infection prx activity significantly decreased and lpo increased. figure 4. tem observations on hemocytes of fifth instar larvae of b. mori showing subcellular variations after infection with n. bombycis. hemocytes (a) showed degranulation by exocytosis (arrow) to the microsporidian infection locale; (b) plasmatocyte (pc) and granulocytes (gc) showed membrane disintegration (arrows) after infection with n. bombycis. sspore; gsghost spore; sgstructured granules; nnucleus. figure 5 a-b. lipid peroxidation in hemocytes of b. mori larva induced after infection by n. bombycis indicated by the variation in malondialdehyde which is a bye-product of lipid peroxidation (a): lipid peroxidation was at significantly higher level from initial stages of infection and at peak level from day 11 onwards. (b): variation in ascorbate peroxidase (prx) activity in hemocytes of b. mori larva induced after infection by n. bombycis: prx activity was significantly higher after infection with peak activity on day 2 and 10 followed by decline from day 11 onwards. 100 hungund p. shambhavi et al. hydrogen peroxide assay in order to quantify reactive oxygen species level in hemocytephagocytes, level of hydrogen peroxide (h2o2) in the hemocytes was tested at 0, 2, 6, 8 and 10 days after the infection using hemocyte lysate extracted in pbs. in hemocytes of n. bombycisinfected larvae, h2o2 levels did not show significant variation from control (data not provided). expression of phagocytosisassociated genes humoral immune responses of hemocytes are initiated with recognition of pathogen followed by signalling and effector action. expression of genes encoding recognition proteins βglucan recognition proteins (bgrp2 and bgrp4), opsonins ctype lectin (ctl11 and ctl 17), hemocy te aggregation factor humoral lectin, phagocytosis enhancer apolipophorin, cytoskeletal protein βactin, hemocyte mediated complement like protein that bind and kill plasmodium berghei in aedes aegypti thioester containing protein (tep-1) and bacterial phagocytosis associated eater from drosophila melanogaster was analysed by rtpcr (table 1) and qpcr (fig. 6). rt-pcr profile and qpcr revealed down regulation of βgrp2 and bgrp4 expression after n. bombycis infection. ctl genes showed low level of expression in earlier days of n. bombycis infection whereas 0.334 fold increase in expression was noticed on 10th day after infection (fig. 6 b). humoral lectin enhanced relative expression on day 6 (0.79 fold) after infection followed by down regulation (-1.97 fold) on day 10. after infection by n. bombycis, apolipophorin showed increase in expression by 1.96 fold on 6th day and by 2.59 fold on 10t h day. expression of βactin showed stable increase from early to late stages of infection with strong positive correlation (r 2 = 0.65) with age after infection (fig. 6 c). in the dipterans a. aegypti and d. melanogaster, tep1 (blandin et al. 2004) and eater (kocks et al. 2005; juneja and  lazzaro 2010) respectively are closely associated with phagocytosis however these genes are not reported from b. mori. primers derived from tep of a. aegypti and eater of drosophila was used for amplification with template cdna from hemocytes of b. mori after infection with n. bombycis. in this study tep did not show expression in hemocytes of b. mori whereas eater showed enhanced relative expression on day 2 and 6 after infection followed by significant (p < 0.005) decrease (fig. 6 d). figure 6 a-d. modulation in expression of immune genes in hemocytes of b. mori larva after infection with n. bombycis: (a) rt-pcr profile of gene expression of different genes including cytoskeletal protein gene βactin and the house keeping gene encoding ribosomal protein resolved from hemocytes collected from control and infected larvae at 2, 6, 8 and 10 days after infection. mmassruler dna marker (thermo). (b) qpcr showed relative expression of different immune genes in hemocytes at 6 and 10 h after infection. (c) βactin, the cytoskeletal protein gene showed gradual increase in expression represented by densitometric units. increase in expression was correlated with age after infection shown by allometric line. the linear regression formula and correlation coefficient are inserted. (d) relative expression pattern of the phagocytosis associated gene eater like after standardization with expression of the house keeping gene, ribosomal protein and after elimination of control value. figure 7. alignment of nucleotide sequence (a) of amplification product from heterologous expression profile of eater gene of drosophila (query) with nucleotide sequence (subject) of the most similar gene, b. mori uncharacterized transcript variant x2 (accession no. xm_004928120.3) revealed by ncbi-blastn analysis, showed 98 % similarity. (b) protein sequence of the uncharacterized transcript variant (subject) is aligned with translated sequence of the eater like gene (query) showing 97% similarity. 101phagocytosis and lipid peroxidation in hemocytes of b. mori induced by microsporidian infection sequence analysis in order to confirm presence of eater like gene in b. mori, amplification products were ligated, cloned and sequenced. the nucleotide sequence was analysed by ncbi-blast. eater like sequence showed 98 % similarity with b. mori uncharacterized transcript variant x2 (accession no. xm_004928120.3) with an expect value 7e-110. the translated sequence of eater like (3’5’ frame 2) showed 97 % similarity with translated amino acid sequence of bm uncharacterized protein (bmucp) bmucp loc101736235 isoform x2 (h9jfy7_bommo of uniprot; bgibmga008434 of b. mori) revealing existence of eater like sequence in b. mori genome (fig. 7). this sequence showed orthology in the lepidopterans heliconius melpomene and danus plexippus with unknown function (eggnog 4.5.1). however complete sequencing of the gene has to be performed for gene structure confirmation. discussion in the initial stages of microsporidian infection in b. mori, spores were not detected microscopically for six days after infection. in the mid and later stages, exponential increase in spore count was recorded. in insects host responses in hemocytes initiated with activation of cell surface receptors and signal transduction (lamprou et al 2007; tsakas and marmaras 2010). hemocytes recognize pathogens entered in larval body with assistance from recognition proteins. the proteins that recognize nosema sporoplasm / spores have not been identified in b. mori though microbial recognition proteins such as peptidoglycan recognition proteins (pgrps) have role in host responses of honey bees against infection by n. ceranae through toll / imd pathways (li et al 2017). β grp (β-1,3-glucan recognition proteins) are recognition proteins in toll/ dif pathway (gobert et al 2003) and are associated with detection of bacterial endotoxin in drosophila (kim et al. 2000) and phenol oxidase activation in b. mori (yoshida et al. 1986) indicating multiple role associated with immune reactions. following recognition, hemocytes initiated cellular immune events such as cell aggregation, nodulation, cytokine release, melanization and encapsulation depend on size of the pathogen / parasite (lavine and strand 2002). similarly, hemocytes initiate phagocytosis against bacteria and fungi particularly against those with size less than five microns (pech and strand 1996; scapigliati and mazzini 2009). notably, n. bombycis spores infecting b. mori larva are of 2.6 to 3.8 microns (breadth x length) (rao et al. 2007) which could be phagocytosed by hemocytes though mechanism of parasite destruction is not clearly known. phagocytic uptake of koh treatedor cold storaged nosema spores is found in insect cell line (cai et al. 2012) however phagocytosis of live nosema spores by larval hemocytes in vivo had not been reported in b. mori. after n. bombycis infection in b. mori larva, tem observation showed symptoms of phagocytosis in granulocytes such as formation of pseudopodia and appearance of phagosomes with lysosomal bags. spores and ghost spores were observed within phagosomes of hemocytes indicative of lysosomal activity on spores. phagosomes enclosing spore / meront were found in granulocytes of b. mori as noticed in a. aegypti after infection by plasmodium gallinaceum (hillyer et al. 2003) and in the coleopteran rhynchophorus ferrugineus infected with the yeast saccharomyces cerevisae (manachini et al. 2011). in the coleopteran flower chafers protaetia brevitarsis seulensis, development of autophagic vacuoles observed in association with phagocytosis by granulocytes indicative of autophagic elimination of pathogens (kwon et al. 2014). after n. bombycis infection, granulocytes showed cytoplasmic projections, pseudopodia, microvilli, membrane porosity and disruption as characteristics of phagocytic cells (castillo et al. 2006; williams 2007). moreover granulocytes showed degranulation by exocytosis in spore ‘locales’ indicating active transportation of structural granules to the plasma membrane and degranulation in the site of infection by the spores as observed in mouse models (dias et al. 2018). hemocytes with extended pseudopodia, cy toplasmic projections and phagosomes were observed in anopheles quadrimaculatus infected with nematode, romanomermis culicivorax (shamseldean et al. 2006), culex quinquefasciatus infected by wuchereria bancrofti (brayner et al. 2007) and in plasmatocytes of the tick rhipicephalus sanguineus infected with leishmania infantum (feitosa et al. 2015). stable increase in expression of the cytoskeletal protein gene βactin with age was noticed in hemocytes of b. mori after n. bombycis infection indicated continuous requirement of actin to redistribute the cytoskeletal protein during formation of pseudopodia and microvilli after n. bombycis infection and to meet rearrangement of cytoskeletal proteins for engulfment (moore et al. 1992; kwon et al. 2014). variation in actin protein content and its critical role was reported in association with formation of pseudopodia in other models also (may and machesky 2001; baranov et al. 2016). lpo and peroxidase activity associated with phagocytosis infection with n. bombycis increased malondialdehyde production in hemocytes revealed increased lipid 102 hungund p. shambhavi et al. peroxidation (lpo). lipoprotein structure of the membrane is disrupted by lpo, which caused membrane porosity and disintegration in association with phagocytosis. lpo activity significantly increased with age in early and mid stages of infection however it was higher in later stages of infection. moreover spore count is increased exponentially after six days of infection and lipid peroxidation increased simultaneously showing correlated increase. during lipid peroxidation, carbon carbon double bonds present in the polyunsaturated fatty acids of plasma membrane are attacked (yin et al. 2011; wong-ekkabut et al. 2007) which is initiated with oxidation of few lipid molecules and subsequently continued as a chain reaction leading to disintegration of cell membrane (mylonas and kouretas 1999; ayala et al. 2014). though infection induced oxidative stress caused lipid peroxidation (milei et al. 2007; pooja et al. 2017), increment in reactive oxygen species (h2o2) was not observed in hemocytes of b. mori larva after n. bombycis infection indicating a direct effect of lpo on hemocyte membrane integrity probably through accumulated toxic products (clark et al 1987). similar direct impact of lipid peroxidation on tissue damage was reported in liver of the fish pimephales promelas infested by liver trematode ornithodiplostomum  sp (stumbo et al. 2012). accumulation of lpo toxic products could suppress the phagocytic action of hemocytes which defend parasite survival. a possibility for less h2o2 content observed in the infected hemocytes might be due to relatively shorter half life induced by its reactivity with biomolecules (lennicke et al. 2015). in order to protect the cells from peroxidation, enzymatic antioxidant peroxidases are activated (brigelius-flohe and maiorino 2013; jablonska et al. 2015). ascorbate peroxidase removed lipid peroxides in the lepidopteran helicoverpa zea (mathews et al. 1997) through ascorbate recycling system (summers and felton 1993; krishnan and kodrik 2006; lukasik et al. 2009). after n. bombycis infection, in hemocytes of b. mori, peroxidase activity was significantly higher in early and midstages of infection which regulated lpo activity to comparatively lower level. in the later stages of infection, peroxidase activity significantly reduced, in contrast, lipid peroxidation increased significantly indicating negative interaction between lipid peroxidation and ascorbate peroxidase activity, corroborating with the negative relation noticed between lpo and peorxidase activity in humans under diseased conditions (mccay et al. 1976; motghare et al. 2001). the peroxidase regulation of lpo in association with phagocytic events is unknown in invertebrates after parasitic infection. modulation in gene expression after infection in order to enhance immune reactions after n. bombycis infection, genes encoding proteins associated with humoral and cellular immune response were activated before eliciting the host responses (brown and gordon 2003; manachini et al. 2011). notably β-grp genes did not show significant variability in expression after infection showing an ambiguity in its role in immune reactions against n. bombycis infection. on the other hand ctl genes showed upregulated expression on day 6 after infection and its role was suggested to be in spore recognition and signal trasnsduction (ma et al. 2013). ctl-11 and 17 implicated in opsonising blastospores of the fungus, beauveria bassiana to make fungal spores susceptible to phagocy tosis (pendland et al. 1988). notably, taxonomic position of n. bombycis is shifted from protozoan to fungus (han and weiss 2017) based on molecular phylogeny. ctl proteins activated during infection by other fungus could be effective during infection by n. bombycis probably due to activation of similar immune mechanisms against different species of fungi. gene encoding hemocyte adhesive factor humoral lectin (kotani et al. 1995) and the phagocytosis enhancer apolipoprotein iii (whitten et al. 2004) enhanced expression after infection. apolipophorin iii together with i/ ii help in pattern recognition as well as enhances phagocytic action of hemocytes in insects (barabas and cytryńska 2013; whitten et al. 2004). in the dipterans aedes and drosophila, thioester containing protein (tep1) (blandin et al. 2004) and eater (kocks et al. 2005) respectively are associated with phagocytosis however these genes have not reported from b. mori. though tep1 did not show expression, eater like showed heterologous expression in hemocytes of b. mori larva on day 2 and 6 after infection. both nucleotide sequence and translated amino acid sequence of eater like amplicon showed 98% similarity with that of an uncharacterized transcript variant from b. mori indicating activation of eater like gene in b. mori in association with hemocy temediated phagocy tosis against n. bombycis. in drosophila, eater is a transmembrane protein involved in binding and internalization of bacteria in the phagosomes (stuart et al. 2005; kocks et al. 2005; chung and kocks 2011) however role of eater like protein in b. mori immune responses is unknown. n. bombycis infection induced subcellular variations in hemocytes of b. mori including demorphed nucleus, activation of phagocytosis including formation of pseudopodia, microvilli, porous plasma membrane and formation of phagosomes. in addition, lipid peroxidation was increased in hemocytes with increase in age after 103phagocytosis and lipid peroxidation in hemocytes of b. mori induced by microsporidian infection infection. simultaneous increase in peroxidase reduced the lpo activity. in the later stage, peroxidase activity reduced and lpo activity increased showing negative relation. moreover infection by n. bombycis induced modulation of phagocytosisassociated genes where heterologous expression of eater also observed indicating activation of phagocytic events and associated events against n. bombycis infection in b. mori larva which are potential novel targets for developing new control measures. the proteinbased targets could be used to develop antibodybased mechanisms for early detection of microsporidian infection. acknowledgements the authors thank anonymous reviewers for valuable suggestions, central silk board, bangalore for the facilities and department of biotechnology (government of india), new delhi for financial support in the form of a research project to arp (bt/pr6355/pbd/19/236/2012 dated 08/01/2013). sph and pm were supported by research fellowships from the project. conflict of interest the authors declare no conflict of interest. references ayala a, munoz mf, arguelles s. 2014. lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. oxid med cell longev. article id 360438; 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c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. and mathematical analysis cihangir alaca1, ali özdemir1, bahattın bozdağ2, canan özdemir2,* clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 75(2): 53-58, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1418 caryologia international journal of cytology, cytosystematics and cytogenetics citation: chandra bhanu singh, vijay kumar singhal, manish kapoor (2022) first record of nucleus migration in premeiotic antherial cells of saccharum spontaneum l. (poaceae). caryologia 75(2): 53-58. doi: 10.36253/caryologia-1418 received: september 29, 2021 accepted: may 20, 2022 published: september 21, 2022 copyright: © 2022 chandra bhanu singh, vijay kumar singhal, manish kapoor. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid cbs: 0000-0001-8337-0629 vks: 0000-0002-7109-7685 mk: 0000-0002-8349-8910 first record of nucleus migration in premeiotic antherial cells of saccharum spontaneum l. (poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* 1 university department of botany, tilka manjhi bhagalpur university, bhagalpur – 812 007, bihar, india 2 department of botany, punjabi university, patiala-147 002, punjab, india *corresponding author. e.mail: jdmanishkapoor@yahoo.com, jdmanishkapoor@pbi. ac.in abstract. the occurrence of nucleus migration is reported for the first time in a clone (2n = 64) of ‘thatch’ grass (saccharum spontaneum l.) of the family poaceae. usually, its premeiotic antherial cells are thin walled, uninucleate and without any trace of chromosome individuality. however, the cells of those anthers that had been affected from flood water stress conditions were anucleated to hexanucleated in varying frequencies. out of 2567 cells analyzed, two and three cells were noticed to be connected to each other through a well-defined cytoplasmic channel. the nuclei were observed at various stages of their migration in interconnected cells. the remaining cells exhibited a mosaic of anucleate to hexanucleate cells in varying frequencies with a dominance of binucleated condition (43.75%). the anucleate ‘ghost’ cells were much smaller in size than the uninucleate, binucleate and multinucleate cells showing insignificant variation among themselves. the anucleate, binucleate and multinucleate cells appeared to be resulted due to nucleus migration through cytoplasmic channels between two cells. the presence of a nucleus in donor cell united with recipient cell having four nuclei of different sizes, diminutive anucleate cell in the neighbourhood of uninucleate/trinucleate cell or connected with cytoplasmic channel/pentanucleate cell, and disorganizing cytoplasmic channel attached with binucleate/ tetranucleate cell witnessed the accomplishment of nucleus migration. this rare phenomenon of nucleus migration seemed to be triggered by flood water induced stress and facilitated by feeble cell wall. the variation in sizes of nuclei in multinucleate cells might be due to the transfer of nucleus/nuclei of different size(s). the prominent features of nucleus migration distinguishing it from the cytomixis have been discussed in detail. the syncytes resulted due to nucleus migration might have generated the pollen grains with different genetic constitution resulting into the origin of new intraspecific aneuploids/ polyploids for better adaptability. keywords: cytomixis, nucleus migration, premeiotic cells, saccharum spontaneum. introduction flooding stress has been considered as the strong driver of adaptive evolution (jackson and colmer 2005). in the disturbed habitat of diara land, it 54 chandra bhanu singh, vijay kumar singhal, manish kapoor seems to act as the main stress that can trigger syncyte formation leading to the production of new polyploids/ aneuploids. cytomixis (inclusive of nuclear migration) that operates as the most common pathway of syncyte formation is an important process of evolutionary significance (kravets 2012, mandal et al. 2013, mursalimov et al. 2013). the phenomenon is considered to be an efficient mechanism for the production of unreduced (2n) pollen grains and thereby the origin of intraspecific polypoids in several plant species (falistocco et al. 1995, ghaffari 2006, kim et al. 2009, sheidai et al. 2009, fadaie et al. 2010, singhal et al. 2010, 2011, 2016, kaur and singhal 2012). the polyploids with more adaptability often thrive better in harsh and disturbed environments (ramsey and schemske 1998, otto and whitton 2000, madlung 2013, de storme and mason 2014, de peer et al. 2021) the occurrence of nucleus migration has been observed in the premeiotic cells of anthers of a clone of saccharum spontanuem l. (english thatch grass, vernacular kans, family poaceae) growing under flood water stress conditions. this clone (2n=64) grows profusely in bhagalpur (bihar) diara land of ganga basin, where flood is almost a perpetual annual feature (singh et al. 2018). this is the favourite fodder of buffaloes. besides, the species is very useful in thatching of roofs and in making various items such as cordage, ropes, mats, baskets, brooms, etc. (singh 1997). the indian sub-continent including bihar is considered as the centre of greatest evolutionary activity of s. spontanuem l. (panje and babu 1960). the occurrence of nucleus migration in the premeiotic cells is the first record for this species, the most valuable germplasm in sugarcane breeding. fifty-two years ago, chromosome and nucleus migrations were observed during microsporogenesis of some radiation-induced mutants of pisum sativum l. (gottschalk 1970). the present communication aims to clear, enrich and elaborate the concept of nucleus migration. materials and methods the spikelets of a clone of saccharum spontaneum l. (voucher specimen number 2180 of herbarium of university department of botany, tilka manjhi bhagalpur university, bhagalpur) growing profusely in bhagalpur diara land constituted the experimental material of the present investigation. these were collected every alternate day from both normal (flood free) and submerged conditions. the collected spikelets from these environments were fixed separately in carnoy’s fluid (6 ethanol: 3 chloroform: 1 glacial acetic acid). the fixative was changed daily for 15 days to clear the cytoplasm. subsequently, the material was preserved in 70% ethanol and refrigerated at 0ºc until use. anthers were squashed in 2% acetocarmine and preparations were studied to record abnormalities in premeiotic cells. photomicrographs of unusual premeiotic cells were taken from the temporary preparations. the diameters of visible nuclei were determined from their enlarged photographs (at constant magnification) and the volumes of nuclei were calculated using the formula 4/3 for sphere; where r is the radius (i.e., half of diameter). results the premeiotic cells from young anthers of a diara clone of ‘thatch’ grass (saccharum spontaneum l.) had thin wall and a distinct nucleus. these cells were constantly uninucleate in the anthers of normal plants. however, wide variations in number of nuclei were observed in the cells from the anthers of flood affected plants. out of a total 2567 cells analyzed, two and three cells were found to be connected to each other through a conspicuous cytoplasmic channel respectively, in nineteen (figure 1) and two (figure 2) instances. the nuclei were seen at various stages of their migration in the united cells (figures 2-3). the remaining cells exhibited a mosaic of anucleate to hexanucleate cells (figures 4-8) in varying frequencies (table1) with dominance of binucleate condition (43.75%). among the multinucleate cells, the trinucleate ones were the most frequent, while the pentanucleate were the least common. with respect to size, the anucleate ‘ghost’ cells were much smaller than the nucleate (uninucleate, binucleate and multinucleate) cells which did not show significant variation among themselves. the anucleate, binucleate and multinucleate cells appeared to be the resultant products of nucleus migration, commencing with the development of a welldefined cytoplasmic channel between two cells (figures 1-6). the possession of one nucleus in the donor cell, which is united with a recipient cell containing four nuclei of different sizes (figure 3) showed that the cytoplasmic channel might have paved the way for gradual transference of nucleus/nuclei through it. the presence of a diminutive anucleate donor cell in the neighborhood of a trinucleate cell (figure 1) or uninucleate cell (figure 2) or connected prominently with a cytoplasmic channel (figure 2) or pentanucleate cell (figure 6) and the disorganizing cytoplasmic channel attached with a binucleate cell (figure 4) or a tetranucleate cell (figure 5), suggested towards the completion of nucleus migra55first record of nucleus migration in premeiotic antherial cells of saccharum spontaneum l. (poaceae) tion. the phenomenon of nuclear migration seemed to be induced by abiotic stress due to flood water conditions and facilitated by feeble wall character of premeiotic cells resulting into syncyte formation. the shapes of cells were noted to be specific to some extent to their nuclear status as exclusively spherical for anucleate (in free condition) to trinucleate; spherical or ellipsoidal for tetranucleate; spherical or irregular for pentanucleate and solely irregular for hexanucleate conditions (table 1). the uninucelate and binucleate cells displayed the presence of only large sized nucleus and nuclei accordingly in them. irrespective of their shapes, the multinucleate cells had nuclei of different sizes in them (table 2). these nuclei were arbitrarily classified as large (above 50 µm3), medium (above 20 µm3 and up to 50 µm3) and small (up to 20 µm3). the presence of equal sized nuclei in binucleate cells and unequal sized nuclei in multinucleate cells seems to have been derived from similar and dissimilar types of cells. this became evident from the existence of an anucleate ghost cell and a binucleate cell that was connected with a trinucleate cell (having one nucleus of each category) through a cytoplasmic channel for nuclear migration (figure 1). the size of nucleus contained in uninucleate cells showed approximation to the dimension of large sized nuclei possessed by tetranucleate cells, whose medium sized nuclei were almost of the magnitude of medium sized nuclei of trinucleate cells. figure 5 indicates the presence of a tetranucleate cell possessing different sized nuclei, which might have resulted due to nucleus migrations from different types of donor cells. discussion ever since the first report of syncytes by gates and reese (1921), these multinucleate cells have been reportfigures 1-8. premeiotic antherial cells of saccharum spontaneum l. showing nucleus migration and its products. 1. two cells connected through a conspicuos cytoplasmic channel (arrowed) in between and a small anucleate ghost cell (arrowhead) present on upper side. 2. two distinct uninucleate cells linked through a cytoplasmic channel (arrowed) and a small deformed anucleate ghost cell (arrowhead) united mid way with the channel – discharged nucleus (double arrowed) entering into a cell to make it binucleate; another small spherical free anucleate ghost cell (arrowhead) and a large binucleate cell discernible on lower side. 3. two cells interconnected with each other through a cytoplasmic channel (arrowed): donor cell possessing one nucleus and recipient cell containing four unequal sized nuclei; a binucleate cell visible above. 4. uni, bi and trinucleate cells; a binucleate cell united with a disorganizing cytoplasmic channel (arrowed). 5. tetranucleate cells showing marked variation in the size of nuclei and one tetranucleate cell showing attachment with a disorganizing channel (arrowed). 6. a tetranucleate cell in left and an elongated cum curved anucleate ghost cell (arrowhead) attached with a pentanucleate cell (having uneven sized nuclei) in right. 7. a deformed pentanucleate cell having nuclei of different sizes. 8. bi and hexanucleate cells: curved shape of hexanucleate cell remarkable. bar = 20μm. table 1. type, shape, and frequency of flood affected premeiotic antherial cells* of saccharum spontaneum l. cell type shape number and frequency (%) anucleate spherical 31(01.21) uninucleate spherical 234(09.12) binucleate spherical 1123(43.75) trinucleate spherical 954(37.16) tetranucleate spherical/ ellipsoidal 165(06.43) pentanucleate spherical/ irregular 16(00.62) hexanucleate irregular 44(01.71) *among 2567 cells, two and three cells connected to each other through a conspicuous cytoplasmic channel respectively in nineteen and two cases for nucleus migration. 56 chandra bhanu singh, vijay kumar singhal, manish kapoor ed in several plant species by different workers. the syncytes in plants are usually formed through archesporial error, cell fusion and nuclear migration. out of these, nuclear migration occurs through the transfer of whole chromatin material or nucleus from one cell to an adjacent cell. the migration of partial or total chromosome, referred to as “cytomixis”, is a widespread phenomenon and has been reported in a large number of plant species (kravets 2012, mandal et al. 2013, mursalimov et al. 2013, rana et al. 2014, 2015, kumar et al. 2015, reis et al. 2015, bhat et al. 2017, mandal and nandi 2017, singhal et al. 2018, paez et al. 2021). however, nucleus migration has been observed only in pollen mother cells of radiation-induced ‘pea’ (gottschalk 1970). the occurrence of nucleus migration through a conspicuous cytoplasmic channel as observed in the present study appears to be closely allied with the wellknown phenomenon of cytomixis through cytomictic channel. both these phenomena seem to be homologous but indeed separate from each other at least in mode of operation. cytomixis involves chromatin transfer between proximate cells (kamra 1960, omara 1976, belluci et al. 2003, singhal et al. 2009, 2018, himshikha et al. 2010, rana et al. 2013, kumar and singhal 2016, kumar et al. 2016, 2017, bhat et al. 2017, mandal and nandi 2017). owing to this unique feature, it is also called as the phenomenon of intercellular chromatin transmigration (kumar and naseem 2013, kumar and choudhary 2016, dwivedi and kumar 2018, khan et al. 2018, kumar and singh 2020). akin to cytomixis, nucleus migration requires the passage of a whole nucleus from donor cell to recipient cell (gottschalk 1970, patra et al. 1987). according to kihara and lilienfeld (1934), the term “cytomixis” should be used to designate only the transit of structureless chromatin drops (“uebertritte structurloser chromatintrophen”). keeping in view the above-mentioned limitation placed on the use of this term, gottschalk (1970) designated the phenomenon of moving chromosome and nucleus with normal structure as “chromosome and nucleus migration” rather than cytomixis. the denotation of migration of chromosomes and nuclei, respectively as nuclear chromosome migration and migration of nucleus by patra et al. (1987) may be a circumstantial consideration that cytomixis and nucleus migration are apart from each other. there appears no hitch in corroborating the event of shift of structured nucleus as evident in the present case as “nucleus migration”. the uniformity in the size of uninucleate, binucleate and multinucleate cells in the presently studied grass suggests that only nuclei pass through cytoplasmic channels. unlike this, cytomixis involves the transfer of both chromatin/ chromosome(s) along with cytoplasm as well as other cell organelles through cytomictic channels (risueno et al. 1969, romanov and orlova 1971, mursalimov and deineko 2011, kumar and choudhary 2016, kumar and singh 2020). this becomes apparent from the increase in the size of cytomictic products/ recipient cells, as has also been recorded by sarbhoy (1980) and singh et al. (1989, 1990). thus, nucleus migration has been treated here as a separate process different from cytomixis. the possible causes of syncyte formation in plants include effect of chemicals, x-rays, temperature, moisture stress, viral infection, culture conditions or genetic factors. in the presently studied grass the syncyte formation could be attributed to flood water induced stress conditions prevailing in the diara land. the products of table 2. data on number of nucleus or nuclei per cell and dimension of different sized nuclei in flood affected premeiotic antherial cells of saccharum spontaneum l. nucleus or nuclei/ cell different sized nuclei / cell diameter of nucleus mean ± s.d. (µm) volume of nucleus (µm3) 1 large 1 6.01 ± 0.20 118.89 2 large 2 5.04 ± 0.68 67.06 3 large 1 5.68 ± 0.70 95.99 medium 1 3.47 ± 0.18 21.89 small 1 2.33 ± 0.37 06.63 4 large 2 6.25 ± 0.28 127.88 medium 2 3.48 ±0.29 23.43 5 large 3 4.68 ± 0.45 53.69 small 2 2.17 ± 0.21 05.35 6 medium 3 3.80 ± 0.39 28.74 small 3 2.26 ± 0.33 06.05 57first record of nucleus migration in premeiotic antherial cells of saccharum spontaneum l. 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(poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 125-132, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-147 citation: r. partovi, a. iaranbakhsh, m. sheidai, m. ebadi (2020) population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers. caryologia 73(1): 125-132. doi: 10.13128/caryologia-147 received: january 9, 2019 accepted: february 23, 2020 published: may 8, 2020 copyright: © 2020 r. partovi, a. iaranbakhsh, m. sheidai, m. ebadi. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iranbakhsh1,*, masoud sheidai2, mostafa ebadi3 1 department of biology, science and research branch, islamic azad university, tehran, iran 2 faculty of life sciences & biotechnology, shahid beheshti university, tehran, iran 3 department of biology, islamic azad university, damghan branch, damghan, semnan province, iran *correspondign author. e-mail: iranbakhsh@iau.ac.ir abstract. olive is an important horticultural plant having both cultivated and wild forms. the aim of the present study was investigating genetic diversity of 13 wild olive trees belonging four geographical populations in iran using srap neutral molecular markers as well as cp-dna rpl intergenic sequences and its region. genetic diversity parameters determined for 76 srap loci within the studied olive populations identified the most variable loci. population differentiation parameters determined for srap loci, identified 13 srap loci with gst value of 1, that means they differentiate the studied trees. pcoa analysis based on srap data separated olive trees from each other due to genetic difference. distribution of the samples in pcoa plot indicated that the population 1 are more spread due to population genetic variability. however, the srap result reveals that these molecular markers can be used in population genetic investigations and germ plasm analysis. amova showed significant genetic difference among the studied olive populations. cp-dna analysis produced 366 bp long sequences, out of which 224 sites were segregating among the studied plants. the mean nucleotide diversity was 0.32. tcs network based on cp-dna separated most of the studied populations. therefore, it seems that cp-dna rpl sequences is a suitable barcode molecular marker for population genetic studies. phylogenetic tree of its data could partially differentiate wild olive population. in conclusion, a combined use of sraps and cp-dna sequences are suggested for wild olive population genetic investigation. keywords. srap, cp-dna, its, population genetic, olive. introduction olive tree (o. europaea l.) of the genus olea (o. europaea subsp. europaea var. europaea) is one of the most important horticultural crop plants. it is an ancient plant species with grate economic value (zohary and hopf 2000), and has both cultivated and wild forms. oleaster (o. europaea sub126 rayan partovi et al. sp. europaea var. sylvestris miller) is the mediterranean wild olive and is possibly the progenitor of the cultivated olive. the non-mediterranean wild olives are geographically isolated from the oleaster and show different morphological characters. green (2002) grouped all morphological forms of wild olive in a single aggregate i.e. olea europaea subsp. cuspidata, but the other investigators consider these intra-specific forms as ecotypes both in africa and iran (besnard et al. 2002; sheidai et al. 2010). the occurrence of natural hybridization has been reported between different sub spices within the genus olea. this holds true also for o. cuspidate and o. africana (besnard and bervill 2000). moreover, (omranisabbaghi et al. 2007) suggested hybridization of subsp. cuspidata and the cultivated olive in south africa and iran and (sheidai et al. 2010) identified a population with intermediate morphological and molecular (rapds) characteristics. the wild relatives of crop plants (cwrs) constitute an important resource for improving agricultural production and for maintaining sustainable agro-ecosystems. genetic material from cwrs has been utilized by humans for to improve the quality and yield of crops. for example, wild maize (zea mexicana) is routinely grown alongside maize to promote natural crossing and improve yields. more recently, plant breeders have utilized cwr genes to improve a wide range of crops like rice (oryza sativa), tomato (solanum lycopersicum) and grain legumes (hajjar and hodgkin 2007). therefore, a cwr can be defined as “a wild plant taxon that has an indirect use derived from its relatively close genetic relationship to a crop. the cwrs comprise a wonderful gene pool for future crop breeding programs. since natural populations of cwrs are at risk and are threatened by habitat loss, deforestation, etc., population genetic study of these natural populations is important task as it provides insight about the genetic variability, population genetic structure, gene flow versus population fragmentation as well population genetic differentiation. the obtained information can be utilized in both breeding as well as conservation strategies of the cwrs. recent population genetic studies use different molecular markers to investigate the genetic diversity as well as other population genetic features. this is also true for olive (bracci et al. 2011), for example, random amplified polymorphic dna (rapds) (sheidai et al. 2010) microsatellite (simple sequence repeat; ssrs) and inter simple sequence repeat; issr markers, amplified fragments length polymorphic markers (aflps) (baldoni et al. 2006), cp-dna (besnard et al. 2011). in the present study, genetic diversity, genetic divergence and genetic structure of four populations of o. europaea subsp. cuspidata from different localities are investigated using nrdna its (internal transcribed spacer) and srap (sequence-related amplified polymorphism) markers. since, srap marker technique combines easiness, reliability, high variability, moderate throughput ratio and superficial sequencing of the selected bands, we used this technique to amplify coding regions of dna to target open reading frames. materials and methods thirteen specimens belonging to four geographical populations of subspecies olea europaea subsp. cuspidata l. were collected from different localities that were placed between three provinces bakhtiari, boyer-ahmad and khuzestan. details of geographical populations are given in table 1. dna extraction and pcr reactions dna was extracted from dried leaf specimens (approximately 0.5 g material per sample) using ctab (cetyl trimethyl-ammonium bromide) activated charcoal protocol (krizman et al. 2006 and sheidai et al. 2013). extracted dna was run on 0.8% agarose gel. pcr reactions were carried in a 25μl volume containing 10 mm tris-hcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 mg genomic dna and 1 u of taq dna polymerase (bioron, germany). table 1. list of 13 specimens of olea europaea subsp. cuspidata l. from four populations accompanied by their distribution, altitude, longitude and herbarium number. no. localities altitude latitude longitude voucher no. 1 chaharmahal and bakhtiari province, dehedz – lordgan, iran 1713 31°31’18” 50°28’26” hsbu2018700 2 kohgiluyeh and boyer-ahmad province, khersaan road, iran 1380 31°26’59” 50°28’57” hsbu2018705 3 chaharmahal and bakhtiari province, lordgan, monj, gachahan, iran 1151 31°26’48” 50°32’19” hsbu2018711 4 chaharmahal and bakhtiari province, lordgan, monj, gachahan, iran 1592 35°55’41” 57°41’53” hsbu2018712 127population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers srap study five sequences related amplified polymorphism (srap) primer pairs including forward primers: me1, me2, me3, me4, me5 and reverse primers: em1, em2, em3, em4, em5 were used (feng et al. 2014). pcr reactions were carried in a 25μl volume containing 10 mm tris-hcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 ng genomic dna and 1 u of taq dna polymerase (bioron, germany). following programs used for amplification of srap region in a pcr reaction: 94°c, 1 min, 35°c, 1 min, and 72°c, 1 min for first five cycles then 5 min initial denaturation step 94°c, followed by 40 cycles of 1 min at 94°c; 1 min at 55°c and 2 min at 72°c and a final extension at 72°c for 7-10 mi its study the complete its region was amplified using forward its5 (5’gga agt aaa agtcgt aac aag g3’) and reverse primers its4 (5’tcc gct tat tga tat gc 3’) (white et al. 1990). following program used for amplification of nuclear region in a pcr reaction: 5 min initial denaturation step 94°c, followed by 40 cycles of 1 min at 94°c; 1 min at 53.5°c and 2 min at 72°c. the reaction was completed by final extension step of 7 min at 72°c. cpdna study the intergenic spacer of chloroplast genome rpl16 was amplified and sequenced with universal primers following the methodology of (shaw et al. 2005; timme et al. 2007). each 20 µl of pcr tube contained 10 µl of 2x pcr buffer, 0.5 mm of each primer, 200 mm of each dntp, 1 unit of taq dna polymerase (bioron, germany), and 1 µl of template genomic dna at 20 ng µl-1 . the amplification reaction was performed in techne thermocycler (germany) with the following program: 2 min 94°c, 1 min at 94°c; 1 min at 54°c and 1min at 72°c. the reaction was completed by final extension step of 6 min at 72°c. data analyses srap bands were coded as binary characters (presence = 1, absence = 0) and used for genetic diversity analysis. data obtained were analyzed for the genetic diversity parameters like, nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism (weising et al. 2005; freeland et al. 2011). principal coordinate analyses (pcoa) were performed using past ver. 2.17 (hammer et al. 2012). nei’s genetic distance was used among populations. amova (analysis of molecular variance) test (with 1000 permutations) as implemented in genalex 6.4 (peakall and smouse 2006). the phylogenetic methods used to investigate the species relationships were maximum parsimony (mp), maximum likelihood (ml), networking and bayesian approaches. the its sequences were firstly aligned and used to test the proper nucleotide substitution model as applied in mega 7. program (tamura et al. 2012). networking was performed using splits tree 4 program (huson and bryant 2006) and bayesian analysis was done using beast software v1.6.1 (drummond et al. 2012a, b). results in total, 76 srap bands were obtained in olive trees studied. some of these bands were common while, few bands were private in these trees. for example, srap bands 51, 52 and 76 occurred only in trees of population 4, while srap band 7 happened only in one of the trees in population 1. similarly, srap band 4 was observed in the tree of population 3. genetic diversity parameters determined for all srap loci within the studied olive populations identified the most variable loci. the loci with highest value of gene diversity (h) and shanon information index (i) are the most diverse srap loci (table 2). the mean value obtained for h = 0.34, while i = 0.52. population differentiation parameters determined for srap loci in the studied olive trees (table 3), identified the loci with highest migration/ exchange value (nm) and also srap loci with the highest differentiation value (gst). in total, 13 srap loci had gst value = 1, that means they differentiate the studied trees. similarly, srap loci with nm>1 are considered highly migrated among the populations. pcoa analysis of the studied olive trees after 99 times permutation, based on srap data is presented in figure 1. pcoa plot clearly separates olive trees of the studied populations from each other due to genetic difference. distribution of the samples in pcoa plot indicates that olive trees of population 1 are more spread due to within population genetic variability. however, in general, the srap result reveals that these molecular markers can be used in population genetic investigations and germ plasm analysis of olive. nei, genetic distance determined among olive trees based on srap data (table 4), revealed that the genetic distance among trees of the population 1 varies from 128 rayan partovi et al. 0.30 to 0.54, while it varies from 0.46 to 0.76 in olive trees of population 2. amova showed significant genetic difference (phipt = 0.43, p =0.01) among the studied olive populations. it also revealed that 43% of total genetic variation was due to among population genetic difference, whereas, 57% occurred due to within population genetic variability. cp-dna analysis we obtained 366 bp long sequences, out of which 224 sites were segregating among the studied plants. the mean nucleotide diversity (p) was 0.32. tcs network of the studied olive trees figure. 2) separated most of the studied populations. for instance, trees of the population 2 and the population 4 were grouped together, while trees of population 1 were scattered in between these two populations. therefore, it seems that cp-dna (rpl16) sequences are a suitable barcode molecular marker for population genetic studies of olive. there has been no report of rpl16 sequences for the cultivates olive. therefore, we could not compare these two forms together. table 2. genetic variability parameters for srap loci studied in olive populations. locus sample size ne h i 5 13 1.9187 0.4788 0.6718 8 13 1.9299 0.4818 0.6749 30 13 1.9683 0.4920 0.6851 36 13 1.9882 0.4970 0.6902 39 13 1.8989 0.4734 0.6663 53 13 1.9882 0.4970 0.6902 54 13 1.8943 0.4721 0.6650 55 13 1.9928 0.4982 0.6913 57 13 1.9562 0.4888 0.6819 58 13 1.9216 0.4796 0.6726 59 13 1.8943 0.4721 0.6650 64 13 1.8943 0.4721 0.6650 65 13 1.9865 0.4966 0.6898 67 13 1.9562 0.4888 0.6819 72 13 1.8943 0.4721 0.6650 mean 13 1.5918 0.3492 0.5242 st. dev 0.2911 0.1268 0.1506 ne = effective number of alleles. h = nei’s (1973) gene diversity. i = shannon’s information index [lewontin (1972)]. table 3. genetic differentiation parameters in the olive trees studied based on srap loci. locus sample size ht hs gst nm 2 13 0.2633 0.2381 0.0958 4.7202 3 13 0.3921 0.3145 0.1981 2.0240 4 13 0.3750 0.0000 1.0000 0.0000 7 13 0.0514 0.0472 0.0813 5.6481 10 13 0.2633 0.2381 0.0958 4.7202 13 13 0.3750 0.0000 1.0000 0.0000 14 13 0.1669 0.1162 0.3036 1.1472 17 13 0.2000 0.1746 0.1270 3.4365 22 13 0.0514 0.0472 0.0813 5.6481 23 13 0.3750 0.0000 1.0000 0.0000 24 13 0.1064 0.0873 0.1791 2.2910 28 13 0.1794 0.1508 0.1595 2.6340 31 13 0.2086 0.1634 0.2164 1.8105 32 13 0.3750 0.0000 1.0000 0.0000 34 13 0.1000 0.0944 0.0557 8.4721 36 13 0.5000 0.0000 1.0000 0.0000 39 13 0.3750 0.0000 1.0000 0.0000 41 13 0.1064 0.0873 0.1791 2.2910 43 13 0.2086 0.1634 0.2164 1.8105 44 13 0.5000 0.0000 1.0000 0.0000 45 13 0.3750 0.0000 1.0000 0.0000 47 13 0.1794 0.1508 0.1595 2.6340 48 13 0.1669 0.1162 0.3036 1.1472 50 13 0.3750 0.0000 1.0000 0.0000 51 13 0.3750 0.0000 1.0000 0.0000 52 13 0.3750 0.0000 1.0000 0.0000 53 13 0.5000 0.0000 1.0000 0.0000 58 13 0.4226 0.3434 0.1875 2.1669 62 13 0.3625 0.2744 0.2431 1.5564 68 13 0.2086 0.1634 0.2164 1.8105 69 13 0.1000 0.0944 0.0557 8.4721 75 13 0.5000 0.0000 1.0000 0.0000 76 13 0.3750 0.0000 1.0000 0.0000 mean 13 0.3680 0.1308 0.6445 0.2758 st. dev 0.0172 0.0085 figure 1. pcoa plot of olive trees based on srap data revealing genetic separation of populations. 129population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers its sequence analysis we obtained 183 bp long sequences in its region with 150 variable sites. the analysis revealed the presence of 8 haplotypes in its with haplotype diversity, hd: 0.80. an olive tree 7, 9, 11 and 13 had similar sequences and forms a single haplotype group. the nucleotide distance (p distance) of the studied trees (table 5), revealed that p distance among olive trees of population 1 varied from 0.36 to 0.54, while the same value in population varied from 0.01 to 0.49. maximum likelihood phylogenetic tree (ml) (figure 3) of the studied olive trees based on its sequences revealed that, trees of population 1, differ in their its sequences and were grouped in a separate clade. however, trees of populations 2, 3, and 4 were placed together in a single unresolved clade. this result indicates that its sequences can be used along with cp-dna barcodes in olive population genetic studies. comparing phylogenetic trees of srap markers, cp-dna and its sequences produced quart let distance = 0.56 and test performed based on the most agreeable sub-trees (mast) (figure. 4) revealed that, these markers do differentiate some of the olive trees and place them in distinct clades. joint phylogenetic its analysis of wild populations and randomly selected cultivated olives (figure. 5) revealed the genetic separation of these olives from each other. its sequences could differentiate different olive trees of wild populations but not the cultivars from each other. discussion genetic structure analysis of both cultivated and wild olive is important for breeding and conservation purposes (baldoni et al. 2006). olive cultivars can be considered as varieties of unknown origin, currently propagated vegetative by cutting or grafting. analysis of nuclear and cytoplasmic dna polymorphisms in mediterranean oleaster populations has shown that eastern oleaster populations differ greatly from those of the west mediterranean (besnard et al. 2001), while the genettable 4. nei genetic distance among the studied olives. pop 1 2 3 4 5 6 7 8 9 10 11 12 2 0.30 3 0.39 0.42 4 0.44 0.46 0.33 5 0.54 0.40 0.52 0.42 6 0.65 0.54 0.57 0.62 0.40 7 0.56 0.62 0.66 0.64 0.65 0.54 8 0.76 0.70 0.81 0.77 0.71 0.44 0.69 9 0.65 0.64 0.68 0.73 0.67 0.49 0.57 0.57 10 0.60 0.53 0.60 0.68 0.50 0.41 0.48 0.55 0.43 11 0.60 0.55 0.73 0.60 0.57 0.65 0.80 0.82 0.69 0.53 12 0.41 0.43 0.57 0.62 0.63 0.60 0.72 0.65 0.56 0.65 0.67 13 0.47 0.46 0.57 0.52 0.63 0.63 0.68 0.69 0.56 0.68 0.71 0.17 figure 2. tcs network of olive trees based on cp-dna sequences revealing almost separation of the studied populations. 130 rayan partovi et al. ic diversity of cultivated populations shows a complex patchy pattern (owen et al. 2005). the present investigation also revealed genetic difference between iranian wild populations and the cultivated olive forms. based on the frequency and distribution of polymorphisms, several authors suggested that many olive cultivars have been produced from naturally cross-bred genotypes (besnard et al. 2001), while, others, due to the great genetic distance between populations of wild olives and cultivars, suggested that many local cultivars may have an allochthonous origin (angiolillo et al. 1999; bronzini de caraffa et al. 2002). genetic diversity of both cultivated and wild olives has been investigated by using different molecular markers (bracci et al. 2011), revealing the genetic structure of these olive forms. in the present study, srap and cp-dna rpl sequences could be used in wild olive diftable 5. p nucleotide distance among olive plants based on its sequences. 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 0.53 3 0.52 0.43 4 0.56 0.50 0.36 5 0.54 0.51 0.46 0.45 6 0.49 0.36 0.25 0.36 0.33 7 0.48 0.35 0.24 0.35 0.32 0.01 8 0.48 0.35 0.24 0.35 0.33 0.01 0.00 9 0.48 0.35 0.24 0.35 0.33 0.01 0.00 0.00 10 0.48 0.35 0.24 0.35 0.33 0.01 0.01 0.01 0.01 11 0.48 0.35 0.24 0.35 0.33 0.01 0.00 0.00 0.00 0.01 12 0.48 0.35 0.24 0.35 0.32 0.01 0.00 0.00 0.00 0.01 0.00 13 0.48 0.35 0.24 0.35 0.32 0.01 0.00 0.00 0.00 0.01 0.00 0.00 figure 3. ml phylogenetic tree of the studied olive trees based on its sequences. figure 4. most agreeable sub-trees (mast) plot showing the common clades differentiated by its, cp-dna and issr trees. 131population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers ferentiation. cp-dna polymorphisms is used for phylogeographic, population genetic and forensic analyses in plants, but detecting cp-dna variation is sometimes challenging, limiting the applications of such an approach (besnard et al. 2011). according to our knowledge rpl16 sequences were only used in genetic variability assessment of tissue culture regenerated olive plants (kangarloo et al. 2016) and not in olive population genetic investigation. therefore, our study is the first time report on application of the cp-dna sequences for wild olive population differentiation. besnard et al. (2001) used eight complete sequences of cp-dna genomes of olea in their study. the reported low nucleotide divergence between olive cp-dna lineages, not exceeding 0.07%. based on these sequences, markers were developed for studying two single nucleotide substitutions and length polymorphism of 62 regions (with variable microsatellite motifs or other indels). they used these markers to study the cp-dna variation in cultivated and wild mediterranean olive trees. the discriminating power of cp-dna variation was particularly low for the cultivated olive tree with one predominating haplotype, but more diversity was detected in wild populations. this is almost in agreement with present study findings. besnard et al. (2001) and pérez-jiménez et al. (2013) suggested that cp-dna markers will have applications for a comparative study of the dynamic of wild olive tree populations in different environments, such as archipelagos and saharan mountains. such information may be relevant for defining appropriate strategies of prospection and in situ conservation of the wild olive tree. in conclusion, the present study revealed that a combination of neutral molecular markers, like sraps and cp-dna sequences are powerful markers to differentiate wild olive populations. acknowledgments this work was funded by islamic azad university, science and research branch and shahid beheshti university for providing the laboratory equipment for this investigation. references angiolillo a, mencuccini m, baldoni l. 1999. olive genetic diversity assessed using amplified fragment length polymorphisms. theoretical and applied genetics. 98: 411–421. azadi r. 2005. family oleaceae. flora of iran, no. 48. 1st ed. tehran, iran: research institute of forest and range lands, pp. 36. baldoni l, tosti n, ricciolini c (2006) genetic structure of wild and cultivated olives in the central mediterranean basin. annals of botany. 98(5):935-942. besnard g, baradat p, berville´ a .2001. genetic relationships in the olive (olea europaea l.) reflect multilocal selection of cultivars. theoretical and applied genetics. 102: 251–258. besnard g, hernández p, khadari b, dorado g, savolainen v .2011. genomic profiling of plastid dna variation in the mediterranean olive tree. bmc plant biology 11:80. bracci tm, busconi m, fogher c, sebastiani l .2011. molecular studies in olive (olea europaea l.): overview on dna markers applications and recent advances in genome analysis. plant cell reports. 30: 449-462. bronzini de caraffa v, maury j, gambotti c, breton c, berville´ a, giannettini j .2002. mitochondrial dna variation and rapd mark oleasters, olive and feral olive from western and eastern mediterranean. theoretical and applied genetics. 104: 1209–1216. drummond aj, rambaut a, suchard ma .2010. beast (v1.6.1). http://beast.bio.ed.ac.uk . accessed 15 october 2010. drummond aj, rambaut a, xie w (2010) beauti (v1.6.1) [computer program]. http://beast.bio.ed.ac. uk. accessed 15 october 2010. green ps .2002. a revision of olea l. 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cytosystematics and cytogenetics volume 73, issue 1 2020 firenze university press karyotypic investigation concerning five bromus species from several populations in iran sara sadeghian, ahmad hatami, mehrnaz riasat high genetic diversity and presence of genetic structure characterise the endemics ruta corsica and ruta lamarmorae (rutaceae) marilena meloni1, caterina angela dettori2, andrea reid3, gianluigi bacchetta2,4,*, laetitia hugot5, elena conti1 cytogenetic effects of c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. and mathematical analysis cihangir alaca1, ali özdemir1, bahattın bozdağ2, canan özdemir2,* clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 75(2): 109-117, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1514 caryologia international journal of cytology, cytosystematics and cytogenetics citation: suphat prasopsin, nawarat muanglen, sukhonthip ditcharoen, chatmongkon suwannapoom, alongklod tanomtong, weera thongnetr (2022) first report on classical and molecular cytogenetics of doi inthanon benttoed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand. caryologia 75(2): 109-117. doi: 10.36253/caryologia-1514 received: november 30, 2021 accepted: november 30, 2021 published: september 21, 2022 copyright: © 2022 suphat prasopsin, nawarat muanglen, sukhonthip ditcharoen, chatmongkon suwannapoom, alongklod tanomtong, weera thongnetr. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* 1 research academic supports division, mahidol university, kanchanaburi campus, saiyok, kanchanaburi, thailand 2 department of fisheries, faculty of agricultural technology, sakon nakhon rajabhat university, sakon nakhon, thailand 3 division of biology, faculty of science and technology, rajamangala university of technology thanyaburi, khlong luang, pathum thani, thailand 4 department of fishery, school of agriculture and natural resources, university of phayao, muang, phayao, thailand 5 program of biology, faculty of science, khon kaen university, muang, khon kaen, thailand 6 walai rukhavej botanical research institute, mahasarakham university, kantharawichai, maha sarakham, thailand *corresponding author. e-mail: weeraatah@hotmail.com abstract. this study analyzed the karyotype of cyrtodactylus inthanon kunya et al., 2015 from doi inthanon, chiang mai province, northern thailand. the metaphase and meiotic chromosome preparations were obtained by squash technique from bone marrow and testes, respectively. the chromosomes were stained by giemsa staining, ag-nor banding and molecular cytogenetics with fluorescence in situ hybridization (fish) using microsatellites d(ca)15, d(gc)15, d(cag)10 and d(gaa)10 as probes. the results showed the diploid chromosome number (2n) of 40. the chromosome types of metacentric, submetacentric, acrocentric and telocentric chromosomes were 12-4-2-22, respectively. the ag-nors banding technique provides the pair of nucleolar organizer regions (nors) on the telomeric region of the long arm of acrocentric pair 12. there are no sex differences in karyotypes between males and females. we also found that during metaphase i on meiosis of c. inthanon, the homologous chromosomes appeared synapsis of 20 bivalents. the microsatellite d(ca)15 signals were located on the sub-centromeric region of the metacentric pair 10, whereas the d(gc)15, d(cag)10 and d(gaa)10 repeats are highly accumulated throughout almost all entire chromosomes. the karyotype formula is as follows: c. inthanon (2n = 40), lm2 + lsm4 + lt6 + mm2 + mt8 + sm8 + sa2 + st8. keywords: bent-toed gecko, cyrtodactylus inthanon, ag-nor staining, karyotype, fish. 110 suphat prasopsin et al. introduction cyrtodactylus is a genus of bent-toed geckos which is widely distributed across south asia to melanesia (wood et al. 2012, grismer et al. 2020, 2021). this genus is the most species group of gekkotans, with approximately 306 species currently recognized (uetz et al. 2021). the gekkonidae found in thailand are highly diverse and include approximately 90 species. among these species, 38 species of bent-toed geckos (cyrtodactylus) are the most well-documented and show wide distribution in thailand (uetz et al. 2021). although a high number of cyrtodactylus species have already been discovered and many more explorations are anticipated based on the rapid discovery rate of new cyrtodactylus species, several are defined as endemic including c. sanook, c. saiyok and c. phuketensis (panitvong et al. 2014, pauwels et al. 2013, sumontha et al. 2012). recently, c. inthanon, doi inthanon bent-toed gecko was discovered as a new species from doi inthanon region, chiang mai province, northern thailand by kunya et al. (2015). it was a novel reptile endemic to doi inthanon strengthening the high significance of this mountain in terms of biodiversity preservation. in terms of endemic species, they are at higher risk of extinction because their habitat is limited or unique. additionally, they have been reported in recent years that the rate of destruction of cyrtodactylus habitat has increased due to both forest land encroachment and wildfires, especially in southeast asia where they are mainly distributed (chomdej et al. 2021). however, cytogenetic studies are still quite scarce within cyrtodactylus, mostly restricted to classical protocols. studies applying molecular cytogenetic approaches (i.e. chromosomal mapping of microsatellite sequences) where done only in two species of c. jarujini and c. doisuthep (thongnetr et al. 2021). up to date only seven species from over 306 recognized species have been cytogenetically examined (table 1). the diploid number among gekkonid lizards ranges from 2n=16 to 2n=46 with most of the karyotypes composed of 28-46 chromosomes (gorman 1973, schmid et al. 1994). the typical karyotype consists of a gradual series of acrocentric chromosomes which there is no difference between macro and microchromosomes (molavi et al. 2014). the occurrence of techniques related to dna are encouraging for the advance of the comprehension of the animal genome structure and evolution (martins et al. 2011; barreto et al. 2021). microsatellites are repetitive (in tandem) dna sequences of one to six nucleotides found in all eukaryotic organisms (cioffi and bertollo 2012; lópez-flores and garrido ramos 2012). in several species, these sequences can be found in long repetitions associated with heterochromatic regions (martins 2007; cioffi et al. 2011). this information on chromosomes is considered important along with other information for the identification of the species (campiranont 2003). the cytogenetic analysis of the higher molecular chromosome structure can provide invaluable insight for the management of threatened species, where dna alone could not address all genetic risks and threats to populations (potter and deakin 2018). the distribution of microsatellites on chromosomes could help on the elucidation of evolutionary processes that lead to a karyotypic macrostructure differentiation and even to the origin of sex chromosomes systems (cioffi et al. 2011), where the investigation of the sex-determining system was finished only one species, the bornean endemic cyrtodactylus pubisulcus through traditional cytogenetics (ota et al. 1992; keating et al. 2021) accordingly, the present study is the first cytogenetic study on c. inthanon from thailand accomplished with classical and molecular cytogenetic techniques. table 1. karyotype reviews in the genus cyrtodactylus. species 2n nf karyotype nor locality reference c. consobrinus 48 50 2bi-arm+46t malaysia ota et al. (1992) c. doisuthep 34 56 14m+6sm+2a+12t p9, 13 thailand thongnetr et al. (2021) c. interdigitalis 42 52 4m+2sm+4a+32t p12 thailand thongnetr et al. (2019a) c. inthanon 40 58 12m+4sm+2a+22t p12 thailand present study c. jarujini 40 56 8m+4sm+4a+24t p13, 14 thailand thongnetr et al. (2021) c. kunyai 40 52 8m+4sm+6a+22t p12 thailand thongnetr et al. (2019a) c. pubisulcus 42 44 2bi-arm+40t malaysia ota et al. (1992) c. saiyok 42 42 42t p15 thailand thongnetr et al. (2019b) remarks: 2n = diploid chromosome number, nors = nucleolus organiser regions, nf = fundamental number (number of chromosome arms), bi-arm = bi-armed chromosome, m = metacentric, sm = submetacentric, a = acrocentric, t = telocentric chromosome, p = chromosome pair and = not available. 111first report on classical and molecular cytogenetics of cyrtodactylus inthanon in thailand data provided here will increase our knowledge of cytogenetic information which can be used as a basis to comprehensively examine the taxonomy and evolutionary relationship of cyrtodactylus species and other gekkonids. materials and methods sample collection and chromosome preparation five male and five female specimens of c. inthanon were collected from the doi inthanon reinforces, chiang mai province, northern thailand (fig. 1). all bent-toed geckos were transferred to the laboratory and kept under standard conditions for one day prior to the experimentation. chromosomes were directly prepared in vivo (ota et al. 1990) by 0.1% colchicine were injected into the geckos’ intramuscular and abdominal cavity and then left for 8-10 hours. bone marrow (in male and female) and testis (male) were cut into small pieces and then mixed with 0.075 m potassium chloride (kcl). after discarding all large cell pieces, 15 ml of cell suspension was transferred to a centrifuge tube and incubated for 30-40 minutes, then centrifuged at 3,000 rpm for 8 minutes. the cell suspension was fixed in fresh cool fixative of methanol:glacial acetic acid (3:1) and gradually made up to 8 ml before centrifuging again at 3,000 rpm for 8 minutes, whereupon the supernatant was discarded. fixation was repeated until the supernatant was clear and the pellet was mixed with 1 ml fixative. giemsa’s staining, ag-nor banding technique and chromosome analysis a drop of the mixture was added to a clean and cold slide by micropipette followed by the air-dry technique. the slide was conventionally stained with 20% giemsa solution for 30 minutes (patawang et al. 2014). then, the slides were rinsed thoroughly with running tap water to remove excess stain. two drops of each 50% silver nitrate and 2% gelatin were droped on slides, respectively. then it was sealed with cover glasses and incubated at 60 °c for 5-10 minute. there after that it was soaked in distilled water until cover glasses are separated. (howell and black 1980). ten clearly observable metaphase cells with well spread chromosomes of each male and female were selected and photographed. the length of short arm chromosome (ls) and long arm chromosome (ll) were measured and the length of total arm chromosome (lt, lt = ls+ll) was calculated. the relative length (rl), the centromeric index (ci) and standard deviation (sd) of rl and ci were analysed according to the chromosome classification of chaiyasut (1989) and turpin and lejeune (1965). chromosome types were described as metacentric (m), submetacentric (sm), acrocentric (a) and telocentric (t) chromosomes, respectively. the fundamental number (nf, number of chromosome arms) was obtained by assigning a value of two to metacentric, submetacentric and acrocentric chromosomes and one to telocentric chromosomes. all parameters were used in karyotyping and idiograming. fluorescence in situ hybridization (fish) the use of microsatellite probes described by kubat et al. (2008) was followed here with slight modifications. the microsatellite probes: d(ca)15, d(gc)15, d(cag)10 and d(gaa)10 were directly labelled with cy3 at the 5 -́terminal during synthesis by sigma (st. louis, mo, usa). fluorescence in situ hybridization (fish) was performed under highly stringent conditions on mitotic chromosome spreads (pinkel et al. 1986). after denaturation of chromosomal dna in 70% formamide/2×ssc (saline sodium citrate) at 70 °c, spreads were incubated in 2×ssc for 4 minutes at 70 °c. the hybridization mixture (2.5 ng/μl each probe, 2 μg/μl salmon sperm dna, 50% deionized formamide, 10% dextran sulphate) was dropped on the slides and the hybridization was performed overnight at 37 °c in a moist chamber containing 2×ssc. the post hybridization wash was carried out with 1× ssc for 5 minutes at 65 °c. a final wash was performed at room temperature in 4×ssctween for 5 minutes. finally, the chromosomes were counterstained figure 1. general characteristic shape of the cyrtodactylus inthanon, the most prominent feature in the top view. 112 suphat prasopsin et al. with dapi (1.2 μg/ml), mounted in antifading solution (vector, burlingame, ca, usa) and analyzed in fluorescence microscope nikon eclipse. results diploid chromosome number (2n), fundamental number (nf) and karyotype the diploid number and nf in c. inthanon are 40 and 58, respectively. the karyotype of c. inthanon is composed of 12 metacentrics, 4 submetacentrics, 2 acrocentrics and 22 telocentrics there are exhibited no sex differences in karyotypes between males and females. (table 2 and fig. 2a-d). nucleolar organizer region and meiotic cell characteristics the determination of chromosome marker for this species is firstly obtained by using the ag-nor banding technique. the nucleolar organizer regions (nors) are observed on the telomeric region of the acrocentric chromosome pair 12 both male and female (fig. 2 e-h). the meiotic cell of c. inthanon reveals the diplotene phase (fig. 3), which shows synapsis between two the homologous and compacted chromosomes. the metaphase i (meiosis i, reductional division) can be defined as the 20 bivalents patterns of microsatellite the microsatellite d(ca)15 presents the signals highly accumulated at the interstitial subcentromeric region on short arms of metacentric chromosome pair 10 (fig. 4a) whereas, the microsatellite d(gc)15, d(cag)10 and d(gaa)10 are distributed weak signals throughout the whole chromosomes (fig. 4c-d). discussion from the previous reports, the chromosome exhibited various number in the cyrtodactylus, ranging from 34 to 48, however, the most frequent numbers were 40 and 42. the present study showed that the chromosome number of c. inthanon were 40. this result revealed accordance with other species that have been reported, such as c. jarujini and c. kunyai (thongnetr et al. 2019a, 2021). moreover, the diploid chromosome number (2n) differed from c. table 2. mean length of short arm chromosome (ls), length of long arm chromosome (ll), length of total chromosomes (lt), relative length (rl), centromeric index (ci) and standard deviation (sd) from 10 metaphases of male and female cyrtodactylus inthanon, 2n=40. chr. pair ls ll lt ci±sd rl±sd chr. size chr. type 1 3.568 4.695 8.263 0.567±0.026 0.101±0.008 large metacentric 2 2.325 4.177 6.503 0.640±0.019 0.079±0.003 large submetacentric 3 2.059 3.799 5.858 0.648±0.023 0.072±0.002 large submetacentric 4 0.000 5.921 5.921 1.000±0.000 0.072±0.005 large telocentric 5 0.000 5.894 5.894 1.000±0.000 0.072±0.003 large telocentric 6 0.000 5.330 5.330 1.000±0.000 0.065±0.004 large telocentric 7 0.000 4.827 4.827 1.000±0.000 0.059±0.003 medium telocentric 8 0.000 4.302 4.302 1.000±0.000 0.052±0.002 medium telocentric 9 0.000 3.994 3.994 1.000±0.000 0.049±0.002 medium telocentric 10 1.688 2.476 4.164 0.595±0.021 0.051±0.002 medium metacentric 11 0.000 3.786 3.786 1.000±0.000 0.046±0.002 medium telocentric 12* 1.069 2.431 3.500 0.705±0.070 0.043±0.003 small acrocentric 13 0.000 2.785 2.785 1.000±0.000 0.034±0.003 small telocentric 14 0.000 2.707 2.707 1.000±0.000 0.033±0.001 small telocentric 15 0.000 2.334 2.334 1.000±0.000 0.028±0.004 small telocentric 16 1.202 1.698 2.900 0.584±0.035 0.036±0.004 small metacentric 17 0.000 2.166 2.166 1.000±0.000 0.027±0.003 small telocentric 18 1.087 1.512 2.599 0.582±0.025 0.032±0.003 small metacentric 19 0.916 1.284 2.200 0.586±0.016 0.027±0.003 small metacentric 20 0.796 0.999 1.795 0.557±0.075 0.022±0.001 small metacentric remark: chr. = chromosome; * = satellite chromosome (nucleolar organizer region, nor). 113first report on classical and molecular cytogenetics of cyrtodactylus inthanon in thailand figure 2. metaphase chromosome plates and karyotypes of male and female cyrtodactylus inthanon, 2n=40 by conventional staining (a-d) and ag-nor banding technique (e-h). scale bars indicate 5 micrometers. the arrows indicate nucleolar organizer regions/nor. 114 suphat prasopsin et al. doisuthep (2n=34), c. interdigitalis, c. pubisulcus, c. saiyok (2n=42) and c. consobrinus (2n= 48) (ota et al. 1992, thongnetr et al. 2019a, 2019b, 2021). the different cyrtodactylus species underwent an extremely diversified karyotype evolution, considering the numerical and structural aspects of their complements, with the nf that varied from 42 to 58. the karyotype of c. inthanon is composed of 12 metacentric, 4 submetacentric, 2 acrocentric and 22 telocentric chromosomes. the karyological characteristics of c. inthanon obtained in the present study is the first report of chromosome sizes and the chromosome types. in other species of cyrtodactylus, the different karyological structure can be found as well. however, the karyotype of c. inthanon resemble other cyrtodactylus and other gekkonids, which comprised many gradient mono-armed (telocentric) and few bi-armed chromosomes (metaor submetacentric). the proximity of chromosome number and karyotype feature within genus cyrtodactylus represents a close evolutionary line in the group. (trifonov et al. 2011). this analysis was performed to highlight the combined importance of the different chromosome rearrangements in the evolutionary modeling of their karyotypes, such as robertsonian rearrangements or centric fission (ueno and takai 2000) fusion and especially, pericentric inversions (jacobina et al. 2011). the nucleolar organizer regions (nors) represent the location of genes that have a function in ribosome synthesis (18s and 28s ribosomal rna). nors of c. inthanon exhibited a single pair of ag-nors located on the telomeric region on the long arm of the acrocentric and are similar to the previous reports of the genus cyrtodactylus, e.g., c. interdigitalis, c. kunyai (thongnetr et al. 2019a, 2021). the metaphase i (meiosis i, reductional division) was found in c. inthanon, which showed as the 20 bivalents (fig. 3). no metaphase i cell with partially paired bivalents, which are speculated to be male heteromorphic sex chromosomes in this species. from this result, the behavior and number of chromosomes in metaphase i confirmed of each other’s accuracy and also verified the accuracy of diploid chromosomes in somatic cells. microsatellites or simple sequence repeats (ssrs) are oligonucleotides of 1-6 base pairs in length, forming excessive tandem repeats of usually 4 to 40 units (tautz and renz 1984, ellegren 2004, chistiakov et al. 2006). they showed abundant distribution throughout eukaryotic genomes, being dispersed or clustered both in euchromatin or heterochromatin. they are highly polymorphic regarding copy number variations (ellegren 2004). in our present study in c. inthanon exhibited the microsatellite d(ca)15 was revealed that the signals highly accumulated at the interstitial subcentromeric region on short arms of metacentric chromosome pair 10 (fig. 4a), whereas, the microsatellite d(gc)15, d(cag)10 and d(gaa)10 were distributed weak signals throughout the whole chromosomes (fig. 4c-d). in previous study, microsatellite pattern (the dinucleotides d(a)20, d(cag)10, d(cgg)10, d(gaa)10 and d(ta)15) on c. jarujini and c. doisuthep accumulated exclusively in telomeric and subtelomeric chromosomal regions bearing dispersed over the whole genomes including chromosomes and some had strong signals on only a pair of homologous chromosomes (thongnetr et al. 2021). however, the results clearly indicate that the microsatellite repeats are in high copy number on some chromosome pairs, according to previous reports on reptile groups (pokorná et al. 2011; matsubara et al. 2013). the first cytogenetic study of the c. inthanon has enabled us to delineate the process of chromosomal reorganization in this group chromosome system and figure 3. metaphase i chromosome plate and karyotypes of cyrtodactylus inthanon, n=20 by conventional staining technique. scale bars indicate 5 micrometers. 115first report on classical and molecular cytogenetics of cyrtodactylus inthanon in thailand fish mapping. the results obtained here can be used to support the further investigation on taxonomy, biodiversity conservation and evolutionary relationship among the genus cyrtodactylus and others. acknowledgements this research project was financially supported by mahasarakham university 2021 and unit of excellence 2022 on biodiversity and natural resources management, university of phayao (ff65-uoe003). references barreto cav, peixoto ma, leite de souza k, travenzoli mn, feio rn, dergam, ja. 2021. further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae). caryologia. 74(3): 169–175. campiranont a. 2003. cytogenetics (2nd ed). department of genetics, faculty of science, kasetsart university, bangkok. thai. chistiakov da, hellemans b, volckaert fam. 2006. microsatellites and their genomic distribution, evolution, function and applications: a review with special reference to fish genetics. aquaculture. 255: 1–29. chomdej s, pradit w, suwannapoom c, pawangkhanant p, nganvongpanit k, poyarkov na, che a, gao y, gong s. 2021. phylogenetic analyses of distantly related clades of bent-toed geckos (genus  cyrtodactylus) reveal an unprecedented amount of cryptic diversity in northern and western thailand. sci rep. 11: 2328. cioffi mb, bertollo lac. 2012. chromosomal distribution and evolution of repetitive dnas in fish. genome dyn. 7: 197–221. cioffi mb, kejnovsky e, bertollo lac. 2011. the chromosomal distribution of microsatellite repeats in the genome of the wolf fish hoplias malabaricus, focusing on the sex chromosomes. cytogenet genome res. 132(4): 289–296. ellegren h. 2004. microsatellites: simple sequences with complex evolution. nat rev genet. 5: 435–445. gorman gc. 1973. the chromosome of the reptilia, a cytotaxonomic interpretation. in: chiarelli a b and cappana e, editors. cytotaxonomy and vertebrate evolution. new york (ny): academic press; 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(poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(1): 135-150, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1041 caryologia international journal of cytology, cytosystematics and cytogenetics citation: a. okorie asita, s. magama, t. mahali moahloli, s. baholo (2021) evaluation of extracts of wild cannabis sativa l. for genotoxicity and phytochemical composition. caryologia 74(1): 135-150. doi: 10.36253/caryologia-1041 received: august 03, 2020 accepted: january 23, 2021 published: july 20, 2021 copyright: © 2021 a. okorie asita, s. magama, t. mamoroesi moahloli, s. baholo. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. evaluation of extracts of wild cannabis sativa l. for genotoxicity and phytochemical composition asita okorie asita*, sibusisiwe magama, thato mamoroesi moahloli, selometsi baholo department of biology, national university of lesotho, p.o. roma 180, maseru 100, lesotho, southern africa email address: aoasita@yahoo.co.uk, ao.asita@nul.ls; sibusisiwe.magama@gmail.com; kimanethato@gmail.com; selometsibaholo@gmail.com. *corresponding author. abstract. cannabis sativa l. is used as medicine and narcotic in lesotho. phytochemical composition and total phenolics content (tpc) for hexane, chloroform, ethyl acetate and methanol extracts of aerial parts of c. sativa were determined. ethyl acetate extract (0.1875, 0.375 and 0.75 mg ml-1) and methanol extract (0.75, 1.5 and 3.0 mg ml-1) were evaluated for cytotoxicity, genotoxicity and modulation of cyclophosphamide (cp, 1.25 mg ml-1)and ethylmethane sulphonate (ems, 0.25 mg ml-1)-induced genotoxicity using allium cepa root meristem assay. cp or ems did not reduce mitotic index (mi) of cells, hence not cytotoxic when compared with negative control using the t-test (p>0.05), but genotoxic. both extracts were genotoxic with methanol extract also being cytotoxic. genotoxicity was the number of aberrant cells per 100 mitotic cells. modulatory effect (me) was obtained by comparing mutagen-induced genotoxicity with mixture-induced genotoxicity and expressed as the number of units of mutagen-induced genotoxicity that equalled the mixture-induced genotoxicity. me was either positive or negative and significant only if me = ≥ 2. both extracts were genotoxic with methanol extract also being cytotoxic. aberrations observed were sticky chromosomes, c-metaphase, anaphase and telophase bridges, chromosome fragments and laggards. mixture of methanol extract with cp or ems was more genotoxic (+me range = 1.61-11.89) than the mutagen or extract alone which suggested synergistic interaction. mixture of ethyl acetate extract with cp induced insignificant +me. mixture of ethylacetate extract with ems was significantly more genotoxic (+me = 2.20) than ems only at high extract concentration. the methanol and ethylacetate extracts of c. sativa were not anti-genotoxic to cpor emsinduced genotoxicity. tpcs for hexane, chloroform, ethyl acetate and methanol extracts were 39831.46, 2544.94, 2438.20 and 56601.12 mg gae/gram dry weight respectively. the differences in the cytotoxicity and mes of the extracts were attributed to differences in phytochemical composition of extracts. keywords: medicinal cannabis, phenolics, modulatory effects, cyclophosphamide, ethyl methanesulphonate, lesotho. 136 asita okorie asita, sibusisiwe magama, thato mamoroesi moahloli, selometsi baholo 1. introduction different human civilizations have depended for many centuries on plants and plant products for their medicinal (balandrin et al. 1985) and recreational (siegel 1977) needs. the scientific basis for the use of plants in traditional medicine, has been attributed largely, to secondary metabolites (sms) which have been shown to possess various biological activities (bourgaud et al. 2001); therefore much of the protective and therapeutic effects of plants have been attributed to phytochemicals such as alkaloids, terpenoids, tannins, phenolics, etc. (harborne 1998; hertog et al.1993; zhang et al. 2001). the concoctions used in traditional medicine are usually crude extracts in water, alcohol, distillates or essential oils, which contain many sms from various structural groups and their activity is often due to synergistic interactions of the sms present (eid et al. 2012; mulyaningsih et al. 2010). at high concentrations, sms change membrane f luidity and increase permeability. therefore, many lipophilic sms exhibit antimicrobial and cytotoxic activities and are responsible for the apparent broad-spectrum activity of concoctions used in traditional medicine (van wyk and wink 2015; wink 2015). in lesotho, as in many other countries in the world, a system of traditional medicine based on the use of plants, birds, animals, their products and their combinations to treat a broad spectrum of communicable and noncommunicable diseases is still being practiced (shale et al. 1999; padmanabhan and sujana, 2008). one plant species commonly used in traditional medicine in lesotho and southern africa is cannabis sativa (c. sativa), (ranotsi et al. 2012). other names for cannabis are marijuana, weed, dagga and “matekoane” in sesotho (ranotsi et al. 2012; bloomer 2019). this plant has been used for multiple purposes (medicinal, recreational, seed oil and industrial fiber, etc.) for thousands of years (elsohly and gul, 2014). in lesotho, c. sativa is used as medicine for all kinds of ailments such as heart burn, blood pressure and “nerves” as a recreational drug, and as part of religious rites (laniel 1999). a concoction of cannabis is a complex mixture of active compounds (phytochemicals) of which about 545 have been identified, 104 are cannabinoids or phytocannabinoids (as they originate from the plant) as well as 22 noncannabinoid constituents (turner et al. 1980; elsohly and slade 2005; elsohly and gul, 2014). the cannabinoids include δ9-tetrahydrocanabinol (thc), cannabidiol (cbd), cannabigerols (cbg), cannabichromenes (cbc), cannabinol (cbn) and cannabinodiol (cbdl) (el-alfy et al. 2010) found in the flowers, to a lesser extent the leaves, and minimally in the stems, and seeds (elsohly and gul 2014). thc is known as the major psychoactive component of cannabis that is responsible for causing addiction to marijuana (ashton, 2001; national institute on drug abuse (nida), 2018). the importance of plants as sources of medicines not withstanding, investigations have revealed that many plants which are used as food or in traditional medicine have mutagenic, cytotoxic and genotoxic effects in in vitro and in vivo assays (higashimoto et al. 1993; schimmer et al. 1994; kassie et al. 1996; çelik and aslantürk 2007). in a review by marselos and karamanakos (1999), they concluded that there was no consensus on the induction of point mutations by cannabinoids, while some experimental results suggest that cannabinoids may cause chromosomal damage (zimmerman and zimmerman 1990) and act as tumour promotors in animals. in addition, the extracts of some plant species have been observed to induce both mutagenic and antimutagenic effects on known mutagens in different test systems (debisri et al. 1996). the content of active compounds in plant species also vary according to their genetics, climatic factors, soil characteristics and the time of harvesting (ramelet 2015); and when plant materials are extracted with solvents of different polarities, often the different solvent fractions contain different biomolecules (herrera-ruiz et al. 2008). studies on agents that modulate carcinogen-induced genotoxic effects in experimental animals provide end points that can be used for assessing the antimutagenic or anticarcinogenic properties of putative chemopreventive compounds and for predicting their protective efficacy in humans (khaidakov et al. 2001). in view of the foregoing therefore, the aim of this study was to evaluate hexane, methanol, ethyl acetate and chloroform extracts of wild cannabis sativa for phytochemical composition, genotoxicity and the modulation of cyclophosphamide (cp)and ethyl methanesulphonate (ems)induced genotoxicity using the allium cepa chromosome aberration assay system. the allium cepa l assay is an in vivo assay and one of the established plant bioassays, validated by the international programme on chemical safety (ipcs, who), as an efficient and standard test for chemicals screening, in situ monitoring of the genotoxicity of environmental substances (leme and marin-morales 2009) and to evaluate the genotoxic potential of medicinal plants (camparoto et al. 2002; knoll et al. 2006; fachinetto et al. 2007; lubini et al. 2008; fachinetto et al. 2009). the allium cepa l assay tests genotoxicity using chromosomes and therefore detects chromosome structural and numerical alterations (tedesco and laughinghouse 2012; bonciu et al. 137evaluation of extracts of wild cannabis sativa l. for genotoxicity and phytochemical composition 2018). cp is an antineoplastic indirect-acting (promutagen) alkylating agent (mohn and ellenberger 1976; hales 1982) while ems is a direct-acting mutagen, tetratogen, and brain carcinogen (stubbs et al. 1997). 2. materials and methods 2.1 test organism onion (a. cepa) seeds of the variety, texas grano 502 p.r.r., a product of sakata seeds, lanseria 1748, republic of south africa were purchased from maseru garden centre, lesotho in southern africa. 2.2 mutagens and chemicals cyclophosphamide (cp) and ethyl methanesulfonate (ems) are products of fluka (biochemika, germany). methanol (absolute) is a product of associated chemical enterprises (pty) ltd (johannesburg, south africa); hydrochloric acid glacial and acetic acid are products of unilab (krugerdorp, south africa); acetocarmine stain was obtained from carolina biological supply company, burlington, north carolina, usa. 2.3 plant material collection and preparation aerial parts of the female plant of wild c. sativa were collected from the thaba bosiu area, some 12 km from the national university of lesotho (nul) campus, in the maseru district of lesotho where they grow in a location with the following geographical coordinates: latitude: 29°22’49˝s, longitude: 27°33’13” e and at an altitude of 1600 m. the aerial parts of the sample were dried in a fanned labcon oven at 37°c to a constant weight and brittle, about 48 hours. thereafter, the pieces were segmented and ground to a fine powder using a pulveriser (kenwood) and the powder was stored in sealed amber bottles in the dark at room temperature. 2.4 preparation of the crude c. sativa extracts sequential solvent extraction of the ground powder was done according to the method outlined in razak et al. (2014); padhi and panda (2015); fayera et al. (2018) with slight modifications. all crude extracts (hexane, chloroform, ethyl acetate and methanol) were stored at 4°c until further investigation for genotoxicity, modulatory effects on mutagen-induced genotoxicity and phytochemical profiling. 2.5 qualitative phytochemical screening of crude extracts of c sativa. the crude extracts of c. sativa prepared with hexane, chloroform, ethyl acetate and methanol were subjected to a qualitative screening for the presence of major phytochemical classes using standard phytochemical methods and the appropriate reagents and chemicals according to the modified methods of trease and evans (1984); trease and evans (2002); soni and sheetal (2013); nwaoguikpe et al. (2014) and uddin et al. (2014). each reaction mixture was visually assessed as in lu et al. (2014), for precipitate formation, foam formation, colour changes and colour intensity according to the following key: (+), low intensity of colour and/or precipitate; (++), moderate intensity of colour and/or precipitate; (+++), strong intensity of colour and/or precipitate (-), not detected (either absent or below the detection limit). the list of screening tests that were carried out is shown in table 1. 2.6 determination of the total phenolic content of c. sativa extracts using the folin-ciocalteau assay determination of the total phenolic content for each of the extracts was done by the method of mcdonald table 1. phytochemical screening tests. phytochemical name of test colour for positive test 1. flavonoids shinoda pink 2. alkaloids wagner blue-black 3. tannins ferric chloride blue-black/green 4. terpenoids salkowski reddish-brown 5. saponins foam test foam formation 6. simple phenols ferric chloride green 7. polyphenols ferric chloride blue 8. anthocyanins naoh blue-green betacyanins naoh yellow quinones hcl green phlobatannins hcl red precipitate anthraquinones hcl+chloroform+ammonia rose-pink/violet coumarins naoh+chlroform yellow phytosterols salkowski red cardiac glycosides and cardenolides keller-kiliani’s brown-red ring reducing sugars benedict’s red precipitate proteins biuret violet amino acids ninhydrin purple fatty acids diethyl ether transparent stain 138 asita okorie asita, sibusisiwe magama, thato mamoroesi moahloli, selometsi baholo et al. (2001) with slight modifications.the total phenolic content of each extract was recorded in milligram gallic acid equivalents (gae) per gram of dry weight of extract from the gallic acid standard curve (wong et al., 2012; moyo et al. 2013; magama et al., 2018). the total phenolic content in each extract determined as milligram gallic acid equivalents (gae) per gram of dry weight of extract was calculated using the following formula: (1) where t is the total phenolic content of the extracts in mg gae per gram of dry weight of extract, c is the concentration of the gallic acid established from the calibration curve in mgml-1, v is the volume of extract in ml and m is the mass of the plant extract in g. 2.7 genotoxicity experiments the preliminary assay to select concentrations of mutagens and plant extracts to use and genotoxicity assay (including the treatment of allium cepa seedlings with test agents, root harvest, slide preparation and scoring of slides) were conducted according to the methods of asita et al. (2017). due to insolubility of the hexane and chloroform extracts of c. sativa only the methanol and ethyl acetate extracts were evaluated for genotoxicity using 2.5% acetone (v/v in distilled water) as the solvent. the 2.5% acetone was not toxic or genotoxic to the onion root meristem cells. from the results of the preliminary assays to select the concentrations of mutagens and plant extracts to use, the following concentrations of plant extracts (in mgml-1); methanol extract (0.75, 1.5 and 3.00) and ethyl acetate extracts (0.750, 0.375, 0.1875 and 0.09375); cp (1.25.00) and ems (0.250) were assessed for cytotoxicity that is, mitotic index (mi), genotoxicity (gt) and the modulatory effect (me) of plant extracts on mutagen-induced genotoxicity. the aberrations assessed were: sticky chromosomes (s), c-metaphase (c-mit), lagging chromosomes (l), chromosome bridges at anaphase and telophase (a.b) and chromosome fragment (f). for calculating the gt, only aberrant mitotic cells were considered. 2.8 analysis of slide preparations 2.8.1 cytotoxicity the mitotic index (mi) was expressed as the number of dividing cells per 100 cells scored according to the formula: mi = number of dividing cells/ total number of cells scored x 100. (2) the mi was used as a measure of cytotoxicity (ct). the mi of each treatment group was compared with that of the negative control group using t-test at a probability level of 0.05, using the spss for windows, version 11.0 software. 2.8.2 genotoxicity genotoxicity (gt) was expressed as the number of aberrant mitotic cells (amc) per 100 mitotic cells [i.e amc + normal mitotic cells (nmc)] scored according to the formula: frequency of gt = amc/ (amc + nmc) x 100 (3) the mean gt of each group of three slides per concentration of test agent was compared with that of the negative control group using t-test. p values less than 0.05 (p < 0.05) were considered as indicative of significance. 2.8.3 modulatory effect (me) of plant extracts on mutagen-induced genotoxicity the modulatory effect (me) of plant extract on cp or ems-induced genotoxicity (gt) was calculated using the formula of asita et al. (2017): me = (b – c) – (a – c) / (a – c) (4) where ‘a’ is the genotoxicity induced by the mutagen (cp or ems) alone, i.e. mutagen-induced genotoxicity; ‘b’ is the genotoxicity induced by mixture of plant extract and mutagen, i.e. mixture-induced genotoxicity and ‘c’ is the genotoxicity induced by negative control, such as tap water alone. the modulatory effect (me) was thus obtained by comparing the mutagen-induced genotoxicity (a) with the mixture-induced genotoxicity (b). the me value indicated the number of units of the mutagen-induced genotoxicity (a) that equaled the mixture-induced genotoxicity (b). me was significant only if me was ≥ 2, i.e mixture was at least twice (200%) more (+) or less (-) genotoxic than mutagen alone. a positive me (+me) indicated that the mixture was more genotoxic (increased gt) than the mutagen and if mixture is also more genotoxic than the genotoxic plant extract alone then a synergistic interaction is inferred. 139evaluation of extracts of wild cannabis sativa l. for genotoxicity and phytochemical composition but mutagen-potentiation is inferred if mixture is less genotoxic than the non-genotoxic plant extract alone a negative me (-me) indicated that the mixture was less genotoxic (reduced gt) than the mutagen alone. if mixture is less genotoxic than the mutagen and the genotoxic plant extract then antagonism is infered. however, if mixture is less genotoxic than mutagen and also, moreor less genotoxic than the non-genotoxic plant extract then it is antimutagenicity. 2.8.4 data analysis in the determination of total phenolics content, data was expressed as means ± standard deviations of three replicate determinations using microsoft excel 2016. differences between controls and treatment groups were determined using student’s t-test. p-values of less than 0.05 (p<0.05) were considered statistically significant using the ibmspss statistics, version 20 software. regression equations and graphs were used for the determination of milligram gallic acid equivalents (mggae equivalents) per gram of dry extract and the concentration of extract needed to inhibit oxidation by 50% (ic50). for the genotoxicty assays, the mean value of each group of three slides per concentration of test agent was compared with that of the negative (solvent) control group using student’s t-test and the chi square test. p-values less than 0.05 (p < 0.05) were considered as indicative of significance. 3. results 3.1 qualitative biochemical profile of c. sativa solvent extracts in table 2 is presented the qualitative phytochemical profile of different solvent extracts of c. sativa obtained from the various tests. the methanol extract contained the highest number of the different phytochemical classes (15/19), followed by hexane and chloroform (9/15 each) and ethyl acetate (7/15). polyphenols as a class and the polyphenols (namelyanthocyanins, betacyanins, coumarins, and flavonoids), quinones (aromatic ketones), simple phenols were detected in trace amounts in the hexane, chloroform and methanol extracts but not detected in the ethylacetate extract. coumarins (also polyphenols) were present in all extracts, though in traces only in the ethyl acetate extract. traces of amino acids were also detected in all the solvent extracts. terpenoids were detected only in the hexane and methanol extracts. flavonoids (also polyphenolic), alkaloids, saponins (steroid and terpenoid glycosides) and phlobatannins (also polyphenolic), were detected in the methanol extract only. phytosterols (unsaturated steroid alcohols) were detected only in the hexane extract.cardiac glycosides and cardenolides, proteins and fatty acids were not detected in any of the solvent extractives. 3.2 quantitative determination of total phenolics content in fig. 1, is presented the gallic acid calibration curve for determination of total phenolic content of the different solvents (hexane, chloroform, ethyl acetate and methanol) extracts, where y was the mean absorbance of the sample at 760nm and x the concentration established from the gallic acid calibration curve. the regression equation was y = 0.0712x – 0.1055; the total phenolic content of the hexane extract, x, with y = 0.0363 was found to be 39 831.46mg gae/gram dry weight. the total phenolic content of the chloroform extract, x, with y = 0.0757 was 2544.94 mg gae/gram dry weight. the total phenolic content of the ethyl acetate extract, x, with y = 0.0681 was 2438.20mg gae/gram dry weight. the table 2. phytochemical screening test results for solvents extracts of wild c. sativa. test c. sativa crude plant extracts hexane chloroform ethylacetate methanol (95%) flavonoids ++ alkaloids +++ tannins + +++ terpenoids +++ +++ saponins + simple phenols + + + polyphenols +++ +++ +++ +++ anthocyanins +++ +++ ++ +++ betacyanins +++ ++ ++ ++ quinones +++ +++ +++ +++ phlobatannins + anthraquinones +++ coumarins ++ + + +++ phytosterols +++ cardiac glycosides and cardenolides reducing sugars + ++ +++ proteins amino acids + ++ ++ +++ fatty acids key: low intensity = “+”; moderate intensity = “++”; strong intensity = “+++”; not detected or negative = “-“(lu et al., 2014). 140 asita okorie asita, sibusisiwe magama, thato mamoroesi moahloli, selometsi baholo total phenolic content of the methanol extract, x, with y = 0.0960 was 56 601.12 mg gae/gram dry weight. 3.3 cytotoxicity and genotoxicity 3.3.1 figures and tables photographs of the most representative pictures of normal mitotic cells and cells containing the different types of chromosome aberrations that were observed and scored are presented in figure 2. the results of the cy totoxicity and genotoxicity experiments with the methanol and ethyl acetate extracts of c. sativa are presented in tables 3 and 4 respectively. y = 0,0712x 0,1055 r² = 0,9219 -0,1000 -0,0500 0,0000 0,0500 0,1000 0,1500 0,2000 0,2500 0,3000 0,3500 0,4000 0,4500 0 0,015625 0,03125 0,0625 0,25 0,5 1 a bs or ba nc e (7 60 nm ) concentration (mg/ml) gallic acid calibration curve figure 1. gallic acid calibration curve for determination of total phenolic content for hexane and methanol extracts. a b c d e f g h i j k l m n o p q r s t u v figure 2. photomicrographs of cells of allium cepa showing untreated cells in normal division stages and chromosomal aberrations (arrowed) in cells treated with methanol and ethyl acetate extracts of cannabis sativa or mixture of extracts with ems or cyclophosphamide. (a) interphase (b) normal prophase (c) normal metaphase (d) normal metaphase (e) early anaphase (f ) late anaphase (g) telophase (h) pyknotic interphase nuclei with micronucleus (i) prophase with sticky chromosomes (j) metaphase with sticky chromosomes (k) metaphase with sticky chromosomes (l) c-metaphase (m) metaphase with dislocated chromosome (n) late anaphase with dislocated chromosome (o) anaphase with sticky and scattered chromosomes (p) late anaphase with chromosome bridge (q) telophase with lagging chromosome (r) telophase with sticky chromosomes and bridge (s) telophase with chromosome bridge and lagging (t) telophase with chromosome bridge and fragment (u) telophase with chromosome bridge (v) telophase with chromosome fragment and lagging. magnification is 1000 x. 141evaluation of extracts of wild cannabis sativa l. for genotoxicity and phytochemical composition 3.3.2 results for methanol (95%) extract experiments in table 3 in table 3 are the results of cytotoxicity and genotoxicity experiments with methanol (95%) extracts of c. sativa and the mutagens, cp and ems. (p+m)/(a+t) ratio: examination of the (p+m)/(a+t) ratio in column 8 of table 3 shows that only the treatment with the lowest concentration (0.75 mgml-1) of c. sativa extract alone or in a mixture with ems (0.75 mgml-1) induced a significant change in (p+m)/(a+t) ratio, when compared with the solvent (2.5% acetone) treated negative control group (p < 0.05). cytotoxicity: examination of the mi in column 9 of table 3 shows that cp (1.25 mg ml-1) and ems (0.25 mg ml-1) were not toxic. all the concentrations of the methanol extract of c. sativa (0.75, 1.5, 3.0 mg/ml) and their mixtures with cp (1.25 mg ml-1) or ems (0.25 mg ml-1) induced significant reduction of the mi (was toxic) when compared to the solvent (2.5% acetone) treated negative control (p<0.05). genotoxicity (gt): examination of induction of genotoxicity in column 10 of table 3 shows that cp (1.25 mg ml-1), ems (0.25 mg ml-1), methanol extract (0.75, 1.5, 3.0 mg/ml) and the mixtures of cp (1.25 mg ml-1) or ems (0.25 mg ml-1) with each contration of c. sativa table 3. cytotoxic and genotoxic effects of methanol extracts of c. sativa, ems and cp on meristem cells of onion root tip and the modulatory effects (me) of the methanol extract of c. sativa on emsor cp-induced genotoxicity. treatment (tc in mg/ml) conc. inter. cells total number of cells in the stages of mitosis in 2000 cells scored total number of cells scored (p+m)/ (a+t) mi genotoxicity modulatory effect n abn total (n + abn) extract on cp extract on ems acetone (2.5%) mean 1790.00 208.00 2.00 210.00 2000 2.60 10.50 1.05     sd 75.54 75.54 0.00 75.54 0.00 0.09 3.78 0.41     cp (1.25) mean 1856.00 135.33 8.67 144.00 2000 2.56 7.20 7.31#     sd 44.80 48.91 6.11 44.80 0.00 0.33 2.24 7.06     ems (0.25) mean 1815.33 171.00 13.67 184.67 2000 3.86 9.23 5.50#     sd 99.36 86.63 16.50 99.36 0.00 1.60 4.97 6.42     c. sativa (0.75) mean 1954.33 34.33 11.33 45.67 2000 8.87 ɉ 2.28* 24.78#     sd 1.53 0.58 1.15 1.53 0.00 0.99 0.08 1.80     c. sativa (1.5) mean 1984.33 11.33 4.33 15.67 2000 3.47 0.78* 27.58#     sd 1.15 0.58 0.58 1.15 0.00 0.92 0.06 1.58     c. sativa (3.0) mean 1942.00 39.67 18.33 58.00 2000 3.59 2.90* 31.17#     sd 16.09 9.71 6.51 16.09 0.00 2.16 0.80 2.69     c. sativa (0.75) + cp mean 1989.00 2.33 8.67 11.00 2000 2.50 0.55* 77.91# 11.27+†   sd 2.00 1.53 2.89 2.00 0.00 0.90 0.10 14.34     c. sativa (1.5) + cp mean 1991.00 1.67 7.33 9.00 2000 4.67 0.45* 81.76# 11.89+†   sd 1.00 0.58 0.58 1.00 0.00 2.31 0.05 5.09     c. sativa (3.0) + cp mean 1981.00 10.00 9.00 19.00 2000 3.88 0.95* 47.59# 6.43+†   sd 4.00 2.65 1.73 4.00 0.00 2.51 0.20 4.65     c. sativa (0.75) + ems mean 1959.00 24.00 17.00 41.00 2000 4.81 ɉ 2.05* 41.55#   8.10+† sd 12.49 8.00 5.29 12.49 0.00 0.97 0.62 4.76     c. sativa (1.5) + ems mean 1950.00 43.67 6.33 50.00 2000 2.83 2.50* 12.67#   1.61+† sd 0.00 0.58 0.58 0.00 0.00 0.54 0.00 1.15     c. sativa (3.0) + ems mean 1964.33 30.33 5.33 35.67 2000 3.14 1.78* 15.00#   2.14+† sd 15.89 13.58 2.31 15.89 0.00 0.48 0.79 0.33     key: tc = test compound; n = normal mitotic cells (comprising prophase, metaphase, anaphase and telophase); abn = aberrant mitotic cells; sd = standard deviation; cp = cyclophosphamide; ems = ethylmethane sulphonate; c.s = cannabis sativa; mi = mitotic index; ɉ = p+m/a+t ratio (significant increase in ratio compared to negative control, p<0.05 in the t-test, n = 3); * = tc is toxic (mi treatment significantly different from negative control, p<0.05 in the t-test, n = 3); # = tc is genotoxic (significant difference from negative control, p<0.05 in the t-test, n = 3); +† = c.s + mutagen mixture more genotoxic than mutagen or c.s alone (synergism); +‡ = c.s + mutagen mixture less genotoxic than mutagen or c.s alone (antagonism); † = c.s + mutagen mixture more genotoxic than mutagen alone but less than c.s alone; ‡ = c.s + mutagen mixture less genotoxic than mutagen alone (antimutagenicity) but more than c.s alone. 142 asita okorie asita, sibusisiwe magama, thato mamoroesi moahloli, selometsi baholo extract used were all genotoxic to the root meristem cells of a. cepa when compared to the solvent (2.5% acetone) treated negative control (p<0.05). modulatory effect (me) of methanol (95%) extract of c. sativa on cp or ems-induced genotoxicity (gt): examination of the modulatory effect (me) in column 11 of table 3 shows that the mixture of each of the three concentrations of c. sativa (0.75, 1.5 or 3.0 mg/ml) methanol extract with cp (1.25 mgml-1) was significantly (> twofold or 200%) more genotoxic than cp or c. sativa extract alone. the mixture of each concentration of c. sativa extract with cp thus induced a positive and significant (> twofold) value of me (11.27, 11.89 and 6.43 respectively) and synergism or synergistic interaction between the c. sativa extracts and cp, was inferred. examination of the modulatory effect (me) in column 12 of table 3 shows that the mixture of each of the three concentrations of c. sativa (0.75, 1.5 or 3.0 mg/ ml) methanol extract with ems (0.25 mg/ml) was more genotoxic than ems alone. the mixture of c sativa (0.75 mg/ml) was also more genotoxic than c. sativa extract alone. the mixtures of c. sativa (0.75 or 3.00 mg/ml) extract with ems induced a positive and significant (> twofold) value of me (8.10 and 2.14 respectively) and table 4. cytotoxic and genotoxic effects of ethylacetate extracts of c. sativa, ems and cp on meristem cells of onion root tip and the modulatory effects (me) of the ethylacetate extract of c. sativa on emsor cp-induced genotoxicity. treatment (tc in mg/ml) conc. inter. cells total number of cells in the stages of mitosis in 2000 cells scored total number of cells scored (p+m)/ (a+t) mi genotoxicity modulatory effect n abn total (n + abn) extract on cp extract on ems acetone (2.5%) mean 1790.00 208.00 2.00 210.00 2000 2.60 10.50 1.05     sd 75.54 75.54 0.00 75.54 0.00 0.09 3.78 0.41     cp (1.25) mean 1856.00 135.33 8.67 144.00 2000 2.56 7.20 7.31 #     sd 44.80 48.91 6.11 44.80 0.00 0.33 2.24 7.06     ems (0.25) mean 1815.33 171.00 13.67 184.67 2000 3.86 9.23 5.50 #     sd 99.36 86.63 16.50 99.36 0.00 1.60 4.97 6.42     c. sativa (0.1875) mean 1828.67 93.67 77.67 171.33 2000 6.58 ɉ 8.57 45.72 #     sd 21.13 22.03 11.59 21.13 0.00 0.61 1.06 7.86     c. sativa (0.375) mean 1805.00 180.67 14.33 195.00 2000 2.28 9.75 7.01 #     sd 21.00 11.59 11.59 21.00 0.00 0.55 1.05 5.57     c. sativa (0.75) mean 1824.67 171.00 4.33 175.33 2000 3.17 8.77 2.37     sd 39.63 37.59 2.08 39.63 0.00 1.55 1.98 0.76     c. sativa (0.1875) + cp mean 1786.33 190.67 23.00 213.67 2000 2.87 10.68 10.26 # 0.47†   sd 86.05 74.51 12.29 86.05 0.00 0.74 4.30 2.91     c. sativa (0.375) + cp mean 1862.33 133.33 4.33 137.67 2000 3.12 6.88 4.16 # -0.50+‡   sd 50.96 53.35 2.52 50.96 0.00 1.79 2.55 4.12     c. sativa (0.75) + cp mean 1817.00 178.00 5.00 183.00 2000 3.50 9.15 2.75 # -0.73‡   sd 20.66 20.42 1.00 20.66 0.00 1.18 1.03 0.55     c. sativa (0.1875) + ems mean 1807.00 189.33 3.67 193.00 2000 3.09 9.65 1.88   -0.81+‡ sd 23.64 22.85 2.08 23.64 0.00 0.91 1.18 0.94     c. sativa (0.375) + ems mean 1820.67 174.67 4.67 179.33 2000 2.32 8.97 2.67 #   -0.64+‡ sd 33.01 33.31 1.15 33.01 0.00 0.45 1.65 0.84     c. sativa (0.75) + ems mean 1898.33 99.33 2.33 101.67 2000 3.27 5.08 15.28 #   2.20+† sd 94.50 95.00 0.58 94.50 0.00 0.67 4.73 23.89     tc = test compound; n = normal mitotic cells (comprising prophase, metaphase, anaphase and telophase); abn = aberrant mitotic cells; sd = standard deviation; cp = cyclophosphamide; ems = ethylmethane sulphonate; c.s = cannabis sativa; mi = mitotic index; ɉ = p+m/ a+t ratio (significant increase in ratio compared to negative control, p<0.05 in the t-test, n = 3); * = tc is toxic (mi treatment significantly different from negative control, p<0.05 in the t-test, n = 3); # = tc is genotoxic (significant difference from negative control, p<0.05 in the t-test, n = 3); +† = c.s + mutagen mixture more genotoxic than mutagen or c.s alone (synergism); +‡ = c.s + mutagen mixture less genotoxic than mutagen or c.s alone (antagonism); † = c.s + mutagen mixture more genotoxic than mutagen alone but less than c.s alone; ‡ = c.s + mutagen mixture less genotoxic than mutagen alone (antimutagenicity) but more than c.s alone. 143evaluation of extracts of wild cannabis sativa l. for genotoxicity and phytochemical composition synergism or synergistic interaction between the c. sativa extracts and ems at those concentrations were inferred. 3.3.3 results for ethylacetate extract experiments in table 4 in table 4 are the results of cytotoxicity and genotoxicity experiments with ethylacetate extracts of c. sativa and the mutagens, cp and ems. (p+m)/ (a+t) ratio: examination of the (p+m)/ (a+t) ratio in column 8 of table 4 shows that only the treatment with the lowest concentration (0.1875 mg/ ml) of c.sativa extract induced a significant change in (p+m)/(a+t) ratio, when compared with the solvent (2.5% acetone) treated negative control group (p < 0.05). cytotoxicity: examination of the mi in column 9 of table 4 shows that none of the treatments induced a reduction of the mi when compared to the solvent (2.5% acetone) treated negative control (p<0.05) and were adjudged none toxic to the root meristem cells of a. cepa i.e. cp (1.25 mg ml-1), ems (0.25 mg ml-1 and all concentrations of the ethylacetate extract of c. sativa (0.1875, 0.375, 0.75 mg/ml) and the mixtures of the individual concentrations of the c. sativa extract with cp (1.25 mg ml-1) or ems (0.25 mg ml-1) were all none toxic. genotoxicity (gt): examination of induction of genotoxicity in column 10 of table 4 shows that cp (1.25 mg ml-1) and ems (0.25 mg ml-1) were genotoxic to the root meristem cells of a. cepa, when compared to the solvent (2.5% acetone) treated negative control (p<0.05). the two lowest concentrations of ethylacetate extracts of c. sativa (0.1875 and 0.375 mg ml-1) were also genotoxic. the mixture of each concentration (0.18750, 0.375 or 0.75 mg/ml) of c. sativa extract with cp (1.25 mg ml-1) was also genotoxic. the mixture of each concentration of the two higher concentrations (0.375 or 0.75 mg/ml) of c. sativa extract with ems was also genotoxic, but not the mixture of the lowest concentration (0.1875 mg/ml) with ems. modulatory effect (me) of ethylacetate extract of c. sativa on cp or ems-induced genotoxicity (gt): examination of the modulatory effect (me) in column 11 of table 4 shows that the mixture of the lowest concentration of the ethylacetate extract of c. sativa (0.1875 mg/ml) with cp (0.25 mg/ml) was none significantly (< twofold, me = 0.47) more genotoxic than cp alone but not the ethylacetate extract (0.1875 mg/ml) of c. sativa alone. each mixture of c. sativa extract (0.375 or 0.75 mg/ml) with cp was none significantly (< twofold, me = -0.50 and -0.73 respectively) less genotoxic than cp or c.s extract alone. each mixture of c. sativa extract (0.1875 or 0.375 mg/ml) with ems was none significantly (< twofold, me = -0.81 and -0.64 respectively) less genotoxic than ems or c.s extract alone. however the mixture of the highest concentration of c.s extract (0.75 mg/ml) with ems was significantly (> twofold (200%), me = 2.20) more genotoxic than ems or c.s extract alone, and synergism or synergistic interaction between the ems and c. sativa extract, at that concentration, was inferred. 4. discussion in this study, hexane, chloroform, ethyl acetate and methanol extracts of the aerial parts of cannabis sativa (c. sativa) growing in lesotho and used in traditional medicine to treat some diseases and for recreational purposes were evaluated for phytochemical composition, genotoxicity and modulatory effects on emsand cp – induced genotoxicity using the onion (allium cepa l.) chromosome aberration assay system. the results of the phytochemical screening tests are presented in table 2 while the results of the genotoxicity tests are presented in tables 3 and 4. in the present qualitative study presented in table 2, based on the intensity of the colour in the colorimetric tests and (or) the appearance of precipitates, during the identification reactions, methanol extract contained the highest number of the different phytochemical classes (15/19) followed by hexane and chloroform (9/19 each) and ethylacetate (7/19). such tests allow a semi-quantitative evaluation for the presence of secondary metabolites in extract solutions (chukwudi and yusha’u 2016). the phytochemicals detected in the extracts of c. sativa in the present study which have been detected in extract of c. sativa previously include flavonoids, sterols and alkaloids (pollastro et al. 2018) and flavonoids, alkaloids, sterols, saponins, tannins and terpenoids in extracts of cannabis indica (pollastro et al. 2018). cardiac glycosides and cardenolides, proteins and fatty acids were not detected in any of the solvent extractives. a study by audu et al. (2014) using c. sativa l. procured from the national drug law enforcement agency (ndlea) in nigeria revealed a high presence of cardiac glycosides in petroleum ether (a non polar solvent) crude extract of c. sativa leaves. the absence of these biomolecules from the extracts of the solvents used in the present study to differences in the strain of c. sativa used and the diffences in the climate, soil and topography between lesotho (temperate climate with two-thirds of the terrain being mountainous and over 80% of soils in the lowlands being acidic) and nigeria (tropics) where the plants were grown, and the different extraction methods used, petro144 asita okorie asita, sibusisiwe magama, thato mamoroesi moahloli, selometsi baholo leum ether (audu et al. 2014) and methanol, hexane, chloroform and ethylacetate in the present study (ramelet 2015).the most ubiquitous classes were the polyphenols, anthocyanins, betacyanins, quinones, coumarins and amino acids which were detected in all the solvent extracts. these compounds were also detected in petroleum ether extracts of leaves of c. sativa in the study by audu et al. (2014). the flavonoids, alkaloids, saponins, phlobatannins and anthraquinones were detected at different colour intencies only in the methanol extract while phytosterols were present at high intensity only in the hexane extract, since hexane is a non polar solvent. terpenoids were detected only in the hexane and methanol extracts. constituents such as carotenoids, terpenoids, ascorbates, reducing sugars and tocopherols are known to contribute to the antioxidant, antiviral, anticancer and anti-inflammatory properties of phenolic compounds (bang et al. 2015; elsohly et al. 2017; andre et al. 2016). in another study by elsohly et al. (2017) they identified the quinone 2-geranyl-5-hydroxy-3-npentyl-1,4-benzoquinone in extracts of leaves and buds of c. sativa using several chromatographic techniques. the differences in biological activities of different solvent fractions have been demonstrated by other researchers. mihailović et al. (2013) studied the antioxidant and antigenotoxic activities of chloroform, ethyl acetate and n-butanol fractions obtained from the methanolic extract of gentiana asclepiadea l. roots. among all fractions, the ethyl acetate fraction exhibited the highest antioxidant activity, as well as total phenolics (146.64 gae/g), flavonoids, flavonols and gallotannins contents. only the total phenolics content of crude hexane, chloroform, ethylacetate and methanol extracts of c. sativa were determined in this study. the determined value of total phenolics (figure 1) of the hexane, chloroform, ethylacetate and methanol extracts were, 39831.46, 2544.94, 2438.20 and 56601.12 mg gae/gram dry weight respectively. the methanol extract, being the most polar, had the highest content of phenolics. in a study conducted by mkpenie et al. (2012), the polyphenol content of the acetone and methanolic extracts of c. sativa was found to be in the range of 0.090 – 0.556 mg gae/g dry weight. we therefore attribute the higher content of total phenolics observed in the present study to the different extraction times; 2, 8 and 18 hours in the study by mkpenie et al. (2012) compared to the extraction time of 48 hours in the current study. as shown in tables 3 and 4, the concentrations of cp (1.25 mg ml-1) and ems (0.25 mg ml-1) used in the present study did not reduce the mitotic index (mi) of meristem cells of the treated roots compared with the negative control, and were adjudged not cytotoxic. the concentrations of cp and ems used however induced genotoxicity in the root meristem cells of a. cepa. in a study by çelik and aslantürk (2010), ems at a concentration of 2x10-2 m (0.2484 mg ml-1) was both toxic and mutagenic to root meristem cells of a. cepa. cp at a concentration of 1% (1 mg ml-1) was also both toxic and clastogenic to onion root meristem cells (akeem et al. 2011). the results of the assessments of the cytotoxic and genotoxic effects of the methanol and ethyl acetate extracts of c. sativa are presented in tables 3 and 4. only the lowest concentration (0.75 mg ml-1) of the methanol extract and its mixture with ems (0.25 mg ml-1) (table 3) and the lowest concentration (0.1875) of the ethyl acetate extract (table 4), significantly reduced the (p+m)/(a+t) ratio. a decrease in the proportion of dividing cells in a+t is an indication of metaphase arrest due to the poisoning of the spindle fibers, akin to the action of the well documented spindle poison, colcemid (parry et al. 1999). the chemotherapeutic agents taxol, vincristine, vinblastine and nocodozole act in a similar manner (alberts et al. 2008). these compounds act by binding to and stabilizing microtubules, inhibiting their dynamic instability and cuasing various genetic disruption, including the induction of cell cycle arrest (alberts et al. 2008; zhang et al. 2015). in the present study, it seems that cell cycle arrest at the metaphase/ anaphase junction by these extracts depended on concentration as only the lowest concentration (0.75 mgml1) of the methanol extract and the lowest concentration (0.1875 mg/ml) of the ethylacetate extract induced mitotic cell arrest. all three concentrations (0.75, 1.5 and 3.0 mg ml-1) of the methanol extract of c. sativa and their individual mixtures with cp or ems (table 3) tested were cytotoxic to the onion root meristem cells. none of the three concentrations (0.1875, 0.375 and 0.75 mg ml-1) of the ethylacetate extract of c. sativa alone or in mixtures with cp or ems (table 4) was cytotoxic to the onion root meristem cells. plant secondary metabolites, such as the ones detected in the extracts of c. sativa in the present study, are the key drivers of the pharmacological actions of medicinal plants and have been shown to possess various biological effects including antibiotic, antifungal, antiviral and cytotoxic effects and therefore are able to protect plants from pathogens (asche 2005; hussein and el-anssary 2018, priyanka et al. 2019). the toxicity of the extracts observed in the present study was therefore attributed to the presence of cytoxic secondary plant metabolites in the solvent extracts. in the ames assay with extracts of c. sativa diluted with olive oil as well as the extracts produced with an isopropanol 145evaluation of extracts of wild cannabis sativa l. for genotoxicity and phytochemical composition and supercritical co2 extraction method, toxicity was evident for strains ta 98, ta 1535, ta 1537 and e. coli wp2 uvra at≥50 μg/plate, with and without s9, in the plate incorporation and/or pre-incubation tests (dziwenka et al. 2020). these results are similar to results of other researches that demonstrated cytotoxicity of plant extracts including betel and tobacco leaf extracts and some nigerian folk medicines to root-tip cells of a. cepa (sopova et al. 1983; abraham and cherian 1978). regarding the genotoxicity of the extracts, all three concentrations (0.75, 1.5 and 3.0 mg ml-1) of the methanol extract of c. sativa and their individual mixtures with cp or ems (table 3) tested were genotoxic to the onion root meristem cells. the 0.1875 and 0.375 mg ml-1 dilutions of the ethyl acetate extract and their individual mixtures with cp and the mixture of 0.75 mg/ml ethyl acetate extract with cp were also genotoxic. in addition, the mixture of each concentration (0.375 or 0.75 mgml-1) of the ethyl acetate extract with ems was also genotoxic. the chromosomal abnormalities observed following treatment of the root tip cells of a. cepa with methanol and ethyl acetate extracts of c. sativa alone or in mixture with cp or ems included sticky chromosomes, c-mitosis, chromosome largards, chromosome fragments, and anaphase and telophase bridges. genotoxic effects of different medicinal herbs in a. cepa have been demonstrated (soliman 2001; bidau et al. 2004; çelik and aslantürk 2007; akinboro and bakare 2007; akintonwa et al. 2009; oyedare et al. 2009). marijuana smoke condensates were mutagenic to the ta98 strain in the ames salmonella/microsome bioassay but only in the presence of liver homogenates (busch et al. 1979). however a supercritical co2 extract of the aerial parts of the c. sativa, was not mutagenic in the ames bacterial reverse mutation test, in vitro mammalian chromosomal aberration test, or in an in vivo mouse micronucleus study (marx et al. 2018). in another assessment of extracts of hemp (c. sativa) using the ames reverse mutation assay, the extracts produced with an isopropanol and supercritical co2 extraction methods were diluted with olive oil and the undiluted extract formulated as a solution in dmso; no mutagenic effect was observed in the four strains of salmonella typhimurium (ta98, ta100, ta1535 and ta1537) and one strain of e. coli (wp2 uvra) that were used (dziwenka et al. 2020). in the present study, methanol and ethyl acetate extracts of the areal parts of c. sativa dissolved in 2.5% acetone as solvent, induced genotoxiciy in the a. cepa root meristem cells. the modulatory effects (me) of the extracts on mutagen-induced genotoxicty are presented in columns 11 and 12 of tables 3 and 4 for the methanol and ethyl acetate extract respectively. the me indicated the type of interaction between the extract and the mutagen. the mixture of each concentration (0.75, 1.5 and 3.0 mg ml-1) of the methanol extract with cp, table 3, was significantly (>two-fold) more genotoxic than the mutagen (cp) alone with +me values of (11.27, 11.89 and 6.43) respectively. the mixtures were also more genotoxic than the c. sativa extract alone at each concentration. the mixtures of the different concentrations (0.75, 1.5 and 3.0 mg ml-1) of the methanol extract of c. sativa with ems were also more genotoxic than the mutagen (ems) alone with positive me (+me) values of (8.10, 1.61 and 2.14) respectively. only the +me values of the mixture of ems with the lowest (0.75 mgml-1) or highest (3.0 mgml-1) concentration were significant, i.e. >two-fold the genotoxicity induced by the mutagen alone. theses results indicated a synergistic interaction between each of the three concentrations of the methanol extract of c. sativa with cp and between two concentrations of the methanol extract c. sativa with ems. the mixture of 0.1875 mgml-1 of the ethylyacetate extract of c. sativa and cp (table 4) was insignificantly more genotoxic (me = 0.47) than the mutagen (cp) alone; the mixture of 0.375 or 0.75 mgml-1 of the ethylacetate extract with cp was insignificantly less genotoxic (me = -0.50 and -0.73 respectively) than cp alone and therefore no interaction between cp and ethylacetate extract was inferred. each mixture of 0.1875 or 0.375 mgml-1 of the ethylacetate extract with ems was insignificantly less genotoxic than ems alone with negative me (-me) values of -0.81 and 0.64 respectively. the mixtures were also less genotoxic than the ethylacetate extract alone. the mixture of 0.75 mgml-1 of the ethyl acetate extract with ems was significantly more genotoxic (+me value of 2.20) than ems or c. sativa extract alone. the significant +me value indicated a synergistic interaction between the ethyl acetate extract of c. sativa with ems at the highest concentration only. these results demonstrated that the methanol and ethylacetate extracts of c. sativa did not exert any anti-genotoxic effects on cpor emsinduced genotoxicity. lack of or differential anti-genotoxic activity of different solvent extracts have been demonstrated in other test systems. mihailović et al. (2013) studied the antioxidant and antigenotoxic activities of chloroform, ethyl acetate and n-butanol fractions obtained from the methanolic extract of gentiana asclepiadea l. roots and found no significant difference in the antigenotoxic effect of the different fractions against ems-induced dna damage in the in vivo sex linked recessive lethal mutations assay in drosophila melanogaster males (mihailović et al., 2013). the differential effects of different concentrations of 146 asita okorie asita, sibusisiwe magama, thato mamoroesi moahloli, selometsi baholo plant extracts or plant derivatives on mutagens-induced genotoxicity have been demonstrated in many test systems. in mice, an increase in the anticlastogenic activity of cp-induced clastogenicity by β-carotene at lower doses and an absence of a protective effect at higher concentrations were observed (salvadori et al. 1992). salvadori et al. (1992) interpreted the observations to mean different mechanisms of β-carotene modulation and a possible alteration of the balance of cp activation/detoxification mechanism of the promutagen. while no study on the antimutagenic activity of c. sativa extract was found, however, two pure terpenes that are found in cannabis (bedini et al. 2016), d-linalool (lnl) and myrcene (myr), were efficient against t-butyl hydroperoxide (t-booh) induced genotoxicity in the reverse mutation assay with e. coli and oxyr mutants and in the comet assay using cultured human hepatoma hepg2 and human b lymphoid nc-nc cells, which was predominately mediated by direct radical scavenging activity (mitić-ćulafić et al. 2009). another pure terpene, found in cannabis, bisabolol (bisa), caused a reduction in the levels of aβ-induced chromosomal damage and apoptosis in pc12 cells (shanmuganathan et al. 2018). it is now well accepted that the health benefits of fruits, vegetables and other plant foods are due to the synergy or interactions between the different bioactive compounds or other nutrients present in the whole foods, and not to the action of a sole compound (liu, 2013). similarly, we attribute the differences in the actions between the ethyl acetate and methanol extracts in the induction of cytotoxicity, genotoxicity and modulatory effects at different extract concentrations observed in this study to the synergistic or antagonistic interactions between various phytochemicals present in the extracts. according to efferth and koch (2011) cannabis-based therapeutics in humans, exert their pharmacological effects via synergistic or antagonistic interactions between the various phytochemicals it contains. 5. conclusion the order of effectiveness at extracting, from the aerial parts of a. sativa, the 19 different phytochemicals investigated in the present study was 95% methanol (15/19), followed by hexane and chloroform (9/19 each) and ethylacetate (7/19). total phenolics content, in mg gae/gram dry weight of extract was 95% methanol (56601.12) > ethylacetate (2438.20) > chloroform (2544.94) > hexane (39831.46). the methanol extract was both cytotoxic and genotoxic to the a. cepa root meristem cells, but the ethyl acetate extract was not cytotoxic but genotoxic. mixtures of methanol extract (0.75, 1.5 and 3.0 mg ml-1) with either cp or ems were more genotoxic than the cp, ems or extract alone which demonstrated a synergistic interaction between the methanol extract of c. sativa with cp and between two concentrations of the methanol extract with ems. mixtures of ethyl acetate extract of c. sativa (0.1875, 0.375, 0.75 mg/ml) with either cp or ems were generally insignificantly (p<0.05) less genotoxic than cp, ems or extract alone. thus no interaction was observed between all three concentrations and the two lower concentrations of ethylacetate extract with cp or ems. there was however a synergistic interaction between the highest concentration of the ethylacetate 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(fabaceae) comprises approximately 87 different species of herbs and shrubs widespread from the mediterranean to central asia. medicago polymorpha is a herbaceous legume that can be a useful pasture plant, in particular, in regions with a mediterranean climate. it had aroused great interest due to high nutritious quality, highly palatability and n-fixing plan in neutral soil. there is no information on its population genetic structure, genetic diversity, and morphological variability in iran. due to the medicinal importance of this species, a genetic variability and populations’ structure study is performed studying 15 geographical populations of medicago polymorpha. therefore, we used six inter-retrotransposon amplified polymorphism (irap) markers and 15 combined irap markers to reveal within and among population genetic diversity in this plant. amova test produced significant genetic difference (phipt = 0.46, p = 0.010) among the studied populations and also revealed that, 66% of total genetic variability was due to within population diversity while, 34% was due to among population genetic differentiation. mantel test showed positive significant correlation between genetic distance and geographical distance of the studied populations. structure analyses and population assignment test revealed some degree of gene flow among these populations. pcoa plot of populations was in agreement with upgma clustering of molecular data. these results indicated that geographical populations of medicago polymorpha are well differentiated based on (irap) markers. keywords: gene flow, irap, medicago polymorpha, population differentiation. introduction knowledge of spatial genetic structures provides a valuable tool for inferring the evolutionary forces such as selective pressures and drift (bi et al, 2021; cheng et al, 2021; khayatnezhad and gholamin, 2020, 2021a, 2021b). low gene flow due to spatial isolation of populations may even increase the degree of local differentiation (karasakal et al, 2020a, 2020b; huang et al, 2021; hou et al, 2021, guo et al, 2021). nevertheless, phenotypic plasticity rather than genetic differentiation may be an alternative way of matching 132 huang jing, somayeh esfandani-bozchaloyi genotypes to environment; indeed increasing environmental variation favors higher levels of plasticity (ma et al, 2021; peng et al, 2021; si et al., 2021; sun et al., 2021; miao et al 2018; zou et al, 2019; wang et al 2020; xiaolong et al, 2021; hou et al, 2021). the genus medicago l. (fabaceae) comprises approximately 87 different species of herbs and shrubs widespread from the mediterranean to central asia (small, 2010), including the widely cultivated forage crop and weedy species m. sativa l. (commonly named alfalfa or lucerne) and the legume model species m. truncatula gaertn. (steele et al., 2010). the annuals species collectively known as “medics” are naturally distributed over a very wide range of environmental conditions in the mediterranean basin. some medics have been introduced to regions of australia, chile, south africa and united states with mediterranean-type climate. medics, as well as other annual pasture legumes, have a high feeding quality, determined by higher protein, mineral and vitamin contents (keivani et al., 2010). due to their capacity to fix atmospheric nitrogen and improve soil fertility in symbiosis with soil bacteria collectively known as ‘rhizobia’, medicago species do not need costly and polluting chemical nitrogen fertilizer (small and jomphe, 1989). the genus medicago in iran has been revised by different authors. boissier (1872), in his flora orientalis, published 11 medicago species for iran. parsa (1948), moussavi (1977) and heyn (1984) recognized 14, 16 and 11 species in iran, respectively. mehregan & al. (2001) reported 18 species of the genus medicago from iran. two main reasons can be accounted for the disagreements over the taxonomic status of this genus in iran: (1) incomplete collecting; and (2) taxonomic confusions encountered in medicago. medicago polymorpha l. is an annual herbaceous and can be a useful pasture plant, in particular, in regions with a mediterranean climate, self-compatible and diploid (2n = 14) (salhi hannachi et al., 1998). it had aroused great interest due to high nutritious quality, high palatability and n-fixing capability in neutral soil (abdelkefi et al., 1996). m. polymorpha is a species of mediterranean origin, but its species range is wide spread throughout the world. the wide diffusion and adaptability can be explained by its low sensitivity to photoperiod and vernalization (aitken, 1981). three botanical varieties of this species were identified by heyn (1963): brevispina; polymorpha and vulgaris. in iran, m. polymorpha grows in a range of environments from humid to arid. in recent years, molecular marker systems such as randomly amplified polymorphic dna (rapd), amplified fragment length polymorphism (aflp), inter simple sequence repeat (issr), simple sequence repeat (ssr) and inter-retrotransposon amplified polymorphism (irap) have been used to measure genetic variation and relationships in cultivars and landraces of medicago species. for instance, genetic diversity among and within 10 populations of iranian alfalfa, from different areas of azarbaijan was analyzed by screening dna from seeds of individual plants and bulk samples (mohammadzadeh et al, 2011). morpho-phenological diversity among natural populations of medicago polymorpha of different tunisian ecological areas (badri et al, 2016). their results from analysis of variance (anova) showed that differences among populations and lines existed for all traits, with population explaining the greatest variation for measured traits. genetic relationships of 98 alfalfa (medicago sativa l.) germplasm accessions examined using morphological traits and ssr markers from europe, usa, australia, new zealand and canada ( cholastova, knotova, 2012). moreover, due to extensive morphological variability of this species in the country, there is possibility of having infra-specific taxonomic forms. therefore, we carried out population genetic analysis and morphometric study of 15 geographical populations for the first time in the country. for genetic study, we used the inter-retrotransposon amplified polymorphism (irap) method that displays insertional polymorphisms by amplifying the segments of dna between two retrotransposons. it has been used in numerous studies of genetic diversity (smykal et al., 2011). the objectives of this research were to study genetic diversity among medicago polymorpha cultivars/population with a different geographical origin by inter-retrotransposon amplified polymorphism (irap) method, to determine genetic variation among and within materials using irap markers. materials and methods plant materials a total of 89 individuals were sampled representing 15 natural populations of medicago polymorpha in east azerbaijan, lorestan, kermanshah, gilan, mazandaran, golestan and ardabil provinces of iran during julyagust 2019-2020. fresh leaves of 5-8 individuals from each population, were collected, and immediately dried in silica gel. different references were used for the correct identification of species (medicago polymorpha) (boissier ,1872; parsa 1948). 133genetic diversity and gene-pool of medicago polymorpha l. based on retrotransposon-based markers dna extraction and irap assay fresh leaves were used randomly from 5-10 plants in each of the studied populations. these were dried by silica gel powder. ctab activated charcoal protocol was used to extract genomic dna. the quality of extracted dna was examined by running on 0.8% agarose gel. a set of six outward-facing ltr primers (smykal et al., 2011; table 1) were used for irap analysis. we also used 15 different combinations of outward-facing ltr pair primers. pcr reactions were carried in a 25μl volume containing 10 mm tris-hcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). the thermal program was carried out with an initial denaturation for 1 min at 94°c, followed by 40 cycles in three segments: 35 s at 95°c, 40s at 47°c and 55s at 72°c. final extension was performed at 72°c for 5 min. the amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated by using a 100 bp molecular size ladder (fermentas, germany). molecular analyses the irap profiles obtained for each samples were scored as binary characters. parameter like nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism were determined (weising et al., 2005). nei’s genetic distance among populations was used for neighbor joining (nj) clustering and neighbornet networking (huson and bryant, 2006). mantel test checked the correlation between geographical and genetic distance of the studied populations (podani, 2000). these analyses were done by past ver. 2.17 , darwin ver. 5 (2012) and splitstree4 v4.13.1 (2013) software. amova (analysis of molecular variance) test (with 1000 permutations) as implemented in genalex 6.4 (peakall and smouse, 2006), and nei,s gst analysis as implemented in genodive ver.2 (2013) (meirmans and van tienderen, 2004) were used to show genetic difference of the populations. moreover, populations, genetic differentiation was studied by g’st est = standardized measure of genetic differentiation (hedrick, 2005), and d_est = jost measure of differentiation (jost, 2008). the genetic structure of populations was studied by bayesian based model structure analysis (pritchard et al. 2000), and maximum likelihood-based method of k-means clustering of genodive ver. 2. (2013). for structure analysis, data were scored as dominant markers. the evanno test was performed on structure result to determine proper number of k by using delta k value (evanno et al., 2005). in k-means clustering, two summary statistics, pseudo-f, and bayesian information criterion (bic), provide the best fit for k. gene flow was determined by (i) calculating nm an estimate of gene flow from gst by popgene ver. 1.32 (1997) as: nm = 0.5(1 gst)/gst. this approach considers equal amount of gene flow among all populations. (ii) population assignment test based on maximum likelihood as performed in genodive ver. in genodive ver. 2. (2013). the presence of shared alleles was determined by drawing the reticulogram network based on the least square method by darwin ver 5. (2012). results populations, genetic diversity genetic diversity parameters determined in 15 geographical populations of medicago polymorpha are presented in table 2. the highest value of percentage polymorphism (53.75%) was observed in ardabil, khalkhalasalem road (population no.1) which shows high value for gene diversity (0.32). and shanon, information index (0.39). population kermanshah: ghasre-shirin, 5 km from paveh to nusod (no.9) has the lowest value for percentage of polymorphism (31.43%) and the lowest value for shanon, information index (0.030), and he (0.011). population genetic differentiation amova (phipt = 0.74, p = 0.010), and gst analysis (0.367, p = 0.001) revealed significant difference among the studied populations (table 3). it also revealed that, 66% of total genetic variability was due to within population diversity and 34% was due to among population genetic differentiation. pairwise amova produced significant difference among the studied populations. table 1. m. polymorpha irap primers based on smykal et al. (2011) study. irap sequence (5´-3´) gu735096 accccttgagctaacttttggggtaag gu980589 agcctgaaagtgttgggttgtcg gu929878 gcatcagcctggaccagtcctcgtcc gu735096 cacttcaaattttggcagcagcggatc gu929877 tcgaggtacacctcgactcagg gu980590 attctcgtccgctgcgcccctaca 134 huang jing, somayeh esfandani-bozchaloyi moreover, we got high values for hedrick standardized fixation index after 999 permutation (g’st = 0.367, p = 0.001) and jost, differentiation index (d-est = 0.176, p = 0.001). these results indicate that the geographical populations of medicago polymorpha are genetically differentiated from each other. populations, genetic affinity in upgma tree, plant samples of each populations, were grouped together and formed separate cluster. in the studied specimens we did not encounter intermediate forms. these results showed that irap data can differentiate the populations of medicago polymorpha in two different major clusters or groups (figure 1). the first major cluster that was supported with significant bootstrapping values of higher than 50%, was divided into two main sub-clusters so that plants of ardabil,meshkin shahr, hatam forest, ardabil, meshkin shahr, sabalan mt, shahbil, qotursooi villageand and ardabil: germi, 20 km from germi to pars-abad (p8p 9, 12; province ardabil) and west azerbaijan, kaleybar and azarbaijan (e): ahar, 45 km from meshkin-shahr table 2. genetic diversity parameters in the studied populations medicago polymorpha (n = number of samples, na= number of different alleles; ne = number of effective alleles, i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations). pop na ne i he uhe %p pop1 0.341 1.058 0.39 0.32 0.31 53.75% pop2 0.455 1.077 0.277 0.24 0.22 55.05% pop3 0.499 1.067 0.24 0.23 0.24 49.26% pop4 0.555 1.020 0.22 0.25 0.28 43.53% pop5 0.431 1.088 0.20 0.22 0.25 41.53% pop6 0.255 1.021 0.25 0.28 0.22 47.15% pop7 0.261 1.024 0.292 0.23 0.23 43.15% pop8 0.886 1.183 0.184 0.116 0.122 44.29% pop9 0.686 1.157 0.030 0.011 0.022 31.43% pop10 0.643 1.173 0.154 0.102 0.109 30.00% pop11 0.243 1.033 0.026 0.018 0.029 34.29% pop12 0.400 1.087 0.076 0.051 0.057 40.29% pop13 0.286 1.046 0.040 0.027 0.032 37.14% pop14 0.400 1.112 0.090 0.062 0.069 35.71% pop15 0.576 1.144 0.122 0.083 0.095 39.18 figure 1. upgma clustering of populations in medicago polymorpha based on irap data. bootstrap value from 1000 replicates are indicated below branches (population numbers are according to table 1). table 3. analysis of molecular variance (amova) of the studied species. source df ss ms est. var. % φpt among pops 20 216.576 21.327 9.082 66% 66% within pops 59 114.767 9.530 1.530 34% total 79 321.342 10.613 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance; φpt: proportion of the total genetic variance among individuals within an accession, (p < 0.001). 135genetic diversity and gene-pool of medicago polymorpha l. based on retrotransposon-based markers to ahar (p10,15; province west azerbaijan ) comprised the first sub-cluster due to morphological similarity, while the plants of ardabil, khalkhal-asalem road (p1) formed the second sub-cluster. similarly, the second major cluster included two sub-clusters too: the first subcluster contained lorestan: khorram-abad, 60 km from pol-dokhtar to khorram-abad (p11) and kermanshah: paveh, paveh shahid kazemi forest park (p3) , while plants of gilan, mazandaran and golestan provine (northen iran) (p24,5,6,7,13,14) were grouped into the second sub-cluster. genetic divergence and separation of populations lorestan (p11) and kermanshah (p3) as well as p8p 9, 12 (province ardabil) from the other populations is evident in pcoa plot of irap data after 900 permutations (figure.3). the other populations showed close genetic affinity. mantel test after 5000 permutations produced significant correlation between genetic distance and geographical distance in these populations (r = 0.48, p = 0.001). therefore, the populations that are geographically more distant have less amount of gene flow, and we have isolation by distance (ibd) in medicago polymorpha. populations genetic structure k = 3 reveal the presence of 3 genetic group. similar result was obtained by evanno test performed on structure analysis which produced a major peak at k = 3 (figure.3). both these analyses revealed that medicago polymorpha populations show genetic stratification. structure plot based on k = 3, revealed genetic difference of populations 11 and 12 (differently colored), as well as 13 and 14 (figure.4). but it showed genetic affinity between populations 1-10 and 15 (similarly colored). the mean nm = 0.654 was obtained for all irap loci, which indicates low amount of gene flow among the populations and supports genetic stratification as indicated by k-means and structure analyses. population assignment test also agreed with nm result and could not identif y significant gene flow among these populations. however, reticulogram obtained based on the least square method (figure not included), revealed some amount of shared alleles among populations 1 and 5, and between 13 and 6 and 7, also between 8, and 9. this result is in agreement with grouping we obtained with pcoa plot, as these populations were placed close to each other. as evidenced by structure plot based on admixture model, these shared alleles comprise very limited part of the genomes in these populations and all these results are in agreement in showing high degree of genetic stratification within medicago polymorpha populations. in total 120 irap bands (loci) were obtained, out of which 34 bands were private. populations 1-7, 8, 14 and 15 contained 1-4 private bands. figure 2. pcoa plot of populations in medicago polymorpha based on irap data. 136 huang jing, somayeh esfandani-bozchaloyi discussion population genetics analyses are important in genetic and breeding studies (kizzie-hayford et al 2021; wasana et al 2021; sawadogo et al., 2021; paul et al 2021; mieso & befa et al 2020). they provide information on the levels of genetic variation, partitioning of genetic variability within/between populations, inbreeding or outcrossing, effective population size and population bottleneck (gholamin and khayatnezhad, 2020a; 2020b, 2020c). the advent of molecular markers has greatly improved population genetic studies. these markers have been used to identify potentially novel genotypes among the many medicago polymorpha accessions. in recent years, molecular marker systems such as randomly amplified polymorphic dna (rapd), amplified fragment length polymorphism (aflp), inter simple sequence repeat (issr), simple sequence repeat (ssr) and inter-retrotransposon amplified polymorphism (irap) have been used to measure genetic variation and relationships in cultivars and landraces (ren and khayatnezhad 2021; khayatnezhad and nasehi 2021, i et al., 2021; jia et al, 2021). transposable elements, particularly retrotransposons, comprise most of plant genomes. their replication generates genomic diversity and makes them an excellent source of molecular markers (smykal et al., 2011). the inter-retrotransposon amplified polymorphism (irap) method displays insertional polymorphisms by amplifying the segments of dna between two retrotransposons. it has been used in numerous studies of genetic diversity (smykal et al., 2011). in china a population genetic study of two species, m. lupulina and m. ruthenica, reported that these types germplasm were valuable resources for improving medicago forage crops (badri et al., 2011). this information has different applications, and from pure understanding of biology of the species to conservation of endangered species, choosing of proper parents for hybridization and breeding and phylogeography and mechanism of invasion. in this study, we investigated the genetic diversity of m. polymorpha populations. the main aim of our study was to evaluate the genetic diversity of m. polymorpha genotypes. to reach this objective, and to be able to detect segregating populations, we used the available inter-retrotransposon amplified polymorphism (irap) marker. the results of diwan et al. (2000) study showed that ssr markers produced by m. truncatula are valuable genetic markers for the genus medicago. these markers will be useful in establishing the genomic relationships important forage such as alfalfa and other annual medics. among the 120 studied lines of m. polymorpha, there was no spineless line. our studies showed that the average number of 6.7 alleles per locus may be due to the high level of homozygous nature of m. polymorpha. acording to flajoulot et al. (2005) the number of allels per locus ranging were 3_24 in medicago sativa. in contrast to work by baquerizo et al. (2001) used six simple sequence repeat to analyse the genetic diversity and relationships between individuals of medicago truncatula, showed to be highly diverse with an average of 25 alleles per locus. as a result, our studies emphasize that genetic variation has figure 3. evanno test of medicago polymorpha populations based on k = 3 of irap data. figure 4. structure plot of medicago polymorpha populations based on k = 3 of irap data. (population numbers are according to table 1). 137genetic diversity and gene-pool of medicago polymorpha l. based on retrotransposon-based markers been effective in determining population relationships and the amova results was level of among populations diversity (66%) is higher than within populations diversity (34%). the markers used in this study were highly effective in detecting the level of genetic diversity in the polymorphic and studied populations. also the ardabil, khalkhal-asalem road population was high gene diversity and high polymorphism percentage. min et al. (2017) investigated the extensive development of genes with micro-rna-based ssr markers in m. trunculata. the mean value of information content of their polymorphisms was 0.71, indicating a high level of information. in other study the average of polymorphism information was 78.75% in m. trunculata and a total of 24 alleles were amplified with an average of 3 alleles per locus (jafari et al. 2013). also in other study reported informations polymorphism by ssr marckers indicating a high level of polymorphism (> 70%) for m. trunculata for m. trunculata and other annual medics (diwan et al. 2000). genetic diversity is of fundamental importance to the survival of a species (sun and khayatnezhad 2021; tao et al, 2021; wang et al, 2021; xu et al., 2021; yin et al., 2021; zhang et al, 2021). degree of genetic variability within a species is highly correlated with its reproductive mode, the higher degree of open pollination/cross breeding generally producing higher levels of genetic variability. according to hamrick and godt (2012) species that have selfing or mixed mating systems have lower levels of genetic variability then predominantly outcrossed species and 51% of their total genetic diversity is apportioned between populations in comparison to 10% for outcrossed species. our study indicated a low level of heterozygosity (he = 0.01-0.32) in m. polymorpha. the substantially higher selfing rate in m. polymorpha likely contributed to a lower overall level of estimated heterozygosity. like this our study a low level of he reported 0.246 in m. lupulina (badri et al., 2011). the our study degree average of selfing rate ( 18.78) levels outcrossing (-10.78). the mean nm = 0.654 was obtained for investigated irap loci, which indicates low amount of gene flow among the populations and supports genetic stratification as indicated by structure analyses. by examining the biological results, it can be observed that the smaller the genetic distance between populations, they are more similar to each other, because of the shape of the seed of the species studied, it is light and easy to move and propagated by wind and other factors. this confers diversity, which results in amova analysis showing that percentages within and among populations are relative, and since m. polymorpha is a selfing plant and regeneration occurs within the species population, which causes. among-population differentiation in phenotypic traits and allelic variation can be the result of drift, founder effects and local selection. according to badri et al. 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application of probability decision system and particle swarm optimization for improving soil moisture content. water supply. caryologia. international journal of cytology, cytosystematics and cytogenetics 75(2): 81-88, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1544 caryologia international journal of cytology, cytosystematics and cytogenetics citation: weera thongnetr, surachest aiumsumang, alongklod tanomtong, sumalee phimphan (2022) classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand. caryologia 75(2): 81-88. doi: 10.36253/caryologia-1544 received: january 23, 2022 accepted: july 07, 2022 published: september 21, 2022 copyright: © 2022 weera thongnetr, surachest aiumsumang, alongklod tanomtong, sumalee phimphan. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* 1 walai rukhavej botanical research institute, mahasarakham university, kantharawichai, maha sarakham 44150, thailand 2 biology program, faculty of science and technology, phetchabun rajabhat university, phetchabun 67000, thailand 3 program of biology, faculty of science, khon kaen university, muang, khon kaen 40002, thailand *corresponding author. e-mail: sumalee.phi@pcru.ac.th abstract. the objectives of this study were to examine size, shape, diploid number (2n), fundamental number (nf), nors region, and distribution of microsatellite by using fluorescence in situ hybridization technique (fish) and to establish the karyotype and standard idiogram of sandstone geckos (gekko petricolus taylor, 1962). sandstone gecko distributed in the sandstone mountains in laos, cambodia, and thailand. five male and five female specimens were collected from ubon ratchathani and mukdahan provinces, thailand. the metaphase cells were directly prepared from the bone marrow cells. chromosomes were stained by conventional staining, nors-banding and fish techniques. the results found that the diploid number was 38 chromosomes. the fundamental number was 54. the karyotype composed of 4 large metacentric, 4 large acrocentric, 2 large telocentric, 4 medium acrocentric, 2 medium telocentric, 2 small submetacentric, 2 small acrocentric and 18 small telocentric chromosomes. no morphological difference was identified between sex chromosomes of male and female specimens. the nors appeared to telomere of the long arm of chromosome pair 17. the study displayed that the distribution of microsatellite using (ca)15 and (gaa)10 probes distributed throughout the genome. however, (ca)15 sequences concentrated in the telomere. the karyotype formula g. petricolus is as follow: 2n (38) = lm4+la4+lt2+ ma4+mt2+ssm2+sa2+st18. keywords: karyotype, gekko petricolus, ag-nor, fish microsatellite. introduction the sandstone gecko (gekko petricolus) belongs to family gekkonidae, a genus mainly found in subtropical limestone areas, near the tropic of cancer, such as southern china, india, myanmar, thailand, vietnam, malaysia, indonesia, and other countries in southeast asia (li et al. 1996). 82 weera thongnetr et al. the genus gekko comprises over 30 species and a few undescribed species distributed in east and southeast asia, and western oceania (kluge 2001; toda 2008). the gekko petricolus group currently contains 10 species, namely g. boehmei, g. badenii, g. canaensis, g. flavimaritus, g. grossmanni, g. lauhachindai, g. petricolus, g. russelltraini, g. takouensis, and g. vietnamensis. (ngo et al. 2009; panitvong et al. 2010; ngo and gamble 2011; rösler et al. 2011; luu et al. 2015; rujirawan et al. 2019). karyological analyses in gekko have differentiated species based on mitotic metaphase chromosomal morphology while sporadic reports have based the species differentiation on meiotic metaphase chromosomal morphology. the chromosome study of gekkonid that have been reported such as; hemidactylus: diploid number (2n) ranging from 40-56 and mostly 40 or 46 (de smet 1981; patawang and tanomtong 2015b), gehyra: mostly 44 (king, 1984), ptychozoon: 2n = 34 and 42 (ota and hikida, 1988), paroedura: diploid number ranging from 31-38 and mostly 36 (aprea et al. 2013; koubova et al. 2014), phelsuma: 2n = 36 (aprea et al., 1996), dixonius: 2n = 42 (ota et al., 2001), and genus gekko, there are several reports on cytogenetic studies of g. gecko, namely, g. gecko: 2n=38 (singh 1974; wu and zhao 1984; solleder and schmid, 1984; trifonov et al. 2011; qin et al. 2012; patawang et al. 2014), g. hokouensis: 2n=38 (chen et al. 1986; shibaike et al. 2009; kawai et al. 2009), g. japonicus: 2n=38 (yoshida and itoh, 1974; shibaike et al., 2009; trifonov et al., 2011), g. shibatai and g. vertebralis: 2n=38 (shibaike et al. 2009), g. vittatus and g. ulikovskii: 2n=38 (trifonov et al. 2011), g. tawaensis: 2n=38 (ota, 1989a; shibaike et al. 2009), g. taylori: 2n=42 (ota and nabhitabhata 1991), g. monarchus: 2n=44 (ota et al. 1990), g. yakuensis, g. petricolus and g. smithii: 2n=38-42 ( ota, 1989), g. kikuchii: 2n=44 ( ota, 1989a), g. chinensis: 2n=40 (lau et al. 1997), g. subpalmatus: 2n=38 (wu and zhao 1984), g. swinhonis: 2n=38 (chen et al. 1986) (table 1). most gekkonid chromosome complements consist of acrocentric or telocentric chromosomes which gradually decreases in size whereas the karyotype evolution within the group is accompanied by fusions, robertsonian fissions and pericentric inversions (gorman 1973). from the previous reports, there are no studies of g. petricolus chromosome or karyotypic analyses. the present study of the cytogenetics of g. gecko provides the first report on the conventional staining, ag-nor banding and fluorescence in situ hybridization techniques. data provided here can gain us the knowledge of cytogenetic information which can be used as a basis to comprehensively examine the taxonomy and evolutionary relationship of gekko species. matherial and methods sample collection we obtained five male and five female specimens of g. petricolus (fig. 1) that were collected from ubon ratchathani and mukdahan provinces, northeastern thailand. chromosome preparation chromosomes were directly prepared in vivo (ota 1989a; qin et al. 2012) using the following methods. the gecko intramuscular was inoculated colchicine solution then left for 12 h. after that cut testis samples and bone marrow into small pieces. then squashed and mixed with 0.075 m kcl. after discarding all large piece tissues, 15 ml of cell sediments were transferred to a centrifuge tube and incubated for 25–35 min. the kcl was discarded from the supernatant after centrifugation again at 3,000 rpm for 8 min. in fresh cool fixative, cells were fixed (3 methanol : 1 glacial acetic acid), gradually added to make a volume of 8 ml, before being centrifuged again at 3,000 rpm for 8 min. afterward the supernatant was expelled. fixation was repeated until the supernatant was clear whereas the pellet was mixed with 1 ml fixative. the mixture was dropped onto a clean and cold slide by micropipette followed by air-drying technique. chromosome staining with 20% of giemsa solution, the slides were conventionally stained for 30 minutes (patawang et al. 2014). after that, to remove excess stain, the slides were rinsed thoroughly with running tap water and were placed in air-dry at room temperature. ag-nor banding was analysed according to the method of howell and black (1980). two drops each of 50% silver nitrate and 2% gelatine solutions were added to slides, respectively. then, they were sealed with cover glasses and incubated at 60°c for 5-10 minutes. they were also soaked in distilled water until the cover glasses were separated. finally, the slides were placed in air-dry at room temperature. they were observed under microscope. chromosome checks metaphase figures were analysed following the chromosome classification of turpin and lejeune (1965). chromosomes were categorized as submetacentric (sm), 83classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand metacentric (m), telocentric (t), and acrocentric (a). the fundamental number (nf: number of chromosome arms) was gained by assigning a value of two to metacentric, submetacentric and acrocentric chromosomes and one to acrocentric chromosomes. fluorescence in situ hybridization technique the use of microsatellite investigations which were described by kubat et al. (2008) was followed here with slight modifications. these sequences were directly labeled with cy3 at the 5´-terminal during synthesis by sigma (st. louis, mo, usa). fluorescence in situ table 1 the karyotype reviews among the genes gekko (gekkonidae, squamata). species 2n nf karyotype formulas nors fish locations references gekko gekko 38 50 6m+4sm+2a+26t p4 thailand patawang (2014) 38 44 6bi-arms+32uni-arms singh (1974) 38 wu and zhao (1984) 38 46 8bi-arms+30uni-arms solleder and schmid (1984) 38 trifonov et al. (2011) 38 50 8 m+2sm+2st(a)+26t laos qin et al. (2012) 38 48 8 m+2sm+28t china qin et al. (2012) g. hokouensis 38 56 4 m+6sm+20t+8bi-arms* p19 china, chen et al. (1986) 38 58/59 2 m+8sm+10a+16t+z(t)+w(a) japan kawai et al. (2009) 38 58 4 m+8sm+18t+8bi-arms* p19 taiwan shibaike et al. (2009) 38 58/59 4 m+8sm+16t+8bi-arms*+z(t)w(sm) p19 japan shibaike et al. (2009) 38 42 2m+18sm+16a+zw p19 fish mapping thailand srikulnath (2015) g. japonicus 38 yoshida and itoh (1974) 38 58 4m+8sm+8st+18t p17 japan shibaike et al. (2010) 38 trifonov et al. (2011) g. shibatai 38 58 4m+8sm+18t+8bi-armed p19 japan shibaike et al. (2009) g. vertebralis 38 62 4m+14sm+14t+6bi-arme* p19 japan shibaike et al. (2009) g. vittatus 38 trifonov et al. (2011) g. ulikovskii 38 trifonov et al. (2011) g. tawaensis 38 58 4m+8sm+18t+8bi-armed p19 japan shibaike et al. (2009) 38 56 p19 japan ota (1989a) g. taylori 42 thailand ota and nabhitabhata (1991) g. monarchus 44 46 malaysia ota et al. (1990) g. yakuensis 38 56 p19 japan ota (1989a) g. petricolus 38 54 thailand ota (1989a) g. kikuchii 44 50 taiwan ota (1989a) g. chinensis 40 46 6bi-armed+34uni-armed china lau et al. (1997) g. smithii 38 48 ota (1989a) g. subpalmatus 38 58 wu and zhao (1984) g. swinhonis 38 66 chen et al. (1986) g. petricolus 38 54 4m+2sm+10a+22t p17 (ca)15, (gaa)10 thailand present study remarks: 2n = diploid chromosome number, nors = nucleolar organizer regions, nf = fundamental number (number of chromosome arms), bi-arm = bi-armed chromosome, m = metacentric, sm = submetacentric, a = acrocentric, t = telocentric chromosome, *=small biarms chromosome, and = not available. figure 1. general characteristic of g. petricolus (scale bar = 5 cm). 84 weera thongnetr et al. hybridization (fish) was performed under highly rigorous conditions on mitotic chromosome spreads (pinkel et al. 1986). after denaturation of chromosomal dna in 70% formamide/ 2×ssc at 70 °c, spreads were incubated in 2×ssc for 4 min at 70 °c. the hybridization mixture (2.5 ng/μl probes, 2 μg/μl salmon sperm dna, 50% deionized formamide, 10% dextran sulphate) was dropped on the slides, and the hybridization was performed overnight at 37 °c in a moist chamber containing 2×ssc. the post hybridization wash was fulfilled with 1×ssc for 5 min at 65 °c. a final wash was operated at room temperature in 4×ssct for 5 min. finally, the chromosomes were counterstained with dapi (1.2 μg/ml), mounted in antifading solution (vector, burlingame, ca, usa), and analyzed in fluorescence microscope nikon eclipse. results and discussion mitotic chromosome features from giemsa staining karyomorphology of the g. petricolus revealed that the number of diploid chromosome (2n) was 38 and the fundamental number was 54. the karyotype composed of 4 large metacentric, 4 large acrocentric, 2 large telocentric, 4 medium acrocentric, 2 medium telocentric, 2 small submetacentric, 2 small acrocentric and 18 small telocentric chromosomes. (table 2 and figs. 1a-b). the karyotype formula of g. petricolus 2n (38) = lm4+la4+lt 2+ma4+mt2+ssm2+sa2+st18. the diploid chromosome number is following previous studies of gekkos. however, overall karyotypes of g. petricolus was similar to other gekko, diploid number ranging from 38-42 and mostly 38 (singh 1974; wu and zhao 1984; solleder and schmid 1984; yoshida and itoh 1974; ota 1989a; ota et al. 1990; ota and nabhitabhata 1991; lau et al. 1997; chen et al. 1986; kawai et al. 2009; shibaike et al. 2009; trifonov et al. 2011; qin et al. 2012; patawang et al. 2014). proximity of chromosome number and karyotype feature within genus gecko represent a close evolutionary line in the group. this species exhibit no sex differences in karyotypes between males and females (figs. 2a-b). no cytologically distinguishable sex chromosome was observed to be similar to g. shibatai, g. tawaensis, g. yakuensis, g. vertebralis, g. japonicas, g. vittatus, g. ulikovskii, g. chinensis, g. kikuchii, g. monarchus, g. petricola, g. smithii, g. subpalmatus, g. swinhonis and g. taylori (shibaike et al. 2009) and other lizards in thailand (satrawaha and ponkanid, 1988, wongwattana et al., 2001). this study is different from the study of kawai table 2. karyomorphological details of the g. petricolus, 2n = 38. chro. pair ls (µm) ll (µm) lt (µm) rl±sd ci±sd sizes types 1 2.350 3.284 5.563 0.116±0.006 0.589±0.017 large metacentric 2 2.237 2.906 5.143 0.107±0.005 0.556±0.024 large metacentric 3 0.967 3.130 4.047 0.085±0.003 0.767±0.033 large acrocentric 4 0.833 2.986 3.790 0.079±0.003 0.781±0.029 large acrocentric 5 0.000 3.294 3.229 0.067±0.004 1.000±0.000 large telocentric 6 0.000 3.085 3.050 0.063±0.002 1.000±0.000 medium telocentric 7 0.656 2.354 2.953 0.062±0.002 0.792±0.025 medium acrocentric 8 0.536 2.269 2.787 0.058±0.003 0.813±0.034 medium acrocentric 9 0.000 2.442 2.420 0.050±0.002 1.000±0.000 small telocentric 10 0.000 2.272 2.236 0.047±0.002 1.000±0.000 small telocentric 11 0.000 1.972 1.941 0.041±0.003 1.000±0.000 small telocentric 12 0.000 1.817 1.774 0.037±0.002 1.000±0.000 small telocentric 13 0.000 1.696 1.653 0.034±0.002 1.000±0.000 small telocentric 14 0.000 1.544 1.515 0.031±0.002 1.000±0.000 small telocentric 15 0.000 1.455 1.415 0.029±0.002 1.000±0.000 small telocentric 16 0.374 1.049 1.388 0.029±0.002 0.748±0.039 small acrocentric 17* 0.466 0.831 1.266 0.027±0.002 0.656±0.059 small submetacentric 18 0.000 0.971 0.969 0.020±0.002 1.000±0.000 small telocentric 19 0.000 0.838 0.857 0.018±0.002 1.000±0.000 small telocentric remarks: ls=short arm, ll=long arm, lt=total chromosome length, ci=centromeric index, rl=relative length, *=nors bearing chromosomes (satellite chromosome) 85classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand figure 2. metaphase chromosome plate and karyotypes of g. petricolus (a, b) by conventional technique, (c, d) by ag-nor banding technique (a, b) by fish technique of microsatellite probe (ca)15, (g, h) by microsatellite probe (gaa)10. scale bars = 10 µm. 86 weera thongnetr et al. et al. (2009) and shibaike et al. (2009) which revealed a zw system (sex-chromosomes) of g. hokouensis in japan. geckos represent an interesting group regarding the evolution of sex determination mechanisms and include species possessing either environmental or genetic sex determination systems. gecko populations without males were first discovered followed by seven parthenogenetic species (smith 1935; ota 1989b; volobouev et al. 1993), including triploid (3n) forms in some populations (moritz 1984). a zw system was recently revealed in g. hokouensis, and the genetic content of its sex chromosomes was similar to that of the avian z chromosome (kawai et al. 2009; shibaike et al. 2009). nucleolar organizer region the first cytogenetic study of g. petricolus carried out by ag-nor banding technique was obtained from this research. we found the observable nors on the region adjacent to telomere of long arm of the submetacentric chromosome pair 17th (figs 2c-d). the objective of the ag-nor banding technique is to detect nors which represent the location of genes that have a function in ribosome synthesis (18s and 28s ribosomal rna). nors produce numerous gene expressions and comprise non-histone proteins more than other chromosome regions. accordingly, the specific dark band (norpositive) is induced by the reduction of organic silver by these proteins that change silver to be dark (sharma et al., 2002). this study according to g. japonicas had two nors on the long arm near telomere of small bi-arms chromosome pair 17 (shibaike et al. 2009). the nor regions compared with other geckos, most showed two nors appearing near telomeric region of small biarmed or small mono-armed chromosome. an example of the previous reports of the genus gekko had two nors on one pair of small bi-arms chromosomes. g. gecko had two nors on the near telomere of mono-arms chromosome pair 4 (patawang 2014), while there were g. shibatai, g. yakuensis, g. hokouensis, g. tawaensis, g. vertebralis and g. yakuensis which had two nors on the long arm near telomere of small bi-arms chromosome pair 19 (ota 1989b; chen et al. 1986; shibaike et al. 2009). the use of nors in explaining kinships depends a large extent on the uniformity of this characteristic and the degree of variety within a taxon (yüksel and gaffaroğlu 2008). microsatellite pattern microsatellites or simple sequence repeats (ssrs) are oligonucleotides of 1–6 base pairs in length, forming excessive tandem repeats of usually 4 to 40 units (tautz and renz 1984; ellegren 2004; chistiakov et al. 2006). they show ample distribution throughout eukaryotic genomes, being scattered or clustered both in euchromatin and heterochromatin. they are highly polymorphic regarding copy number deviation (ellegren 2004). fluorescence hybridization indicate the (ca)15 repeat showing abundance at the telomeric ends of most chromosomes (figs. 2e-f), verifying the findings from other gekko groups investigated to date (srikulnath 2015). hybridization signals of (gaa)10 repeats were studied at all chromosomes (figs. 2g-h), while the results clearly showed that the microsatellite repeats are in high copy number on some chromosome pairs, according to previous reports on reptile groups (pokorná et al. 2011; matsubara et al., 2013). in this study, a comparison of the cytogenetic maps of g. hokouensis, enabled us to describe the processes of chromosomal reorganization in gekkota. these cytogenetic data could also be a substantial prerequisite the reptiles’ genome projects in the future. conclusions this study, the first cytogenetic study of g. petricolus. karyotyping from metaphase spreads of g. petricolus showed a chromosome number of 2n = 38, nf=54. the karyotype formula is 2n (38) = lm4+la4+lt2+ma4+mt 2+ssm2+sa2+st18. data obtained in this study can increase the knowledge of cytogenetic which can be used as a basis to comprehensively examine the taxonomy and evolutionary relationship of gekko species and other gekkonid. acknowledgments this research project was financially supported by mahasarakham university, phetchabun rajabhat university, khon kaen university and phetchabun rajabhat university. figure 3. idiogram represents the microsatellite probes (caa)10, (ca)15 and ag-nor banding on the chromosomes of g. petricolus. 87classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand references aprea g, odierna g, capriglione t, caputo v, morescalchi a, olmo e. 1996. heterochromatin and nor distribution in the chromosomes of six gekkonid species of the genus phelsuma (squamata: gekkonidae). african zoology 110 (5), 341–349. chen j, peng x, yu d. 1986. studies on the karyotype of three species of the genus gekko. acta herpetologica sinica 5, 24–29. de smet who. 1981. description of the orcein stained kariotypes of 27 lizard species (lacertilia: reptilia) belonging to the familias iguanidae, agamidae, hamaeleontidae and gekkonidae (ascalabota). acta zoologica et pathologica antverpiensia 76: 35–72. https://books.google.com/books/about/ a c t a _ z o o l o g i c a _ e t _ p a t h o l o g i c a _ a n t v e r p i e n . html?id=upuraqaaiaaj gorman g.c. 1973. cytotaxonomy and vertebrate evolution. new york: academic press. the chromosomes of the reptilia, a cytotaxonomic interpretation; 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(poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(4): 3-13, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1592 caryologia international journal of cytology, cytosystematics and cytogenetics citation: laleh malekmohammadi, masoud sheidai, farrokh ghahremaninejad, afshin danehkar, fahimeh koohdar (2022). avicennia genus molecular phylogeny and barcoding: a multiple approach. caryologia 75(4): 3-13. doi: 10.36253/caryologia-1592 received: march 02, 2022 accepted: october 20, 2022 published: april 28, 2023 copyright: © 2022 laleh malekmohammadi, masoud sheidai, farrokh ghahremaninejad, afshin danehkar, fahimeh koohdar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. avicennia genus molecular phylogeny and barcoding: a multiple approach laleh malekmohammadi1, masoud sheidai1,*, farrokh ghahremaninejad2, afshin danehkar3, fahimeh koohdar1 1 department of plant sciences and biotechnology, faculty of life sciences and biotechnology, shahid beheshti university, tehran, iran 2 department of plant sciences, faculty of biological sciences, kharazmi university, tehran, iran 3 department of environmental sciences, faculty of natural resources, university of tehran, karaj, iran *corresponding author. e-mail: msheidai@sbu.ac.ir abstract. the genus avicennia contains of 8 species which show a great extent of morphological and genetic variability, which make taxonomy of the genus difficult. molecular barcoding along with advancement in computational approaches may be proper methods to investigate and assess the efficiency of different molecular genetic regions in avicennia species delineation and also produce data on species evolution and divergence. the aims of present study were to utilize multiple genetic data for the species delineation and study the phylogeny of the genus. moreover, we developed a hypothesis on biogeography of these species with respect to barcode divergence. the results showed that both internal transcribed spacer (its) and trnhg–psba intergenic spacer (trnhg-psba) sequences may be used in avicennia species delineation. barcode gap analysis and nucleotide difference of the studied taxa showed significant fst for pairwise species comparison and the role of nucleotide changes in avicennia speciation. keywords: avicennia, barcode-gap analysis, genetic differentiation, nucleotide difference speciation. introduction dna barcoding is applied to plant and animal species with the aim to improve organismal identification and taxonomic clarification. the main principles of dna barcoding are standardization, minimalism, and scalability, which means selection one or a few standard loci that can be sequenced routinely and reliably in very large and diverse sample sets, and obtaining a reliable and conveniently comparable data to differentiate the species in question from one another (hollingsworth et al. 2011). controversy exits on the use and choosing the plant molecular barcode markers. different researches resulted in general agreement that several different marker combinations produce equivalent performance, and that none of the proposed barcodes is perfect in every respect (seberg and petersen 4 laleh malekmohammadi et al. 2009). utilizing a multiple approach for a better species differentiation has been suggested by several authors (see for example, fazekas et al. 2008). in most of the studies, researchers use of a common, easily amplified and aligned region such as rbcl, trn l-f spacer regions, mat k, trnhg-psba, nrits1, nrits2 or the full its1-5.8s-its2 (nrits), as suggested by the cbol plant working group and bold (cbol 2009; ratnasingham and hebert 2007). the genus  avicennia is composed of eight species of mangrove trees which grow in intertidal zones in tropical and temperate regions of the world. these plant species are economically important as they are extensively used as medicinal plants. in fact, different parts of these plants have ethno medicinal applications for treatment of various diseases such as cancer, diabetes, malaria, rheumatism, asthma, small pox and ulcer (hrudayanath et al. 2016). these species show variation and are taxonomically complex due to vast geographical distribution and introgressive hybridization (mori et al. 2015). therefore, the aims of present study are: 1assessment of different molecular markers in avicennia species delineation through barcode analysis, 2species relationships based on molecular markers, and 3biogeographical distribution of these species with regard to dna sequence divergence. materials and methods in this study, we used published data on trn l-f, trnhg-psba and its sequences for a number of avicennia species which are reported from different parts of the world in ncbi site (table 1, 2 and 3). data analyses dna sequences obtained were initially aligned by muscle program and cured accordingly. the total length, polymorphic sites, average of p distance, genetic diversity within the studied species, the fst values for the sequences and maximum liklihod phylogenetic tree based on these sequences as well as tajimas’d test was performed as implemented in mega ver. 7 (kumar et al. 2016). mantel test performed with 1000 permutations, for showing significant association between nucleotide difference of the studied species and population to the geographical longitude and altitude. the range of bayesian probability value obtained for species with mr. bayes analysis (ronquist et al. 2012). barcoding analysis and windows sliding of nucleotides were performed by barcodingr, and spider package, while skyline plot and mismatch distribution of nucleotides were determined by ape, and pegas package in r. monophyletic phylogenetic tree and its statistical test of rosenberg (2007), as implemented in r package. we used rasp (reconstruction of ancestral states in phylogeny) program ver. 4.2 in the rasp-bayesian analyzed phylogenetic tree. results species delimitation and barcode gap analysis we used published data on trn l-f, trnhg-psba and its sequences for a number of avicennia species which are reported from different parts of the world (table 1). in the first step, we evaluated the efficiency of these molecular markers in species delineation, and finally, we extracted different evolutionary information from the one with the highest degree of efficiency. details of the studied sequences with regard to avicennia species differentiation and species phylogeny are presented below. available data on trn l-f sequence is very limited and is available for only 21 samples of avicennia officinalis, a. marina, and a. alba. these sequences had a total length 296 bp, with only 14 polymorphic sites, and average p distacne = 0.006. the ml phylogetable 1. voucher information and genbank accession numbers of taxa sampled for the genus avicennia based on trnl-f data. sp accession number avicennia officinalis kt074999.1 avicennia officinalis mh215695.1 avicennia officinalis mh215694.1 avicennia officinalis mh215693.1 avicennia officinalis mh215692.1 avicennia officinalis mh215691.1 avicennia officinalis mh215690.1 avicennia officinalis mh215689.1 avicennia alba mh215683.1 avicennia alba mh215682.1 avicennia alba mh215681.1 avicennia alba mh215680.1 avicennia alba mh215679.1 avicennia marina kt074998.1 avicennia marina mh215688.1 avicennia marina mh215687.1 avicennia marina mh215686.1 avicennia marina mh215685.1 avicennia marina mh215684.1 avicennia marina km888791.1 avicennia marina jq728990.1 5avicennia genus molecular phylogeny and barcoding: a multiple approach netic tree based on these sequences (fig. 1), revealed that only the samples of a. alba can be differentiated from the other two species. analyses performed by bayesian method of species barcoding as implemented in barcoding package in r program indicated that only 25% of the studied samples have success in species identification, and the others may not be differentiated. in this analysis also the higher degree of bayesian probability was obtained for a. alba (0.50-0.97). the probability value obtained for the other species was about 0.06 only. sliding windows of the mini-barcodes within trn l-f barcode sequence 9 (fig. 2), also showed genetic distance in mini-barcodes between a. alba and the other species studied. trnhg-psba sequence efficiency in species delineation and barcoding trnhgpsba sequence data is available for 26 sample in avicennia species (table 2) namely, a. officinalis, a. marina, a. bicolor, a. germinans, a. alba, and a. rumphiana. these samples have also been reported from different parts of the world. preliminary analysis of trnhgpsba sequences indicated that the total length of the studied sequences is 167 bp, with 52 polymorphic sites, and average p distance = 0.09. table2 . voucher information and genbank accession numbers of taxa sampled for the genus avicennia based on trnhg-psba data. sp accession number avicennia officinalis kt161361.1 avicennia officinalis mn117565.1 avicennia officinalis mn117564.1 avicennia officinalis mn117563.1 avicennia officinalis mn117562.1 avicennia officinalis mn117561.1 avicennia officinalis mn117560.1 avicennia officinalis mn117559.1 avicennia alba mn117553.1 avicennia alba mn117552.1 avicennia alba mn117551.1 avicennia alba mn117550.1 avicennia alba mn117549.1 avicennia alba jx448690.1 avicennia marina kt161360.1 avicennia marina mn117558.1 avicennia marina mn117557.1 avicennia marina mn117556.1 avicennia marina mn117555.1 avicennia marina mn117554.1 avicennia marina jx448688.1 avicennia germinans kc420634.1 avicennia germinans kj426610.1 avicennia germinans hg963703.1 avicennia bicolor kc420633.1 avicennia rumphiana jx448689.1 figure 1. ml phylogenetic tree of avicennia species based on its sequences. figure 2. mini-barcode sliding windows of trnl-f sequence in avicennia species, showing genetic distance of avicennia alba with the other studied taxa. 6 laleh malekmohammadi et al. table 3. voucher information and genbank accession numbers of taxa sampled for the genus avicennia based on its data. sp accession number avicennia alba ef540977.1 avicennia alba af365980.1 avicennia alba mh243937.1 avicennia alba mh243936.1 avicennia alba mh243935.1 avicennia alba mh243934.1 avicennia alba mg880036.1 avicennia alba mg880035.1 avicennia alba mg880034.1 avicennia alba mg880033.1 avicennia alba mg880032.1 avicennia alba mg880031.1 avicennia alba mg880030.1 avicennia alba mg880029.1 avicennia alba mg880028.1 avicennia alba eu528876.1 avicennia alba kx641594.1 avicennia alba kj784551.1 avicennia alba kf848261.1 avicennia bicolor ef540988.1 avicennia bicolor ef540988.1 avicennia bicolor ef540987.1 avicennia bicolor eu352151.1 avicennia bicolor eu352150.1 avicennia bicolor eu352149.1 avicennia bicolor af365977.1 avicennia bicolor eu528877.1 avicennia germinans ef540990.1 avicennia germinans ef540985.1 avicennia germinans ef540984.1 avicennia germinans ef540983.1 avicennia germinans ef540982.1 avicennia germinans ef540981.1 avicennia germinans ef540980.1 avicennia germinans eu352146.1 avicennia germinans eu352147.1 avicennia germinans kx641596.1 avicennia germinans mg880047.1 avicennia germinans mg880046.1 avicennia germinans mg880045.1 avicennia germinans mg880041.1 avicennia germinans mg880040.1 avicennia germinans mg880039.1 avicennia germinans mg880038.1 avicennia germinans mg880037.1 avicennia germinans dq469846.1 avicennia germinans dq469845.1 avicennia germinans dq469860.1 avicennia germinans dq469859.1 sp accession number avicennia germinans dq469858.1 avicennia germinans dq469857.1 avicennia germinans dq469856.1 avicennia germinans dq469855.1 avicennia germinans dq469854.1 avicennia germinans dq469853.1 avicennia germinans dq469852.1 avicennia integra kx641598.1 avicennia officinalis mh243949.1 avicennia officinalis mh243948.1 avicennia officinalis mh243947.1 avicennia officinalis mh243946.1 avicennia officinalis mh243945.1 avicennia officinalis mh243944.1 avicennia officinalis mh243943.1 avicennia officinalis mg880054.1 avicennia officinalis mg880053.1 avicennia officinalis mg880052.1 avicennia officinalis mg880051.1 avicennia officinalis mg880050.1 avicennia officinalis kx641597.1 avicennia officinalis kj784553.1 avicennia officinalis kf848263.1 avicennia rumphiana kx641595.1 avicennia schaueriana ef540986.1 avicennia schaueriana dq469862.1 avicennia schaueriana ab861412.1 avicennia schaueriana ab861385.1 avicennia schaueriana ab861382.1 avicennia schaueriana ab861365.1 avicennia schaueriana ab861357.1 avicennia schaueriana ab861354.1 avicennia schaueriana ab861345.1 avicennia schaueriana ab861327.1 avicennia schaueriana ab861326.1 avicennia schaueriana ab861325.1 avicennia schaueriana ab861307.1 avicennia schaueriana ab861306.1 avicennia schaueriana ab861305.1 avicennia schaueriana ab861287.1 avicennia schaueriana ab861286.1 avicennia schaueriana ab861285.1 avicennia schaueriana ab861284.1 avicennia schaueriana ab861280.1 avicennia schaueriana ab861270.1 avicennia schaueriana ab861266.1 avicennia schaueriana ab861265.1 avicennia schaueriana ab861263.1 avicennia schaueriana ab861257.1 avicennia schaueriana ab861251.1 avicennia schaueriana ab861246.1 7avicennia genus molecular phylogeny and barcoding: a multiple approach bayesian method analysis of barcoding for trnhg psba sequences, reveals that about 53% of the samples are identified with success. bayesian probability value obtained for a. officinalis samples, ranged from 0.24 to 0.96. the same value for a. germinans, ranged from 0.50.95, for a. marina and a. alba the value ranged from was 0.2 to 0.96. monophyletic phylogenetic tree and its statistical test of rosenberg (2007), as implemented in r package is presented in fig. 3. the red circles on the nodes of this phylogenetic tree indicates that monophyly has passed the significant test at p = 0.05. therefore, we observe the presence of significant monophyletic groups for some of the sample in a. officinalis, a. alba, a. germinans, and a. marina. we have also some cases of admixture between the studied species which makes that clade nonmonophyletic. trnhgpsba nucleotide difference in the studied avicennia species is presented in fig. 4. this plot shows a great difference of the trnhgpsba nucleotides, which is a significant difference according to chi-square and snn test of hudson (hudson, 2000). the fst values for trnhgpsba sequences in the studied species ranged from 0.40-0.65. inter-specific genetic differentiation estimates obtained for the trnhgpsba nucleotide produced chi-square = 63.321, with p-value = 0.0012. similarly, hudson snn after 1000 permutations produced snn = 0.86, p<0.01. these values indicate significant difference among the studied samples. pair-wise mismatch plot of these sequences (fig. 5), revealed that, almost all the studied species-pairs differ significantly in their trnhgpsba nucleotides. sp accession number avicennia schaueriana ab861245.1 avicennia schaueriana ab861244.1 avicennia schaueriana ab861240.1 avicennia schaueriana ab861231.1 avicennia schaueriana ab861226.1 avicennia schaueriana ab861225.1 avicennia schaueriana ab861224.1 avicennia schaueriana ab861222.1 avicennia schaueriana ab861220.1 avicennia marina mf063712.1 avicennia marina mf063711.1 avicennia marina mf063710.1 avicennia marina mf063709.1 avicennia marina mf063708.1 avicennia marina ef540978.1 avicennia marina af477771.1 avicennia marina af477770.1 avicennia marina mn883387.1 avicennia marina mn883386.1 avicennia marina mn883385.1 avicennia marina mn883384.1 avicennia marina mh243942.1 avicennia marina mh243941.1 avicennia marina mh243940.1 avicennia marina mh243939.1 avicennia marina mh243938.1 avicennia marina mg880049.1 avicennia marina mg880048.1 avicennia marina mk027295.1 avicennia marina eu528879.1 avicennia marina km652500.1 avicennia marina kf848262.1 avicennia marina dq469861.1 avicennia marina subsp. marina kx641593.1 avicennia marina subsp. eucalyptifolia kx641592. avicennia marina subsp. australasica kx641591. avicennia marina subsp. australasica af365978.1 figure 3. monophyly analysis of avicennia species based on trnhg-psba sequences. athe red circles on the nodes indicate that the clade is significant at p = 0.05. figure 4. trnhg-psba nucleotide difference among avicennia species. 8 laleh malekmohammadi et al. mini-barcode analysis by windows sliding (fig. 6), also showed that trnhgpsba sequences contain mini-barcodes which differs among avicennia species. therefore, trnhgpsba sequences could be utilized for avicennia. species delineation. mini-barcode analysis by windows sliding (fig. 7), also showed that trnhgpsba sequences contain minibarcodes which differs among avicennia species. therefore, trnhgpsba sequences could be utilized for avicennia species delineation. its sequences species delineation and barcoding its sequences (table3) obtained had total length 494 bp, with polymorphic sites = 88, and average p dist = 0.038. bayesian method analysis revealed that about 42% of the samples are identified correctly. bayesian probability value obtained for avicennia alba ranged from 0.12 to 0.99, for a. bicolor ranged from 0.20 to 0.99, and for a. germinans, ranged from 0.16 to 1.00. almost the same ranges were obtained for other species. ml phylogenetic tree of 135 species samples (fig. 8), profigure 5. pair-wise mismatch plot of trnhg-psba sequences in avicennia species. figure 6. window sliding of trnhg-psba sequences in avicennia species, showing that these species differ in mini-barcodes. figure 7. trnhg-psba sequences in avicennia species, showing mini-barcodes which differentiate the studied taxa. figure 8. ml phylogenetic tree of avicennia species based on its sequences. 9avicennia genus molecular phylogeny and barcoding: a multiple approach duced almost distinct separate clades for the studied species, but some degree of admixtures was observed too. for the test of monophyly, we kept randomly some of the replicates of each species. the result of monophyly and its statistical test of rosenberg is presented in fig. 9. the red circles on some of the tree nodes indicates the monophyletic clade which is significant at p = 0.05. we also obtained monophyletic clades for some of the sample in a. officinalis, a. alba, a. germinans, a. bicolor and a. marina. we have also some cases of admixture between the studied species which makes some of the clade non-monophyletic. the nucleotide difference in its sequences of the studied avicennia species is presented in fig. 10. the plot shows a great difference, which is a significant difference according to chi-square and snn test of hudson. the fst values for its sequences ranged from 0.430.99. the inter-specific genetic differentiation estimates obtained for the its nucleotide produced chi-square = 90.26, with p-value = 0.001. similarly, hudson snn after 1000 permutations produced snn = 0.82, p<0.001. these values indicate significant difference among the studied samples. mismatch plot of its sequences (fig. 11), revealed that, almost all the studied species-pairs differ significantly in their trnhgpsba nucleotides. moreover, tajimas’d obtained was 0.32, which indicates the presence of a positive selection on its sequences and therefore its changes may be in some way related to speciation events in the genus avicennia. windows sliding of its sequences also revealed the occurrence of mini-barcodes in these sequences which also differed greatly among the studied species. for example, barcode sequences in some of the species are provided in figs 12 and 13. genetic diversity within the studied species ranged from 0.03 in a. alba to 0.12 in a. germinans, while interspecific genetic distance, 0.04 between a. alba and a. officinalis, and 0.08 between a. marina and a. integra, to 0.43, between a. germinans and a. alba. dna barcoding gap analysis of its sequences is presented in fig. 14. both intraand inter-specific sequence gaps, supports the use of its sequences for delineation of avicennia species. mantel test performed with 1000 permutations, showed no significant association between nucleotide difference of the studied species and population to the geographical longitude and altitude (correlation r = 0.044, p = 0.1456). details of avicennia species diversification based on its sequences and in relation with geographical distrifigure 9. monophyly test of the studied avicennia species based on its sequences. athe red circles on the. nodes indicate a significant monophyletic clade. figure 10. the nucleotide difference in its sequences among avicennia species. figure 11. mismatch plot of its sequences in avicennia species. figure 12. its barcode sequences differentiating avicennia alba and a. bicolor. 10 laleh malekmohammadi et al. bution of these species is presented in the rasp-bayesian analyzed phylogenetic tree (fig. 15). two main clades are present in this phylogenetic tree. the species of a. alba, a. officinalis, a. marina, a. rumphiana, and a. integra, comprised the first major clade, while, a. schueriana, a. germinnas, and a. bicolor formed the second major clade. looking at details of each major clade points out some interesting results. for example, most of a. marina samples were grouped together due to sequence similarity. though mantel test revealed no association between nucleotide difference and geographical longitude and latitude of the studied taxa, some interesting relationships between a. marina geographical populations can be seen figure 13. barcode its sequences differentiating avicennia alba and a. germinans. figure 14. barcode gap analysis of its sequences revealing both intraas well as inter-specific sequence difference in avicennia species studied. figure 15. rasp bayesian tree of its data, placing the species studied in two major clades. 11avicennia genus molecular phylogeny and barcoding: a multiple approach when we plot these studied specimens on the world map. for example, a. marina specimens studied from saudiarabia and egypt show sequence affinity and are placed close to each other in the phylogenetic tree. moreover, the specimens studied from madagascar, is placed close to the above said specimens. madagascar is some-what close to and connected by indian ocean to saudi-arabia and egypt. similarly, avicennia marina samples from china and australia, also show sequence similarity and form a separate sub-clade from the other specimens studied. the studied specimens from india stand in a separate sub-clade, far from the sub-clade of china-australia. if we consider all the samples studied, biogeographical distribution reveals that the species of a. marina, a. alba, and a. officinalis are mostly found in asia and australia region (denoted a. in fig. 16), while a. terminals, a. bicolor, and a. schauriena, are distributed in south america (denoted b in fig. 16). this may indicate speciation events in avicennia in two different regions of the world. we have also provided barcodes for geographical regions a and b, which shows nucleotide changes possibly associated / or the outcome of speciation in these two areas (fig 16). discussion the present study showed that based on its sequence analysis, a. alba and a. officinalis show close affinity and comprise sister-clades. a. marina joins these two with some distance. a. germinans samples form three separate clades, which indicates a potential presence of infra-specific taxon rank within this species. this is in accordance with earlier consideration which propose three different varifigure 16. biogeographical distribution of avicennia marina populations based on its sequences. 12 laleh malekmohammadi et al. eties for this species, which were later on were merged into a single species with no variety. a. bicolor showed close relationship to one of the clades of a. germinans. sample of a. schaueriana comprise a single distinct clade based on its data. however, the species relationships were partly distorted by trnhgpsba sequence data. the three species of a. officinalis, a. marina, and a. alba, were placed inter-mixed to some degree. close affinity bet ween a. germinans and a. bicolor are similar to its results. the close affinity between a. officinalis, a. marina, was also indicated by li et al. (2016). these authors showed closer relationship between a.  rumphiana and  a. alba, which in agreement with our trnhgpsba tree, and to some extent also with its-baes phylogenetic tree. according to li et al. (2016), even though the first fossil record of  avicennia in the iwp region dates back to the late eocene of southwest australia,  avicennia speciation was active during the miocene. similarly, they suggest that distribution of ancestral  avicennia was likely to have been similar to its present location, extending from japan to borneo and from the marshall islands to the red sea. therefore, the materials studied in this project, may have migrated from japan, china or india, through red sea and reached to the countries like iran, egypt, and sauidi-arabia. in these migration path, avicennia speciation may have resulted in formation of a. marina, a. officinalis and a. alba, as well as a. integra, and a. rumpiana. if we consider our its-based phylogenetic tree, we observe the species of the region b, viz. a. bicolor, a. germinans, a. schaueriana. are related through a. rumpiana and a. integra, to the species of region a. therefore, we may suggest a preliminary hypothesis that through migration of either or both of a. rumpiana and a. integra, new speciation events resulted in the formation of other species found in south america countries. we believe that more works are required to second this raw and immature hypothesis. mangroves in general, have a broad distributional patterns, ability in long-distance dispersal and can adapt to rigorous environmental constraints associated with regular seawater inundation. however, the present day distributions of individual taxa show several instances of finite dispersal limitations, especially across open water. these dis-continuities, in the absence of current dispersal barriers, may be explained by persistent past barriers (duke et al. 2002). in present study we report genetic diversity both with the studied avicennia species and between these taxa. a high levels of genetic diversity were also reported among the central populations of many mangrove species including avicennia in the indo-west pacific (iwp) (mantiquilla et al. 2021). mori et al. (2015), suggest that a. bicolor, a. germinans  and  a. schaueriana  are three evolutionary lineages that present historical and ongoing hybridization. they also consider gene flow between a. germinans,  and  a. schaueriana by propagules rather than pollen in a. schaueriana. we also reported distinct inter-specific genetic distance and significant fst value among different species both within those species distributed in the a geographical region (australia, india, china), and those distributed in the b region (south america in general). in a similar investigation performed presence of a strong genetic structuring resulting in divergence among mangrove populations of indian ocean and south china sea, as well as between south china sea and southwestern pacific was reported (mantiquilla et al. 2021). conclusion with regard to avicennia species taxonomy and the presence of high level of genetic diversity within these species, we provided distinct molecular barcodes for species delineation. we suggest it is suitable to utilize a combination of its nuclear sequences along with trnhgpsba spacer region of chloroplast genome for taxonomic purpose. list of abbreviations its: internal transcribed spacer ml: maximum liklihod rasp: reconstruction of ancestral states in phylogeny mega: molecular evolutionary genetics analysis acknowledgements we thank the iran national science foundation (insf), for partial financial supportof this project (no.4002299). author contribution statement masood sheidai: conceptualization of the project and corresponding author, farrokh ghahremaninejad: conceptualization of the project and data collection, afshin danehkar: conceptualization of the project and plant collection, fahimeh koohdar: conceptualization of the project and lab work, laleh malekmohammadi: data collection and lab work and data analysis. 13avicennia genus molecular phylogeny and barcoding: a multiple approach references cbol plant working group (2009) a dna barcode for land plants. proc natl acad sci 106, 12794±12797 fazekas aj, burgess ks, kesanakurti pr, graham sw, newmaster sg, husband bc, percy dm, hajibabaei m, barrettsch (2008) multiple multilocus dna barcodes from the plastid genome discriminate plant species equally well. plos one 3: e2802. hollingsworth pm, graham sw, little dp (2011) choosing and using a plant dna barcode. plos one 6(5): e19254 hudson rr (2000) a new statistic for detecting genetic differentiation. genetics 155: 2011-2014 hrudayanath t, dibyajyoti s, swagat kd (2016)  the genus  avicennia, a pioneer group of dominant mangrove plant species with potential medicinal values. front life sci 9: 267-291. mantiquilla ja, shiao msh, shih hch, chen wh, chiang ych (2021) a review on the genetic structure of ecologically and economically important mangrove species in the indo-west pacific. ecol genet genom 18: 100078. kumar s, stecher g, tamura k (2016) mega7: molecular evolutionary genetics analysis version 70 for bigger datasets. mol biol evol 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large model space syst biol61(3): 539–542. rosenberg n a (2007) statistical tests for taxonomic distinctiveness from observations of monophyly. evolution 61 (2): 317-323 seberg o, petersen g (2009) how many loci does it take to dna barcode acrocus?. plos one 4: e4598 caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 3 2022 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 75(2): 71-80, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1523 caryologia international journal of cytology, cytosystematics and cytogenetics citation: surachest aiumsumang, p a t c h a r a p o r n c h a i y a s a n , k a n khoomsab, weerayuth supiwong, alongklod tanomtong sumalee phimphan (2022) comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes). caryologia 75(2): 71-80. doi: 10.36253/ caryologia-1523 received: december 02, 2021 accepted: may 20, 2022 published: september 21, 2022 copyright: © 2022 surachest aiumsumang, patcharaporn chaiyasan, kan khoomsab, weerayuth supiwong, alongklod tanomtong sumalee phimphan. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* 1 biology program, faculty of science and technology, phetchabun rajabhat university, phetchabun 67000, thailand 2 program of biology, faculty of science, khon kaen university, muang, khon kaen 40002, thailand 3 education science program, faculty of science and technology, phetchabun rajabhat university, phetchabun 67000, thailand 4 applied science program, faculty of interdisciplinary studies, khon kaen university, nong khai campus, muang, nong khai 43000, thailand *corresponding author. e-mail: sumalee.phi@pcru.ac.th abstract. the present study focused on the repetitive dna of the chromosome in four minnow fishes from the genera danio hamilton, 1822, devario heckel, 1843 and rasbora bleeker, 1859. chromosomes were analysed using fluorescence  in situ  hybridization (fish) with microsatellite probes including (ca)15, (cac)10, (cgg)10, (gc)15 and (ta)15 staining. all species retained the diploid chromosome number 2n = 50 in male and female. the microsatellite sequences were mapped in the chromosomes of danio albolineatus (blyth, 1860), devario regina (fowler, 1934), rasbora aurotaenia tirant, 1885 and r. paviana tirant, 1885. in most cases, the microsatellite was dispersed in the chromosome with conspicuous markings in the telomeric region and the whole genome, which suggests that sequences contribute to chromosome structure and may have played a role in the relationship of this fish group. the comparative genome mapping data presented here provide novel information on the structure and organisation of the repetitive dna region of the minnow’s genome and contribute to a better understanding of the genomes of these minnows. keywords: cyprinidae, danioninae, fish, microsatellite. introduction the level of molecular cytogenetics plays an important role in precise characterisation of the structure of fish genomes (cioffi and bertollo 2012). the family cyprinidae is the most abundant and globally widespread family of freshwater fish, comprising 3,000 species are one of the largest groups (eschmeyer and fong 2015). thailand is one of the important areas for fish fauna in terms of both diversity and endemicity. the total number of fish spe72 surachest aiumsumang et al. cies in thai waters is over 2,700 with about 2,000 marine and 720 freshwater species (vidthayanon 2005). danio albolineatus (blyth, 1860), devario regina (fowler, 1934), rasbora aurotaenia tirant, 1885 and r. paviana tirant, 1885 are four of the species of minnows, belonging to the family cyprinidae (subfamily danioninae-danionini). they are tropical freshwater fish of minor commercial importance, which are native in thailand. their distributions include the mekong, chao phraya, and meklong basins (froese and pauly 2012) and they can be easily found in large and small rivers, ponds, ditches, lakes, paddy field, and swamps. it rarely occurs in low oxygen waters (brittan 1954; 1971; 1998). they could be used to assess if they were sensitive to change in environmental problems and aquatic pollution (blazer 2002; frame and dickerson 2006; raskovic et al. 2010; yenchum 2010; reddy and rawat 2013). however, cytogenetic studies in minnows are quite scarce, in which only conventional technique reported to determine chromosome number and karyotype composition has been performed. up to the present time, cytogenetic studies on subfamily danioninae (cyprinidae, osteichthyes) with 20 genera and about 335 species have been undertaken for only 21 species from three genera (danio, devario and rasbora) as yet, only conventional cytogenetics have been applied to determine chromosome numbers and karyotype complements. diploid chromosomes number (2n) varies between 48-50. cytogenetic studies of the genera danio, devario and rasbora have been reported in table 1. from the previous reports, in most cases, more descriptions of karyotypes seem to be inconclusive when they are not used in combination with other methods to produce more accurate chromosome markers. more recently, molecular cytogenetics begun to be implemented in a finer-scale characterization of karyotype structures in certain cyprinid taxa (spoz  et al. 2014; saenjundaeng et al. 2018; phimphan et al. 2020). more specifically, fish-based repetitive dna mapping, multiple dna copies of repetitive dnas are a large substantial portion of the genome of eukaryotes that can be generally classified into two main classes: tandem repeats, such as the multigene families and satellite dnas; also, there are dispersed elements, such as transposons and retrotransposons, known as transposable elements (tes) (jurka et al. 2005). repetitive dna sequences display a high degree of polymorphism because of the variation in the number of repetitive units, which results from specific evolutionary dynamics. the taxonomic status by dna genome as well as the phylogenetic relationships of danioninae species is confirmed and well established based on previous study (rüber et al., 2007; tang et al., 2010). recently, molecular cytogenetic studies, using fluorescence in situ hybridization (fish) for mapping of repetitive dna sequences, have provided important contributions to the characterisation of biodiversity and evolution of divergent fish groups (cioffi and bertollo 2012). moreover, some microsatellite repeats are species-specific characters amongst some fish groups (cioffi et al. 2015). an important role of repetitive dnas in genome evolution has been reported for different fish groups (cioffi and bertollo 2012; cioffi et al. 2010, 2015; moraes et al. 2017; 2019; sassi et al. 2019; terencio et al. 2013; yano et al. 2014). thus, the present study is the report on chromosomal characteristics of fish mapping of the microsatellites repeats in da. albolineatus, de. regina, r. aurotaenia and r. paviana by using molecular cytogenetic protocols. the knowledge gained can provide cytogenetic information potentially useful for further study in this family. matherial and methods individuals from both sexes of the four minnows were collected for analyses from river basins in, thailand (table 2 and fig. 1). the fishes were transferred to laboratory aquaria and kept under standard conditions for three days before the experiments. the procedures followed ethical protocols, as approved by the ethics of animal experimentation of the national research council of thailand u1-04498-2559. preparation of fish chromosomes was from kidney cells (phimphan et al. 2020). the chromosomes were stained with giemsa’s solution for 10 min. metaphase figures were analysed according to the chromosome classification of levan et al. (1964). chromosomes were classified as metacentric (m), submetacentric (sm), subtelocentric (st) or acrocentric (a). the fundamental number, nf (number of chromosome arms) is obtained by assigning a value of two (2) to metacentric and submetacentric chromosomes and one (1) to subtelocentric and acrocentric chromosomes. fish was performed under stringent conditions on metaphase chromosome spreads with microsatellite (ca)15, (cac)10, (cgg)10, (gc)15 and (ta)15 probes (kubat et al. 2008; liehr 2009) which were directly labelled with cy3 at 5´terminal during synthesis by sigma (st. louis mo, usa). fish, under stringent conditions on mitotic chromosome spreads (pinkel et al. 1986), was carried out by previous protocols as reported by supiwong et al. (2019) and yano et al. (2017). the hybridzation signals were checked and analysed on an 73comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) table 1. reviews of cytogenetic reports in the genera danio, devario and rasbora. (2n = diploid number, m = metacentric, sm = submetacentric, st = subtelocentric, a = acrocentric, nors = nucleolar organizer regions, nf = fundamental number, + = positive = not available). genus/species 2n nf karyotype formula fish reference danio rerio 48 post (1965) 50 100 50m endo and ingalls (1968) 50 100 10m + 40sm fontana et al. (1970) 50 100 10m + 12sm +28a rishi (1976) 50 100 16m + 32sm + 2a schreeb et al. (1993) 50 100 12m + 26sm + 12a pijnacker and ferwerda (1995) 50 100 4m + 16sm + 30a daga et al. (1996) 50 100 4m + 16sm + 30a gornung et al. (1997) 50 100 f: 7m + 7sm + 36a m: 6m + 8sm + 36a sharma et al. (1998) 50 100 12m + 26sm + 12a ueda and naoi (1999) 50 100 100 4m + 30sm + 16a or* 4m + 20sm + 26a amores and postlethwait (1999) 50 100 4m + 16sm + 30a + phillips and reed (2000) 50 100 4m + 16sm + 30a + sola and gornung (2001) da. roseus 50 100 8m+34sm+8a kaewtip et al. (2021) da. albolineatus 50 99 10m+39sm+1a arai (2011) 50 100 8m + 14sm + 28a + present study devario laoensis 50 50 100 96 6m+10sm+34a 6m+20sm+20a+4t aiumsumang et al. (2021) kaewtip et al. (2021) de. aequipinnatus 50 96 6m+34sm+6st+4t sukham et al. (2013) de. regina 50 100 6m+12sm+32a aiumsumang et al. (2021) 50 100 6m+12sm+32a + present study rasbora agilis 50 100 24m+26sm donsakul et al. (2009) r. aurotaenia 50 92 14m+26sm+2a+8t seetapan and moeikum (2004) 50 98 8m+16sm+24a+2t aiumsumang et al. (2012) 50 98 8m+16sm+24a+2t + present study r. borapetensis 50 88 24m+14sm+12t donsakul et al. (2005) r. buchanani 50 100 30m+18sm+2a manna and khuda-bukhsh (1977) r. caudimaculata 50 98 20m+26sm+2a+2t donsakul and magtoon (2002) r. daniconius 50 80 18m+6sm+6a+20t khuda-bukhsh et al. (1979) 50 92 32m+8sm+2a+8t donsakul et al. (2005) r. dorsiocellata 50 92 18m+24sm+8t donsakul et al. (2009) r. einthovenii 50 94 6m+30sm+8a+6t donsakul et al. (2005) 50 100 16m+18sm+16a yeesaem et al. (2019) r. heteromorpha 48 post (1965) 48 74 14m+10sm+2a+22t donsakul et al. (2005) r. myersi 50 90 20m+14sm+6a+10t donsakul and magtoon (2002) r. paviei 50 100 10m+24sm+16a donsakul and magtoon (2002) r. paviana 50 98 8m+16sm+24a+2t aiumsumang et al. (2021) 50 98 8m+16sm+24a+2t + present study r. retrodorsalis 50 88 26m+10sm+2a+12t donsakul and magtoon (2002) r. rubrodorsalis 50 82 16m+16sm+18t donsakul et al. (2009) r. sumatrana 50 94 26m+16sm+2a+6t donsakul and magtoon (1995) r. trilineata 48 post (1965) 50 94 26m+16sm+2a+6t donsakul et al. (2005) 74 surachest aiumsumang et al. epifluorescence microscope olympus bx50 (olympus corporation, ishikawa, japan). results diploid number and fundamental number of da.  albolineatus, de. regina, r. aurotaenia and r. paviana the four minnow fishes have the same diploid number of 2n = 50. although the minnow fishes analysed share the same 2n, there are differences in the fundamental number (nf) i.e. da. albolineatus and de. regina nf = 100, while for the two rasbora, nf = 98, the karyotype complements of d. albolineatus composed of 8m + 14sm + 28a, d. regina was m6+sm10+a34, while r. aurotaenia and r. paviana were 8m+16sm+24a+2t (figs. 2a-d). patterns of the microsatellite repeat in the genomes of da. albolineatus, de. regina, r. aurotaenia and r. paviana the result of the mapping of the microsatellite repeats (ca)15 show that hybridization signals are abundantly distributed on telomeric regions in all species (figs. 3e-h), (cac)10 showing moderate abundance in da. albolineatus and r. paviana, while de. regina and r. aurotaenia were not detected (figs. 3i-l). the region hybridized (cgg)10 of da. albolineatus and r. paviana identified a partial genome, de. regina has a hybridization pattern throughout the genomes and r. paviana was not detected (figs. 3m-p). (gc)15 hybridized in da. albolineatus and r. paviana as whole genomes, da. albolineatus as telomeric regions and r. paviana was not detected (figs. 3q-t). the microsatellite (ta)15 probe showed hybridization on the whole genomes of da. albolineatus and de. regina, while two rabora were not detected (figs. 3u-x) (table 3). discussion diploid number of and fundamental number da. albolineatus, de. regina, r. aurotaenia and r. paviana da. albolineatus had 2n = 50 which is in accordance with one single previous report (arai 2011) and the same in other species in genus danio (post 1965; endo and ingalls 1968; fontana et al. 1970; rishi 1976; schreeb et al. 1993; pijnacker and ferwerda 1995; daga et al. 1996; gornung et al. 1997; sharma et al. 1998; ueda and naoi 1999; amores and postlethwait 1999; phillips table 2. collection sites of the analyzed species show the sample number. species number of specimens in site sampling mae khong basin sirindhorn peat swamp forest pasak basin chi basin chao phaya basin songkhram basin danio albolineatus 10 ♀ 10 ♂ 04 ♀ 05 ♂ 05 ♀ 05 ♂ devario regina 05 ♀ 06 ♂ 06 ♀ 08 ♂ rasbora aurotaenia 08 ♀ 07 ♂ 05 ♀ 08 ♂ rasbora paviana 05 ♀ 08 ♂ 03 ♀ 04 ♂ 05 ♀ 07 ♂ 04 ♀ 05 ♂ figure 1. map showing the collection sites of danio albolineatus [red circles], devario regina [blue circles], rasbora aurotaenia [green circles] and rasbora paviana [yellow circles] for studied herein. 75comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) and reed 2000; sola and gornung 2001), da. roseus: 2n=50 (kaewtip et al. 2021) and da. albolineatus: 2n=50 (arai 2011). the 2n of de. regina is the same as that of de. laoensis pellegrin & fang, 1940 (aiumsumang et al. 2021; kaewtip et al. 2021) and de. aequipinnatus reported by sukham et al. (2013). r. aurotaenia has 2n=50 according to seetapan and moeikum (2004) and aiumsumang et al. (2012). the diploid chromosome number of r. paviana was 50, which is the same as that from the previous study by aiumsumang et al. (2021). this 2n is considered the same as for the other species of subfamily danioninae (danio, devario and rasbora). patterns of microsatellite repeats in the genomes of da. albolineatus, de. regina, r. aurotaenia and r. paviana the chromosomal distribution of repetitive dna elements revealed remarkable differences amongst the analysed species. in this study, the number and distribution of microsatellite sequence (ca)15, (cac)10, (cgg)10, (gc)15 and (ta)15 were not conserved among four analyzed species. the microsatellite sequence (ca)15 was mapped on chromosomes of da. albolineatus, de. regina, r. aurotaenia and r. paviana, and abundantly located and distributed in all chromosomes, usually in telomeric regions and a few chromosome pairs showed the strongest signal intensities, these not really being suited to serve as chromosomal markers. microsatellites are usually located in the heterochromatic regions (telomeres/centromeres) of fish genomes, where a significant fraction of repetitive dna is localized (cioffi and bertollo, 2012). indeed, this distribution pattern is found in epalzeorhynchos frenatum (fowler, 1934), puntigrus partipentazona (fowler, 1934), scaphognathops bandanensis boonyaratpalin & srirungroj, 1971 (phimphan et al, 2020); catlocarpio siamensis boulenger, 1898 and probarbus jullieni sauvage, 1880 (saenjundaeng et figure 2. karyotype of danio albolineatus [a], devario regina [b], rasbora aurotaenia [c], rasbora paviana [d], m = metacentric, sm = submetacentric, a = acrocentric and t = telocentric chromosomes. table 3. molecular cytogenetic studies on four minnow fishes. [2n = diploid chromosome number, nf fundamental number (number of chromosome arm)]. species 2n nf (ca)15 (cac)10 (cgg)10 (gc)15 (ta)15 da. albolineatus 50 100 telomere partial genome partial genome whole genome whole genome de. regina 50 100 telomere not detected whole genome telomere whole genome r. aurotaenia 50 98 telomere not detected not detected partial genome not detected r. paviana 50 98 telomere partial genome partial genome whole genome not detected 76 surachest aiumsumang et al. figure 3. fish using dapi (a-d), [ca]15 (e-h), [cac]10 (i-l), [cgg]10 (m-p), [gc]15 (q-t), [ta]15 (u-z) of danio albolineatus, devario regina, rasbora aurotaenia and rasbora paviana, respectively. scale bar 5 µm. 77comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) al. 2018); clarias species (maneechot et al. 2016) and channa micropeltes (cuvier, 1831) (cioffi et al. 2015).this distribution pattern are differences known for channa gachua (hamilton, 1822), c. lucius (cuvier, 1831), c. striata (bloch, 1793) (cioffi et al. 2015), hemibagrus wyckii (bleeker, 1858) (supiwong et al. 2017) and monopterus albus (zuiew, 1793) (supiwong et al. 2019). for the mapping of (cac)10 repeats in da. albolineatus and r. paviana, partial genomes were identified, while de. regina and r. aurotaenia were not detected. in da. albolineatus, de. regina, r. aurotaenia and r. paviana, most microsatellites (cgg)10 and (cg)15 were abundantly distributed in all chromosomes with the exception of (cgg)10 in r. aurotaenia, this not being a signal probe. nevertheless, comparative analyses among the species indicate that the microsatellites have also preferential zones of accumulation at telomeric heterochromatin. for de. regina the accumulation of microsatellites (gc)15 in all chromosomal pair could point out a species–specific type of heterochromatin is similar to previous reports in channa micropeltes (cioffi et al. 2015). in da. albolineatus r. aurotaenia and r. paviana the same microsatellites are not only located at the telomeric region of some chromosomes, but they are also found at the centromeres of the chromosome pair. (ta)15 repeats in da. albolineatus and de. regina, displaying high accumulations at the whole genome, while not detected in the rasbora group. repetitive dna sequences display a high degree of polymorphism because of the variation in the number of repetitive units, which results from a specific evolutionary dynamic. amongst these elements, microsatellites (or short tandem repeats) are the most polymorphic and consist of short sequences of one to six nucleotides repeated in tandem throughout the dna (tautz and renz 1984). due to their supposed neutral evolution, these molecular markers have been widely used in population genetics, to identify taxonomic limits and in hybridization and forensic studies (goldstein and pollock 1997; filcek et al. 2005; racey et al. 2007; mccusker et al. 2008) and can be used to spot genomic evolution as previously been reported for different fish groups (cioffi et al. 2010; cioffi and bertollo 2012; terencio et al. 2013; yano et al. 2014; cioffi et al. 2015; moraes et al. 2017; 2019; sassi et al. 2019). the comparative study on four species showed that the diploid chromosome is the same, but the patterns of microsatellite repeat on chromosomes have differences amongst them. thus, the molecular cytogenetic data may be a tool for classification of fish species where there is similar morphology, such as the stripe. overall, it is believed that the microsatellites have specific zones of accumulation in genomes, preferentially in heterochromatic regions (supiwong et al. 2014). in fact, microsatellites are located in the heterochromatic regions (telomeres, centromeres and in the sex chromosomes) of fish genomes (cioffi and bertollo 2012, including the present study). however, the distribution of microsatellites was not only restricted to heterochromatin, but also dispersed in euchromatic regions of the chromosomes (getlekha et al. 2016). additionally, such variability is also reinforced by the dynamism of repetitive elements in the genome, especially by the differential distribution and accumulation of rdna sequences amongst chromosomes (cioffi et al. 2015). although not yet completely understood, this marked diversity is likely linked to the lifestyle of these fishes and to population fragmentation, as already identified for other fish species. conclusions our study is the first one to offer reliable chromosomal data for da. albolineatus, de. regina, r. aurotaenia and r. paviana by molecular cytogenetic protocols, this study were useful tools in highlighting the remarkable chromosomal diversification which was characterised in the four minnow fishes. besides, data from comparative genomic hybridization experiments also highlighted an advanced stage of repetitive dna divergence, evidencing their evolutionary diversification. acknowledgments this study was supported by the national research council of thailand under the phetchabun rajabhat university (grant no. frb640052/04) and the postdoctoral training program from research affairs and graduate school, khon kaen university, thailand (grant no. 59255). references aiumsumang s, phimphan s, suwannapoom c, chaiyasan p, supiwong w, tanomtong a. 2021. a comparative chromosome study on five minnow fishes (cyprinidae, cypriniformes) in thailand. caryologia 74(1): 87–94. https://doi.org/10.36253/caryologia-1017 amores a, postlethwait jh. 1999. banded chromosomes and the zebrafish karyotype. methods in cell biology 60: 323–338. https://doi.org/ 10.1016/s0091679x(08)61908-1 78 surachest aiumsumang et al. arai r. 2011. fish karyotypes: a check list. springer japan, tokyo. https://doi.org/10.1007/978-4-431-53877-6 blazer vs. 2002. histopathological assessment of gonadal tissue in wild fishes. physiology biochemistry 26: 85–101. https://doi.org/10.1023/a:1023332216713 brittan mr. 1954. a revision of the indo-malayan fresh-water fish genus rasbora. institute  of  science and technology. manila monogr 3: 1–224. brittan mr. 1971. rasbora: a revision of the indomalayan fresh-water fish genus rasbora. t.f.h. publications, neptune city. brittan mr. 1998. rasboras: keeping and breeding them in captivity. t.f.h. publications, neptune city. cioffi mb, bertollo lac. 2012. distribution and evolution of repetitive dnas in fish. in: garrido-ramos, m. a. 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[in thai] caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 2 2022 firenze university press cytogenetic studies of six species in family araceae from thailand piyaporn saensouk1, surapon saensouk2,*, rattanavalee senavongse2 effect of ag nanoparticles on morphological and physio-biochemical traits of the medicinal plant stevia rebaudiana sherzad r. abdull, sahar h. rashid*, bakhtiar s. ghafoor, barzan s. khdhir morphometric analysis and genetic diversity in hypericum l. using sequence related amplified polymorphism wei cao1, xiao chen2,*, zhiwei cao3 population differentiation and gene flow of salicornia persica akhani (chenopodiaceae) xiaoju zhang1, li bai2,*, somayeh esfandani-bozchaloyi3 scot molecular markers are efficient in genetic fingerprinting of pomegranate (punica granatum l.) cultivars shiva shahsavari1, zahra noormohammadi1,*, masoud sheidai2,*, farah farahani3, mohammad reza vazifeshenas4 first record of nucleus migration in premeiotic antherial cells of saccharum spontaneum l. (poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(2): 89-101, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1058 caryologia international journal of cytology, cytosystematics and cytogenetics citation: amanda de souza machado, samantha kowalski, leonardo marcel paiz, vladimir pavan margarido, daniel rodrigues blanco, paulo cesar venere, sandra mariotto, liano centofante e orlando moreira-filho, roberto laridondo lui (2021) comparative cytogenetic analysis between species of auchenipterus and entomocorus (siluriformes, auchenipteridae). caryologia 74(2): 89-101. doi: 10.36253/caryologia-1058 received: august 21, 2020 accepted: july 20, 2021 published: october 08, 2021 copyright: © 2021 amanda de souza machado, samantha kowalski, leonardo marcel paiz, vladimir pavan margarido, daniel rodrigues blanco, paulo cesar venere, sandra mariotto, liano centofante e orlando moreira-filho, roberto laridondo lui. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid asm:0000-0002-1253-1357 sk: 0000-0002-4507-5714 lmp: 0000-0002-4761-8321 vpm: 0000-0002-0823-6646 drb: 0000-0003-1619-2417 pcv: 0000-0001-7236-8857 sm: 0000-0003-4007-3100 lc: 0000-0003-0712-8149 omf: 0000-0001-5137-0122 rll: 0000-0003-4310-4865 comparative cytogenetic analysis between species of auchenipterus and entomocorus (siluriformes, auchenipteridae) amanda de souza machado, samantha kowalski, leonardo marcel paiz, vladimir pavan margarido, daniel rodrigues blanco, paulo cesar venere, sandra mariotto, liano centofante, orlando moreira-filho, roberto laridondo lui* centro de ciências biológicas e da saúde, universidade estadual do oeste do paraná, cascavel, paraná, brazil; departamento de biologia geral, universidade estadual de londrina, centro de ciências biológicas, londrina, paraná, brazil; universidade tecnológica federal do paraná, santa helena, paraná, brazil; universidade federal de mato grosso, cuiabá, mato grosso, brazil; instituto federal de educação, ciência e tecnologia de mato grosso, cuiabá, mato grosso, brazil; universidade federal de são carlos, são carlos, são paulo, brazil *corresponding author. e-mail: roberto.lui@unioeste.br abstract. according to auchenipteridae initial morphological data, auchenipterus and entomocorus have been considered phylogenetically close, and cytogenetic analyses are limited only to auchenipterus osteomystax. herein, we provide the first cytogenetic results about auchenipterus nuchalis from araguaia river and entomocorus radiosus from paraguay river. these data were generated in order to contribute to the investigation of the auchenipterus chromosomal diversity and to attempt to better understand the phylogenetic relationship of these auchenipterinae genera, mainly due to the existence of incongruous characters between entomocorus and centromochlinae. the two species presented 2n=58 chromosomes and had different karyotype formulas. the heterochromatin distribution was primarily shown in terminal regions, along with interstitial and/or pericentromeric blocks in submetacentric/subtelocentric pairs in a. nuchalis and e. radiosus. single and terminal agnors were confirmed by 18s rdna for the analyzed species, differing from a. osteomystax (cited as a. nuchalis) from upper paraná river. the variation in the number of 5s rdna between species and its equilocality in e. radiosus suggest that the dispersion of the gene associated with the amplification of heterochromatic regions in the interphase, possibly promoted by the rabl model system. the differences found between the species of auchenipterus can work as species-specific characters and assist in studies of these taxa, which historically have been wrongly identified as a single species with wide distribution throughout the neotropical region, when they are actually different species. furthermore, there are cytogenetic similarities between e. radiosus and members of centromochlinae like pointed out by recent morphological and molecular analyses in the family. keywords: centromochlinae, equilocality, species-specific characters, rabl, 5s rdna. 90 amanda de souza machado et al. introduction vertebrates comprise more than 60.000 described species and about 32.000 of them are fish (nelson 2016). in south america, a great ichthyofaunal diversity is reported, estimated to be over 9.100 species, which approximately 56% is from freshwater systems (reis et al. 2016). the emergence and evolution of the freshwater ichthyofauna in the neotropical region is large due to the humid tropical regions favorable for aquatic life (albert et al. 2011). furthermore, extensive geological events such as the formation of the guiana shield, the brazilian shield and the uplift of the andes allowed the formation of important drainage axes that resulted in several speciation processes within and between the basins, thus reflecting the rich taxonomic composition of the freshwater ichthyofauna in the region (reis et al. 2016). auchenipteridae, endemic to the neotropical region, is subdivided into centromochlinae and auchenipterinae and consists of 25 genera and 127 species (fricke et al. 2021). moreover, it includes fishes known as inseminating and with external development (calegari et al. 2019), just like in other siluriformes families, such as scoloplacidae and astroblepidae (spadella et al. 2006, 2012). this characteristic is directly associated with the sexual dimorphism related to modification of fins or barbels, which makes the internal insemination as a reproductive strategy in the group possible (baumgartner et al. 2012; calegari et al. 2019). auchenipterinae comprises 18 genera, including auchenipterus valenciennes, 1840 and entomocorus eigenmann, 1917 (fricke et al. 2021). according to morphological data, these taxa are considered sister-groups and constituting a clade with other groups. the phylogenetic relationships propositions between these genera of auchenipteridae have undergone changes over time (e.g., britski 1972; ferraris 1988; royero 1999; akama 2004; calegari et al. 2019). entomocorus is composed of 4 species, entomocorus benjamini eigenmann, 1917 distributed in the upper madeira river basin; entomocorus gameroi mago-leccia, 1984 distributed in the drainages of the orinoco river; entomocorus malaphareus akama and ferraris, 2003 found in portions of the lower and middle amazon river and entomocorus radiosus reis and borges, 2006 endemic to the paraguay river basin, the latter is described for the pantanal region (reis and borges 2006; fricke et al. 2021). currently, the clade is reinforced by 41 molecular synapomorphies and 19 morphological synapomorphies (calegari et al. 2019), a number that increased considerably after the previous review by reis and borges (2006), which presented 8 morphological synapomorphies for the genus. auchenipterus is reinforced by 9 morphological synapomorphies (calegari et al. 2019) and is currently composed of 11 species widely distributed in the south american continent throughout the east of the andean region (fricke et al. 2021). unlike most species of the genus, auchenipterus nuchalis spix and agassiz, 1829 has a more restricted distribution and occurs only in a few portions of the amazon river basin and low portions of the tocantins river (ferraris and vari 1999); although it differs from more recent records in some locations (e.g., fricke et al. 2021). on the other hand, auchenipterus osteomystax miranda ribeiro, 1918 has a greater distribution from the lower amazon river basin, tocantins river and the prata river basin (fricke et al. 2021). according to ferraris and vari (1999), these two species have already been wrongly identified in different hydrographic systems, as is the case of records of specimens of a. osteomystax identified as a. nuchalis in portions of the paraná river, in the region of itaipu reservoir, and in porto rico (pr, brazil) (e.g., agostinho et al. 1993; cecilio et al. 1997; ravedutti and júlio jr. 2001). regarding the type species a. nuchalis (type locality: amazon river), synonymization problems of new species in different locations overestimated its distribution (ferraris and vari 1999). auchenipterus nuchalis was the first species described for auchenipterus valenciennes, 1840, however, it was initially classified as hypophthalmus nuchalis spix and agassiz, 1829 (birindelli 2014). after the genus description, a. nuchalis was included and kept in auchenipteridae since then, mainly due to the presence of sexual dimorphism (miranda ribeiro 1968), a character that proves to be very informative for the family (calegari et al. 2019). on the other hand, entomocorus was a target for some phylogenetic inconsistencies until a consensus was reached on its relationship with other close groups. according to britski (1972), auchenipterus was initially considered sister-group of the clade composed of epapterus and pseudepapterus (auchenipterus (epapterus, pseudepapterus)), whereas entomocorus was allocated close to trachelyichthys and pseudauchenipterus in a clade that is also made up of genera that currently belong to centromochlinae (trachelyichthys (entomocorus (pseudauchenipterus (centromochlus, glanidium)))). subsequently, auchenipterus and entomocorus were relocated to the same clade (entomocorus (auchenipterus, epapterus)), this closeness was reinforced by 14 morphological synapomorphies (ferraris, 1988). subsequent studies by royero (1999) and akama (2004) also kept entomocorus and auchenipterus close although, for these authors, the group (entomocorus, auchenipterus) has divergences in comparison with the epapterus and pseudepapterus taxa. 91comparative cytogenetic analysis between species of auchenipterus and entomocorus (siluriformes, auchenipteridae) this clade has remained allocated in auchenipterini tribe bleeker, 1862, initially created to contain auchenipterus valenciennes, 1840 and, currently with the addition of pseudauchenipterus, it is supported by 6 molecular synapomorphies and 9 morphological synapomorphies (pseudauchenipterus (entomocorus (pseudepapterus (epapterus, auchenipterus))) (calegari et al. 2019). nonetheless, entomocorus shares characters with centromochlinae and other siluriforms and diverges by some diagnostic characteristics of auchenipteridae (reis and borges 2006; calegari et al. 2019). this set of characteristics shared among members of the clade and other groups of catfish, according to birindelli (2014), is what could explain this group (entomocorus (auchenipterus (epapterus)) as basa l in the family, as proposed by royero (1999). regarding the relationship between entomocorus and centromochlinae, bayesian inference analyses (bi) based on molecular characters reinforced its inclusion in the subfamily, besides entomocorus shares the genital tube anteriorly to the anal fin base and separated from its first rays like seen in members of centromochlinae (calegari et al. 2019). however, calegari et al. (2019) still suggest that this relationship may be the result of events of genetic homoplasy (independent evolution) and not a common ancestry between the groups. regarding cytogenetic analyses in species of this clade, only a. osteomystax (cited as a. nuchalis) from the upper paraná river basin (e.g., ravedutti and júlio jr. 2001) was studied and, together with data from some other species of the family (e.g., fenocchio and bertollo 1992; fenocchio et al. 2008; lui et al. 2009, 2010, 2013a, 2013b, 2015; kowalski et al. 2020) (table 1) have contributed to the understanding of evolutionary relationships and diversification mechanisms in auchenipteridae. due to the absence of chromosomal data about a. nuchalis and e. radiosus, this study aimed (1) to investigate the chromosomal characteristics of a. nuchalis from the araguaia river basin, in search of species-specific characters that help to understand the diversity in auchenipterus, considering the history of incongruences related to its taxa using morphological data, and (2) searching for chromosomal characters in entomocorus and auchenipterus that can add information to the evolutionary understanding between auchenipteridae genera, specifically to the clade involving auchenipterus and entomocorus, since there are characters of morphological nature that approach entomocorus to some centromochlinae species. material and methods chromosomal analyses were performed on four specimens of auchenipterus nuchalis (figure 1a), two males and two females, from the araguaia river basin, between aragarças (go) and barra do garças (mt) (gps: 15°53’03,9”s; 52°06’17,9”w); and eleven specimens of entomocorus radiosus (figure 1b), six males and five females, from the paraguay river basin, poconé (mt) (gps: 16°25’40,9”s; 56°25’07,4”w) (permanent license sisbio 10538-1). the specimens of a. nuchalis e e. radiosus were deposited in the zoology museum of the university of são paulo, under the respective vouchers: mzusp 110805 and mzusp 109791. the specimens were euthanized with a clove oil overdose (griffthis 2000) to remove the anterior kidney and prepare the mitotic chromosome suspensions as described by bertollo et al. (1978) and foresti et al. (1993), according to committee of ethics in animal experimentation and practical classes from unioeste – (protocol 13/09 ceeaap/unioeste). the mitotic chromosomes were stained with giemsa 5% diluted in phosphate buffer (na2hpo4 x 12h2o + kh2po4 x 12h2o), ph = 6.8, for 7 minutes and classified according to levan et al. (1964) in metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (a). the c-banding technique followed the protocol according to sumner (1972) with modifications suggested by lui et al. (2012) and the detection of agnors through silver nitrate impregnation, according to howell and black (1980). the analysis of metaphases was done sequentially. fluorescent in situ hybridization (fish) was performed according to the methodology of pinkel et al. (1986) with modifications suggested by margarido and moreira-filho (2008), using the probes rdna 18s (hatanaka and galetti jr. 2004) and rdna 5s (martins et al. 2000). the rdna 18s probe was labeled with biotin-16-dutp by nick translation (biotin nick translation mix roche), with detection and amplification with avidin-fitc and anti-avidin biotin (sigma) for both species. the 5s rdna probe was labeled with digoxigenin-11-dutp by nick translation (dig 11 nick translation mix roche) and detected with anti-digoxigenin-rhodamine for a. nuchalis and labeled with fluorescein-12-dutp (fitc) by pcr for e. radiosus, using primers a (5’-tac gcc cga tct cgt ccg atc-3 ‘) and b (5’-cag gct ggt atg gcc gta agc-3’) (pendás et al. 1994). hybridizations were performed with 77% stringency (200 ng of each probe, 50% formamide, 10% dextran sulfate, 2xssc; ph 7.0 7.2). fish slides were analyzed using an epifluorescence photomicroscope olympus bx60 under an appropriate filter. 92 amanda de souza machado et al. ta bl e 1. c yt og en et ic d at a in a uc he ni pt er id ae . su bf am ily /s pe ci es lo ca lit y fn 2n k ar yo ty pi c fo rm ul a a gn o r s/ 18 s rd n a 5s r d n a r ef . c en tr om oc hl in ae g la ni di um r ib ei ro i ig ua çu r iv er , r es . s al to c ax ia s, p r 11 2 58 28 m +1 6s m +1 0s t+ 4a pa ir 1 7, p , i , s m 1 ig ua çu r iv er , r es . s eg re do , p r 10 6 58 22 m +1 6s m +1 0s t+ 10 a pa ir 1 3, p , i s m 2 ig ua çu r iv er , r es . s al to o só ri o, p r 10 6 58 22 m +1 6s m +1 0s t+ 10 a pa ir 1 3, p , i s m 2 ig ua çu r iv er , c ap an em a, p r 11 0 58 22 m +2 0s m +1 0s t+ 6a pa ir 1 4, p , i , s m pa ir 1 6, q , i , s m 3 ta tia n ei va i m ac ha do r iv er , d en is e, m t 11 6 58 26 m +2 6s m +6 st pa ir 2 8, p , t , s t pa ir 4 , p , i , s m / p ai r 21 , p , t , s m / pa ir 2 2, q , i , s m 4 ta tia ja ra ca tia ig ua çu r iv er , c ap an em a, p r 11 6 58 20 m +2 6s m +1 2s t pa ir 2 8, p , t , s t pa ir 4 , p , i , m / p ai r 18 , p , t , s m / pa ir 1 9, q , i , s m / p ai r 29 , p , t , s m 4 c en tr om oc hl us h ec ke lii so lim õe s r iv er , m an au s, a m 72 46 15 m +6 sm +5 st +2 0a ( z w ) 14 m +6 sm +6 st +2 0a ( z z ) pa ir z w , p , t , m -s t pa ir 2 0, p , t , a 9 a uc he ni pt er in ae ty m pa no pl eu ra a tr on as us (c ite d as a ge ne io su s at ro na su s) so lim õe s r iv er , m an au s, a m 10 0 56 16 m +1 6s m +1 2s t+ 12 a q, i, s m 5 a ge ne io su s in er m is (c ite d as a ge ne io su s br ev ifi lis ) so lim õe s r iv er , m an au s, a m 10 2 56 20 m +1 6s m +1 0s t+ 10 a p, t, s m 5 a ge ne io su s in er m is a ra gu ai a r iv er , a ra ga rç as , g o 10 8 56 32 m +1 6s m +4 st +4 a pa ir 2 0, p , t , s m pa ir 4 , p , i , m 6 a uc he ni pt er us o st eo m ys ta x (c ite d as a uc he ni pt er us n uc ha lis ) pa ra ná r iv er , p or to r ic o, p r 10 6 58 24 m +1 4s m +1 0s t+ 10 a pa ir 1 5, p , i , s m 1 a uc he ni pt er us n uc ha lis a ra gu ai a r iv er , a ra ga rç as , g o 11 0 58 22 m +1 6s m +1 4s t+ 6a pa ir 1 4, p , t , s m pa ir 2 2, p , t , s t 10 en to m oc or us r ad io su s pa ra gu ai r iv er , p oc on é, m t 10 6 58 22 m +1 2s m +1 4s t+ 10 a pa ir 2 1, p , t , s t pa ir 1 2, p , t , s m / p ai r 13 , p , t , s m / pa ir 1 4, p , t , s m / p ai r 15 , p , t , s m / pa ir 1 6, p , t , s m / p ai r 18 , p , t , s t / pa ir 1 9, p , t , s t 10 tr ac he ly op te ru s ga le at us (c ite d as p ar au ch en ip te ru s ga le at us ) pa ra ná r iv er , p or to r ic o, p r 98 58 22 m +1 2s m +6 st +1 8a pa ir 2 3, p , t , a 1 pa ra ná r iv er , t rê s la go as , m s 10 8 58 24 m +1 8s m +8 st +8 a pa ir 2 5, p , t , s t pa ir 1 6, p , i , s m / p ai r 17 , q , i , s m 7 pi um hi r iv er , c ap itó lio , m g 10 8 58 20 m +1 6s m +1 4s t+ 8a pa ir 2 4, p , t , s t pa ir 1 5, p , i , s m / p ai r 16 , q , i , s m 7 sã o fr an ci sc o r iv er , l ag oa d a pr at a, m g 10 8 58 22 m +1 6s m +1 2s t+ 8a pa ir 2 3, p , t , s t pa ir 1 6, p , i , s m / p ai r 17 , q , i , s m 7, 8 fn : f un da m en ta l n um be r; 2 n: d ip lo id n um be r; r es .: r es er vo ir ; a m : a m az on as ; g o : g oi ás ; p r : p ar an á; m s: m at o g ro ss o do s ul ; m g : m in as g er ai s; r n : r io g ra nd e do n or te ; m t: m at o g ro ss o; r ef .: r ef er en ce s; m : m et ac en tr ic ; s m : s ub m et ac en tr ic ; s t: su bt el oc en tr ic ; a : a cr oc en tr ic ; p : s ho rt a rm ; q : l on g ar m ; i i nt er st iti al ; t : t er m in al ; r ef er en ce s: 1 r av ed ut ti an d jú lio j r. (2 00 1) ; 2 fe no cc hi o et a l. (2 00 8) ; 3 lu i et a l. (2 01 5) ; 4 lu i et a l. (2 01 3a ); 5 fe no cc hi o an d b er to llo ( 19 92 ); 6 l ui e t al . ( 20 13 b) ; 7 lu i et a l. (2 01 0) ; 8 lu i et a l. (2 00 9) ; 9 k ow al sk i e t a l. (2 02 0) ; 1 0 p re se nt s tu dy . 93comparative cytogenetic analysis between species of auchenipterus and entomocorus (siluriformes, auchenipteridae) results auchenipterus nuchalis araguaia river basin the diploid number (2n) found for a. nuchalis was 58 chromosomes, 22 metacentric chromosomes, 16 submetacentric chromosomes, 14 subtelocentric chromosomes and 6 acrocentric chromosomes and fundamental number (fn) of 110 (figure 2a). the heterochromatin distribution pattern showed blocks mainly in the terminal regions, as well as a pericentromeric block on the short arm of submetacentric pair 14 and an interstitial block on the long arm of submetacentric pair 16 and subtelocentric pair 20 (figure 2b). single agnors were detected in terminal position on the short arm of submetacentric pair 14 (figure 2a, in box), and confirmed by fluorescent in situ hybridization (fish/18s rdna) (figure 3a). the 5s rdna sites were found in the terminal position on the short arm of the subtelocentric pair 22 (figure 3a). entomocorus radiosus paraguay river basin the diploid number (2n) found for e. radiosus was 58 chromosomes, 22 metacentric chromosomes, 12 submetacentric chromosomes, 14 subtelocentric chromosomes and 10 acrocentric chromosomes and fundamental number (fn) of 106 (figure 2c). the heterochromatin distribution pattern showed blocks mainly in terminal regions, as well as strongly marked blocks in the pericentromeric position of submetacentric pair 13, subtelocentric pairs 18, 19 and 23 and acrocentric pairs (figure 2d). single agnors were detected in terminal position figure 1. (a) specimen of auchenipterus nuchalis (total length = 18.5 cm); (b) specimen of entomocorus radiosus (total length = 4.96 cm). figure 2. karyotypes of auchenipterus nuchalis (a, b) and entomocorus radiosus (c, d) stained with giemsa (a, c) and submitted to c-banding (b, d). agnors presented in boxes. the presence of only one marked chromosome (fig 2a, in box) during the silver nitrate impregnation technique (agnor3) in a. nuchalis suggests that the nucleolus organizer region (nor) on its corresponding chromosome was inactive during the previous interphase or even in due the region is small. 94 amanda de souza machado et al. in the short arm of subtelocentric pair 21, confirmed by fluorescent in situ hybridization (fish/18s rdna) (figure 3b, in box). multiple sites of 5s rdna were found in terminal position on the short arm of the submetacentric pairs 12, 13, 14, 15 and 16 and subtelocentric pairs 18 and 19 (figure 3b). discussion in auchenipteridae, c y togenetic ana lyses are restricted to few species and most of them present diploid number of 58 chromosomes (e.g., ravedutti and júlio jr. 2001; fenocchio et al. 2008; lui et al. 2009, 2010, 2013a), except ageneiosus and tympanopleura with 56 chromosomes (fenocchio and bertollo 1992; lui et al. 2013b) and centromochlus with 46 chromosomes (kowalski et al. 2020) (table 1), caused by fusion events confirmed by the presence of its (interstitial telomere sequence) (lui et al. 2013b). in doradidae, sistergroup of auchenipteridae (e.g., pinna 1998; sullivan et al. 2006, 2008; birindelli 2014; calegari et al. 2019), the most frequent diploid number is also 58 chromosomes (milhomen et al. 2008; takagui et al. 2017, 2019), which reinforces it as a basal condition for both families and it is also corroborated by the data obtained in the species figure 3. karyotypes of auchenipterus nuchalis (a) and entomocorus radiosus (b) hybridized with rdna 18s probes (pair 14 of a. nuchalis and pair 21 in box of e. radiosus, green signal) and rdna 5s probes (red signal in the pair 22 of a. nuchalis and green signal in the pairs 12, 13, 14, 15, 16, 18 and 19 of e. radiosus), counterstained with dapi. rdna = ribosomal dna and dapi = 4’,6-diamidino-2-phenylindole. 95comparative cytogenetic analysis between species of auchenipterus and entomocorus (siluriformes, auchenipteridae) of this study. in neotropical fish, the variation of karyotypic formula among different populations of a given species or among species of the same family with maintenance of 2n is a common process resulted of chromosomal rearrangements, such as inversions or translocations (ravedutti and júlio jr. 2001; fenocchio et al. 2008; lui et al. 2009, 2013a), as seen in t. galeatus (cited as p. galeatus) and g. ribeiroi (lui et al. 2010, 2015). the terminal heterochromatin distribution found in a. nuchalis and e. radiosus follows the pattern observed in auchenipteridae (lui et al. 2015), as well as for a. osteomystax (cited as a. nuchalis) (e.g., ravedutti and júlio jr. 2001). however, interstitial and/or pericentromeric heterochromatins in some pairs in two species in this study (figure 2b, 2d) diverge from what is more common to the family (e.g., lui et al. 2009, 2010, 2015). auchenipterus osteomystax (cited as a. nuchalis) from the upper paraná river (ravedutti and júlio jr. 2001), the only species of this genus previously studied, presented only pale blocks in terminal and centromeric regions, in contrast to a. nuchalis, with some interstitial heterocromatins. on the other hand, similar markings have also been observed in e. radiosus, these heterochromatin data show greater similarity among species of different genera than between the two species of auchenipterus. these small inconsistencies in the detection of heterochromatins are common among works performed by different authors and may be the result of artifacts of techniques, as observed between a. nuchalis from the araguaia river and a. osteomystax (cited as a. nuchalis) from the upper paraná river, which used propidium iodide and giemsa for the staining of the c-banding, respectively. according to lui et al. (2012), the use of some nonspecific fluorescent dyes such as propidium iodide promote a greater contrast between heterochromatic and euchromatic regions, due to its greater interaction/ absorbance in more compacted regions of the dna (heterochromatin) and less interaction/absorbance in the dna degraded during the c-banding process (euchromatin). this possibly explains that such inconsistencies between the populations of auchenipterus may be due to the use of different dyes, since studies that use iodide has shown that the interstitial and/or pericentromeric markings found in a. nuchalis and e. radiosus can occur in other species of auchenipteridae, from both subfamilies, such as ageneiosus, tatia and centromochlus (e.g., lui et al. 2013a, 2013b; kowalski et al. 2020). the nors in the two species (figure 2) resemble the heterochromatic pattern found in the family, such as a. inermis, g. ribeiroi, t. galeatus, t. neivai (e.g., lui et al. 2009, 2013a, 2013b, 2015) and closer taxa like doradidae (e.g., eler et al. 2007; takagui et al. 2017, 2019; baumgärtner et al. 2018) and aspredinidae (e.g., ferreira et al. 2016). single and terminal agnors/18s rdna in submetacentric (a. nuchalis) and subtelocentric (e. radiosus) pairs (figure 2, in boxes) coincided with those found in some species of the family, as in t. galeatus (subtelocentric pairs) (lui et al. 2009), a. inermis (submacentric pair) (fenocchio e bertollo 1992; lui et al. 2013b), t. jaracatia and t. neivai (subtelocentric pairs) (lui et al. 2013a) (table 1), as well as for most doradidae species (e.g., fenocchio et al. 1993; eler et al. 2007; milhomen et al. 2008; takagui et al. 2017, 2019; baumgärtner et al. 2018). recently, data about c. hechelli demonstrated the first case of multiple and terminal nors (acrocentric and zw pairs) in auchenipteridae (table 1), an event that the authors propose to be the result of translocation between pairs during the interphase (e.g., kowalski et al. 2020). nevertheless, these results reinforce the presence of single and terminal nors as the basal characteristic of the group, refuting data about a. osteomystax (cited as a. nuchalis) from the upper paraná river, which presented single and interstitial nors (table 1), initially suggested as standard in auchenipteridae (ravedutti and júlio jr. 2001). despite the differences related to the morphology of the pair carrying the 18s rdna and the position of these cistrons on the chromosome among the auchenipteridae species, we can suggest correspondence of this pair in the family, considering the similar size and the absence of multiple nors for most auchenipteridae species (table 1), as well as for the pairs a. nuchalis and e. radiosus from this paper. variations in the morphology and chromosome pair number in the karyotype must be related to chromosomal rearrangements, such as pericentric inversions or translocations (lui et al. 2009, 2010, 2013a), as also observed in other families of neotropical fishes, such as doradidae (e.g., eler et al. 2007; milhomem et al. 2008), loricariidae (e.g., mariotto et al. 2019) and rhamphichthyidae (e.g., cardoso et al. 2011; fernandes et al. 2019). comparing the two species of auchenipterus, it is possible to notice that both have nors in submetacentric pairs and on the short arm, however in a terminal position in a. nuchalis and interstitial position in a. osteomystax (cited as a. nuchalis) (table 1), representing a specific chromosomal marker between them. thus, this difference may be useful in future studies of other populations these species, since there are some inconsistencies regarding the real geographic distribution of these species, especially as for a. nuchalis, which may be due to synonymizations and identification errors within the genus (ferraris and vari 1999). 96 amanda de souza machado et al. rega rd ing repet it ive sequence mapping data in auchenipteridae, rdnas are the most common, although limited to few species (lui et al. 2009, 2010, 2013a, 2013b, 2015). variations in the number of 5s rdna sites in the family, from single to multiple, were observed in centromochlinae and auchenipterinae. centromochlinae, t. jaracatia and t. neivai had multiple sites (lui et al. 2013a), while g. riberoi had a single site (lui et al. 2015) (table 1). in auchenipterinae, t. galeatus presented multiple sites (lui et al. 2009) and a. inermis had only one pair containing the 5s rdna (lui et al. 2013b) (table 1). compared to close groups, the same scenario is observed for doradidae (e.g., baumgärtner et al. 2016, 2018; takagui et al. 2017, 2019); while aspredinidae, sister-group of doradoidea (auchenipteridae + doradidae) (sullivan et al. 2006, 2008; calegari et al. 2019), presents 5s rdna mapping data only for a species of the family with multiple sites (ferreira et al. 2016, 2017). there is still difficulty in determining the plesiomorphic condition related the 5s rdna in auchenipteridae, mainly due to (1) these variations (simple sites: multiple sites) in doradoidea are distributed in an approximate ratio of 1:1, both in auchenipteridae (table 1) and in doradidae (e.g., baumgärtner et al. 2016, 2018; takagui et al. 2017, 2019); and (2) analyzing the outgroup of doradoidea (aspredinidae), there is not enough data to understand the evolution of this gene in the groups, since there is only one species studied, which has polymorphic multiple condition related to the number of sites (ferreira et al. 2016, 2017). however, despite these complicating factors, it would be coherent and parsimonious to hypothesize that single 5s rdna sites are plesiomorphic in doradoidea, or at least in auchenipteridae. according to martins and galetti jr. (1999), this is probably the ancestral condition for fish, as observed in cichlidae (e.g., nakajima et al. 2012; paiz et al. 2017) and pimelodidae (e.g., girardi et al. 2018). on the other hand, the occurrence of multiple sites in different subfamilies of auchenipteridae would be a result from independent dispersion events during the diversification of these species, just as the presence of transposition/translocation in species of pimelodus is suggested (girardi et al. 2018). considering the distribution of 5s rdna in the terminal position of the short arm of the chromosome pairs in both species of this study (table 1, figure 3), it is possible to raise discussions about the dispersing mechanism of these sites in the genome of e. radiosus, which showed a significant higher number of chromosomes carrying this gene compared to the rest of the family. as a result, it would be possible to hypothesize that the dispersion these genes could (1) be associated with the distribution of heterochromatin or (2) be associated with transposing elements present in the genome (e.g., gouveia et al. 2017; glugoski et. al 2018; primo et al. 2018). however, based on the arrangement of these sites, the hypothesis of dispersion related to the heterochromatic regions seems to be more likely because these genes have shown to correspond to terminal heterochromatins and are distributed evenly (equilocal) in the species genome, as already reported for cyprinidae species (e.g., saenjundaeng et al. 2020). according to schweizer and loidl (1987), this arrangement could explain the dispersion of sequences through transfer and amplification to other regions by proximity or physical contact between these stretches during the interphase nucleus. furthermore, such movements could be favored because they are associated with heterochromatic regions (schweizer and loidl 1987) like already identified as recombination hotspots (gornung 2013; saenjundaeng et al. 2020). this characteristic corresponds to observed for e. radiosus from this study. during the interphase, these mitotic chromosomes are organized into chromosomal territories (cremer et al. 2018; szalaj and plewczynski 2018; stam et al. 2019), thus they maintain their individuality during this phase and establish different and stable patterns with territories adjacent to each metaphasic cycle (cremer et al. 1982; fritz et al. 2015, 2019). these territories are designed from primary chromatin beams that depart from specific centromeric regions of the nucleus and extend, together with secondary and tertiary filaments, to the nuclear envelope until the telomeres, also called “rabl model” (cremer and cremer 2010). this arrangement would allow the spatial organization of equilocal telomeric regions proposed by schweizer and loidl (1987), facilitating the proximity and/or contact between homologous and non-homologous chromosomes and consequently the transfer and amplification of these regions in the genome (e.g., prestes et al. 2019; suaréz et al. 2019; saenjundaeng et al. 2020; takagui et al. 2020). this organization would explain the high number of terminal sites of 5s rdna in entomocorus which seems to be an apomorphy of the genus, or at least in e. radiosus. although, these hypotheses need to be further investigated due to the lack of ribosomal analysis in auchenipterus, as in a. osteomystax (e.g., ravedutti and júlio jr. 2001) or other species of entomocorus. so far, t. jaracatia and t. neivai have a greater number of 5s rdna sites after e. radiosus in auchenipteridae (table 1). these data can be interpreted in a similar way to what is proposed by calegari et al. (2019) about the presence of possible homoplasies, it would explain 97comparative cytogenetic analysis between species of auchenipterus and entomocorus (siluriformes, auchenipteridae) the proximity of entomocorus to members of centromochlinae, supported mainly by bayesian inference (bi) analyses. however, the monophyly of auchenipterinae and centromochlinae is well supported by maximum parsimony (mp) analyses of combined data (264 morphological characters and 1082 molecular sites), and they keep entomocorus and the members of centromochlinae phylogenetically distant (calegari et al. 2019). therefore, these similarities related to the number of 5s rdna sites should not be considered as a common ancestry among these groups. however, it is interesting to mention that such phylogenetic inconsistencies generated by bi analyses, both of morphological and molecular data, can also be recognized through chromosomal markers. in summary, differences in the karyotypic formula, fundamental number (fn), position of the nors (table 1) and distribution of heterochromatins can be pointed out as species-specific characters for the populations/ species of auchenipterus from the araguaia and upper paraná river basins. at the moment, there is no data about 5s rdna for a. osteomystax (cited as a. nuchalis) (ravedutti and júlio jr. 2001), which would be useful and interesting to add to the data from the classic analyses, since this marker proves to be very informative for the group. its variation in the group, mainly related to the number of sites, shows potential as a cytotaxonomic marker and raises discussions about its dynamics in the genomes of the group, like pointed out in this study for the equilocality in e. radiosus, suggesting to be related to scattering events associated with amplification of heterochromatic regions in the interphase. furthermore, for this level of cytogenetic analysis, no apomorphies were found that reinforce the phylogenetic proximity between a. nuchalis and e. radiosus, resulting from two aspects: (1) the high similarity of the karyotype macrostructure observed by classical chromosomal markers, compared to others auchenipteridae groups; and (2) absence of molecular chromosomal markers for the group, which considering the potential of 5s rdna, should be better explored, since in the family some taxonomic/phylogenetic conflicts remain throughout history due to the lack of research beyond morphological diagnosis. geolocation information auchenipterus nuchalis from the araguaia river basin, between aragarças (goiás state) and barra do garças (mato grosso state) (gps: 15°53’03,9”s; 52°06’17,9”w), and entomocorus radiosus from the paraguay river basin, poconé (mato grosso state) (gps: 16°25’40,9”s; 56°25’07,4”w). acknowledgments the authours are grateful to the universidade estadual do oeste do paraná (unioeste)/campus cascavel for the infrastructure for research development. we are grateful dr. heraldo antonio britski for the identification of 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genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers. caryologia 75(2): 119-127. doi: 10.36253/caryologia-1540 received: january 17, 2022 accepted: july 06, 2022 published: september 21, 2022 copyright: © 2022 qin zhao, zitong guo, minxing gao, wenbo wang, lingling dou, sahar h. rashid. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. evaluation of genetic diversity and genepool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 1.school of chemistry and chemical engineering, xianyang normal university, xianyang 712000, shaanxi, china 2.technical college of applied science, sulaimani polytechnic university, iraq *corresponding author. e-mail: zhaoqin2018@aliyun.com abstract. pistachio genetic variety includes a wide range of female variations and male genotypes, and iran is regarded as one of the critical sites for this diversity in the world. the genus pistacia consists of eleven species that only have edible nuts and are commercially important. four important species of pistachios include pistacia vera, p. khinjuk stocks, p. eurycarpa yalt. (p. atlantica subsp. kurdica zoh.), and p. atlantica dsef are found in iran. genetic diversity is one aspect of biological diversity that is extremely important for conservation strategies, especially in rare and narrowly endemic species. in iran, there is no knowledge concerning the genomic organization of the population, genetic diversity, or phenotypic variations of the species. pistacia khinjuk has eight distinct regional populations, all of which were studied for genetic variation and demographic organization because of the species’ therapeutic value. for this reason, we employed six inter-retrotransposon amplified polymorphism (irap) indicators and 15 mixed irap indicators to highlight genomic variation in this plant both within and across populations in this study. it was discovered that 73% of overall genomic variability was related to within-population variety and 27% was attributable to inter-population genomic divergence using the amova test among the examined populations (phipt = 0.49, p = 0.010). it was discovered by the mantel analysis that there was a substantial positive association between genomic isolation and geographic distance among the tested populations. structure analyses and population assignment tests revealed some degree of gene flow among these populations. there was consistency between the mds plots of communities and the nj grouping of molecular information. based on (irap) indicators, these findings demonstrated that regional communities of the plant pistacia khinjuk are well distinct. keywords: gene flow, irap, pistacia khinjuk, population differentiation. introduction according to current estimates, the pistacia genus has at least twelve species and has existed for around eighty million years (karimi et al. 2009). pistacia vera is the sole commercially viable species throughout this genus 120 qin zhao et al. (fares et al. 2009). according to previous theories, the pistacia genus originated in europe and north africa, but recent research suggests that it probably originated in central asia. pistacia species have been reported to have spread over the world, based on initial research. one theory concentrates on the mediterranean region of europe, northern africa, and the middle east. the eastern portion of the zagros mountains (iran) and the caucasus regions stretching from crimea to the caspian sea are further options (zohary 1952). four important species of pistachios include p. vera, p. khinjuk stocks, p. eurycarpa yalt. (p. atlantica subsp. kurdica zoh.), and p. atlantica dsef are found in iran (karimi et al. 2009). pistacia vera, p. khinjuk, and p. atlantica are three of the most important wild pistacia species that thrive in iran. in central asia, which includes turkmenistan, afghanistan, and northeast iran, wild p. vera has grown in an area of approximately 75,000 hectares. in the sarakhs region, p. vera grows in an area of approximately 17,500 hectares (behboodi 2003). with the biggest area under cultivation, iran is the world’s leading pistachio exporter, although recent years have seen poor yields relative to other nations, notably the united states and turkey (ahmad et al. 2003a). pistachio plants are long-living with a juvenile period of approximately 5–10 years. in addition, wild pistacia species have edible seeds. they are used as rootstock seed sources for cultivated p. vera, and sometimes, fruit consumption, oil extraction, soap production, and as forest trees (katsiotis et al. 2003). pistacia genetic diversity has been the subject of severa l investigations that have been conducted on the basis of examination of morphological, physiological, and metabolic properties (tayefeh aliakbarkhany et al. 2013). a number of these methods have been employed to characterize pistachio cultivars across time, with r apd (williams et al., 1990) being the most extensively utilized (kaf kas et a l., 2002; katsiotis et al., 2003). to examine the evolutionary connection between pistacia species and cultivars, aflp and ssr approaches have also been utilized on pistachio (katsiotis et a l., 2003; ibrahim basha et a l., 2007; ahmad et al., 2003; ahmad et al., 2005; ahmadi afzadi et al., 2007). pistachio pollination difficulties may be solved by identif ying the genetic variety of male cultivars and genotypes in iran because there is not enough data surrounding their genetic characteristics (ahmad et al., 2005). most of the taxonomic and nomenclatura l ambig uit y in european species has been cleared up thanks to the later research. to examine the genetic diversity and connections among pistacia khinjuk cu ltivars and landraces, randomly amplif ied poly morphic dna (r apd), amplif ied fragment length polymorphism (aflp), inter simple sequence repeat (issr), simple sequence repeat (ssr), and inter-retrotransposon amplif ied poly morphism (ir a p) were some molecu la r ma rker tech niques employed during recent years. there is also the potential that this species might have infra-specific taxonomic variants owing to the wide range of morphological variation throughout the nation. as a result, we conducted the first-ever nationwide demographic genetic evaluation and morphometric examination of eight distinct regional groups. through amplifying the segments of dna between two retrotransposons for genomic analysis, we employed the inter-retrotransposon amplif ied poly morphism (irap) approach to detect insertional polymorphisms. it has been employed in various investigations on genomic variation (smykal et al., 2011). the objectives of this research were to study genetic diversity among pistacia khinjuk cultivars/populations with a different geographical origin by inter-retrotransposon amplified polymorphism (ir ap) method to determine genetic variation among and within materials using ir ap markers. materials and methods plant materials during the months of july and august of 2019-2020, a number of 40 participants from eight natural communities of pistacia khinjuk were collected in the iranian provinces of fars, kerman, sistan and baluchestan, and hormozgan (table 1). fresh leaves of 3-6 individuals from each population were collected and immediately dried in silica gel (table 1). the accurate recognition of species was achieved through the utilization of numerous sources (pistacia khinjuk) (kafkas et al., 2002; katsiotis et al., 2003). table 1 list the locations where samples were taken. dna extraction and irap examination three to six plants from each group were randomly selected to collect fresh leaves. the silica gel powder was used to dry them. genomic dna was extracted using a ctab stimulated charcoal technique (esfandani-bozchaloyi et al., 2019). by passing the isolated dna across a 0.8% agarose gel, the purity of the dna was determined. the irap assessment was conducted using a collection of six outward-facing ltr primers (smykal et al., 2011; 121evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers table 2). outward-facing ltr paired primers were additionally utilized in 15 distinct mixtures. pcr reactions were carried in a 25μl volume containing 10 mm trishcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). an initial denaturation during 1 minute at 94°c was continued by 40 rounds divided into three sections, including  35 s at 95°c, the 40s at 47°c, and the 55s at 72°c, which comprised the thermal schedule. the final extension was performed at 72°c for 5 min. in order to see the amplification results, the gels were first to run on a 1 percent agarose solution and then stained with ethidium bromide. a molecular size ladder with a step size of 100 bp was used to determine the fragment size (fermentas, germany). data analyses the irap profiles obtained for each samples were scored as binary characters. for grouping of the plant specimens, ordination methods such as mds (multidimensional scaling) analysis were also performed (podani 2000). multivariate and all the necessary calculations were done in the past software, 2.17 (hammer et al. 2012). parameter like nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism were determined (freeland et al., 2011). nei’s genetic distance among populations was used for neighbor joining (nj) clustering and neighbor-net networking (freeland et al., 2011). mantel test checked the correlation between geographical and genetic distance of the studied populations (podani, 2000). these analyses were done by past ver. 2.17 (hammer et al., 2012), darwin ver. 5 (2012) and splitstree4 v4.13.1 (2013) software. amova (analysis of molecular variance) test (with 1000 permutations) as implemented in genalex 6.4 (peakall and smouse, 2006), and nei,s gst analysis as implemented in genodive ver.2 (2013) were used to show genetic difference of the populations. moreover, populations, genetic differentiation was studied by g’st est = standardized measure of genetic differentiation, and d_est = jost measure of differentiation. the genetic structure of populations was studied by bayesian based model structur e analysis (pritchard et al. 2000), and ma ximum likelihoodbased method of k-means clustering of genodive ver. 2. (2013). for structure analysis, data were scored as dominant markers (falush et al. 2007). the evanno test was performed on structure result to determine proper number of k by using delta k value. in k-means clustering, two summary statistics, pseudo-f, and bayesian information criterion (bic), provide the best fit for k. gene flow was determined by (i) calculating nm an estimate of gene flow from gst by popgene ver. 1.32 (1997) as: nm = 0.5(1 gst)/gst. this approach considers equal amount of gene flow among all populations. (ii) population assignment test based on maximum likelihood as performed in genodive ver. in genodive ver. 2. (2013). the presence of shared alleles was determined by drawing the reticulogram network based on the least square method by darwin ver 5. (2012). results genetic variation across communities. table 3 displays the genetic variation characteristics of pistacia khinjuk collected from eight different geographic locations. fars, shiraz (population no. 1) exhibited the largest polymorphism  percentage  (53.75 percent) and the maximum  scores for gene variation (0.39) and shanon data indicator  (0.40). hormozgan, bandar abbas, and genow (no.6) populations had the minimum polymorphism rate (17.15%) and the minimum values for shanon, data score (0.15), and he (0.18). table 1. populations studied their locality and ecological features. pop.no locality 1 fars, shiraz 2 fars, 60 km south of shiraz at the vicinity to shiraz-bushehr 3 fars, arjan lake 4 hormozgan, bandar lengeh 5 hormozgan, bandar abbas 6 hormozgan, bandar abbas, genow 7 kerman, hamun-e jaz murian 8 sistan and baluchestan, iranshahr table 2. irap primers based on smykal et al. (2011) study. irap sequence (5´-3´) gu735096 accccttgagctaacttttggggtaag gu980589 agcctgaaagtgttgggttgtcg gu929878 gcatcagcctggaccagtcctcgtcc gu735096 cacttcaaattttggcagcagcggatc gu929877 tcgaggtacacctcgactcagg gu980590 attctcgtccgctgcgcccctaca 122 qin zhao et al. population genetic differentiation amova (phipt = 0.49, p = 0.010), and gst analysis (0.844, p = 0.001) revealed significant difference among the studied populations (table 4). within-population variation accounted for 27% of overall genomic variation, whereas among-population genomic divergence accounted for 73% of variations. there were substantial variations in the communities analyzed using pairwise amova analysis. moreover, we got high values for hedrick standardized fixation index after 999 permutation (g’st = 0.844, p = 0.001) and jost, differentiation index (d-est = 0.116, p = 0.001). pistacia khinjuk has been shown to be genetically distinct across its geographical communities, according to these findings. populations, genetic affinity there were different clusters of plants from each population in the nj tree. no transitional stages were found throughout the samples that we examined. these results showed that irap data could differentiate the populations of pistacia khinjuk in three different major clusters or groups (figure 1). the first significant cluster supported with significant bootstrapping values of 94% so that plants of fars, shiraz (no.1) comprised the first cluster due to morphological similarity. in contrast, the plants of hormozgan, bandar abbas pop 5 (b=94%), formed the second cluster and finally, the population 2 (fars, 60 km south of shiraz at the vicinity to shirazbushehr) with 97% of support. while plants of hormozgan, bandar lengeh (pop 4), hormozgan, bandar abbas, genow (pop6), kerman, hamun-e jaz murian (pop7), sistan and baluchestan, iranshahr (pop 8) showed genetic affinity and intermixture. genetic divergence and separation of populations fars, 60 km south of shiraz at the vicinity to shirazbushehr (no.2) as well as hormozgan, bandar abbas (no.5) and hormozgan, bandar abbas, genow (no.6) from the other communities is obvious in mds design of irap information following 900 permutations (figure.2). the other groups were genetically related to each table 3. genetic diversity parameters in the studied populations pistacia khinjuk (n = number of samples, na= number of different alleles; ne = number of effective alleles, i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations). pop n na ne i he uhe %p pop1 5 0.241 1.158 0.40 0.36 0.39 53.75% pop2 6 0.355 1.077 0.377 0.34 0.32 35.05% pop3 4 0.449 1.167 0.24 0.23 0.24 19.26% pop4 4 0.535 1.020 0.22 0.25 0.28 43.13% pop5 4 0.231 1.088 0.30 0.22 0.25 31.63% pop6 3 0.355 1.121 0.15 0.18 0.12 17.15% pop7 6 0.538 1.091 0.207 0.23 0.280 23.93% pop8 5 0.291 1.333 0.231 0.333 0.167 21.59% table 4. analysis of molecular variance (amova) of the studied species. source df ss ms est. var. % φpt among pops 55 116.596 22.329 17.077 73% 73% within pops 14 33.757 29.580 33.590 27% total 69 150.342 51.773 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance; φpt: proportion of the total genetic variance among individuals within an accession, (p < 0.001). figure 1. nj tree of populations in pistacia khinjuk based on irap data. bootstrap value from1000 replicates are indicated above branches (population numbers are according to table 1). 123evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers other. a substantial association between genetic isolation and geographic separation was found in these communities after a mantel analysis with 5000 permutations (r = 0.55, p = 0.001). we possess isolation by distance (ibd) in the pistacia khinjuk species because communities that are spatially separated exhibit less genetic exchange. populations genetic structure there are three genetic subgroups present when k = 3. when the evanno examination was run on the structure evaluation, it yielded a comparable outcome, with a large peak appearing at k=3. both studies found genetic differentiation in pistacia khinjuk groups. structure plot based on k = 3 revealed a genetic difference of populations 1-3 (differently colored), as well as 4-6 (figure.3). but it showed genetic affinity between populations 7, 8 (similarly colored). the mean nm = 0.29 was obtained for all irap loci, which indicates a low amount of gene flow among the populations and supports genetic stratification as indicated by k-means and structure analyses. it was also found that there was no substantial genetic exchange between these groups when the demographic allocation experiment was performed. it was found that populations 1 and 5, as well as populations 3 and 6, also 2 and 5  shared certain alleles, according to a reticulogram created using the least square approach (figure not shown). due to the proximity of both communities, our mds map resulted in the same classification. genetic differentiation among pistacia khinjuk communities is clearly evident from the structure plot, which shows that the common figure 2. mds plot of populations in pistacia khinjuk based on irap data. (population numbers are according to table 1). figure 3. structure plot of pistacia khinjuk populations based on k = 3 of irap data. (population numbers are according to table 1). 124 qin zhao et al. genetic alleles throughout these communities represent only a small percentage of the genomes. it was possible to collect 75 irap bands totally; 15 of them were considered exclusive. two to four unique bands were found in communities 3 and 6, and 8. discussion genetic and breeding investigations benefit greatly from population genetics analysis. data on the degrees of genomic diversity, genetic diversity distribution within and across communities, inbreeding and outcrossing, the efficient community size and bottleneck are  presented by these studies (ellis and burke, 2007). demographic genomic research has significantly advanced with the introduction of molecular biomarkers. among the various pistacia accessions, such indicators have been utilized to detect possibly unique genotypes (martin et al.,1997). to examine the genetic diversity and connections among pistacia khinjuk cultivars and landraces, randomly amplified polymorphic dna (rapd), amplified fragment length polymorphism (aflp), inter simple sequence repeat (issr), simple sequence repeat (ssr), and inter-retrotransposon amplified polymorphism (irap) were some molecular marker techniques employed during recent years (wiesnerova and wiesner, 2004; ren and khayatnezhad 2021; khayatnezhad and nasehi 2021, i et al., 2021; jia et al, 2021). the majority of plant genomes are made up of transposable elements, especially retrotransposons. genomic variety is generated through their replication,  rendering  them an ideal repository of molecular indicators (smykal et al., 2011; gholamin and khayatnezhad, 2020a; 2020b, 2020c). through replicating the sections of dna between two retrotransposons, the inter-retrotransposon amplified polymorphism (irap) approach reveals insertional polymorphisms. several genomic investigations have relied on this technique (smykal et al., 2011). iranian pistacia khinjuk’s genomic variation was evaluated during this research in order to help in the preservation of its germplasm. in order to formulate suitable conservation approaches, the data gathered on genetic diversity between and within various groups will be used to establish a solid foundation for future research. iranian pistacia khinjuk is very diverse, according to the results of the current study, which is likely owing to differences in genetic backgrounds across different geographical areas, breeding pressure, and/ or restricted exchange of genomic information. our findings demonstrate the distinct character of the iranian pistacia khinjuk germplasm, hence bolstering the rationale for deploying more intensive characterization, preservation, and reproduction techniques. it was possible to determine the genomic variation of the iranian population employing  irap indicators. the results of this molecular assay in fingerprinting the 8 pistacia khinjuk population are presented in table 3. a total of 75 bands were amplified by the six primers, an average of 8 bands per primer, of which 62 (84%) were polymorphic. the total number of amplified fragments was between 6 to 10, and the number of polymorphic fragments ranged from 5 to 9. nj clustering and mds plot (figs 1–2), of the studied populations did not entirely delimit the studied populations and revealed that some plants in these populations are intermixed. in mds plot, a higher degree of intermixture occurred between populations of 7, 8 and seem to be an area that populations of 1,4 and 7, 8 together with the gene exchange (fig. 2). these results indicate that the geographical populations of pistacia khinjuk are not genetically differentiated from each other. evanno test performed on structure analysis produced the best number of k = 3. this genetic grouping is in agreement with nj clustering result presented before. throughout the semi-arid and dry farming areas of iran, pistachio has significant socioeconomic and environmental implications (kafkas et al., 2006). more than 300 pistachio genotypes have been identified in iran, which is home to a diverse range of pistacia species. pistachio development and preservation efforts may thus benefit from iran’s pistachio germplasm. it is consequently vital to evaluate genomic variation and interactions among cultivars of iranian pistachio employing discriminative and reliable indicators. genetic diversity is of fundamental importance to the survival of a species (sun and khayatnezhad 2021; tao et al, 2021; wang et al, 2021; xu et al., 2021; yin et al., 2021; zhang et al, 2021). there were three primers ultimately chosen for further testing out of the original six employed during issr following initial screening  (kafkas et al., 2006; zheng, et al., 2021; zhu et al, 2021), in  accordance with  the stated findings. the three primers replicated a maximum of 28 bands, with each primer amplifying an average of 9.3 bands  among  13 types  (or 46 percent),  which were polymorphic. approximately seven to 12 pieces of dna were replicated, and three to five segments of dna were polymorphic. between 22 iranian cultivars and wild pistachio varieties, mirzaei et al. (2005) found 80% polymorphism. because of the changes in genotypes and primers between the present research and the previous study, it is possible that variations  in polymorphism are observed. 125evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers 82.41%% polymorphism was discovered by katsiotis and colleagues (2003);  there were 18.2 polymorphic bands out of a total number of 22.11. in a study reported by golan-goldhirsh et al. (2004) in assessing polymorphisms among 28 mediterranean pistacia accessions, twenty-seven selected primers produced 259 total bands (average 9.59), and 86.1 of them were polymorphic. the genotypes investigated by khadivi (2018) showed a significant degree of polymorphism. 18 alleles were produced by seven ssr primer pairs, thirteen among them were polymorphic across the genotypes. averaging 2.57, the polymorphic alleles ranged from one for ptms9, ptms40, ptms41, and ptms42 loci to five for the ptms7 locus. allele lengths ranging from 120 to 250 bp were replicated. the coefficients of genomic homology between two individuals ranged from 0.20 to 0.75. to summarize, it can be concluded that  one of two significant hubs of pistacia variety is iran. genomic variation among several pistacia khinjuk communities employing irap indicators was studied during the current research,  offering  useful data in the effort to conserve the species’ germplasm. because iranian pistacia khinjuk possesses limited genetic variety, its preservation and prospective reproduction initiatives are very vital. preserving, core  collecting, and reproducing the pistacia khinjuk will be made easier thanks to the outcomes of this research. acknowledgements the national natural science foundation of china (31872175);special scientific research project of education department of shaanxi province (21jk0965); key r & d program of shaanxi province (2019ny-103). references ahmad r, ferguson l, southwick sm (2003a) identification of pistachio (pistacia vera l.) nuts with microsatellite markers. j am soc hortic sci 128:898–903 ahmad r, struss d, southwick sm (2003b) development and characterization of microsatellite markers in citrus. j am sochorticsci 128:584–590 ahmad, r., fergusen, l. and southwick, s.m (2005). molecular marker analysis of pistachio rootstocks by simple sequence sepeats and sequence-selated amplified solymorphisms. journal of horticultural science and biotechnology, 80, 382-386. ahmadi afzadi m, seyedtabatabaei be, mohammadi sa, tajabadipur a (2007) comparison of genetic diversity in species and cultivars of pistachio (pistacia vera l.) based on amplified fragment length polymorphism marker. iran j biotechnol 5:147–152 behboodi b (2003) ecological distribution study of wild pistachios for selection of rootstock. options mediterr ser a 63:61–67 bi, d., c. dan, m. khayatnezhad, z. sayyah hashjin, z. y. ma (2021): molecular identification and genetic diversity in hypericum l.: a high value medicinal plant using rapd markers markers. genetika 53(1): 393-405. cheng, x., x. hong, m. khayatnezhad, f. ullah (2021): genetic diversity and comparative study of genomic dna extraction protocols in tamarix l. species.” caryologia 74(2): 131-139. esfandani bozchaloyi, s., m., sheidai, m., keshavarzi, z., noormohammadi (2017a): genetic diversity and morphological variability in geranium purpureum vill. 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(poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(3): 39-46, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1611 caryologia international journal of cytology, cytosystematics and cytogenetics citation: a cius, ca lorscheider, lm barbosa, ac prizon, ch zawadzki, la borin-carvalho, fe porto, alb portela-castro (2022). contributions of species rineloricaria pentamaculata (loricariidae:loricariinae) in a karyoevolutionary context. caryologia 75(3): 39-46. doi: 10.36253/caryologia-1611 received: march 28, 2022 accepted: november 23, 2022 published: april 5, 2023 copyright: © 2022 a cius, ca lorscheider, lm barbosa, ac prizon, ch zawadzki, la borin-car valho, fe porto, alb portela-castro. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. contributions of species rineloricaria pentamaculata (loricariidae:loricariinae) in a karyoevolutionary context a cius¹, ca lorscheider2, lm barbosa¹, ac prizon¹, ch zawadzki3, la borin-carvalho¹, fe porto4, alb portela-castro1,4 ¹ departamento de biotecnologia, genética e biologia celular, universidade estadual de maringá, avenida colombo, 5790, 87020-900, maringá – paraná, brasil 2 colegiado de ciências biológicas, universidade estadual do paraná, campus união da vitória, praça coronel amazônas, s/n, 86400-000, união da vitória paraná, brasil 3 departamento de biologia/núcleo de pesquisas em limnologia, ictiologia e aquicultura (nupélia), universidade estadual de maringá, avenida colombo, 5790, 87020-900, maringá – paraná, brasil 4 departamento de ciências do movimento humano, campus regional do vale do ivaí, universidade estadual de maringá, avenida espanha, s/n, 86870-000 ivaiporã-paraná, brasil 5 núcleo de pesquisas em limnologia, ictiologia e aquicultura (nupélia), universidade estadual de maringá, avenida colombo, 5790, 87020-900, maringá – paraná, brasil *corresponding author. e-mail: ferpsaparolli@uem.br abstract. species of rineloricaria demonstrate an interesting evolutionary history from a cytogenetic point of view, due to the occurrence of extensive variation in diploid number (2n=36 -70 chromosomes), with robertsonian rearrangements mostly responsible for this karyotypic diversity. in this study we present the karyotypic data for a population of rineloricaria pentamaculata, collected in the itiz stream, a tributary of the paraná river basin (paraná, brazil), which exhibited 2n=56 chromosomes distributed in 8m/sm+48st/a (number fundamental equal to 64) and simple nor system revealed by fluorescent in situ hybridization (fish) with 18s rdna probe, silver nitrate and positive c band, located on the first submetacentric chromosome pair (pair 5). in addition, the nor pair showed a size heteromorphism for this region, rich in gc composition (positive cma3). clusters of 5s rdna were located in 14 chromosomes and the fish with a telomeric probe was used to map possible evidence of chromosomal fusions, however, it showed only telomeric sites. these results corroborate the data for the species r. pentamaculata and the genus rineloricaria, showing that they are similar to most of the populations analyzed. about the cytogenetic data of r. pentamaculata, we reaffirm that most populations were conserved, but in those with derived characteristics, robertsonian chromosomal rearrangements probably contributed to the karyotypic evolution of the group. keywords: rineloricaria, chromosomal rearrangements, cytogenetics, paraná river basin, karyotypic evolution. 40 a. cius et al. introduction the subfamily loricariinae contain 252 valid species distributed by basins of central and south america, and although morphologically the group is considered monophyletic, taxonomic problems have been reported, for example, the tribe loricariini several species have similar descriptions (costa-silva 2015; roxo et al. 2019). the genus rineloricaria (bleeker 1982) is the most numerous genus in the subfamily loricariinae (tribe loricariini), currently consisting of 78 valid species (fricke and eschmeyer et al., 2022), distributed throughout the neotropical region, from the panama to argentina, occupying a wide variety of habitats and with restricted information on genetic diversity (rodriguez and reis 2008; vera-alcaraz et al. 2012). rineloricaria is also considered a monophyletic táxon, however, it presents historical taxonomic problems such as some synonymous species (hemiloricaria bleeker 1862, ixiandria regan 1906, fonchiichtys isbrücker and michels 2001 and leliella isbrücker, 2001), species complex (r. heteroptera isbrücker and nijssen 1976, r. lima kner 1853, r. cadeae hensel 1868, r. strigilata hensel 1868 and r. lanceolata günther 1868) and doubts about the identity of the genus, due to loss of specimen data from the type locality (costa-silva 2015; covain et al. 2016; venturelli et al. 2021). rineloricaria pentamaculata was described by langeani and araújo (1994) from specimens collected in the turvo river (ourinhos, sp) in the upper paraná river basin and has been collected in different environments of the upper paraná river basin (table 1). although cytogenetic studies in rineloricaria are scarce and were performed in only 16 species (giulianocaetano 1998; alves et al. 2003; maia et al. 2010; rodrigues 2010; porto et al. 2011; rosa et al. 2012; porto et al. 2014; ventureli 2014, primo et al. 2017; guloski et al 2018, takagui et al. 2020; venturelli et al. 2021), these studies have contributed as an important support for studies in species taxonomically complex. considerable karyotype diversity has been reported in genus, with diploid numbers ranging from 2n=36 in rineloricaria latirostris (giuliano-caetano 1998) to 2n=70 in r. lima (rosa et al. 2012). in addition, chromosomal polymorphisms (structural and numerical) were found in six species, called: r. latirostris (giuliano-caetano 1998), r. pentamaculata (porto et al. 2011; primo et al. 2017), r. lima (rosa et al. 2012), r. lanceolata (porto et al. 2014). robertsoninan chromosomal rearrangements are suggested as the main involved in of karyotypic evolution of rineloricaria, as they would explain the origin of chromosomal polymorphisms and the extensive numerical and structural chromosomal diversity detected in species of genus (alves et al. 2005; porto et al. 2011; porto et al. 2014). this hypothesis has been investigated and supported due to evidence of occurrence of these rearrangements in chromosomes of some species. cytogenetic techniques have supported this proposition, such as fish using 5s rdna and telomeric probes, showed interstitial telomeric sites (its) co-located with 5s rdna sites on specific chromosomes, in addition to detecting transposable elements associated with sequences of 5s rdna. (rosa et al. 2012; porto et al. 2014; primo et al. 2017; guloski et al. 2018). cytogenetic studies carried out in rineloricaria pentamaculata show that most populations have conserved characteristics, in relation to karyotype, location and simple nor system and distribution of constitutive heterochromatin. however, in some populations cytogenetic diversity was observed, due to reports of b chromosomes, intrapopulational and interpopulational karyotypic differences, multiple nor system and variation with respect to the location and amount of chromosomes with 5s rdna sites 5s (porto et al. 20102011; venturelli 2014; primo et al. 2017). this study consists of the cytogenetic characterization of r. pentamaculata from the itiz stream (rio ivaí sub-basin, upper rio paraná basin) located in the city of marialva (paraná-brazil), using classical cytogenetic techniques and physical chromosomal mapping of 18s and 5s rdna, telomeric sequences. thus, we compile and discuss the cytogenetic data available for the species r. pentamaculata, highlighting the similarities and variations found in the different populations with inferences about karyoevolutionary aspects in this group. materials and methods cytogenetic analysis was conducted on 16 specimens (9 females and 7 males) identified as rineloricaria pentamaculata (nup 17750 1), collected from the itiz stream, a small tributary of the ivaí river (basin of the upper paraná river, paraná state, brazil), voucher specimens were deposited in the ichthyological collection of the limnology, ichthyology and aquaculture research center (nupélia) at maringá state university, paraná, brazil. the protocols used in this study were submitted and reviewed by the ethics committee on animal experimentation (protocol 07/2011) of the maringá state university. the mitotic chromosomes of r. pentamaculata were obtained from kidney cells as described by bertollo et al. (1978). chromosomal banding was performed for detection of constitutive heterochromatin by the c-band 41contributions of species rineloricaria pentamaculata (loricariidae:loricariinae) in a karyoevolutionary context technique (sumner 1972) and double staining using the f luorochromes chromomycin a3 (cma3) and dapi, according to schweizer (1976). nucleolus organizer regions were labeled by silver nitrate (ag-no3) staining as described by howell and black (1980). fluorescent in situ hybridization (fish) using 18s and 5s rdna probes was performed based on pinkel et al (1986). the 18s rdna probes were obtained from cloned and amplified fragments of prochilodus argenteus spix and agassiz, 1829 (hatanaka and galetti 2004), the 5s rdna probe was isolated from the genomic dna of leporinus elongatus valenciennes, 1850 (martins and galetti 1999). in this study, we also used a telomeric dna probe amplified by pcr, free of dna, from primers (ttaggg)n and (ccctaa)n, based on the method of ijdo et al. (1991). the rdna probes were labeled by nick translation with biotin-16-dutp and 5s and telomeric digoxigenin-11-dutp. fluorescent signals were detected with avidin-fitc (for 18s rdna) and with digoxigenin-rhodamine for 5s rdna probes and telomeric probe. the metaphases were photographed in an zeiss axioskop microscope with image capture and epifluorescence system. the morphology of the chromosomes was established according to the ratio of arms (rb), according to the proportions proposed by levan et al. (1964), classifying them as metacentric rb from 1.00 to 1.70), submetacentric (rb from 1.71 to 3.00), subtelocentric (rb from 3.01 to 7.00) and acrocentric (rb greater than 7.00). to calculate the fundamental number (nf), metacentric (m) and submetacentric (sm) chromosomes were considered to have two arms, while subtelocentric (st) and acrocentric (a) chromosomes were single-armed. results the specimens of rineloricaria pentamaculata had a diploid number of 2n= 56 chromosomes, with 8 m/ sm+48st/a and fundamental number (nf) of 64, in both sexes (figure 1a). ag-nor sites were found the entire short arm of the first pair of subtelo-acrocentric chromosomes (pair 5), showed a heteromorphism in the size of the secondary constriction (figure 1a, in box), confirmed by fish with an 18s rdna probe (figure 1b). fish using 5s rdna probe revealed 14 subtelo/acrocentric chromosomes with sites (pairs: 6, 7, 8, 10, 11, 12 and 14) located in the terminal regions of these chromosomes (figure 1b). hybridization with a telomeric probe revealed markings in the telomeric regions of all chromosomes and absence of interstitial telomeric sites (its) (figure 1c). few blocks of heterochromatin were detected in some chromosomes, however, the first pair of subtelo/ acrocentric chromosomes presented blocks conspicuous associates the nor sites (figure 2d), which also revealed double staining with cma3 /dapi and, therefore, rich in cg at the sites ag-nor (figure 2e). discussion the data obtained in the present study showed similarities to those observed for most populations of r. figure 1. karyotype for rineloricaria pentamaculata of the itiz stream after: a) conventional staining by giemsa and ag-nor located on par nº 5 (in box); b) double-fish using 18s rdna (green) and 5s rdna (red) probes; c) fish with telomeric probe. note the absence of the its. figure 2. karyotype of rineloricaria pentamaculata from the itiz stream showing: d) the heterochromatin distribution pattern after c-banding; e) cma3/dapi base-specific profile. 42 a. cius et al. pentamaculata, in relation to diploid number (2n=56), karyotypic formula (8m/sm+48st/a), fundamental number (nf=64) and simple nor system located on the first pair of st/a chromosomes (table 1). however, divergent karyotypes were found in the populations from the upper paraná basin (pr), that is, 2n=58 e 2n=54 and distinct karyotypic formulas (table 1). in the population of r. pentamaculata from the barra grande river, primo et al. (2017) registered a karyomorph b (2n=54) and founded traces of its in the centromeric region of a pair of metacentrics (pair 1) suggesting the occurrence of robertsonian fusion that resulted in the reduction of 2n from 56 to 54 chromosomes. in the population of the tauá stream (porto et al. 2011), two karyotypic formulas were detected and the one with 9m/sm +47st/a originated from 8m/sm + 48st/a, and that meiotic nondisjunction and chromosome fusion mechanisms promoted this karyotypic alteration, besides, b microchromosomes (0-3b) were also described for this population, whose origin has been suggested as centric fragments originated from chromosome rearrangements (porto et al. 2010; table 1). therefore, we suggest that the cytogenetic characteristics detected in the present study and in most populations of r. pentamaculata, be considered a primitive condition for the species. in the loricariidae family, from a cytogenetic point of view, the diploid number of 2n=54 chromosomes has been suggested as a plesiomorphic characteristic, and that due possible chromosomal rearrangements such as fusions and fissions occurred throughout the evolution of loricariids, promoted the increase and decrease of diploid numbers (artoni and bertollo 2001; kavalco et al. 2005, mendes-neto et al. 2011; alves et al. 2012). possible promoters of chromosomal rearrangements and consequently of chromosomal polymorphisms were investigated in the species of rineloricaria. interstitial telomeric sites (its) have been related to the centric fusion events, corroborating the hypothesis that these rearrangements the involvement with changes in karyotypic formulas, nf and reduction of diploid number in some populations of rineloricaria, such as in r. lanceolata (porto et al. 2014), r. latirostris (primo et al. 2017) and in two species from the iguaçu river (r. cubatoni and r. maackii, in preparation). primo et al. (2017) conducted cytogenetic analysis on a population of r. pentamaculata from the barra grande river whose specimens showed diploid number of 2n=54 chromosome with the first metacentric chromosome pair bearer of an its. the telomeric sequences located in an interstitial position it has been considered traces of centric fusion occurred between acrocentric chromosomes originating metacentric chromosomese with consequent reduction of the diploid number from 56 to 54 chromosomes and alteration of karyotypic formula (table 1; primo et al. 2017). however, even though its were not observed in the population of the itiz stream and the cytogenetic data show conserved characteristics, this method is not sufficient to postulate the occurrence of centric fusion chromosomal rearrangements. on the other hand, repetitive telomere-like dna sequences that are components of heterochromatin and located in an interstitial position could be misinterpreted as its, these sequences would table 1. cytogenetic data available for rineloricaria pentamaculata. river/basin/state 2n (nf) karyotype formula nor rdna 5s ref ag-nor rdna 18s taquaral river/ paranapanema/ sp 58(62) 4m/sm+54st/a te (1th st/a) te (1th st/a pair 3) 10 sites/te 1 juruba/ tibagi river/ pr 56(70) 14m/sm+42st/a te (1th st/a, pair 3) 12 sites/te 2 barra grande river/ ivaí river / pr 56(70)* 14m/sm+42st/a te (1th st/a, pair 3) 10 sites/te 54(64)** 10m/sm + 44st/a te (1th st/a, pair 4) 8 sites/te tauá stream/ alto paraná river basin / pr 56(65) 9m/sm+47st/a te (1th st/a) te (1th st/a) 3 56(64) 8m/sm+48st/a te (1th st/a) te (1th st/a) tatupeba river/ alto paraná river basin pr 56(64) 8m/sm+48st/a te (1th st/a) te (1th st/a) and 4th st/a) keller river/ alto paraná river basin / pr 56(64) 8m/sm+48st/a te (1th st/a) te (1th st/a) jacucaca river/ alto paraná river basin / pr 56(64) 8m/sm+48st/a te (1th st/a) te (1th st/a) 12 sites 4 água do oito stream/tibagi river/ alto paraná river 56(64) 8m/sm+48st/a te (1th st/a) 5 quexada river/ alto paraná river / pr 56(64) 8m/sm+48st/a te (1th st/a) te (1th st/a) 12 sites 6 itiz stream/ ivaí river / alto paraná river pr 56(64) 8m/sm+48st/a te (1th st/a) te (1th st/a) 14 sites/te 7 subtitles: 2n: diploid number; nf: fundamental number; m: metacentric; sm: submetacentric; st: subtelocentric; a: acrocentric; te: terminal; sp: são paulo; pr: paraná; ref: references: 1rodrigues 2010; 2primo et al. 2017: * karyomorph a and ** karyomorph b;.3porto et al. 2011; 4maia et al. 2010 and venturelli 2014; 5-maia et al. 2010; 6-venturelli 2014; 7present study. 43contributions of species rineloricaria pentamaculata (loricariidae:loricariinae) in a karyoevolutionary context not be involved with centric fusion events (meyne et al. 1990; ocalewicz 2013; bolzán 2017). in addition to its, other repetitive dna sequences are considered hotspot for chromosomal rearrangements, contributing to the understanding of the karyotypic diversity of the genus. in r. lima, 5s rdna sites associated with ttagggn were observed in centromeric position in some meta-submetacentric chromosomes suggesting as susceptible sites for chromosomal breaks (rosa et al. 2012). glugoski et al. (2018) found transposable elements associated with 5s rdna sites and ttagggn sequences in a population of r. latirostris (river of pedras, ventania-pr brazil) and that these elements are probably also involved with chromosomal rearrangements and the karyotypic variability of the species. however, transposable elements were not observed associated with repetitive regions of the genome (5s rdna and/or ttagggn) in populations of r. latirostris (laranjinha river, ventania-pr brazil), r. pentamaculata, r. stellata and r. capitonia (primo et al. 2018) and this presente study, such analysis was not performed. thus, the expansion of molecular cytogenetic studies in the genus are essential for the better understanding of the types of rearrangements that caused chromosomal variability, as well as the mechanisms involved in the karyotypic evolution of rineloricaria. the simple nor system located in the terminal position of the first pair of subtelo-acrocentric chromosomes is a conserved characteristic among the populations of the species and rineloricaria genus, and was also detected in the present study (alves et al. 2003; rodrigues 2010; maia et al. 2010; porto et al. 2011; rosa et al. 2012; porto et al. 2014; primo et al. 2017; venturelli et al. 2021), except for r. pentamaculata from the tatupeba stream, which showed nor system multiple with two pairs of subtelo-acrocentric chromosomes containing 18s rdna sites, one of the pairs being the first pair of subtelo-acrocentric chromosomes (porto et al. 2011; table 1). the nor phenotype detected in the present study and in most populations of r. pentamaculata and in other species of rineloricaria indicates an origin from a common ancestor. in the loricariidae most species, showed that the simple nor system located in terminal position and constitutive heterochromatin generally associated with this region are suggested as an ancestor phenotype. (júlio-jr 1994; artoni and bertollo 1996; ribeiro et al. 2015; prizon et al. 2016; venturelli et al. 2021). however, in species of loricariidae with multiple nor system and the occurrence of b chromosomes, these characteristics were considered apomorphic. (artoni and bertolo 1996; artoni and bertolo et al. 2001; kavalco et al. 2005; porto et al. 2010-2011; rubert et al. 2016). likewise, spécimens of r. pentamaculata from the itiz stream showed similarity to other cytogenetic studies in the pattern of constitutive heterochromatin distribution (maia et al. 2010; porto et al. 2011; venturelli 2014; primo et al. 2017). the association of constitutive heterochromatin and nor, detected in the present study, has been frequently reported in fish and shared by all species of rineloricaria has been interpreted as a synapomorphic trait and comes from a common ancestor. (giuliano-caetano 1998; porto et al. 2011; venturelli et al. 2021). in addition to corroborating the data on the distribution of constitutive heterochromatin in r. pentamaculata, we show the composition of cg-rich heterochromatin (cma3 positive) and emphasize the importance of descriptive and comparative cytogenetics. furthermore, it is observed that most species of rineloricaria, especially r. pentamacula, exhibited low constitutive heterochromatin profiles, with a varied distribution being found in the interstitial and pericentromeric regions, occupying low portions of the long or short chromosome arms (primo et al. 2017). physical mapping of 5s rdna sequences in species of rineloricaria, especially in r. pentamaculata, has increased since 2011 and of the 11 cytogenetically characterized species, seven presented studies of 5s rdna sites, showing variation in location and quantity (7 to 14 sites, table 1). for the genus rineloricaria, these regions are evidenced in more than one pair of chromosomes (rosa et al. 2012; primo et al. 2017; glugoski et al. 2018; venturelli et al. 2021), however, it is not possible to establish a pattern, suggesting that these sites is a species-specific character. information on diploid number and 5s rdna distribution, has been used as a support to distinguish species of rineloricaria that are morphologically similar, with difficulties in characterizing and validating the taxonomic status of the species. (venturelli et al, 2021). a hypothesis that could explain the multiple sites of 5s rdna detected in species of rineloricaria is that tranposable elements promoted the dispersion of copies of these genes throughout the genomes (primo et al. 2018; glugoski et al. 2018). furthermore, according to glugoski et al. (2018) in r. latirostris, showed multiple degenerate 5s rdna, would be involved with the insertion of the transposable element hat. unequal crossover has been suggested to explain the existence of these degenerate sequences, establishing a breakpoint region susceptible to chromosome breakage, non-homologous recombination and robertsonian fusion (rb), and thus corroborating the hypothesis that both 5s rdna sites and transposable elements may be involved with chromosomal polymorphisms and the karyotypic variability 44 a. cius et al. observed in rineloricaria (glugoski et al. 2018). therefore, the present study contributes to aggregate and stimulate cytogenetic studies in the species rineloricaria pentamaculata and others species of rineloricaria. futhermore, we postulate that the diploid number of 2n=56, karyotypic formula 8m/sm + 44st/a, nf=64 and simple nor system located in the first pair of ts/a chromosomes reinforcing this chromosomal structure as representative of this species and probably, a plesiomorphic condition for r. pentamaculata. on the other hand, for those populations that presented apomorphic cytogenetic characteristics (tauá and tatupeba streams and barra grande river, table 1), robertsonian rearrangements could have caused these variations, and that 5s rdna sequences and the transposable elements promoted these rearrangements, contributing for the karyotypic evolution to the species. however, the presence of multiple 5s rdna sites also seems to be a characteristic of the chromosomal structure of r. pentamaculata, constituting an important marker of intraspecific variations in comparative analyzes of this group. acknowledgements the authors are grateful to cnpq (conselho nacional de desenvolvimento científico e tecnológico) for financial support, maringá state university (uem) and limnology, ichthyology and aquaculture research center (nupélia). references alves alc, oliveira c, foresti f. 2003. karyotype variability in eight species of the subfamilies loricariinae and ancistrinae (teleostei, siluriformes, loricariidae). caryologia. 1:57–63. alves al, oliveira c, foresti f. 2005. comparative cytogenetic analysis of eleven species 308 of subfamilies neoplecostominae and hypostominae (siluriformes: loricariidae). genetica. 309(124):127-136. alves al, borba rs, oliveira c, nirchio m, granado a, foresti f. 2012. karyotypic diversity and evolutionary trends in the neotropical catfish genus hypostomus lacépède, 1803 (teleostei, siluriformes, loricariidae). comp cytogen. available from: http://www.pensoft. net/journals/compcytogen. artoni rf, bertollo lac. 2001. trends in the karyotype evolution of loricariidae fish (siluriformes). hereditas. 134:201–210. available from: http://doi/10.1111/ j.1601-5223.2001.00201.x artoni rf, bertollo lac. 1996. cytogenetic studies on hypostominae (pisces, 312 siluriformes, loricariidae). considerations on karyotype evolution in the genus hypostomus. caryologia. 49:81–90. bertollo lac, takahashi cs, moreira filho o. 1978. cytotaxonomic considerations on hoplias lacerda (pisces, erythrinidae). brazilian journal of genetics. 1: 103–120. bolzán ad. 2017. interstitial telomeric sequences in vertebrate chromosomes: origin, function, instability and evolutions. mutation research. 1383-5742(16):30153-3. available from: http://dx.doi. org/doi:10.1016/j.mrrev. costa-silva gj, rodriguez ms, roxo ff, foresti f, oliveira c. 2015. using different methods to access the difficult task of delimiting species in a complex neotropical hyperdiverse group. plos one. 10(9):e0135075. available from: http://doi/10.1371/ journal. pone.0135075 covain r, fish-muller s, oliveira c, mol jh, montoyaburgos ji, dray s. 2016. molecular phylogeny of the highly diversified catfish subfamily loricariinae (siluriformes, loricariidae) reveals incongruences with morphological classification. mol phylogenet evol. 94:492–517. available from: https://doi.org/10.1016/j. ympev.2015.10.018 kavalco kf, pazza r, bertollo lac, moreira-filho. 2005. karyotypic diversity and 370 evolution of loricariidae (pisces, siluriformes). heredity. 4:180-186. fricke r, eschmeyer wn. 2022. a guide to fish collections in eschmeyer’s catalog of fishes [internet]. san francisco: california academy of science. accessed [2022-03-02]. available from: http://researcharchive. calacademy. org/research/ichthyology/catalog/collections.asp. giuliano-caetano l. 1998. polimorfismo cromossômico robertosiano em populações de rineloricaria latirostris (pisces, loricariidae). 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[dissertation]. londrina (pr): universidade estadual de londrina. venturelli nb, takagui fh, pompeo lrs, rodriguez ms, rosa r, giuliano-caetano l. 2021. cytogenetic markers to understand chromosome diversification and conflicting taxonomic issues in rineloricaria (loricariidae: loricariinae) from rio grande do sul coastal drainages. biologia. available from: https:// doi.org/10.1007/s11756-021-00748-3. vera-alcaraz hs, pavanelli cs, zawadzki ch. 2012. taxonomic revision of the rineloricaria species (siluriformes: loricariidae) from the paraguay river basin. neotropical ichthyology. 10:285–311. caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 3 2022 firenze university press chromosome mapping of repetitive dnas in the picasso triggerfish (rhinecanthus aculeatus (linnaeus, 1758)) in family balistidae by classical and molecular cytogenetic techniques kamika sribenja1, alongklod tanomtong1, nuntaporn getlekha2,* chromosome number of some satureja species from turkey esra kavcı1, esra martin1, halil erhan eroğlu2,*, fatih serdar yıldırım3 l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations selma tabur1,*, nai̇me büyükkaya bayraktar2, serkan özmen1 characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques kristel ramírez-matadamas1, elva irene cortés-gutiérrez2, sergio moreno-limón2, catalina garcía-vielma1,* contributions of species rineloricaria pentamaculata (loricariidae:loricariinae) in a karyoevolutionary context a cius¹, ca lorscheider2, lm barbosa¹, ac prizon¹, ch zawadzki3, la borin-carvalho¹, fe porto4, alb portela-castro1,4 cadmium induced genotoxicity and antioxidative defense system in lentil (lens culinaris medik.) genotype durre shahwar1,2,*, zeba khan3, mohammad yunus khalil ansari1 biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts and evaluation of their biosafety potential denisa manolescu1,2, georgiana uță1,2,*, anca șuțan3, cătălin ducu1, alin din1, sorin moga1, denis negrea1, andrei biță4, ludovic bejenaru4, cornelia bejenaru5, speranța avram2 polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel (foeniculum vulgare mill.) egizia falistocco methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. sazada siddiqui*, sulaiman a. alrumman comparative study and genetic diversity in malva using srap molecular markers syamand ahmed qadir1, chnar hama noori meerza2, aryan mahmood faraj3, kawa khwarahm hamafaraj4, sherzad rasul abdalla tobakari5, sahar hussein hamarashid6,* nuclear dna 2c-values for 16 species from timor-leste increases taxonomical representation in tropical ferns and lycophytes inês da fonseca simão1, hermenegildo ribeiro da costa1,2,3, helena cristina correia de oliveira1,2, maria helena abreu silva1,2, paulo cardoso da silveira1,2,* nuclear dna content and comparative fish mapping of the 5s and 45s rdna in wild and cultivated populations of physalis peruviana l. marlon garcia paitan*, maricielo postillos-flores, luis rojas vasquez, maria siles vallejos, alberto lópez sotomayor identification of genetic regions associated with sex determination in date palm: a computational approach zahra noormohammadi1,*, masoud sheidai2, seyyed-samih marashi3, somayeh saboori1, neda moradi1, samaneh naftchi1, faezeh rostami1 comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(1): 141-153, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1444 caryologia international journal of cytology, cytosystematics and cytogenetics citation: haiou xia, tianyu cheng, xin ma (2022) genetic relationships between populations of aegilops tauschii coss. (poaceae) using scot molecular markers. caryologia 75(1): 141-153. doi: 10.36253/caryologia-1444 received: november 3, 2021 accepted: april 20, 2022 published: july 6, 2022 copyright: © 2022 haiou xia, tianyu cheng, xin ma. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. genetic relationships between populations of aegilops tauschii coss. (poaceae) using scot molecular markers haiou xia1,*, tianyu cheng2, xin ma2 1 school of architecture and civil engineering, chongqing metropolitan college of science and technology, yongchuan,chongqing , 402167, china 2 china shipbuilding ndri engineering co., ltd chongqing branch  , yubei,chongqing , 401120, china *corresponding author. e-mail: xho19880526@163.com abstract. the genus aegilops has an important potential utilization in wheat improvement because of its resistance to different biotic and abiotic stresses and close relation with the cultivated wheat. aegilops tauschii grows in iran, westward to turkey and eastward to afghanistan and china with a distribution center in the south of caspian sea. in spite of its very good biochemical characterization, the knowledge about the dna variability is very limited and no dna markers were used to analyses the genomic variability of the populations, up to date. in the present study, genetic diversity of 117 aegilops tauschii, individuals nine populations were studied using 10 start codon targeted (scot) markers. high polymorphic bands (96.33%), polymorphic information content (0.48) and allele number (1.024) showed scot as a reliable marker system for genetic analysis in aegilops tauschii. at the species, the percentage of polymorphic loci [p] was 66.30%, nei’s gene diversity [h] was 0.35, shannon index [i] was 0.33 and unbiased gene diversity [uhe] was 0.37. genetic variation within populations (59%) was higher than among populations (41%) based on analysis of molecular variance (amova). we used scot molecular marker for our genetic investigation with the following aims: 1— investigate genetic diversity both among and with date aegilops tauschii, 2—identify genetic groups within these nine populations aegilops tauschii, and 3—produce data on the genetic structure of date aegilops tauschii populations. the results obtained revealed a high within-population genetic variability. keywords: aegilops tauschii, genetic admixture, gene flow, genetic structure, scot. introduction genetic variability description specifies differences among individuals or populations of the same species and serves as a very good tool for plant breeding and conservation programmes (karasakal et al, 2020a, 2020b; huang et al, 2021; hou et al, 2021, guo et al, 2021). different types of dna markers have been applied in evaluation of genetic diversity of different plants, considering also the effects of the plant growing environment and 142 haiou xia, tianyu cheng, xin ma developmental stage (bi et al, 2021; cheng et al, 2021; khayatnezhad and gholamin, 2020, 2021a, 2021b). crop wild relatives (cwrs) are valuable plant genetic resources (pgr) owing to high affinity to crops, including crop progenitors, to improve several properties of crops following the yield improvement or stability, pest or disease resistance, etc. they appear as a tangible genetic diversity that has been experimentally used for several centuries (maxtend et al. 2015). however, the development in the biotechnological methodologies also allowed the transfer of genes from cwr species as the valuable reservoirs of genetic diversity to improve the crops (hajjar and hodgkin, 2007). iran is located in middle eastern center of cultivated plants that are considered in the higher ranks in terms of conservation priorities for cwrs in the world (saydi and mehrabian, 2019). besides, there are several wild relatives of cereals (e.g., triticum, hordeum, aegilops, secale, etc.) (bor, 1970) that show high potentials to improve the cereals. there exist 22 aegilops and five triticum species in three ploidy levels consisting of diploid (2n=2x=14), tetraploid (2n=4x=28), and hexaploid (2n=6x=42) cytotypes (van slageren, 1994). iran has been known as the main distribution center of wheat’s ancestors and the associated compositions of triticum and aegilops as the richest wheat gene pool detected in this region. many agronomically valuable characteristics including the bread making quality (orth and bushuk, 1973), cold hardiness (marcussen et al., 2014), and salt tolerance (schachtman et al., 1992) are governed by d genome. aegilops tauschii grows in iran, westward to turkey and eastward to afghanistan and china with a distribution center in the south of caspian sea. the natural hybridization of tetraploid wheat and ae. tauschii about 8,000–10,000 years ago led to the formation of hexaploid wheat, with ae. tauschii contributing many genes that extended the climatic adaptation and improved the bread making quality (lagudah et al., 1991). however, much greater genetic diversity is present in this wild donor of d-genome. aegilops tauschii harbors considerable genetic diversity for diseases and abiotic resistance factors relative to the wheat d-genome. hammer (1980) classified ae. tauschii into two subspecies, a. tauschii subsp. tauschii and a. tauschii subsp. strangulata (eig) tzvel. four varieties were identified under subsp. tauschii including var. tauschii, var. meyeri (griseb.) tzvel, var. anathera (eig) hammer, and var. paleidenticulata (gandilyan) hammer. the typical subspecies tauschii is characterized by elongated, cylindrical spikelets. the subsp. strangulata is characterized by more quadrate spikelets with equal length and width. the intermediate forms have also been identified by some scientists (kim et al., 1992). the phenotypic classification of the subspecies, especially the varieties, is challenging. therefore, the phenotypic data often poorly correlate with genetic classification (lubbers et al., 1991). the phenotypic divisions in a. tauschii may not always be distinguishable due to the hybridization and, as a result, the occurrence of intermediate forms (dvorak et al., 1998). furthermore, this indicates that the morphological variations of ae. tauschii should not always be used to predict the genetic variability at the molecular level. t he subsp. strangulata g rows ma i n ly on t he southeastern shores of the caspian sea between rasht and azadshahr, whereas subsp. tauschii is distributed to the east and west of this area (aghaei et al., 2008). the ae. tauschii populations in the southwest of the caspian sea in iran (aghaei et al. 2008) and nearby mountainous areas in azerbaijan are believed to be the d-genome source of t. aestivum. this is because of the distribution of the wa xy bloom alleles in the popu lations occurring in t he regions (tsunewa k i, 1966). the evaluation of esterase isozymes also provided the support in the southwest of the caspian sea, iran as the origin of t. aestivum (nakai 1979). in addition to the ae. tauschii populations found in the south of caspian sea and throughout the alborz mountains, some special populations of this species are also found in fars, hormozgan and kerman provinces in the southern iran. in recent years, a novel marker system termed start codon targeted (scot) markers was developed by collard and mackill (collard and mackill 2009) based on the short-conserved region f lanking the start codon (atg) in plant genes. scot employs long primers (18mers), and can generate polymorphisms that are reproducible. it is considered as a dominant marker system, requiring no prior sequence information, and the polymorphism is correlated to functional genes and their corresponding traits. other excellent characteristics include their simplicity of use, high polymorphism, the use of universal primers, low cost and gene targeted markers. this technique has been successfully used to assess genetic diversity and structure (collard and mackill 2009; ma et al, 2021; peng et al, 2021; si et al., 2021; sun et al., 2021; miao et al 2018; zou et al, 2019; wang et al 2020; xiaolong et al, 2021; hou et al, 2021), construct dna fingerprints, identify qtls, and analyze differential gene expression and screen stress tolerance genes. the present study is the first attempt to use scot markers to assess the level of genetic diversity of aegilops tauschii which were collected from the wild populations. the main objectives of this study were to 143genetic relationships between populations of aegilops tauschii coss. (poaceae) using scot molecular markers assess the genetic diversity and genetic relationship of aegilops tauschii in iran. these results could benefit aegilops tauschii germplasm collection, conservation and future breeding. materials and methods plant materials a total of 117 individuals were sampled representing nine natural populations of aegilops tauschii in east azerbaijan, alborz, mazandaran, guilan, golestan, and ardabil provinces of iran during july-agust 2018 (table 1). for morphometric and scot analysis we used 117 plant accessions (four to eleven samples from each populations) belonging to nine different populations with different eco-geographic characteristics were sampled and stored in -20 till further use. more information about geographical distribution of accessions are in table 1. different references were used for the correct identification of species (aegilops tauschii). environmental variables in this experiment, the data regarding climate variables included elevation, and geographic data (latitude and longitude), and this data was determined at each site using an electronic gps. the climate variable data of mean annual temperature, mean maximum temperature (°c), mean minimum temperature (°c), annual rainfall (mm), number of frost days were downloaded from http://www.worldclim.org. (table 1). dna extraction and scot-pcr amplification fresh leaves were used randomly from four to eleven plants in each of the studied populations. these were dried by silica gel powder. ctab activated charcoal protocol was used to extract genomic dna. the quality of extracted dna was examined by running on 0.8% agarose gel. a total of 25 scot primers developed by collard and mackill (2009), 10 primers with clear, enlarged, and rich polymorphism bands were chosen (table 2). pcr reactions were carried in a 25μl volume containing 10 mm tris-hcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). the thermal program was carried out with an initial denaturation for 1 min at 94°c, followed by 40 cycles in three segments: 35 s at 95°c, 40s at 47°c and 55s at 72°c. final extension was performed at 72°c for 5 min. the amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated by using a 100 bp molecular size ladder (fermentas, germany). data analyses morphological studies in total 19 morphological (19 quantitative) characters were studied. four to twelve samples from each population were randomly studied for morphological analyses (table 2). morphological characters were first standardized (mean = 0, variance = 1) and used to establish euclidean distance among pairs of taxa (podani, 2000). for grouping of the plant specimens, the upgma table 1. populations studied, their locality and ecological features of ae. tauschii in this study. pop no. subspecies locality no. of collected accessions mean maximum temperature (°c) mean minimum temperature (°c) annual rainfall (mm) 1 ssp. strangulata gilan, lahijan 10 40.12 -18.12 325 2 ssp. strangulata mazandaran; chalous 9 35.55 -20.34 378 3 ssp. strangulata mazandaran, kandovan 18 41.34 -10.34 377 4 ssp. strangulata gorgan, ramian 16 39.14 -17.55 390 5 ssp. strangulata mazandaran, behshahr 12 36.88 -11.23 320 6 ssp. tauschii alborz, asara 19 32.55 -22.45 334 7 ssp. tauschii ardabil, fandoghlou 10 30.44 -18.66 229 8 ssp. tauschii azarbaijan, arasbaran, kolaleh 13 32.88 -11.66 210 9 ssp. tauschii azarbaijan, arasbaran, kaleybar 10 20.44 -25.66 478 144 haiou xia, tianyu cheng, xin ma (unweighted paired group using average) and ward (minimum spherical characters) as well as ordination methods of mds (multidimensional scaling) were used (podani, 2000). past version 2.17 (hammer et al. 2012) was used for multivariate statistical analyses of morphological data. molecular analyses excel 2013 was used to calculate the total number of bands (tnb), the number of polymorphic bands (npb), and the percentage of polymorphic bands (ppb). the polymorphism information content (pic) of scot primers was determined using powermarker v3.25. binary characters (presence = 1, absence = 0) were used to encode scot bands and used for further analyses. parameter like nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism (p% = number of polymorphic loci/number of total loci) were determined (freeland et al. 2011). shannon’s index was calculated by the formula: h’ = -σpiln pi. rp is defined per primer as: rp = ∑ ib, were “ib” is the band informativeness, that takes the values of 1-(2x [0.5-p]), being “p” the proportion of each genotype containing the band. the percentage of polymorphic loci, the mean loci by accession and by population, uhe, h’ and pca were calculated by genalex 6.4 software. nei’s genetic distance among populations was used for neighbor joining (nj) clustering and neighbor-net networking (freeland et al. 2011; huson & bryant 2006). mantel test checked the correlation between geographical and genetic distances of the studied populations (podani, 2000). these analyses were done by past ver. 2.17 (hammer et al. 2012), darwin ver. 5 (2012) and splitstree4 v4.13.1 (2013) software. amova (analysis of molecular variance) test (with 1000 permutations) as implemented in genalex 6.4 (peakall & smouse, 2006), and nei,s gst analysis as implemented in genodive ver.2 (2013) were used to show genetic difference of the populations. moreover, populations, genetic differentiation was studied by g’st est = standardized measure of genetic differentiation (hedrick, 2005), and d_est = jost measure of differentiation. to assess the population structure of the aegilops tauschii accessions, a heuristic method based on bayesian clustering algorithms were utilized. the clustering method based on the bayesian-model implemented in the software program structure (falush et al. 2007) was used on the same data set to better detect population substructures. this clustering method is based on an algorithm that assigns genotypes to homogeneous groups, given a number of clusters (k) and assuming hardy-weinberg and linkage equilibrium within clusters, the software estimates allele frequencies in each cluster and population memberships for every individual (pritchard et al. 2000). the number of potential subpopulations varied from two to ten, and their contribution to the genotypes of the accessions was calculated based on 50,000 iteration burn-ins and 100,000 iteration sampling periods. the most probable number (k) of subpopulations was identified following evanno et al. (2005). in k-means clustering, two summary statistics, pseudo-f, and bayesian information criterion (bic), provide the best fit for k. gene flow (nm) which were calculated using popgene (version 1.31) program. gene flow was estimated indirectly using the formula: nm = 0.25(1 fst)/fst. in order to test for a correlation between pair-wise genetic distances (fst) and geographical distances (in km) between populations, a mantel test was performed using tools for population genetic analysis (tfpga; miller, 1997) (computing 999 permutations). this approach considers equal amount of gene flow among all populations. results scot polymorphisms twenty-five scot primers were tested with four aegilops tauschii accessions as dna templates; all primtable 2. evaluated morphological characters in ae. tauschii species. 1 spike length (mm)sl 2 number of spikelets per spike nsp 3 spikelet length (mm) spl 4 spikelet width spw 5 length of upper glumes lug 6 width of upper glumes wug 7 length of upper lemas lul 8 width of upper lemaswul 9 length of lower glumes llg 10 width of lower lemas wlg 11 width of upper lemas wll 12 number of awner spikelets nap 13 longest awns of the upper glumes laug 14 shortest awns of the upper glumes saug 15 middel awns of the upper glumes maug 16 awns number on lower glumes anlg 17 awns number on second glumes ansg 18 awns number on third glumes tntg 19 awns number on forth glumes tntg 145genetic relationships between populations of aegilops tauschii coss. (poaceae) using scot molecular markers ers produced amplification products, and only primers showing clear and reproducible band patterns were selected for further analysis. ten primers were then chosen for species identification and phylogenetic analysis. as shown in table 3, all 10 primers used for scot analysis. the gel electrophoresis pattern obtained using primer scot-14 is illustrated in figure 1. a total of 139 fragments were obtained, and 131 of the fragments were polymorphic. the number of polymorphic fragments for each scot primer ranged from 9 (scot-18, 11) to 17 (scot-19,6), with an average of 13. the percentage of polymorphic fragments was from 88.99% to 100.00%, with an average of 96.33% polymorphism. polymorphism information content (pic) values were 0.33 to 0.67, with an average of 0.48. the number of different alleles was 1.024 at the species (table 4). these results indicated that a high level of polymorphism could be detected among aegilops tauschii accessions using scot markers. populations, genetic diversity genetic diversity parameters determined in nine geographical populations of aegilops tauschii are presented in table 4. the percentage of polymorphic loci (p) and nei’s gene diversity (h) were important parameters for measuring the level of genetic diversity. in table 4, the genetic diversity parameters of the nine populations are shown. the highest value of percentage polymorphism (64.30%) was observed in gilan, lahijan (population no.1) which shows high value for gene diversity (0.35) and shanon, information index (0.33). population alborz, asara (no.6) has the lowest value for percentage of polymorphism (42.15%) and the lowest value for shanon, information index (0.15), and he (0.18). population genetic differentiation amova (phipt = 0.789, p = 0.010), revealed significant difference among the studied populations (table 5). it also revealed that, 59% of total genetic variability was due to within population diversity and 41% was due to among population genetic differentiation. moreover, pair-wise amova revealed significant genetic difference almost among all the studied populations. these results indicate that aegilops tauschii population are genetically differentiated and we can use such genetic difference in future breeding programs of this valuable plant species. table 3. scot primers used for this study and the extent of polymorphism. primer name primer sequence (5’-3’) tnb npb ppb pic scot-1 caacaatggctaccacca 14 13 95.74% 0.67 scot-3 caacaatggctaccaccg 13 12 92.31% 0.54 scot-6 caacaatggctaccacgc 17 17 100.00% 0.47 scot-11 aagcaatggctaccacca 11 9 96.89% 0.43 scot-14 acgacatggcgaccacgc 13 12 95.81% 0.34 scot-15 acgacatggcgaccgcga 12 12 100.00% 0.47 scot-16 ccatggctaccaccggcc 13 12 92.31% 0.34 scot-17 catggctaccaccggccc 11 11 100.00% 0.57 scot-18 accatggctaccaccgcg 9 9 88.89% 0.33 scot-19 gcaacaatggctaccacc 17 17 100.00% 0.49 mean 14 13 96.33% 0.48 total 139 131 tnp: total number of bands; npb: number of polymorphic bands; ppb: percentage of polymorphic bands; pic: polymorphism information content. figure 1. electrophoresis gel of studied ecotypes from dna fragments produced by scot-19(population numbers according to table 1). table 4. genetic diversity parameters in the studied populations ae. tauschii (n = number of samples, na = number of different alleles, ne = number of effective alleles, i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations). pop n na ne i he uhe %p pop1 10 0.288 1.024 0.33 0.35 0.37 64.30% pop2 9 0.499 1.067 0.18 0.271 0.24 49.26% pop3 18 0.261 1.024 0.192 0.26 0.28 43.15% pop4 16 0.555 1.021 0.29 0.29 0.28 43.53% pop5 12 0.431 1.088 0.23 0.22 0.29 57.53% pop6 19 0.255 1.021 0.15 0.18 0.12 42.15% pop7 10 0.258 1.029 0.231 0.28 0.27 45.38% pop8 13 0.452 1.089 0.18 0.29 0.25 45.05% pop9 10 0.333 1.006 0.31 0.27 0.26 43.23% mean 0.355 1.024 0.23 0.284 0.252 45.91% 146 haiou xia, tianyu cheng, xin ma the pairwise comparisons of ‘nei genetic identity’ among the studied populations aegilops tauschii (table not included) have shown a higher a genetic similarity (0.91) between populations mazandaran; chalous (ssp. strangulata; pop. no 2) and mazandaran, kandovan (ssp. strangulate; pop. no 3), while the lowest genetic similarity value (0.733) occurs between mazandaran, kandovan (ssp. strangulate; pop. no.3) and azarbaijan, arasbaran, kaleybar (ssp. tauschii; pop. no. 9). populations, genetic affinity upgma dendrogram and neighbor-net network produced similar results therefore only upgma dendrogram is presented and discussed (figure 2). two major clusters were formed in the upgma tree (fig. 2). the first major cluster contained two sub-clusters: the population of azarbaijan, arasbaran, kolaleh (pop. no. 8, ssp. tauschii) is distinct and remains separate from the other populations with a great distance and comprises the first sub-cluster. the second sub-cluster was formed by the other populations from ssp. tauschii, which showed close genetic affinity. the second major cluster contained only ssp. strangulate, which separated from the other studied populations and joined the others with a great distance. these results show that the plant specimens of each studied subspecies were not grouped together, indicating that the subspecies are delimited based on the scot molecular markers. therefore, this result confirms our morphology results. the nm analysis by popgene software also produced mean nm= 0.734, which is considered a very low value of gene flow among the studied species. mantel test after 5000 permutations produced significant correlation between genetic distance and geographical distance in these populations (r = 0.348, p = 0.001). therefore, the populations that are geographically more distant have less amount of gene flow, and we have isolation by distance (ibd) in aegilops tauschii. this result was similar to the result of the structure analysis at k = 2. the principal coordinate analysis (pcoa) (figure 3) for 9 populations of aegilops tauschii revealed that the populations 1 -5 (ssp. strangulate), as well as populations 6 -9 (ssp. tauschii) are separated from the other populations and also show closer genetic affinity. the results of pcoa were the same from the other cluster analyses as shown above. table 5. analysis of molecular variance (amova) of the studied species. source df ss ms est. var. % φpt among pops 22 333.576 30.327 6.082 41% 41% within pops 40 587.767 9.530 5.230 59% total 62 888.342 11.513 100% figure 2. upgma tree of populations in ae. tauschii based on scot molecular markers, (population numbers are according to table 1). 147genetic relationships between populations of aegilops tauschii coss. (poaceae) using scot molecular markers populations genetic structure the number of genetic groups was determined by two methods of 1—k-means clustering which is based on the maximum likelihood approach, and 2—evanno test which is based on structure analysis and is a bayesian approach based method. k-means clustering, based on pseudo-f and bic (bayesian information criterion) recognized 2 and 4 genetic groups, respectively. this is in agreement with amova result, showing significant genetic difference among date populations of ae. tauschii. evan test based on delta k (figure 4) identified the optimum number of genetic groups 2. we performed structure analysis based on k = 2, to identify the genetic groups (figure 5). in the plot of k = 2, the populations mazandaran, kandovan; gorgan, ramian and azarbaijan, arasbaran, kolaleh (pop. no 3,4,8) (red colored) are placed in the first genetic group, while the other populations of ae. tauschii formed the second genetic group. these different genetic groups may be used in future breeding and hybridization programs of iranian date ae. tauschii. the mean nm = 0.734 was obtained for all scot loci, which indicates high amount of gene flow among figure 3. pcoa plot of populations in ae. tauschii based on scot molecular markers, (population numbers are according to table 1). figure 4. delta k plot of evanno’s test based on structure analysis. 148 haiou xia, tianyu cheng, xin ma the populations and supports genetic stratification as indicated by k-means and structure analyses. this result is in agree with grouping we obtained with pcoa plot, as these populations were placed close to each other. as evidenced by structure plot based on admixture model, these shared alleles comprise very limited part of the genomes in these populations and all these results are in agreement in showing high degree of genetic stratification within aegilops tauschii populations. morphometric analyses in present study we used 117 plant accessions (four to eleven samples from each populations) belonging to nine different populations. in order to determine the most variable characters among the taxa studied, pca analysis has been performed. it revealed that the first three factors comprised over 63% of the total variation. in the first pca axis with 40% of total variation, such characters as spikelet length (mm) spl, middel awns of the upper glumes and awns number on forth glumes have shown the highest correlation (> 0.7), number of awner spikelets nap; shortest awns of the upper glumes; 1st internode length (cm) il1and 2nd internode length (cm)il2 were characters influencing pca axis 2 and 3, respectively. different clustering and ordination methods produced similar results therefore, pca plot of morphological characters are presented here (figure 6). the result showed morphological difference/ divergence among most of the studied populations. this morphological difference was due to quantitative characters only. for example, character (length of upper glumes lug), separated population no. 1-4, character (width of upper lemaswul) separated population no. 6-9. a consensus tree was obtained for both scot and morphological trees (no shown), to reveal the populations that are diverged based on both morphological and molecular features. interesting enough, it showed divergence of almost all populations at molecular level as well as morphological characteristics. discussion the existing genetic variability of the individual species within and among the populations is connected to this species ability to mirror the shortand long-term specific regimes of their living habitats (ren and khayatnezhad 2021; khayatnezhad and nasehi 2021; i et al., 2021; jia et al, 2021). the analysis of the distribution of the genetic variability patterns specific for landscape and ecological parameters is valuable for identification of the taxa most vulnerable to the anthropogenic impacts (amedi et al 2020; das et al 2021; gutierrez-pacheco et al 2021). the coupling of ecological and genetic data will provide the most suitable background for preserving the ability of the biota to respond the rapid environmental changes (sun and khayatnezhad 2021; tao et al, 2021; wang et al, 2021; xu et al., 2021; yin et al., 2021; zhang et al, 2021). the literature reports the following basic factors influencing the distribution of genetic varifigure 5. structure plot of ae. tauschii populations based on k = 2, numbers are according to table 1. figure 6. pca plot of ae. tauschii populations based on morphological characters. numbers are according to table 1. 149genetic relationships between populations of aegilops tauschii coss. (poaceae) using scot molecular markers ation: habitat specify, plant-insect interactions, connectivity and disturbance, dispersal ability, species lifespan, reproductive rates and existing genetic diversity (gholamin and khayatnezhad, 2020a; 2020b, 2020c). genetic diversity when analysed by neutral markers does not correspond to the adaptive ability of plant populations, but these types of markers are very useful for the interpretation of the past landscapes, refugia and gene flow (brandvain et al., 2014). that is, why the selected genes or markers of active parts of plant genomes are used to interprete the plant genome response to the changes to the local climate and environment (hoffman & willi 2008; hindersah et al 2021; jordaan & rooyen et al. 2021; lucena et al. 2021; mieso & befa et al. 2020). molecular-based population genetic data are very useful for determining the ecological and habitat events in the past and for detection of patterns of the recent genetic divergence. this can be achieved using different types dna markers (davey and blaxter, 2010). scot markers are novel molecular markers that target the translation initiation site and preferentially bind to genes that are actively transcribed. these primers have been shown to exhibit relatively high levels of polymorphism [collard and mackill 2009]. it was more informative than irap and issr for the assessment of diversity of plants [collard and mackill 2009]. all of 10 primer pairs from d-genome of common wheat provided the amplification and showed a good polymorphism in ae. tauschii. totally, 150 alleles were recognized. the total number of bands per primer ranged from 9 to 20 polymorphic bands and the mean number of alleles in loci was 13.37, which did not conform to the results of saeidi et al. (2006) who obtained these results: 7.3 mean and 4–12 range, and also according to pestsova et al. (2000) who obtained these results: 18.8 mean and 11–25 range, which were achieved by ssr marker. according to nouri, et al (2021) compared the efficiency of inter-simple sequence repeat (issr) (as an arbitrary technique) and start codon targeted (scot) (as a gene-targeting technique) markers in determining the genetic diversity and population structure of 90 accessions of ae. tauschii. scot markers indicated the highest values for polymorphism information content, marker index and effective multiplex ratio compared to issr markers. their results of the analysis of molecular variance showed that the genetic variation within populations was significantly higher than among them (issr: 92 versus 8%; scot: 88 versus 12%). furthermore, scot markers discovered a high level of genetic differentiation among populations than issrs (0.19 versus 0.05), while the amount of gene flow detected by issr was higher than scot (2.13 versus 8.62). cluster analysis and population structure of scot and issr data divided all investigated accessions into two and four main clusters, respectively. their results revealed that scot and issr fingerprinting could be used to further molecular analysis in ae. tauschii and other wild species. in our study, genetic diversity of 117 aegilops tauschii, individuals nine populations were studied using 10 start codon targeted (scot) markers. high polymorphic bands (96.33%), polymorphic information content (0.48) and allele number (1.024) showed scot as a reliable marker system for genetic analysis in aegilops tauschii. at the species, the percentage of polymorphic loci [p] was 66.30%, nei’s gene diversity [h] was 0.35, shannon index [i] was 0.33 and unbiased gene diversity [uhe] was 0.37. genetic variation within populations (59%) was higher than among populations (41%) based on analysis of molecular variance (amova). jaaska (1981) stated that subsp. tauschii has a higher level of genetic variability than subsp. strangulata. according to tahernezhad et al. (2009), the cluster analysis based on upgma algorithm was calculated for the genotypes. in this group, durum wheat was in a separate class , but subsp. strangulata and subsp. tauschii did not separate from each other. this classification did not conform to the morphological studies and geographical sites of the ae. tauschii accessions. in fact, there was no classification based on subspecies or geographical regions. there was no significant grouping based on the geography of the accessions or subspecies, which conforms to our results. in saeidi et al.’s (2006) ssr marker study, there was also no significant grouping according to the geographical sites or subspecies. the high level of genetic diversity in iran was reported by lubbers et al. (1991), pestsova et al. (2000), and saeidi et al. (2006). the highest level of diversity in ae. tauschii is seen in the north of iran (south of caspian sea). also, based on the morphological traits, there were many genetic diversities in ae. tauschii, which can show the high potential of iran genepool for this species. the issr data could not separate the accessions of tauschii and strangulata subspecies. this may be due to the classification of tauschii and strangulate subspecies. in fact, the gene flow occurred between the two subspecies in iran can lead to a decrease of the genetic differentiation between them. also, kihara et al. (1965) found intermediate and hybrid forms between subspecies. kim et al. (1992) did not distinguish ssp. strangulata genotype from ssp. tauschii genotype by studying a highly conserved region of ribosomal dna in ae. tauschii subspecies. the classification based on the morphological traits did not conform to the classification according to ssr markers and 150 haiou xia, tianyu cheng, xin ma geographical regions. many studies showed t hat t he div ision based on the morphological diversity does not conform to genetic division. therefore, tauschii genepool exists around the strangulata genepool and the classification based on genetical information does not conform to the classification based on the morphological traits. gene flow inversely correlates with the gene differentiation, but it is very important for the population evolution and takes place by pollen and seeds among the populations (song et al., 2010). in the present study, the detected gene flow (nm) among ae. tauschii subspecies was 0.11, showing low genetic differentiation among ae. tauschii subspecies. according to lubbers et al. (1991) and pestsova et al. (2000) studies, one of the important origin sites for ae. tauschii is the southwest of caspian sea. therefore, the study about iranian ae. tauschii, especially in the south of the caspian sea, and the detection of their genetic diversity are very helpful in the breeding programs. this is because the south of the caspian sea is the main origin site of ae. tauschii where bread wheat has evolved (lubbers et al., 1991; pestsova et al., 2000). the study of the d-genome diversity in other d-genome containing polyploid species of the genus aegilops in iran may also lead to interesting results. comparison of results of this study with those based on ssr data (saeidi et al. 2006) shows that the ssrs are suitable markers to study the genetic diversity among closely related populations, but the scot are suitable marker system to demonstrate the genetic diversity at species level, indicating the importance of choosing the suitable marker type for the analysis we need. acknowledgment the authors thank anonymous reviewers for valuable comments on an earlier draft. references amedi, m., dumayiri, m., suhuyini, m.a.-r. 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(malvaceae) is considered one of the most complicated and challenging genera of the middle eastern flora (iljin, 1949; zohary, 1963b; riedl, 1976; townsend, 1980; pakravan, 2001). the irano-turanian floristic region (takhtajan, 1986) stretches from central anatolia to the highlands of central asia and is a main center of diversity for many mediumto large-sized genera. a well-suited system for investigating radiations in the irano-turanian region is the genus alcea. it includes approximately 70 species, which are 78 chnar hama noori meerza et al. mainly of irano-turanian distribution with extensions into the caucasus and the eastern mediterranean (zohary, 1963b). riedl (1976) has reported 39 species in iran, but the number has been reduced to 34 due to taxonomic rearrangement among them, 15 species are endemic (pakravan 2008b). alcea species are mainly annual, biennial or perennial, mostly tall-growing hemicryptophytes. the stem is erect and rarely branched from the base or acaulescent in a few cases. the mucilage that containing the plants of the malvaceae family are sources of carbohydrates, which are used in medicine (azizov et al., 2007). any classifications are hampered by uniformity in many morphological and ecological traits (flower, inflorescence and fruit structure, habitat and life form) combined with a pronounced plasticity in the morphological characters considered important for species identification (indumentum, leaf shape and degree of division, calyx and epicalyx morphology, flower colour; zohary, 1963b, c). due to this plasticity, accurate species identification requires character state combinations of the sequence of change of leaf morphology along the main stem (hereafter leaf sequence), relative proportions of calyx and epicalyx lobes and (mature) mericarp morphology, which are rarely found together on herbarium specimens. additionally, due to political tensions in the middle east and the caucasus, alcea has received only limited attention in recent studies of malvaceae (la duke & doebley, 1995; alverson & al., 1998; nyffeler & al., 2005; tate & al., 2005; escobar garcía & al., 2009). so far, only two infrageneric classification systems have been suggested. boissier (1867) defined two sections, pterocarpae boiss. and apterocarpae boiss., distinguished by winged versus unwinged mericarps. zohary (1963b, c) proposed nine informal groups based on overlapping character combinations (leaf shape and degree of leaf division, mericarp morphology, relative dimensions of calyx and epicalyx, indumentum). previous study on species delimitation and species relationship performed in this genus. badrkhani et al (2014) sequence-related amplified polymorphism (srap) marker was employed to assess the genetic diversity and genetic similarity relationships among 14 species of alcea collected from northwest of iran. two main clusters were detected using upgma, which did not correspond to geographical origin of the species. their study indicates that srap markers could be good candidates for assessing genetic variation in alcea. escobar garcía et al. (2012) examines the phylogeny of alcea using three molecular markers (nrdna its and the plastid spacers psba-trnh and trnl-trnf), their results show that while molecular data unambiguously support the circumscription of alcea inferred from morphology, they prove to be of limited utility in resolving interspecific relationships, suggesting that alceas high species diversity is due to rapid and recent radiation. literature revealed that studies are mainly dealing with taxonomy, seed and pollen morphology, stem and leaf anatomy (arabameri and khodayari 2019; escobar garcía et al. 2012) of alcea species and also, genetic diversity of alcea species have been reported in only some studies by (badrkhani et al (2014)) but there is no attempt to study genetic diversity, ecological adaptation and intraand inter-specific differentiation along with morphometric studies on of iraq. therefore, we performed morphological and molecular study of 80 collected specimens of 10 of alcea species. we try to answer the following questions: 1) is there infra and interspecific genetic diversity among studied species? 2) is genetic distance among these species correlated with their geographical distance? 3) what is the genetic structure of populations and taxa? 4) is there any gene exchange between alcea species in iraq? 2. materials and methods 2.1. plant materials we performed morphological and molecular analysis of 10 alcea species growing in iraq. for morphometric studies we used 80 plant specimens (5-15 samples from each species) and for srap analysis, we used 80. different references were used for the correct identification of species (zohary, 1963b; riedl, 1976; townsend, 1980; pakravan, 2001). details of sampling sites are mentioned (table 1). 2.2. morphological studies in total 36 morphological quantitative characters were studied (supplementary table 2). data obtained were standardized (mean= 0, variance = 1) and used to estimate euclidean distance for clustering and ordination analyses (podani 2000). 2.3. dna extraction and srapassay fresh leaves were used randomly from 5-11 plants in each of the studied species. these were dried by silica gel powder. ctab activated charcoal protocol was used to extract genomic dna. the srap analysis was performed as described by li and quiros (2001). ten srap 79delimiting species using dna and morphological variation in some alcea (malvaceae) species based on srap markers primer combinations (pcs) were used (table 3); these were synthesized by bioneer (daejeon, korea). pcr reactions were carried in a 25μl volume containing 10 mm tris-hcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). the amplifications, reactions were performed in techne thermocycler (germany) with the following program: 5 min initial denaturation step 94°c, followed by 40 cycles of 1 min at 94°c; 1 min at 52-57°c and 2 min at 72°c. the reaction was completed by final extension step of 7-10 min at 72°c. the amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated by using a 100 bp molecular size ladder (fermentas, germany). 2.4. data analyses 2.4.1. morphological studies morphological characters were f irst standardized (mean = 0, variance = 1) and used to establish euclidean distance among pairs of taxa (podani 2000). for grouping of the plant specimens, the upgma (unweighted paired group using average) ordination methods were used (podani 2000). anova (analysis of variance) were performed to show morphological difference among the populations while, pca (principal components analysis) biplot was used to identify the most variable morphological characters among the studied populations (podani 2000). past version 2.17 (hammer et al. 2012) was used for multivariate statistical analyses of morphological data. 2.4.2. molecular analyses srap bands obtained were coded as binary characters (presence = 1, absence = 0) and used for genetic diversity analysis. parameter like nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism were table 1. alcea species and populations, their localities and voucher numbers. sp. a. sachsachanica iljin a. flavovirens (boiss. & buhse) iljin a. rechingeri (zohary) i. riedl a. arbelensis boiss. & hausskn., a. koelzii i. riedl, a. hyrcana (grossh.) grossh. a. peduncularis boiss. & hausskn. a. glabrata alef. a. tabrisiana (boiss. & buhse) iljin a. persarum bornm. table 2. morphological characters in studied species. no characters 1 plant height (mm) 2 length of stem leaves petiole (mm) 3 length of stem leaves (mm) 4 width of stem leaves (mm) 5 length / width of stem leaves (mm) 6 number of segment stem leaves (mm) 7 length of basal leaves petiole (mm) 8 length of basal leaves (mm) 9 width of basal leaves (mm) 10 length / width of basal leaves (mm) 11 number of segment basal leaves 12 calyx length (mm) 13 calyx width (mm) 14 calyx length/ width (mm) 15 petal length (mm) 16 petal width (mm) 17 petal length / width (mm) 18 fruit length (mm) 19 mericarp length (mm) 20 mericarp width (mm) 21 mericarp length/width (mm) 22 seed length (mm) 23 seed width (mm) 24 seed length/ width (mm) 25 stipules length (mm) 26 stipules width (mm) 27 stipules length/ width (mm) 28 bract length (mm) 29 bract width (mm) 30 bract length / width (mm) 31 pedicel length (mm) 32 peduncle length (mm) 33 rostrum length (mm) 34 style length (mm) 35 stamen filament length (mm) 36 number of flowers per inflorescence (flower, inflorescence and fruit structure, habitat and life form) combined with a pronounced plasticity in the morphological characters considered important for species identification (indumentum, leaf shape and degree of division, calyx and epicalyx morphology, flower colour. 80 chnar hama noori meerza et al. determined (freeland et al. 2011). nei’s genetic distance among populations was used for neighbor joining (nj) clustering and neighbor-net networking (freeland et al. 2011, huson & bryant, 2006). mantel test checked the correlation between geographical and genetic distance of the studied populations (podani 2000). these analyses were done by past ver. 2.17 (hammer et al. 2012), darwin ver. 5 (2012) and splitstree4 v4.13.1 (2013) software. amova (analysis of molecular variance) test (with 1000 permutations) as implemented in genalex 6.4 (peakall and smouse 2006), and nei,s gst analysis as implemented in genodive ver.2 (2013) (meirmans & van tienderen 2004) were used to show genetic difference of the populations. moreover, populations, genetic differentiation was studied by g’st est = standardized measure of genetic differentiation (hedrick 2005), and d_est = jost measure of differentiation (jost 2008). the genetic structure of populations was studied by bayesian based model structure analysis (pritchard et al. 2000), and maximum likelihood-based method of k-means clustering of genodive ver. 2. (2013). for structure analysis, data were scored as dominant markers (falush et al. 2007). the evanno test was performed on structure result to determine proper number of k by using delta k value (evanno et al. 2005). in k-means clustering, two summary statistics, pseudof, and bayesian information criterion (bic), provide the best fit for k (meirmans 2012). gene flow was determined by (i) calculating nm an estimate of gene flow from gst by popgene ver. 1.32 (1997) as: nm = 0.5(1 gst)/gst. this approach considers equal amount of gene flow among all populations. (ii) population assignment test based on maximum likelihood as performed in genodive ver. in genodive ver. 2. (2013). the presence of shared alleles was determined by drawing the reticulogram network based on the least square method by darwin ver 5. (2012). results 3.1. species identification and inter-relationship morphometry anova showed significant differences (p <0.01) in quantitative morphological characters among the species studied. in order to determine the most variable characters among the taxa studied, pca analysis has been performed. it revealed that the first three factors comprised over 76% of the total variation. in the first pca axis with 41% of total variation, such characters as seed outline, seed length, stipules length, shape of petal, peduncle and pedicel hair, stem hair, stipules length/ width bract and leaf hair have shown the highest correlation (>0.7). length of bract and peduncle, length of petal, sepal hair, number of flowers per inflorescence were characters influencing pca axis 2 and 3 respectively. different clustering and ordination methods produced similar results. therefore pca plot of morphological characters are presented here (fig. 1). in general, plant samples of each species, were grouped together and formed separate cluster. this result show that morphological characters studied can differentiate the alcea species in two different major clusters or groups. in the studied specimens we did not encounter intermediate forms. the pca plot of morphological characters (fig. 1) separated the species into distinct groups with no intermixing. this is in agreement with upgma tree. 3.2. species identification and genetic diversity all sr ap primer produced polymorphic bands. genetic diversity parameters determined in the studied species (table 3) revealed that a. rechingeri had the highest level of genetic polymorphism (49.13%), while the lowest level of genetic polymorphism (17.22%) occurred in a. sachsachanica. a. glabrata also had the highest values for effective number of alleles (ne = 1.264) and shannon information index (i =0.235). amova test showed significant genetic difference (p = 0.01) among studied species. it revealed that 60% of total variation was among species and 40% was within species. pair-wise fst values showed significant difference among all studied species (table 4). moreover, genetic differentiation of these species was demonstrated by significant nei’s gst (0.89, p = 0.01) and d_est values (0.587, p = 0.01). nj tree based on nei,s genetic distance (fig. 2), showed that a. flavovirens; a. arbelensis; a. persarum are separated from the other studied species and join the others with a great distance. this dendrogram showed close genetic affinity between a. koelzii and a. hyrcana. similarly, a. rechingeri and a. peduncularis were placed close to each other, to which, e. litvinovii was joined with some distance. in general, this indicates that srap molecular markers can be used in alcea species differentiation. this is in agreement with amova and genetic diversity parameters presented before. the species are genetically well differentiated from each other. the nm analysis by popgene software also produced mean nm= 0.78, that is considered very low value of gene f low among the studied species. mantel test with 5000 permutations showed a significant correlation (r = 0.24, p=0.0002) between genetic 81delimiting species using dna and morphological variation in some alcea (malvaceae) species based on srap markers distance and geographical distance, so isolation by distance (ibd) occurred among the alcea species studied. nei’s genetic identity and the genetic distance determined among the studied species (table is not included). the results showed that the highest degree of genetic similarity (0.83) occurred between a. koelzii and a. hyrcana. the lowest degree of genetic similarity occurred between a. arbelensis and a. persarum (0.64). figure 1. alcea species:a, i: a. flavovirens; b: a. sachsachanica; c: a. rechingeri;d, h: a. arbelensis; e: a. koelzii; f, g: a. hyrcana 82 chnar hama noori meerza et al. 3.3. the species genetic structure we performed structure analysis followed by the evanno test to identify the optimal number of genetic groups. we used the admixture model to illustrate interspecific gene flow or / and ancestrally shared alleles in the species studied. structure analysis followed by evanno test produced δk =10. the structure plot (fig. 3) produced more detailed information about the genetic structure of the species studied as well as shared ancestral alleles and / or gene flow among alcea species. this plot revealed that genetic affinity between a. rechingeri and a. arbelensis (similarly colored), as well as a. koelzii and a. hyrcana (similarly colored) due to shared common alleles. this is in agreement with neighbor joining dendrogram presented before. the other species are distinct in their allele composition and differed genetically from each other. the low nm value (0.78) indicates limited gene flow or ancestrally shared alleles between the species studied and supports genetic stratification as indicated by k-means and structure analyses. population assignment test also agreed with nm result and could not identify significant gene flow among members of table 3. genetic diversity parameters in the studied alcea species. (n = number of samples, ne = number of effective alleles, i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations). pop n na ne i he uhe %p a. sachsachanica iljin 13.000 0.178 1.017 0.016 0.002 0.019 17.22% a. flavovirens (boiss. & buhse) iljin 10.000 0.276 1.061 0.053 0.036 0.044 29.68% a. rechingeri (zohary) i. riedl 17.000 0.355 1.145 0.234 0.288 0.211 49.13% a. arbelensis boiss. & hausskn., 10.000 0.301 1.009 0.211 0.154 0.177 43.23% a. koelzii i. riedl, 7.000 0.677 1.087 0.093 0.057 0.099 23.66% a. hyrcana (grossh.) grossh. 10.000 0.699 1.156 0.143 0.094 0.205 27.96% a. peduncularis boiss. & hausskn. 4.000 0.376 1.054 0.055 0.035 0.021 21.83% a. glabrata alef. 5.000 0.452 1.264 0.235 0.039 0.044 24.90% a. tabrisiana (boiss. & buhse) iljin 5.000 0.269 1.021 0.023 0.011 0.023 22.15% a. persarum bornm. 8.000 0.548 1.013 0.029 0.012 0.019 19.68% figure 2. pca plots of morphological characters revealing species delimitation in alcea species 83delimiting species using dna and morphological variation in some alcea (malvaceae) species based on srap markers the studied species. however, reticulogram obtained based on the least square method (figure not included), revealed some amount of shared alleles between species 2 and 1,3,5 and between 9 and 1,3-5 also between 3 and 1, 2, 9-10. as evidenced by structure plot based on admixture model, these shared alleles comprise very limited part of the genomes in species studied and all these results are in agreement in showing high degree of genetic stratification in species studied 4. discussion 4.1. species identification and taxonomic consideration species delimitation is important in different biological disciplines, like ecology, biogeography, and plant conservation (mayr 1982). species delimitation is done by tree-based and non-tree-based approaches (wiens 2007). in the first method, species form distinguishing clades (phylogenetic species concept), whereas in nontree-based method, the species are recognized on the basis of gene flow assessments (biological species concept; pérez-losada et al. 2005). wiens & penkrot (2002), proposed to use dna data, morphological data and character data for species delimitation while, knowles & carstens (2007) addressed how molecular data (i.e., gene trees from dna sequence data) can be used in species delimitation. the latter authors used coalescent simulations to test the species limits and incorporated data from multiple loci. they showed the importance of population genetics in species delimitation. similarly, medrano et al. (2014), applied population genetics methods to the species delimitation problem in narcissus linnaeus (1753: 289) (amaryllidaceae j.st.-hil. nom. cons.) by the help of amplified fragment length polymorphism (aflp) molecular markers. in the present study we used morphological and molecular (srap) data to evaluate species relationship in alcea. morphological analyses of the studied alcea species showed that they are well differentiated from each other both in quantitative measures (the anova test result) and qualitative characters (the pca plot result). in addition, pca analysis suggests that characters like bract length, stipule length, bract shape, calyx shape, petal shape, length and width of stem-leaf, length and width of petal, peduncle and pedicel hair, mericarp hair density, mericarp surface could be used in species groups delimitation. this morphological difference was due to quantitative and qualitative characters. 4.2. genetic structure and gene flow this is the first study on the use of srap markers for genetic diversity, species delimitation and determining genetic relationships among alcea species in iraq. alarcón, et al., (2012) showed that srap technique along with proper statistical tools could be successfully applied to assess the genetic diversity and phylogenetic analysis among alcea species in iraq. our results clearly demonstrated that srap markers can be used in genetic diversity study as well as genetic identification of alcea. moreover, our results indicate a very high efficiency of the srap markers in the identification and delimitation of alcea species. similar efficiency of the srap markers has also been reported by other authors (alarcón, et al., 2012; li, et al, 2021; sun, et al. 2021; xu, et al, 2021; zhang, et al. 2022). amova and structure analysis revealed that the species of alcea are genetically differentiated but have some degree of shared common alleles. several trends in pollination mechanism can be observed in alcea with gradual transition between them. based on rapd markers analysis, kazemi et al. (2011) showed figure 3. structure plot of alcea species based on srap data. 84 chnar hama noori meerza et al. 93% polymorphism level with high variation in genetic similarity (0.31 to 0.75) within a. rosea populations in iran. öztürk et al. (2009) analyzed genetic profile of 18 alcea species using rapd markers and reported wide differentiation (0.13 to 0.69) among them. according to badrkhani et al (2014) sequence-related amplified polymorphism (srap) marker was employed to assess the genetic diversity and genetic similarity relationships among14 species of alcea collected from northwest of iran. seventeen srap primer combinations generated 104 fragments, of which 97 (93%) were polymorphic, with an average of 5.7 polymorphic fragments per primer. percentage of polymorphism ranged from 50% (me2-em6) to a maximum of 100%, and mean polymorphism information content value obtained was 0.3. the lowest genetic similarity (0.17) was observed between a. sophiae and a. flavovirens, while the highest was found between a. digitata and a. longipedicellata (0.68). two main clusters were detected using upgma, which did not correspond to geographical origin of the species. their study indicates that srap markers could be good candidates for assessing genetic variation in alcea. iranian alcea species have only been characterized with morphological data, so far. however, the genus has a complicated taxonomy due to small number of characters. based on study of pakravan (2008) on alcea, only examination of the leaf sequence and configuration of the carpels would represent valuable characters. for example, a. fl avovirens and a. glabrata differ only in the size of the carpel and width of wing (pakravan 2008). our results grouped these two species into two different clusters. the methods we used are indirect estimation of gene flow and if it is identified to occur among species may be either due to ancestral shared alleles or ongoing gene flow. the nm value obtained based on srap data, revealed very limited amount of gene flow among the studied species that was also supported by structure analysis as alcea species mostly had distinct genetic structure. reticulation analysis also showed some degree of gene flow for srap. we did not observe any intermediate forms in our extensive plant collection, but morphological variability within each species did occur to some extent. to conclude, the present study revealed the use of srap molecular markers along with morphological characters in alcea species identification. some degrees of interspecific genetic admixture occur in alcea species, but the studied species are strongly differentiated during the speciation process and invasion in new habitats. genetic drift, strong inbreeding and local adaptation are effective evolutionary forces operating in alcea species and population divergence and adaptation. plant species identification is of central importance in phylogenetic systematics, evolution, biogeography and biodiversity. it is significant to infer patterns and mechanisms of speciation and hybridization, the evolutionary process by which new biological species arise and gene flow between closely related phylogenetic species can occur (al-quran 2008; bi, et al., 2021; duan, et al., 2022; guo, et al, 2021; guo, et al, 2022). isolation by distance, local adaptation and gene flow are different mechanisms responsible for species differentiation and genetic diversity (freeland et al. 2011, frichot et al. 2013). references alarcón, m., vargas, p., sáez, l., molero, j., aldasoro, j.j., 2012. genetic diversity of mountain plants: two migration episodes of mediterranean erodium (geraniaceae) molecular phylogenetics and evolution 63, 866–876 al-quran s. 2008. taxonomical and pharmacological survey of therapeutic plants in jordan. journal of natural products, l (1):10-26. azizov u.m., mirakilova d.b., umarova n.t., salikhov s.a., rakhimov d.a. and mezhlumyan l.g.(2007). chemical composition of dry extracts from alcea rosea. chemistry of natural compounds 43:508-511. alefeld, f.g.c. 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journal of cytology, cytosystematics and cytogenetics volume 75, issue 3 2022 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 75(4): 67-76, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1674 caryologia international journal of cytology, cytosystematics and cytogenetics citation: saeedeh sadat mirzadeh vaghefi, adel jalili (2022). chromosome counts of some species of wetland plants from northwest iran. caryologia 75(4): 67-76. doi: 10.36253/caryologia-1674 received: may 29, 2022 accepted: december 06, 2022 published: april 28, 2023 copyright: © 2022 saeedeh sadat mirzadeh vaghefi, adel jalili. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. chromosome counts of some species of wetland plants from northwest iran saeedeh sadat mirzadeh vaghefi*, adel jalili research institute of forests and rangelands, agricultural research, education and extension organization (areeo), p. o. box 13185-116, tehran, iran *corresponding author. e-mail: myalyssum94@gmail.com abstract. wetlands scatter as microclimates in mountain areas of iran. in this investigation, the chromosome number of species and populations from azerbaijan provinces were studied. after seed germination and root fixation, meristem cells were stained and photographs were taken for cells in the metaphase stage by light microscope. data were analyzed by micromeasure and excel softwares. as many as 28 populations of 24 species were studied. the chromosome numbers of two species (viz. ranunculus kotschyi) were reported for the first time. 11 populations in 9 species were reported for the first time from iranian populations (viz. alisma plantago-aquatica, prunella vulgaris, scrophularia umbrosa). the range of haploid chromosome numbers is between n=6 and n=21. ideograms were depicted for each species. keywords: chromosome number, wetland, ideogram, azerbaijan. introduction wetlands are considerate as microclimates in alpine areas with special species in iran. in the steppic mountain areas of iran are a wide range of wetland vegetation. the wetland habitats are sharply embedded within vegetation of the iranoturanian steppes that are more characteristic of this region and are of interest both in themselves and for wider comparison with eurosiberian wetlands (naqinezhad, 2012). most of the wetlands in the northwest part of iran develop when snowmelt and rain fill the pockmarks left on the landscape by glaciers. this kind of wetlands was categorized in prairie potholes class (epa 2001). in angiosperms, the haploid chromosome number varies between n = 2 and n = 132, but the majority of them show a range between n = 7 and n = 12(sharma, 2009). variation or constancy in the chromosome number within taxa of different categories has been proven to be important characters for taxonomic groupings (sharma, 2009). we studied on chromosome number of 28 accessions of 24 species belonging to 21 genera of 12 families wetland plants of azerbaijan mountains of iran. the aim of this study was to investigate on chromosome number of wetland plants of azerbaijan provinces. 68 saeedeh sadat mirzadeh vaghefi, adel jalili materials and methods plant materials specimens and seeds were collected from natural habitats of azerbaijan provinces (west, east azerbaijan and ardebil) in 2013 and 2014. vouchers are deposited in tari (table 1). the plant samples were identified by flora of iran (assadi et al., 2018) and flora iranica (rechinger, 2005). chromosome counts the seeds were used for chromosome counts. seeds were germinated at 20-25°c on moist filter paper in petri dishes. root tips of seeds had grown 1–1.5 cm in length were stopped from the germinating by pretreating with alpha-bromonaphthalene for two hours. then fixed with acetic alcohol (1:3) for 4 h and stored in 70% alcohol at 2–4°c. then the root tips were rinsed in water and hydrolyzed with 1 n hcl for 10–18 min at 60°c and rinsed in running water for a minimum of 3–5 min. staining of root tips was carried out for 1–2 h and root tips were put in hematoxylin for 5–10 min to improve staining. finally, squash preparations were made. chromosome nomenclature follows levan et al. (1964) and stebbins (1971). the photos were taken by light microscope (bh2 olympus × 1000). we measured the chromosomes using micromeasure (version 3.3 (reeves and tear, 2000). results our goal in this project was to create a chromosome index for the wetland plants of azerbaijan. it is so difficult to discussion about several species from several families that collected for common goal. table 1. list of species examined, family, locality, altitude, voucher number, and habitat. voucher no. alt. locality family species 102715 2392 east azerbaijan, tabriz, arshad chamani alismataceae alisma plantagoaquatica l. 102769 2281 west azerbaijan, salmas, jam valley poaceae alopecurus arundinaceus poir. 102752 1900 aligo village brasicaceae barbarea plantaginea dc. 8228 ---east azerbaijan, bostanabad poaceae catabrosa aquatica (l.) p.beauv. 102750 2266 ardebil province, salmas, jam valley asteraceae erigeron acris l. subsp. pycnotrichus (vierh.) rech. f. 101699 2266 west azerbaijan, dalamper village geraniaceae geranium sylvaticum l. 102751 2266 west azerbaijan, dalamper village hypericaceae hypericum perforatum l. 102807 2266 west azerbaijan, dalamper village asteraceae inula aucheriana dc. 102762 1688 east azerbaijan, hashtroud, zalbil asteraceae mulgedium tataricum (l.) dc. 8239 1819 east azerbaijan, hashtroud, baboneh village poaceae phragmites australis trin. ex steud. 101691 2281 west azerbaijan, salmas, jam valley plantaginaceae plantago atrata hoppe. 101601 2266 west azerbaijan, dalamper village lamiaceae prunella vulgaris l. (dalamper) 8242 ---east azerbaijan, bostanabad lamiaceae prunella vulgaris l. (bostanabad) 101668 2296 ardebil, agh ghabali rannunculaceae ranunculus aquatilis var. diffusus with. 101670 east azerbaijan, varzaghan rannunculaceae ranunculus dolusus fisch . & c. a. mey. 101674 1648 ardebil province, ardebil to bolaghvar rannunculaceae ranunculus kotschyi boiss. 102738 1900 east azerbaijan, bostanabad scrophulariaceae scrophularia umbrosa dumort. 102805 1688 east azerbaijan, hashtroud, zalbil asteraceae senecio pseudoorientalis schischk. (hashtroud) 102761 1648 ardebil province, ardebil to bolaghvar asteraceae senecio pseudoorientalis schischk. )bolaghvar) 101688 2266 west azerbaijan, dalamper village fabaceae trifolium pratense l. 102724 2281 west azerbaijan, salmas, jam valley juncaginaceae triglochin maritima l. (jam valley( 102728 1650 ardebil province, ardebil to bolaghvar juncaginaceae triglochin maritima l. (bolaghvar) 102753 1900 east azerbaijan, bostanabad asteraceae tripleurospermum disciforme sch.bip. (bostanabad) 102784 1900 east azerbaijan, tabriz, arshad chamani asteraceae tripleurospermum disciforme sch.bip. (jam valley) 102713 2392 east azerbaijan, tabriz, arshad chamani scrophulariaceae veronica orientalis miller (arshad chamani) 102716 2281 east azerbaijan, bostanabad scrophulariaceae veronica orientalis miller (bostanabad) 102729 2281 east azerbaijan, bostanabad scrophulariaceae veronica filiformis sm. 101677 1650 ardebil province, ardebil to bolaghvar fabaceae vicia variabilis freyn & sint. ex freyn 69chromosome counts of some species of wetland plants from northwest iran of 24 examined species (table 2), 5 species have two populations (prunella vulgaris, tripleurospermum disciforme, pedicularis sibthorpii, senecio pseudoorientalis and veronica orientalis). another species has just one population. asteraceae (5 species), scrophulariaceae (3 species), poaceae and ranunculaceae (3 species) families have the most species respectively. the chromosomes counts of the taxa previously reported are in table 2. most of them confirm our results. chromosome numbers of ranunculus kotschyi (fig. 2p) and erigeron acris subsp. pycnotrichus (fig. 1f) were reported for the first time. alisma plantago-aquatica (fig. 1a), prunella vulgaris (fig. 2m & n), scrophularia umbrosa (fig. 2q), mulgedium tataricum (fig. 1j), ranunculus aquatilis var. diffusus (fig.1d), erigeron acris subsp. pycnotrichus (fig.1f), geranium sylvaticum (fig.1g), triglochin maritima (fig.3u&v), veronica filiformis (fig. 3y) were reported for the first time of iranian populations. sporophytic count of barbarea plantaginea (fig. 1c) was reported for the first time from iran. the details of each taxon reported as in table 3. table 2. chromosome counts of studied species previously described in the literature. references (viz.) 2n references n species (dobes et al., 1997)(wulff, 1939)(love and love., 1942) 10, 12, 14 (kaur et al., 2011) 14 alisma plantagoaquatica (kuzmanov, 1993)(amosova et al., 2019)(sheidai et al., 2009) 28, 42 (koull and gohil 1991) 14 alopecurus arundinaceus (astanova, 1999)(ørgaard and linde-laursen, 2014) 16 (aryavand, 1977) (ghaffari, 2007) 8 barbarea plantaginea (sawicka, 1991)(lövkvist and hultgård., 1999)(sheidai et al., 2009) 20 catabrosa aquatica. (kaur et al., 2011)(bala and gupta, 2013)(paule et al., 2017) 18 erigeron acris (other subspecies) (petrova and stanimirova, 2001) (lövkvist and hultgård 1999)(dmitrieva, 1986) 28, 24 (clifford odets, 1954) 12 geranium sylvaticum (ciccarelli et. al. 2001)(lövkvist and hultgård, 1999)(krasnikov and schaulo 1990)(kalinka et al., 2014((baltisberger and widmer, 2009((brutovska et al., 2000) 32 (ghaffari, 2006) 16 hypericum perforatum (chehregani and hajisadeghian, 2009) 9 inula aucheriana (probatova, 2004) 36 mulgedium tataricum (lövkvist and hultgård 1999)(panahi., 1979)(gervais et al., 1993) 42,48, 72,96 phragmites australis (petrova and stanimirova, 2001)(lessani and chariatpanahi., 1979) 12, 24 plantago atrata (lövkvist and hultgård 1999((krasnikov and schaulo., 1990) 28 prunella vulgaris (dahlgren and cronberg, 1996) 32,48 ranunculus aquatilis var. diffusus (baltisberger, 1991) (agapova, 1981)(assadi, 1989) (ghasemi et al., 2015) 28, 32 ranunculus dolusus not reported ranunculus kotschyi (javurkova, 1979)(grau, 1979)(vitek et al. 1992) 26,52 (grau, 1979) 13, 26 scrophularia umbrosa (ghaffari, 1999) 20 senecio pseudoorientalis (probatova, 2000) (krasnikov and schaulo, 1990)(zhang et al., 1993)(sheidai et al., 1998) 14, 16 trifolium pratense (lövkvist and hultgård. 1999)(krasnikov, 1991)) rotreklova, 2004( (uchiyama, 1989)(iwatsubo et al., 1998) 48,120 triglochin maritima (ghaffari, 1999) (hayirlioglu-ayaz, 2011) 18 (ghaffari, 1999) (razaq, et al., 1994) 9 tripleurospermum disciforme (ghaffari, 1986) 32 (ghaffari, 1987) 32 veronica orientalis (pogan et al., 1990)(dzhus and dmitrieva, 2001)(albach et al., 2009) 14 (dobes and vitek, 2000) 7 veronica filiformis (hesamzadeh hejazi and rasuli, 2006)(hesamzadeh hajazi and ziaei nasab, 2009) 14 vicia variabilis 70 saeedeh sadat mirzadeh vaghefi, adel jalili figure 1. somatic metaphase chromosome, ideogram of each population is on the right side of the image: a: alisma plantagoaquatica (2n = 14); b: alopecurus arundinaceus (2n = 28); c: barbarea plantaginea (2n = 16); d: ranunculus aquatilis var. diffusus (2n = 32); e: catabrosa aquatica (2n = 20 ); f: erigeron acris subsp. pycnotrichus (2n = 18); g: geranium sylvaticum (2n = 28); h: hypericum perforatum (2n = 32); i: inula aucheriana (2n = 18); j: mulgedium tataricum (2n = 36). scale bar: 1 µm. 71chromosome counts of some species of wetland plants from northwest iran figure 2. somatic metaphase chromosome, ideogram of each population is on the right side of the image: k: phragmites australis (2n = 42); l: plantago atrata (2n = 12); m: prunella vulgaris (dalamper) (2n = 28); n: prunella vulgaris (bostanabad) (2n = 28); o: ranunculus dolusus (2n = 32); p: ranunculus kotschyi (2n = 16); q: scrophularia umbrosa (2n = 52); r: senecio pseudoorientalis (hashtroud) (2n = 40); s: senecio pseudoorientalis )bolaghvar) (2n = 40); t: trifolium pratense (2n = 16). scale bar: 1 µm. 72 saeedeh sadat mirzadeh vaghefi, adel jalili somatic metaphase and ideograms of taxa were shown (figs 1-3). discussion only some interesting results are discussed here. the chromosome number of the most of the species support by former studies (table 2). the variation ranges of haploid chromosome numbers in studied species are between n=6 and n=21. populations in triglochin maritima (fig. 3u & v) comprise different ploidy levels (2n = 24, 12). some species like prunella vulgaris (fig. 2m & n), veronica orifigure 3. somatic metaphase chromosome, ideogram of each population is on the right side of the image: u: triglochin maritima (jam valley) (2n = 24); v: triglochin maritima (bolaghvar) (2n = 12); w: tripleurospermum disciforme (bostanabad). (2n = 18); x: tripleurospermum disciforme (jam valley( (2n = 18); y: veronica filiformis (2n = 14); y1: veronica orientalis (arshad chamani) (2n = 32); y2: veronica orientalis (bostanabad) (2n = 32); z: vicia variabilis (2n = 14). scale bar: 1 µm. 73chromosome counts of some species of wetland plants from northwest iran entalis (fig. 3y1 & y2), senecio pseudoorientalis (fig. 2r & s), tripleurospermum disciforme (fig. 3w & x) have invariant ploidy level in this study. alisma plantago-aquatica n = 7 (fig. 1a), is not particularly variable; nevertheless three different chromosome numbers have been found for it viz. n = 5 (wulff, 1939), 6 ( liehr, 1916; love and love, 1942) and 7 ( love and love, 1942). combining the available chromosome studies, the erigeron has relatively consistent chromosome diversification with a basic number of 9, and most species contain diploid individuals, which suggested erigeron at the initial phase of polyploid diversification (baldwin and speese, 1955). phragmatis australis is represented by many polyploids, cuploids from 3x = 36 to 8x = 96 (without 5 x) and aneuploidies (2n = 42, 44, 46, 49, 50, 51, 52, 54). tetraploidy and octoploidy are in majority (gorenflot, 1979). chromosome numbers of phragmatis australis showed a high degree of aneuploidy and varied between 2n=42 and 2n=59 (gervais et al., 1993). the degree of polyploidy is not in direct relation with the individual’s habitus. meiosis study shows that this complex has already passed the maturity, the diploid forms having disappeared (gorenflot, 1979). base on this study our case is aneuploidy. hypericum perforatum has the smallest chromosomes than other species of this genus. in other studies it had 2n=32 with median centromeric chromosomes as our result (brutovska et al., 2000). most of the subgenera of veronica exhibit only one single basic number, i.e., x = 6, 7, 8, 9, 12, 17, or 20/21. table 3. somatic chromosome number (2n), ploidy level, karyotype formula, ranges of chromosome length and degree of asymmetry according to stebbins (1971) for the studied taxa, figure numbers are related images in fig 1 figure number (figs.1-3) stebbins chromosome range length (µm) karyotype formula ploidy level x 2n taxa a b1 5.3-7.83 2sm+5m 2 7 14 alisma plantagoaquatica b a2 4.1-5.95 8sm+6m 4 7 28 alopecurus arundinaceus c a1 1.06-1.29 2sm+6m 2 8 16 barbarea plantaginea e a1 2.25-3.09 10m 4 5 20 catabrosa aquatica f a1 3.44-5.10 3sm+6m 2 9 18 erigeron acris subsp. pycnotrichus g a1 1.51-2.19 2sm+12m 2 14 28 geranium sylvaticum h a1 0.92-1.37 16m 4 8 32 hypericum perforatum i a1 2.91-3.8 9m 2 9 18 inula aucheriana j a2 2.14-3.30 5sm+13m 4 9 36 mulgedium tataricum k a2 2.8-4.89 10sm+11m 4 an. 42 phragmites australis l a1 6m 2 6 12 plantago atrata m b1 0.95-2.28 14m 2 14 28 prunella vulgaris (dalamper) n a1 0.7-1.11 14m 2 14 28 prunella vulgaris (bostanabad) d a2 2.01-3.31 9sm+7m 4 8 32 ranunculus aquatilis var. diffusus o a2 1.89-2.87a 4sm+12m 4 8 32 ranunculus dolusus p a3 3.67-5.38 5sm+3st 2 8 16 ranunculus kotschyi q a1 1.51-3.15 4sm+22m 4 13 52 scrophularia umbrosa r a1 2.41-4.31 4sm+16m 4 10 40 senecio pseudoorientalis (hashtroud) s a2 2.6-3.66 2sm+18m 4 10 40 senecio pseudoorientalis )bolaghvar) t a1 3.41-4.80 2sm+6m 2 8 16 trifolium pratense u a1 1.88-2.46 3sm+9m 4 6 24 triglochin maritima (jam valley) v a3 4.23-5.16 3sm+3st 2 6 12 triglochin maritima (bolaghvar) w a1 2.14-2.7 9m 2 8 18 tripleurospermum disciforme (bostanabad). x b1 1.81-2.86 9m 2 9 18 tripleurospermum disciforme (jam valley( y1 a1 0.73-1.31 16m 4 8 32 veronica orientalis (arshad chamani) y2 a1 1.38-2.19 16m 4 8 32 veronica orientalis (bostanabad) y a1 0.28-0.48 1sm+6m 2 7 14 veronica filiformis z a3 1.66-2.37 3sm+4m 2 7 14 vicia variabilis an.= aneuploidy. 74 saeedeh sadat mirzadeh vaghefi, adel jalili in this genus, the putative ancestral base number of 9 has been reduced several times to 8 and 7, respectively (aneuploidy/dysploidy), often associated with transition to annual life history. in contrast, no unambiguous increase of chromosome base number has been inferred (albach et al., 2008). a base chromosome number reduction to x = 8 (aneuploidy), seems to have occurred in veronica orientalis (tetraploid, 2n=4x=32). previous results confirm our result (ghaffari, 1986). basic chromosome number(x) the frequency of 2x (53.57%) and 4x (46.43%) in this study are almost identical. 6x and more another nx not found. 2x and 4x are most common in flowering plants (bala and gupta, 2013). the present result is in agreement with the reports of earlier investigators. polyploidy and habit the overall chromosome numbers during present study lie on two different levels of ploidy i.e. 2x, 4x. among these, the diploids are the most common in terms of frequency (53.57 %), followed by tetraploids (46.43 %) (table 3). as reported at least 47% of species have undergone a recent polyploidy event (wood et al., 2009)polyploidy has been recognized as an important phenomenon in vascular plants, and several lines of evidence indicate that most, if not all, plant species ultimately have a polyploid ancestry. however, previous estimates of the frequency of polyploid speciation suggest that the formation and establishment of neopolyploid species is rare. by combining information from the botanical community’s vast cytogenetic and phylogenetic databases, we establish that 15% of angiosperm and 31% of fern speciation events are accompanied by ploidy increase. these frequency estimates are higher by a factor of four than earlier estimates and lead to a standing incidence of polyploid species within genera of 35% (n = 1,506. as ramsey and ramsey,(2014) indicated that; polyploids are able to colonize larger geographic ranges and/or occur in more habitats than related diploids, similar picture occurred in our study: those species with higher ploidy level have wide spread geographical distribution than their related diploid populations or species. for example in iran, some species such as; alopecurus arundinaceus, phragmites australis, catabrosa aquatica, hypericum perforatum, veronica orientalis with higher polyploidy level showed same attitudes (table 3) (alinejad et al., 2017; ghahremaninejad et al.; 2012, khanhasani et al., 2021; safikhani et al., 2018; aref tabad et al., 2016; jalili et al., 2014) karyotype metacentric and submetacentric are commonly observed. karyotype of ten species (35.7%) consists of chromosomes with the centromere in median regions (m). 14 species (50%) have metacentric and submetacentric chromosomes in their formula. only 2 species have  karyotypes with submetacentric and  subtelocentric chromosomes. karyotypes of most species (78.58%) were classified in the 1a and 2a stebbins classes (table 3). acknowledgment this paper was supported by a research project of research institute of forests and rangelands of iran; project title: “study and survey of chromosome numbers and ploidy levels in wetland plants in 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(2n=22) is an herbaceous species native to the mediterranean region and naturalized in many temperate areas around the world. it includes subsp. piperitum and subsp. vulgare which are, respectively, the wild and cultivated forms. fennel is of economic importance both as a vegetable crop and for its wide use in the food and pharmaceutical industries. in recent years, the therapeutic and pharmacological potential of this species has been widely analyzed, its cytogenetic traits have aroused less interest. therefore, the intention of this study was to reduce this gap by investigating some aspects, such as the variations in its chromosome number and the occurrence of polyploidization events, so far neglected. by means of extensive chromosome counting, the presence of tetraploid cytotypes has been discovered both in the wild and cultivated fennel. moreover, the analysis of pollen and pmcs at the tetrad stage provided evidence for spontaneous sexual polyploidization as the most probable origin of the tetraploid cytotypes discovered. the results of this study provide the first evidence of the occurrence of polyploidization events in f. vulgare and suggest that the use of 2n gametes could be a useful approach to genetic improvement of this crop. keywords: foeniculum vulgare mill., fennel, polyploid cytotypes, polyploidization, unreduced gametes. introduction foeniculum vulgare mill., of the apiaceae (syn. umbelliferae) family, is a biennial-perennial herb native to the southern mediterranean region from where it was brought for cultivation throughout the temperate regions of asia, north america and europe. fennel is described as a diploid species (2n=22) characterized by erect, cylindrical, bright green and smooth stems growing up to 2 m in height. its leaves are finely dissected with terminal filiform segments. its bright yellow flowers are contained in terminal compound umbels with a variable number of rays (subramanian 1986; badgujar 2014). two subspecies of f. vulgare are generally recognized: subsp. piperitum and subsp. vulgare which represent the wild and cultivated forms, respec86 egizia falistocco tively. the wild fennel (subsp. piperitum) is abundantly present in the mediterranean flora and commonly found in limey soil near the sea and on river banks. subsp. vulgare includes var. azoricum which is cultivated as a vegetable for its enlarged leaf base and var. dulce which is grown prevalently for its seeds containing essential oil (pignatti 1982). f. vulgare is commonly called fennel and it is denoted locally by more than 100 vernacular names. it is a traditional and popular species with a long history having been appreciated and utilized as a medicinal, aromatic and edible plant at least since roman times up to the present day. in some countries, for example, fennel is currently used to stimulate lactation and to improve the taste of some medicines, while the seeds are widely used as a spice (simon et al. 1984; sheidai et al. 2007; faudale et al. 2008; badgujar et al. 2014; rather et al. 2012). f. vulgare is at present a species of considerable economic interest not only for its wide diffusion as a vegetable crop but also for its pharmaceutical properties which, in recent years, have been re-evaluated and extensively investigated. the species possesses antioxidant, antispasmodic, carminative, diuretic and laxative properties and is useful in the treatment of numerous infectious disorders of bacterial, fungal, viral and protozoal origin (agarwal et al. 2017). fennel is also widely used in the food industry as a flavor additive to meats, salad dressing, breads, pastries, teas and alcoholic beverages (badgujar et al. 2014). despite its economic importance and wide diffusion in the wild, only limited attention has been dedicated to it by cytologists mainly interested in its karyological aspects (garde and garde 1949; das and mallick 1988; lentini et al. 1988; paul and datta 2003¸ subramanian 1986; jovine et al. 2008; ozkan et al. 2017). most of these studies were carried out on only few samples. extensive cytogenetic investigations have been to date very limited. the purpose of this study was to expand the study of f. vulgare and explore areas of cytogenetics so far overlooked, such as the existence of chromosome variants and events of polyploidization. to realize this, investigations on wild and cultivated populations of f. vulgare were carried out by attempting, as first step, an extensive chromosome count. then, because tetraploid cytotypes were identified during this survey, the successive objective was to clarify the contribution of 2n gametes to the origin of the polyploid plants discovered. for this purpose the size of pollen grains and the constitution of sporads were analyzed to verify the occurrence of unreduced gametes and to estimate the frequency of their formation. it has been widely demonstrated that pollen grains and pmcs (pollen mother cells) at the tetrad stage provide essential data on the tendency of plants to produce 2n male gametes and offer information on the origin of polyploids (orjeda et al., 1990; ramsey and schemske 1998; garcia et al. 2020). large pollen is normally 2n pollen because there is a positive correlation between dna content and cell volume, which in turn influences the size of the pollen grain. thus, 2n pollen grains are larger than reduced grains. on the other hand, microsporogenesis offers irrefutable indications of the occurrence of 2n gametes because the presence of dyads and triads in the sporad stage constitutes strong evidence for the formation of unreduced pollen (garcia et al. 2020). this study is the first to document the occurrence of polyploidization events in f. vulgare. it also provides data supporting sexual polyploidization as the possible origin of the polyploid cytotypes discovered. materials and methods plant material seeds and plants of f. vulgare subsp. piperitum and subsp. vulgare were used for this study. seeds of wild fennel (subsp. piperitum) were collected by the author in two different localities of italy: the countryside surrounding the trasimeno lake in central italy (population1), and the north-east coast of sardinia (population 2). seeds of the cultivated varieties azoricum and dulce (subsp. vulgare ) were provided by specialized nurseries (table 1). the seeds were germinated at 20-22 °c in petri dishes on filter paper moistened with distilled water. a part of the seedlings was transplanted to obtain plants for the production of flowers and roots. a total of 400 seeds and 10 plants for each accession were checked for their chromosome numbers. the same plants were also used for the analysis of microsporogenesis and pollen size. chromosome counts roots 5-10 mm long were collected from seedlings and adult plants and immersed in ice-cold water for about 16 h to accumulate metaphases. then they were pre-treated in a 1‰ aqueous solution of a stock solution consisting of 1 ml of a-bromonaphthalene dissolved in 100 ml of absolute ethanol (linde-laursen 1978) for 3 h, fixed in ethanol-glacial acetic acid (3:1) at room temperature overnight and then stored at -20 °c until required. mitotic chromosome preparations were realized according to the protocol described in detail in falistocco (2018). 87polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel the excised roots were washed in distilled water for 10 min and transferred to the enzyme buffer (10mm citric acid/sodium citrate, ph 4.6) for 20 min. root tips were then excised and digested in the enzyme solution (4% cellulase onozuka r10 and 1% pectolyase sigmaaldrich in distilled water) for 45-60 min at 37°c. the cell suspension was pelleted and resuspended in enzyme buffer. after pelleting, the material was washed twice with the fixative and resuspended in the fixative. finally, 20-30 ml of cell suspension was applied to a slide. the slides were air-dried and stained with 2mg/ml dapi (4’, 6-diamidino-2-phenylindole) for the determination of the chromosome numbers. detection of unreduced male gametes pollen analysis pollen from five flowers per plant was spread over five slides and stained with a solution of acetocarmine and glycerol (1:1). the dark colored and regular shaped pollen grains were considered as viable. the pollen size was determined by measuring the major diameter of the grains by using an ocular micrometer. measurements revealed two types of pollen grains which can be considered normal pollen and large pollen. the size of the normal pollen was determined by measuring the grains present in three light vision fields of the microscope for each slide. about 600 grains for each plant were measured. all large pollen grains present in the slides were measured. analysis of pmcs at the tetrad stage inflorescences were collected and immersed in the fixative ethanol-glacial acetic acid (3:1) for 24 hours and then they were transferred to 70% ethanol and stored at 4°c until analysis. for cytological preparations anthers of a single flower were squashed on a glass slide with some drops of 0.5% acetocarmine (merk life science, italy), intensified by ferric oxide. in order to select only anthers containing pmcs at the tetrad stage, preliminary observations were made to assess the meiotic stage of the flowers. one single anther was removed from the floral bud, squashed on a slide as described above and examined. when the anther contained sporads the other anthers of the same flower were used for cytological preparations. five flowers per plant were analyzed. the number of dyads, triads and tetrads detected in four light vision fields for each slide were counted. about 1500 sporads for each plant were examined. estimation of the theoretical frequency of 2n pollen grains was made from the number of observed dyads, triads, and tetrads at the end of microsporogenesis. considering that a dyad evolves into two unreduced pollen grains, a triad produces one unreduced pollen grain and two reduced pollen grains, and each tetrad gives origin to four reduced pollen grains, the frequency of 2n pollen grains was calculated by applying the equation f2n (%) = (2xdy + tr) / (2xdy+ 3x tr + 4x te) x 100, for which dy, tr and te, are the number of dyads, triads and tetrads, respectively (kumar and singhal 2012). chromosome preparations and slides containing pollen and sporads were observed under uv and light illumination, respectively, with a microphot nikon microscope. images were recorded with a digital photocamera sony icx282aq and then processed using adobe photoshop 5.0. results chromosome counts all plants examined resulted diploid having the chromosome number 2n=22 (figure 1a), but polyploid seedlings were detected in each accession of subspp. piperitum and vulgare (figure 1b). three seeds from population 1 (central italy) and four seeds from population 2 (sardinia) were found to be tetraploid with 2n=2x=44 (figure 1b). the frequency of tetraploids discovered in cultivated fennel was greater: five in the cv. azoricum and seven in the cv. dulce (table 1). the tetraploid condition was always clearly distinguishable in all metaphases analyzed. no other ploidy levels were detected. estimation of the occurrence of unreduced male gametes to detect the formation of unreduced male gametes pollen size was measured and the constitution of the sporads analyzed. full pollen viability was generally observed, with very sporadic cases of shriveled and unstained grains present. table 1. list of accessions of f. vulgare subspp. piperitum and vulgare examined and number of tetraploid (2n=44) plants discovered. subspecies accessions origin n. of tetraploid (2n=44) plants piperitum population 1 central italy 3 population 2 sardinia 4 vulgare cv. azoricum commercial source 5 cv. dulce commercial source 7 88 egizia falistocco most of plants examined produced pollen of uniform size, but few plants produced also noticeably larger grains (figure 2a). diameter measurements confirmed the results of visual observation. according to their size, pollen grains were categorized as n, that is normal reduced pollen, with its diameter ranging from 33.0 to 35.0 μm; and 2n, unreduced pollen, measuring from 42.0 to 44.0 μm. large pollen was identified as unreduced 2n pollen according to darlington (1937) who defines as 2n pollen the grains with a size 1,25x larger than the average size of normal pollen. pollen grains of intermediate size were not observed. large pollen grains were detected in two plants of population 1, three plants of population 2, one plant of cv. azoricum and three plants of cv. dulce (table 2). the sporad constitution was analyzed to confirm that large pollen grains effectively represent 2n pollen production. this analysis revealed, in addition to the expected normal tetrads, the presence of dyads in all plants producing large pollen grains (figure 2b,c). the frequency of dyads produced was 2.26 and 4.0% in plants of population 1; 2.44, 3.0 and 4.0% in plants of population 2; 5.0% in the plant of var. azoricum; and 5.00, 7.00 and 8.00% in plants of var. dulce (table 2). dyads or triads were not observed in the remaining plants examined. additionally, abnormal tetrads (that is containing microspores of different sizes, collapsed or with micronuclei) or polyads were not found in any of the plants object of this analysis. by the rule that each tetrad can form four n microfigure 1. mitotic metaphases of f. vulgare. chromosome complements of diploid (a) and tetraploid (b) cytotypes. the bar represents 5μm. figure 2. example of pollen grains and pmcs at the sporad stage observed in plants producing large pollen. pollen sample showing normal and large pollen grains (a); group of tetrads with four n (reduced) microspores (b); dyad containing two 2n (unreduced) microspores (c). the bar represents 10 μm. 89polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel spores and each dyad can form two 2n microspores, the frequency of 2n pollen grains could be estimated for each plant according to the abovementioned formula. the results are reported in table 2. discussion the discovery of tetraploid cytotypes deriving from this study provided the first evidence of polyploidy in f. vulgare. furthermore, the pollen and sporad analyzed indicate spontaneous sexual polyploidization as the most probable origin of such cytotypes. various methods exist to reveal the formation of 2n pollen and one of these is based on the size of the pollen grains. due to the relatively close correlation between large pollen and the 2n status, the presence of large pollen has been frequently used as an indicator for 2n pollen (ghaffari 2006; kumar and singhal 2012). the analysis of pollen size carried out during this study revealed two types of pollen grains which according to the criterion of darlington (1937) were classified as n (normal reduced) and 2n (unreduced) pollen. further evidence that large pollen effectively indicates unreduced pollen formation was provided by the sporad analysis demonstrating that fennel plants producing dyads also produce large pollen grains. the large 2n pollen grains examined were well filled, stained, and apparently fertile; therefore, it is very possible that fertilization by these 2n gametes led to the formation of polyploid cytotypes. the tetraploid constitution of these cytotypes suggests that in fennel unreduced 2n gametes are generated also during the macrosporogenesis process. the formation of 2n pollen grains in f. vulgare has been previously observed in natural populations from iran (sheidai et al. 2007); but they have never been sought in cultivated fennel. the production of 2n gametes in plants is a common phenomenon which may result from a variety of different meiotic irregularities (dewitte et al. 2012). the microsporogenesis analysis carried out during this study was focused on meiocytes at the tetrad stage, so that the meiotic events occurring in the phases preceding the tetrad stage still remain unknown. therefore, no definitive conclusion on the meiotic aberrations responsible for the formation of 2n pollen is possible. the absence of abnormal sporads and the irrelevant number of non viable pollen grains observed would exclude the occurrence of meiotic abnormalities affecting chromosome segregation with the consequent formation of aneuploid gametes. rather, the regularity of microspores would suggest that the origin of unreduced pollen is principally due to the meiotic events connected to a process of nuclear restitution during the first or second division. another interesting point is the higher incidence of tetraploids detected in the cultivars with respect to wild populations. the greater tendency of cultivated plants to produce unreduced gametes could be connected to this phenomenon. however, it may be assumed that the tetraploid condition generates characteristics in plants favoring their selection during the breeding activities. clarification of this and the other hypotheses inherent in this study will come from further investigations. the fact that tetraploid plants were found in all accessions examined suggests that polyploidy in f. vulgare is a rather widespread phenomenon and not a sporadic event. the demonstrated characteristic of fennel plants to produce 2n gametes should be exploited to generate polyploid genotypes. the induction of polyploidy could be a useful method for improving specific traits in crop varieties, such as quality, yield and environmental adaptation. in fennel, this practice has been attempted by colchicine treatments but with scarce results (solanki et al. 2017). polyploid plants obtained by sexual polyploidization may turn out to be a promising approach to breeding programs of this species. funding this work was supported by fondazione cassa di risparmio di perugia (italy), project code: 2019.0317.029. 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(convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 67-73, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-170 citation: e. mitrenina, m. skaptsov, m. kutsev, a. kuznetsov, h. ikeda, a. erst (2020) a new diploid cytotype of agrimonia pilosa (rosaceae). caryologia 73(1): 67-73. doi: 10.13128/caryologia-170 received: february 19, 2019 accepted: february 23, 2020 published: may 8, 2020 copyright: © 2020 e. mitrenina, m. skaptsov, m. kutsev, a. kuznetsov, h. ikeda, a. erst. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* 1 laboratory of herbarium, national research tomsk state university, tomsk, russia 2 south-siberian botanical garden, altai state university, barnaul, russia 3 the university museum, the university of tokyo, tokyo, japan 4 laboratory of herbarium, central siberian botanical garden, siberian branch of the russian academy of sciences, novosibirsk, russia *corresponding author. e-mail: erst_andrew@yahoo.com abstract. a new diploid cytotype of agrimonia pilosa ledebour (rosaceae) collected in china has been revealed. karyotype formula is 2n = 2x = 16 = 14m + 2sm. previously, chromosome numbers in a. pilosa established by other researchers were 2n = 28; 56; 70 with basic chromosome number x = 7. all the other members of genus agrimonia linnaeus have the same basic chromosome number. in the meanwhile, some members of fam. rosaceae have different basic chromosome numbers: x = 8 (e.g., in genera amygdalus l., aphanes l., cerasus mill., etc.), x = 9 (e.g., in genera adenostoma hook. & arn., chamaebatia benth., etc.), x = 17 (e.g., in genera amelanchier medik., chaenomeles lindl., etc.). we suppose that the new basic chromosome number x = 8 was revealed in agrimonia pilosa collected in china. keywords. agrimonia pilosa ledebour, rosaceae, chromosomes, karyotype, new cytotype, flora of china. 1. introduction genus agrimonia linnaeus (fam. rosaceae, subfam. rosoideae) comprises 15 to 25 species and some naturally occurring hybrids distributed mainly in temperate regions throughout europe, asia, north america, central america, the west indies, southern south america, and the southern africa (li et al. 2003; chung 2008; kline and sørensen 2008; angelo and boufford 2012). the genus belongs to the tribe sanguisorbeae dc divided into two subtribes – agrimoniinae j. presl and sanguisorbeae torr. & a. gray. the first subtribe, along with genus agrimonia, includes genera aremonia neck. ex nestl., hagenia j. f. gmel., leucosidea eckl. & zeyh., spenceria trimen (potter et al. 2007). last four genera are monotypic endemics (chung 2008; chung et al. 2012). species of the subtribe display basic chromosome number x = 7 and different levels of ploidy (2x, 4x, 6x, 8x, 10x, 12x) correlating with geographic distribution patterns (chung 2008; rice et al. 2015). 68 elizaveta mitrenina et al. several species in agrimonia (e.g., a. pilosa, a. eupatoria) are used for medicinal purposes. they have been reported to possess antibacterial (muruzović et al. 2016), antiviral (kwon et al. 2005), antitumor (miyamoto et al. 1987; tang et al. 2017), diuretic (giachetti 1986) and antidiabetes properties (swanston-flatt et al. 1990; kuczmannová et al. 2016), antioxidant (chen and kang 2014; muruzović et al. 2016), immunomodulating (bukovsky and blanarik 1994), hepatoprotective (park et al., 2004) and other effects. in flora of china, the genus comprises following species: agrimonia coreana nakai, agrimonia eupatoria linnaeus subsp. asiatica (juzepczuk) skalický, agrimonia nipponica koidzumi var. occidentalis skalický ex j. e., agrimonia pilosa ledebour (agrimonia pilosa var. pilosa and agrimonia pilosa var. nepalensis (d. don) nakai) (li et al., 2003). in the current study, karyotype analysis of a. pilosa collected in china (figure 1) has been conducted. a new diploid cytotype 2n = 16 and a new probable basic chromosome number x = 8 for the genus agrimonia l. were revealed. combination of chromosome investigation with morphological methods, molecular genetics methods and scanning electron microscopy gives possibility to obtain essential data to reach conclusions on plants systematics and phylogeny. 2. materials and methods seeds of a. pilosa for cytological study and herbarium specimens were col lected in china, beijing, yun xiu gu forest park, rocky ledges (40°60’n; 117°41’e, 22 july 2016; collectors: erst a.s., erst t.v., lian l., bing l., shi c) and were collected in russia, west sibiria, tomsk, southern edge of the city, inundation meadow (56°47’n; 85°03’e, 1 sep. 2017; collector: mitrenina e. yu.). all herbarium materials are deposited in novosibirsk (ns). 2.1. karyotype analysis mitotic metaphase chromosomes in root tips of seedlings were studied. seeds were grown at petri dishes with wet sand at room temperature after cold stratification at 3–4° c during 4 months. newly formed roots about 1.0–1.5 cm long were pretreated in a 0.2% colchicine solution during 2 hours at room temperature. fixation was carried out in a mixture of absolute ethanol and glacial acetic acid (3 : 1). root tips were stained in 1% aceto-haematoxylin, and the squashing method was employed for investigating of karyotype (smirnov 1968). chromosomes were counted in 127 mitotic cells of 5 a. pilosa seedlings collected in china and in 25 mitotic cells of 5 a. pilosa seedlings collected in russia. mitotic metaphase chromosome plates were observed by microscope primo star (carl zeiss, germany) and photographed by microscope axioimager a.1 (carl zeiss, germany) with software axiovision 4.7 (carl zeiss, germany) and ccd-camera axiocam mrc5 (carl zeiss, germany) at 1000× magnification at laboratory for ecology, genetics and environmental protection (“ecogene”) of national research tomsk state university. for karyotyping, the software karyotype (altinordu et al. 2016) was used, and for figures, the software adobe photoshop cs5 (adobe systems, usa) and inkscape 0.92 (usa) was used. the measurements were performed on 10 metaphase plates. for analysis of karyotype, the nomenclature of levan, fredgam, and sandberg (1964) has been used. figure 1. agrimonia pilosa ledebour (beijing, china). 69a new diploid cytotype of agrimonia pilosa (rosaceae) 2.2. flow cytometry flow cytometry with propidium iodide (pi) staining was implemented to determine relative dna content. at least 10 seeds from each plant were taken for this study. each seed was analysed separately. seed buffer (matzk et al. 2001) was used for nuclei extraction. seeds was squashed by porcelain pestle and chopped with a sharp razor blade in the nuclei extraction buffer. the samples were filtered through 50-μm nylon membrane into a sample tube. flow cytometry with partec cyflow pa revealed the data on isolated nuclei fluorescence (partec, gmbh) using the laser 532 nm wavelength, while logarithmic fluorescence data representation (logarithmic scale) was used to record the signals. to calculate the mean of peak, at least 1000 nuclei peaks with less than 2.5% cv indicator values were used. the final data did not exceed the dna content of the mean sample by more than 3% (kubešová et al. 2010). as an external standard was used euryops chrysanthemoides (dc.) b. nord, 2c = 2.70 pg, and internal standard glycine max ‘polanka’, 2c = 2.50 pg (doležel et al. 1994; skaptsov et al. 2016). we used the statistica 8.0 software (statsoft inc.), flowing software 2.5.1 (turku centre for biotechnology) and cyview software for the flow cytometer data analysis (partec, gmbh), and for the analysis of our results. the possible effect of secondary metabolites on the binding of the intercalating dye was evaluated by cogrinding in a nuclei extraction buffer of the samples and allium fistulosum l. leaves. the resulting preparation was investigated three times within 10 minutes. in the absence of variations in the average values of the detection channels of the a. fistulosum peak, it was believed that no effect was detected. flow cytometry performed at the laboratory of bioengineering of south-siberian botanical garden, altai state university. 3. results karyotype analysis of a. pilosa collected in china, beijing has been conducted. all 127 investigated cells in 5 seedlings had diploid chromosome number 2n = 16 (figure 2). by the software karyotype (altinordu et al. 2016) morphometric chromosome analysis (total chromosome length, short and long chromosome arms length, arm ratio) has been conducted (table 1). chromosome length ranged from 1.87 ± 0.17 µm to 2.14 ± 0.18 µm. arm ratio varied from 1.03 to 1.81. chromosomes were classified into two groups: seven pairs with median centromeric position (metacentric chromofigure 2. mitotic metaphase chromosomes of agrimonia pilosa ledebour (beijing, china), 2n = 16. scale bar = 10 µm. table 1. karyotype parameters of agrimonia pilosa ledebour, china (2n = 16). chromosome pair total length, µm, ± sd long arm, µm, ± sd short arm, µm, ± sd arms ratio (long/ short) chromosome type 1 2.14 ± 0.18 1.29 ± 0.12 0.85 ± 0.07 1.52 m 2 2.10 ± 0.24 1.10 ± 0.11 1.00 ± 0.13 1.10 m 3 2.04 ± 0.23 1.12 ± 0.13 0.92 ± 0.12 1.22 m 4 2.02 ± 0.24 1.15 ± 0.26 0.87 ± 0.11 1.32 m 5 1.95 ± 0.29 1.06 ± 0.17 0.89 ± 0.13 1.19 m 6 1.88 ± 0.17 1.05 ± 0.22 0.83 ± 0.08 1.27 m 7 1.87 ± 0.17 0.95 ± 0.08 0.92 ± 0.09 1.03 m 8 2.11 ± 0.27 1.36 ± 0.17 0.75 ± 0.10 1.81 sm notes: m – metacentric chromosome; sm – submetacentric chromosome; ± sd – mean length ± standard deviation. 70 elizaveta mitrenina et al. somes, m; arm ratio 1–1.7), and one pair with sub-median centromeric position (submetacentric chromosomes, sm; arm ratio 1.7–3.0). some metacentric pairs were lowdifferentiated. they had almost equal length and arm ratio. karyotype formula is 2n = 2x = 16 = 14m + 2sm (figure 3). karyotype asymmetry degree (stebbins 1971): 1a. secondary constrictions in 1–2 metaphase chromosome pairs were revealed. nucleolus number observed in mitotic interphase were 1–2 per cell. chromosome counting in a. pilosa collected in russia, tomsk has revealed typical octaploidic cytotype for the species: 2n = 8x = 56. by the flow cytometry method, we have found out relative dna content in two agrimonies: 2c = 2.51 pg in a. pilosa (china) with 2n = 16, and 2c = 4.96 pg in a. pilosa (russia) with 2n = 56 (figure 4). 4. discussion according to the data of chromosome counts database (rice et al. 2015), index to plant chromosome numbers and other learned treatise (iwatsubo et al. 1993; chung 2008; angelo and boufford 2012; kumar et al. 2014), diploid chromosome numbers in agrimonia are known as 28; 42; 56; 70 и 84 (table 2). the basic chromosome number in the genus x = 7. currently, within agrimonia only polyploids have been reported. as long as the lowest ploidy levels reported among species of agrimonia are tetraploids, the lineage appears to have an ancient origin where diploids have gone extinct (chung 2008). such a basic chromosome number is common for many members of fam. rosaceae (e.g., genera geum l., potentilla l., rosa l., etc.). at the same time, there are other basic chromosome numbers in rosaceae: х = 8 (e.g., in genera amygdalus l., aphanes l., cerasus mill., exochorda lindl, padus mill., prunus l.), x = 9 (e.g., in genera adenostoma hook. & arn., chamaebatia benth., holodiscus maxim.), x = 17 (e.g., in genera amelanchier medik., chaenomeles lindl., kageneckia ruiz & pav.) (rice et al. 2015). conventionally, subfamily classification was based on a combination of basic chromosome numbers and fruit types (chung et al. 2012). other genera belonging to subtribe agrimoniinae, figure 3. idiogram of agrimonia pilosa ledebour (beijing, china), 2n = 16. m – metacentric chromosome, sm – submetacentric chromosome. figure 4. flow cytometry histograms: a – agrimonia pilosa (tomsk, russia); b – agrimonia pilosa (beijing, china); c – agrimonia pilosa (tomsk, russia), samp. 1 with internal standard (glycine max (l.) merr.), st.; d – agrimonia pilosa (tomsk, russia), samp. 1 with agrimonia pilosa (beijing, china), samp. 2. table 2. chromosome numbers in the genus agrimonia l. (chung 2008; angelo and boufford 2012; rice et al. 2015). species chromosome numbers agrimonia coreana nakai 24; 28 agrimonia eupatoria l. 28; 42; 56; 70; 84 agrimonia grandis andrz. ex c. a. mey. 42 agrimonia gryposepala wallroth 56 agrimonia incisa torr. & a. gray 28 agrimonia japonica (miq.) koidz. 56 agrimonia nipponica koidz. 28 agrimonia parviflora aiton 28 agrimonia pilosa ledeb. 28; 56; 70 agrimonia x nipponica-pilosa murata 42 agrimonia procera wallr. 56 agrimonia pubescens wallroth 28 agrimonia repens l. 28 agrimonia rostellata wallroth 28 agrimonia striata michx. 28; 56 71a new diploid cytotype of agrimonia pilosa (rosaceae) disregarding agrimonia, have following chromosome numbers and ploidy: aremonia – 2n = 5x = 35 and 2n = 6x = 42, hagenia – 2n = 6x = 42, leucosidea and spenceria – 2n = 2x =14 (ikeda et al. 2006; chung et al. 2012; rice et al. 2015). previously, a karyotype of a. pilosa var. japonica was examined by iwatsubo et al. (1993). all studied plants had 2n = 56. chromosomes at metaphase ranged 1.2–2.5 µm in length and 1.0–2.5 in arm ratio. these were classified into two groups: 21 metacentric pairs, and seven submetacentric pairs. one submetacentric pair had a satellite on the short arm. according to other scientific data, somatic chromosome number of a. pilosa are 2n = 28 and 2n = 70 (table 2). we had revealed a new diploid cytotype in a. pilosa collected in china with 2n = 16. apparently, these plants exhibit diploid karyotype and new basic chromosome number x = 8 for the genus and subtribe. that chromosome number 2n = 16 was determined in all 127 investigated root meristematic cells. we suppose there were no b chromosomes for diploid cytotype 2n = 14. dysploidy arising from chromosomes fusions (escudero et al. 2014) is also unlikely, having our data on chromosomes length and morphology are relevant to the results previously obtained on a. pilosa with 2n = 56 by iwatsubo et al. (1993). we suppose that a haploidization of genome took place in a. pilosa specimen collected in china. according to classification developed by kimber and riley (1963), this event relates to aneupolyhaploidia, that is haploidization of polyploid form associated with aneuploidy. in addition to karyotypè s divergence from typical polyploid a. pilosa with 2n = 56, the investigated herbarium specimen exhibits reduced dimensions, fruits, and seeds. this corresponds with the revealed less ploidy level of the plant because polyploidy is followed by «gigas»effect (ramsey and ramsey 2014). we do not exclude the possibility that this specimen could be related to a new taxon. our flow cytometry studies revealed that c-values in two investigated a. pilosa differ at a factor of two, approximately. this result was unexpected to us due to the fact that the number of chromosomes didǹ t correlate with relative dna content in two examined agrimonies. unfortunately, we had no a. pilosa specimen with chromosome number 2n = 28 to determine relative dna content and to compare it with data obtained. the sizes of the monoploid genome were found to be equal 1сх = 1.25 pg for samples with 2n = 16, and 1сх = 0.62 pg for samples with 2n = 56 which indicates a significantly more ancient origin of diploid populations, according to the genome downsizing theory (leitch, bennett 2004). due to other studies reports, dna loss in polyploid series is usually at the level of 15.4% (zenil-fergusson et al. 2016), whereas in our case, such a significant decrease may indicate complex moleculargenetic processes and dna loss during the evolution of the agrimonia pilosa genome. studies of many eukaryotic genomes show that noncoding regions of dna can be lost in the polyploidization process (shaked et al. 2001). some cytological studies show a loss of heterochromatin, whole chromosomes or their segments after polyploidization (gustafson and bennett 1982; song et al. 1995; chen and ni 2006; xiong et al. 2011). thus, our study is correlated with the idea that the reduction of the genome is a frequent biological phenomenon. more detailed investigation of the herbarium specimen of a. pilosa collected in china by molecular cytogenetics and molecular genetics methods with morphological analysis can elucidate the problem of evolution of the genus agrimonia. disclosure statement no potential conflict of interest was reported by the author. funding the research was supported by the scientific programme аааа-а17-117012610055-3 of the central siberian botanical garden, of sb ras (field trip), “the tomsk state university competitiveness improvement programme” under grant no 8.1.09.2018 (karyotyping works); state assignment of the altay state university under grant no fzmw-2020-0003 (genome size works). we would like to thank the reviewers for their valuable comments and suggestions. authors are grateful to roman annenkov for preparing figures 2 and 3. references angelo r, boufford de. 2012. atlas of the flora of new england: rosaceae. phytoneuron. 81:1–56. altinordu f, peruzzi l, yu y, he x. 2016. a tool for the analysis of chromosomes: karyotype. taxon. 65(3):586–592. bukovsky m, blanarik p. 1994. immunomodulative effects of ethanolic-aqueous extracts of herba agrimoniae, flos chamomillae and flos calendulae cum calyce. farm obz. 63(4):149–156. 72 elizaveta mitrenina et al. chen l, kang yh. 2014. antioxidant activities of agrimonia pilosa ledeb: in vitro comparative activities of its different fractions. korean j plant res. 27(6):642–649. chen zj, ni z. 2006. mechanisms of genomic rearrangements and gene expression changes in plant polyploids. bioessays. 28:240–252. chung ks. 2008. a systematic study of genus agrimonia (rosaceae). doctoral dissertation. university of oklahoma, norman. chung, ks, hoang n, elisens w, un oh b. 2012. phylogenetic implication of seed coat sculpturing in subtribe agrimoniinae (rosaceae). korean j pl taxon. 42(4):247–252. escudero m, martín-bravo s, mayrose i, fernándezmazuecos m, fiz-palacios o, hipp al, pimentel m, jiménez-mejías p, valcárcel v, vargas p et al. 2014. karyotypic changes through dysploidy persist longer over evolutionary time than polyploid changes. plos one. 9(1):e85266. doležel j, doleželova m, novak fj. 1994. flow cytometric estimation of nuclear dna amount in diploid bananas (musa acuminata and m. balbisiana). biol plant. 36:351–357. giachetti d, taddei e, taddei i. 1986. diuretic and urisurica of agrimonia eupatoria l. boll soc ital biol sper. 62(6):705–711. gustafson jp, bennett md. 1982. the effect of telomeric heterochromatin from secale cereale on triticale (x triticosecale). i. the influence of the loss of several blocks of telomeric heterochromatin on early endosperm development and kernel characteristics at maturity. can j genet cytol. 24:83–92. ikeda h, hand k, wu sk, ohba h. 2006. a revision of the genus spenceria trimen (rosaceae). j jpn bot. 81(3):154–167. iwatsubo y, mishima m, naruhashi n. 1993. chromosome studies of japanese agrimonia (rosaceae). cytologia. 58:453–461. kimber g, riley r. 2011. the relationships of the diploid progenitors of hexaploid wheat. can j genet cytol. 5:83–88. kline gj, sørensen p. 2008. a revision of agrimonia (rosaceae) in north and central america. brittonia. 60(1):11–33. kubešová m, moravcová l, suda j, jarošík v, pyšek p. 2010. naturalized plants have smaller genomes than their non-invading relatives: a flow cytometric analysis of the czech alien flora. preslia. 82:81–96. kuczmannová a, balažová a, račanská e, kameníková m, fialová s, majerník j, nagy m, gál p, mučaji p. 2016. agrimonia eupatoria l. and cynara cardunculus l. water infusions: comparison of anti-diabetic activities. molecules. 21(5):564. kumar p, rana pk, himshikha, singhal vk, gupta rc. 2014. chromosome numbers, characterization of chromosomal pairing during meiosis, origin and natural propagation in polyploidy cytotypes (4x, 5x and 6x) of agrimonia eupatoria l. 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caterina angela dettori2, andrea reid3, gianluigi bacchetta2,4,*, laetitia hugot5, elena conti1 cytogenetic effects of c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. and mathematical analysis cihangir alaca1, ali özdemir1, bahattın bozdağ2, canan özdemir2,* clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 155-161, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-184 citation: h. sharghi, m. azizi, h. moazzeni (2020) a karyological study of some endemic trigonella species (fabaceae) in iran. caryologia 73(1): 155-161. doi: 10.13128/caryologia-184 received: march 8, 2019 accepted: february 23, 2020 published: may 8, 2020 copyright: © 2020 h. sharghi, m. azizi, h. moazzeni. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 1 department of horticultural sciences, faculty of agriculture, ferdowsi university of mashhad, p.o.box: 917751163, mashhad, iran 2 department of botany, research center for plant sciences, ferdowsi university of mashhad, p.o.box 91779-48974, mashhad, iran *corresponding author. e-mail: azizi@um.ac.ir abstract. karyotypes of nine populations belonging to six endemic species of the genus trigonella (trifolieae/fabaceae) studied in this survey. all studied species are perennial and recorded only from northeast of iran. excluding the karyotype of trigonella subenervis, which was previously reported, all of the other species studied here (six species) for the first time. our results present that all assessed genotypes are diploid with 2n=2x=16 and the chromosomal basis of x=8. in addition to the chromosome counts, length of long and short arms of the chromosome and their ratios analyzed and presented in this study. keywords. chromosome number, cytogenetic, karyotype, khorassan, trifolieae, trigonella. introduction the genus trigonella l. is a member of the tribe trifolieae of the family fabaceae, with about 135 species worldwide, most of which distributed in the dry regions around the mediterranean region extended to west asia, and naturalized in north america. only two species being present in south australia (mabberley 1997). trigonella consists of annual or perennial herbs with pinnately trifoliate leaves, a campanulate or tubular calyx with two large and three small equal lobes, diadelphous stamens, uniform anthers, terminal stigma and ovary with numerous ovules (širjaev 1928-1932; hutchinson 1964; dangi et al. 2016). according to rechinger (1984), the genus represented by 58 species in to flora iranica area. this number has increased to about 66 species as a result of recent researches (hamzeh’ee 2000; janighorban 2004; badrzadeh and ghafarzadeh-namazi 2009; ranjbar, karamian, and hajmoradi 2012; ranjbar and hajmoradi 2012, 2015, 2016). of which, 48 taxa (15 endemics (32%)) accommodated in 12 sections growing in iran; 14 of those are perennial species of trigonella sect. ellipticae (boiss.) sirj. in flora iranica account (rechinger 1984), section ellipticae is represented with seven perennial species in iran. the characteristics of the section 156 hamidreza sharghi, majid azizi, hamid moazzeni ellipticae are: perennial species with entire or dentate stipules, calyx campanulate, petals yellow or rarely white with violet veins, sometimes completely dark violet, standard without interlocking projection, fruit a legume which is different in shape and size, elliptic or lanceolate to oblong, beakless, generally transversely veined, wingless or with thin wing and smooth seeds. several cytological investigations have been conducted on approximately a hundred trigonella species (agarwal and gupta 1983; ahmad et al. 1999; astanova 1981; aykut et al. 2009; bal 1990; bidak and amin 1996; darlington and wylie 1955; dundas et al. 2006; ghosh 1980; kumari and bir 1990; ladizinsky and vosa 1986; martin et al. 2006, 2008, 2010, 2011a, 2011b; pavlova 1996; ranjbar et al. 2011a, 2011b, 2015; singh and roy 1970; singh and singh 1976; tutin and heywood 1964; yilmaz et al. 2009). the reported chromosome numbers of the genus trigonella are 2n=14, 16, 18, 24, 28, 30, 32, 42, 44, 46 and 48. to contribute to the karyological study of the genus, we carried out a karyological study on some perennial endemic species collected from different regions in east and northeast of iran. this study aimed to verify the chromosome numbers of some endemic trigonella species recently reported in iran. in this contribution, we report the somatic chromosome numbers of six taxa (nine populations), that five species are determined for the first time. material and methods the chromosome number were analyzed in nine population of trigonella. the nomenclature of taxa, collection data, and vouchers are given in table 1. the mitotic chromosome numbers were studied in three populations of t. subenervis rech. f., two populations of t. binaloudensis and one population from each of t. lasiocarpa, t. stipitata, t .heratensis and t. torbatejamensis. seed materials were collected between the years of 2016 and 2018 from natural habitats. voucher specimens were deposited at the ferdowsi university of mashhad herbarium (fumh), iran. for karyological observation, to accelerate germination, the seed surfaces were abraded with emery paper. seeds were sown on wet filter paper in petri dishes at room temperature. the seeds germinated after 2-3 days. one cm fresh root tip cells were used to study the somatic chromosomes. the root tips pretreated in an ice-water mixture for 24 hours. afterward, they fixed in carnoy’s fixative (ethanol: acetic acid 3:1) for 24 h at 4 °c (fukui and nakayama 1996). the root tips were washed in distilled water to remove the fixative, then hydrolyzed in 1n hcl for 13–15 minutes at room temperature, and finally stained with 2% aceto-orcein for two hours. the slides were created using the squash method. the prepared slides were slightly heated under an alcohol frame for 1–2 s before observation. photographs of chromosomes were taken using a nikon eclipse ni-u (tokyo, japan) photomicroscope equipped with nikon ds-fi3 digital camera. chromosome counts were made from well-spread metaphases in intact cells, by direct observation, and from photomicrographs. to ensure for chromosome number, a minimum of five cells, at somatic metaphase, observed (figure 1). karyotypic analyses were conducted on ideokar software (version 1.2) to calculate karyotypic parameters and generate ideograms (mirzaghaderi and marzangi 2015). karyotype formulae and nomenclature followed levan et al. (1964), and karyotype asymmetry followed stebbins (1971). table 1. iranian endemic species of trigonella analyzed in this study, their locations, and voucher specimens data. taxa location collection date elevation (m) herbarium number t. binaloudensis* population 1 sw chenaran, ferizi towards binaloud mountains 2016/05/30 1730 46443 population 2 nw sabzevar, w jalambadan, in mnts. near chromite mine 2018/05/29 1770 46323 t .heratensis s fariman, between torbate-heydariyeh & fariman 2016/06/06 1685 45959 t. lasiocarpa* ne birjand, now-qand towards bidesk 2016/05/24 2320 46442 t. stipitata* n mashhad, e chenaran, mian-marq 2016/05/25 1420 25771 t. subenervis* population 1 n torbate-heydariyeh, khomari pass 2016/05/23 1851 46441 population 2 n kashmar, ne hills of chalpu village 2017/06/06 1879 46446 population 3 n shirvan, 12 km from lowjalli toward namanlu village 2017/06/24 1820 45844 t. torbatejamensis* ne torbate-jam, between saleh-abad & gush-laqar, 2016/05/04 750 45958 157a karyological study of some endemic trigonella species (fabaceae) in iran results our investigations comprised nine populations belonging to 6 species of the genus trigonella, of which data for five species reported here for the first time. the chromosomes of these taxa at mitotic metaphase shown in figure 1. detailed karyotypic parameters, formulae, and asymmetry are summarized in table 2. in this study, the basic chromosome number of all taxa is x=8, and all of them are diploid. trigonella binaloudensis ranjbar & karamian population 1 (sw chenaran): the chromosome number was 2n=2x=16 (figure 1e1). haploid chromosome length was 27.73 μm. chromosome length varied from 2.75 to 4.83 μm, and arm ratio from 1.06 to 1.88. the chromosome complement at mitotic metaphase consisted of 14 median region and two submedian region chromosomes. karyotypic asymmetry was 1a. population 2 (nw sabzevar): the chromosome number was 2n=2x=16 (figure 1e2). haploid chromosome length was 24.03 μm. chromosome length varied from 2.15 to 3.72 μm, and arm ratio from 1.030 to 1.82. the chromosome complement at mitotic metaphase consisted of 14 median region and two submedian region chromosomes. karyotypic asymmetry was 1a. trigonella heratensis rech.f the chromosome number of t. heratensis was 2n=2x=16 (figure 1c). haploid chromosome length was 22.98 μm. chromosome length varied from 2.46 to 3.40 figure 1. photographs of somatic metaphase chromosomes of nine populations belonging to six species of the genus trigonella collected from northeast of iran. (a, b) t. binaloudensis from two locality; (c) t. heratensis; (d) t. lasiocarpa; (e) t. stipitata; (f, g, h) t. subenervis from three locality; (i) t. torbatejamensis. scale bars = 10 μm. 158 hamidreza sharghi, majid azizi, hamid moazzeni μm, and arm ratio from 1.02 to 1.69. the chromosome complement at mitotic metaphase consisted of 16 median region chromosomes, and karyotypic asymmetry was 1a. trigonella lasiocarpa ranjbar & z.hajmoradi the chromosome number of t. lasiocarpa was 2n=2x=16 (figure 1a). haploid chromosome length was 28.9 μm. chromosome length varied from 2.44 to 4.44 μm, and arm ratio from 1.09 to 1.51. the chromosome complement at mitotic metaphase consisted of 16 median region chromosomes, and karyotypic asymmetry was 1a. trigonella stipitata ranjbar & joharchi the chromosome number of t. stipitata was 2n=2x=16 (figure 1b). haploid chromosome length was 19.17 μm. chromosome length varied from 1.92 to 3.13 μm, and arm ratio from 1.02 to 1.44. the chromosome complement at mitotic metaphase consisted of 16 median region chromosomes, and karyotypic asymmetry was 1a. trigonella subenervis rech.f population 1 (n torbate-heydariyeh): the chromosome number was 2n=2x=16 (figure 1f1). haploid chromosome length was 19.87 μm. chromosome length varied from 1.94 to 3.00 μm, and arm ratio from 1.01 to 1.94. the chromosome complement at mitotic metaphase consisted of 14 median region and two submedian region chromosomes. karyotypic asymmetry was 1a. population 2 (n kashmar): the chromosome number was 2n=2x=16 (figure 1f3). haploid chromosome length was 20.68 μm. chromosome length varied from 2.03 to 3.04 μm, and arm ratio from 1.01 to 1.52. the chromosome complement at mitotic metaphase consisted of 16 median region chromosomes, and karyotypic asymmetry was 1a. population 3 (n shirvan): the chromosome number was 2n=2x=16 (figure 1f3). haploid chromosome length was 28.04 μm. chromosome length varied from 2.67 to 4.18 μm, and arm ratio from 1.01 to 1.44. the chromosome complement at mitotic metaphase consisted of 16 median region chromosomes, and karyotypic asymmetry was 1a. trigonella torbatejamensis ranjbar the chromosome number of t. torbatejamensis  was 2n=2x=16 (figure 1d). haploid chromosome length was 26.77 μm. chromosome length varied from 2.42 to 4.50 μm, and arm ratio from 1.05 to 2.21. the chromosome complement at mitotic metaphase consisted of 14 median region and two submedian region chromosomes. karyotypic asymmetry was 2a. discussion all taxa in the present study showed the same basic chromosome number x=8 and same polyploidy level, which is congruent with those previously reported by ranjbar et al. (2016) in trigonella subenervis and six other species of the section. ellipticae. this section comprises most of the perennial species of the genus trigonella and widely distributed in iran, afghanistan and middle asia. table 2. somatic chromosome numbers and karyotypes of nine trigonella taxa. hcl: haploid chromosome length, cl: chromosome length, ar: arm ratio(l/s), rl%: relative length of the chromosome, ci: centromeric index. species 2n x hcl (μm) cl μm) ar rl% ci karyotype formulae karyotypic asymmetry (stebbins) t. binaloudensis* (1) 16 8 27.73 2.75-4.83 1.06-1.88 4.96-8.70 0.35-0.49 14m+2sm 1a t. binaloudensis* (2) 16 8 24.03 2.15-3.72 1.03-1.82 4.48-7.74 0.35-0.49 14m+2sm 1a t. heratensis 16 8 22.98 2.46-3.40 1.02-1.69 5.36-7.39 0.37-0.50 16m 1a t. lasiocarpa* 16 8 28.90 2.41-4.44 1.09-1.51 4.17-7.68 0.40-0.48 16m 1a t. stipitata* 16 8 19.17 1.92-3.13 1.02-1.44 5.00-8.15 0.41-0.49 16m 1a t. subenervis* (1) 16 8 19.87 1.94-3.00 1.01-1.94 4.88-7.54 0.34-0.50 14m+2sm 1a t. subenervis* (2) 16 8 20.68 2.03-3.04 1.01-1.52 4.91-7.34 0.40-0.50 16m 1a t. subenervis* (3) 16 8 28.04 2.67-4.18 1.01-1.44 4.75-7.45 0.41-0.50 16m 1a t. torbatejamensis* 16 8 26.77 2.42-4.50 1.05-2.21 4.51-8.41 0.31-0.49 14m+2sm 2a 159a karyological study of some endemic trigonella species (fabaceae) in iran all of the studied taxa in this study, analyzed karyotypically for the first time, covering chromosome length, karyotype formulae, and asymmetry (table and figure 2). in term of karyotypic parameters, our results support the results reported by riasat (2015) about trigonella elliptica, a perennial species from section ellipticae. in later study, the karyotype formulae reported as 14m+2sm, 16m, 8m+8sm, and 12m+4sm in different genotypes. we found the same variations in karyotype formulae and asymmetry among different species and populations that are shown in table 2. karyotypic asymmetry in most of the studied taxa, was as a1 but in t. torbatejamensis which is a2. karyotype formulae in most of the specimens were as 16m and in four specimens (t. torbatejamensis, two populations of t. binaloudensis and one population of t. subenervis) was as 14m+2sm. the incongruences may result from variations among different populations and chromosome preparation treatments. it seems that chromosome variation and evolution of trigonella species need more comparative cytological studies based on more collections from different populations. acknowledgments this work was partly supported by the research center for plant science, ferdowsi university of mashhad. the authors would like to thank the staff assistance of fumh in field and herbarium. we are grateful to miss. s. hosseini for technical assistance. references agarwal k, gupta pk. 1983. cytological studies in the genus trigonella linn. cytologia. 48(4):771-779. ahmad f, acharya s, mir z, mir p. 1999. localization and activity of rrna genes on fenugreek (trigonella foenum-graecum l.) chromosomes by fluorescent in figure 2. haploid ideograms of trigonella taxa. 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vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 75(1): 15-27, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1202 caryologia international journal of cytology, cytosystematics and cytogenetics citation: rajani singh, girjesh kumar (2022) analyzing frequency and spectrum of chlorophyll mutation induced through gamma ray and combination treatment (gamma + ems) on genetic paradigm of artemisia annua l.. caryologia 75(1): 15-27. doi: 10.36253/caryologia-1202 received: january 31, 2021 accepted: march 23, 2022 published: july 6, 2022 copyright: © 2022 rajani singh, girjesh kumar. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid rs: 0000-0001-9782-6754 analyzing frequency and spectrum of chlorophyll mutation induced through gamma ray and combination treatment (gamma + ems) on genetic paradigm of artemisia annua l. rajani singh*, girjesh kumar plant genetics laboratory, department of botany, university of allahabad, india *corresponding author. e-mail: singh.rajani1995@gmail.com abstract. for the development of genetic programs with novel characteristics induced mutagenesis has been used extensively. chlorophyll (chl) mutations are considered as the most dependable indices for assessing the efficiency of different mutagens in inducing the genetic variability in crop plants and are also used as genetic markers in basic and applied research. in the present scenario of high health susceptibility, the global demand for natural medicine derived from plant species has increased enormously. sweet wormwood (artemisia annua linnaeus) – an important medicinal plant species with immense remedial values, was selected for the present study and exposed to gamma rays at 100 gy, 200 gy, 300 gy and combination treatments with 100 gy + 0.1%ems, 200 gy + 0.1% ems, 300 gy + 0.1%ems. meiotic study was also done and various cytological aberrations were observed in m2 generation like stickiness, precocious, scattering, laggard and bridge etc. the frequency of induced chl mutation varied in different mutagen treatments. eight different types of chl mutants namely albina, chlorina, xantha, aurea, viridis, yellow viridis and tigrina etc. were recorded in m2 generation on plant population basis. the frequency of xantha mutants was quite high in both the treatments but in gamma exposed set it was followed by albina whereas in combination treatments viridis was second highest mutant. in different mutants quantitative analysis of chl pigments was also done and content was highest in viridis i.e. 3.86µg/ml fw and lowest in albina i.e. 0 µg/ml fw . although chlorophyll mutations thought to be lethal in nature, but present study has proven to be a milestone in identifying the threshold dose of a mutagen that would increase the genetic variability and induces new trait in artemisia annua linnaeus. keywords: artemisia annua l. (linnaeus), chlorophyll mutants, cytological anomalies, gamma rays, ems. introduction the medicinal plant artemisia annua, also known as sweet wormwood or sweet annie, is one of the top 10 pharmaceutical crops which are getting intensive worldwide scientific consideration as this valuable treasure is the only source for the commercial pharmaceutical production of the ses16 girjesh kumar, rajani singh quiterpene lactone artemisinin (prasad and das 1980). artemisia has been applied in the traditional medicine, for the treatment of diabetes, depression, insomnia and stress, to clear the lymphatic system and in the oncotherapy. the whole plant of a. annua l.is still the most economic source of artemisinin, and the developments of high-producing plants of a. annua l. appear to be the main direction to obtain large quantities of relatively inexpensive artemisinin. for any successful crop improvement programme, genetic variability plays an important role because it provides a spectrum of variants for effective and better selection which can be obtained using mutation, hybridization, recombination and selection processes (dhumal and bolbhat 2012). mutational breeding involves high energy radiation such as x, β and γ-rays, which are electromagnetic radiations that initiate or inhibit the growth and differentiation of plant cells and organs (hasbullah et al. 2012) they could also modify physiological characteristics of plant to create new mutants for production of high amounts of commercially important metabolites. ionizing radiation has been recognized as a powerful technique for plant improvement of medicinal plants (vardhan and shukla 2017). this technique creates genetic variability in plants, which can be screened for desirable characteristics. previously, koobokurd et al. (2008) reported a method for establishing in vitro plantlet variants of a. annua using low-dose gamma irradiation. by using gamma rays many high yielding mutant varieties have been developed world wide, which are resistant to biotic and abiotic stresses with improved quality (iaea 2017). the success of mutation breeding programme largely depends on selection of promising mutants based on phenotypic characters (arisha et al. 2015). ems, as a chemical mutagen, can be used as a supplementary approach to improve desired identifiable characters such as yield related characters (botticella et al. 2011). chemical mutagens are not only mutagenic themselves but also affect mutation in specific ways when combined with radiation (reddy and smith 1981). it produces random point mutations in genetic material. so, mutation frequency, detected using various techniques, displays a wide range of variation in combination treatment where plants seeds exposed to physical mutagen followed by chemical mutagen. during m1 generation, probably identification of recessive character is difficult only mutations of dominant characters can be identified. in the m2 generation, the mutation will segregate to create homozygotes for recessive or dominant alleles (page and grossniklaus 2002). the most effective way to identify the phenotypic mutation is visual screening which can be used as a primary indicator to select plants that have desired characters, for example: disease resistance, f lowering earliness, plant height or growth period (østergaard and yanofsky 2004). gene mutations influencing the green coloration of photosynthetically active parts are among the most common spontaneous or induced alterations arising in higher plants (kolar et al. 2011). although chlorophyll mutations are generally not useful for plant breeding purpose because of not having any economic value due to their lethal nature, their study could be useful in identifying the suitable mutagen and threshold dose of mutagen that would increase the genetic variability and number of economically useful mutations in the segregating generations (wani and anis 2004). the chlorophyll mutation frequency is an indicator to predict the frequency of factor mutations and thus an index for evaluation of genetic effects of mutagens (walles 1973). in addition, chl mutations are important for identifying gene function and elucidation of chl metabolism and its regulation15. the occurrence of chl mutations after treatments with physical and chemical mutagens have been reported in several crops (swaminathan et al. 1962; sharma and sharma 1981; reddy and gupta 1989; mitra 1996; kharkwal 1998; solanki 2005; wu et al. 2007). induced mutations can rapidly create variability in quantitatively and qualitatively inherited traits in crops. genetic variability has been induced through mutagenesis in several plants, but the information available in a. annua l. is meager. in the present study attempt has been made to understand the comparative response of physical and chemical mutagens on a. annua, with a view to determine the mutagen and treatment causing maximum chl mutations in m2 generation and also on cytological parameter. materials and methods plant materials m2 seeds generated from the m1 generation of variety ec-415012, were used in this study. the m1 seeds were produced by exposing separate 1000 dry seed samples (for each dose) to 100gy, 200gy and 300gy at a dose rate of 15.48 gy/min of gamma radiation using a 60co (cobalt 60) gamma source under ambient conditions at the national botanical research institute (nbri), lucknow and for combination treatment concentration of ethyl methyl sulphonate (ems) solution of 0.1% was prepared. ems solution was settled in a 0.1m phosphate buffer at ph 7.0 to avoid rapid hydrolysis (bosland 2002). gamma ray treated (100gy, 200gy, 300gy) seeds were presoaked in water for 6h 17analyzing frequency and spectrum of chlorophyll mutation induced through gamma ray and combination treatment then treated with the above-mentioned concentration of ems at 20°c with orbital shaking(110rpm) along with control (untreated) seeds. seeds were then thoroughly washed under running water then transferred to petridishes containing wet filter paper and kept in a growth chamber at 25°c in the seed germinator for germination (at 2 days after the treatment).control seeds were exposed to the same conditions except for the ems treatment. experimental plan and procedure the experiments were carried out in the first week of the month january at roxburg botanical garden, department of botany. the m1 plants are individually harvested and sown as m2 families. according to the pedigree method; the m1 plants are individually harvested and plants with probable mutants following phenotypic observations as plant habit variation in leaves (chl mutants), early plant vigour (poor, good and very good), plant height (short stature, up to top of the plant), sown as m2 families .sweet wormwood m2 lines were grown in the field (geographical location is 25o27’43.01”n, 81o51’10.42”e) in randomized complete block design (rcbd) and allowed to produce the m2 seeds.. the net plot size was 4 m _ 4 m, with nine rows (each 4 m long) with a 45 cm distance between two rows and approximately 20 cm distance between two plants. the untreated seeds (control) were planted in the first row of each plot. for weed control plots were irrigated during vegetative growth and the plants were harvested individually at full maturity. germination (%) taken after 7 days and plant survival (%) was recorded after 14 days for each mutagenic treatment as well as control in m2 generation. after a month six phenotypic traits were analysed and recorded as plant height (cm), internodal length (cm), leaf area(per m2), no. of primary branches, days to 50% flowering and days to maturity etc. cytological investigations for the cytological analysis young floral capitula of control and variant plant of artemisia annua l. with appropriate size were fixed in carnoy’s fixative (alcohol 3: glacial acetic acid 1) for 24 hrs and then transferred in 90% alcohol to preserve the capitula for meiotic study. anthers were teased and stained in 2% acetocarmine, followed by squash preparation. slides were observed under the microscope and pollen fertility was evaluated by acetocarmine stainability test. the snapshots of chromosomes were captured by the help pinnacle pctv software. for pollen fertility, mature capitula having pollen grains were dusted over glass slide and stained with acetocarmine and mounted with glycerine. then observed under optical microscope to count the frequency of fertile and sterile pollen grains. pollen fertility (%) = no. of fertile pollen × 100 total no. of pollen quantification of photosynthetic pigments photosynthetic pigment was quantified according to lichtanthelar and welburn (1983)method. 20mg of leaves were taken and dissolved in 5ml of 80%acetone. solution was extracted and were centrifuged at 15000 rpm for 10min at 10°c. the supernatant volume was diluted with 80% acetone. o.d. was taken at three different wavelength i.e. 470nm, 663nm and 646nm in the spectrophotometer and finally chl a, chl b calculated. observations recorded and statistical procedure the m2 generation was screened for phenotypic variations from germination to harvesting. the frequency of the mutant plants out of the total number of individuals in m2 generation was calculated. the mutagenic frequency was estimated as the percentage of segregating m1 plant progenies. chl mutations were classified into various types based on the method followed by gustafsson(1940). for statistical analysis in the table, three replicates for each treatment were used. statistical analysis was performed using the spss 16.0 software. a oneway analysis of variance (anova) and duncan’s multiple range test (dmrt, p < 0.05) was conducted for mean separation and the graph was plotted by using sigma plot 10.0 software. actual mean and standard error were calculated and the data was subjected to analysis of variance. results germination and survival percentage fig. 1 shows that germination percentage and plant survival were significantly declined as the mutagenconcentration increased. conspicuous variations were recorded after both the treatment in sweetworm wood. athigher doses germination and survival percentage was found to be 76.60%, 61.76% (in gamma ray) and 33.83%,24.66% (in gamma+ ems) respectively. 18 girjesh kumar, rajani singh chl leaf variations in the present appraisal, the frequency of chl mutants calculated as percent of m1 plant progenies and m2 plants basis were presented in table 1. it was observed that the frequency of induced chl mutant was increased with an increase in the dose of gamma ray and combination treatment but at the maximum doses mutation frequency was decreased. at the 300gy mutation frequency was recorded as 3.60%(in m1 generation) and 0.98%(in m2 generation)in gamma ray treatment while it was 2.60%(in m1 generation) and 0.79% (in m2 generation) in combination treatment. it was observed that the mutation frequency in m2 generation was more in combination treatment of 100gy+0.1% ems( 2.82%) than gamma ray alone (1.09 %) m2 generation depicts presence of broad chl mutantspectrum, comprising total 8 type mutants. the spectrum of m2 chl mutants included albino, xantha, chlorina,maculata, tigrina, auria and viridis are presented in table 2. in both the treatment, frequency of xantha mutants (in fig. 2) was maximum (all total) 92.75% (in gamma) and 103.78% (in combined treatment), followed by viridis, albino and yellow viridis in gamma treatment while in gamma+ems, it was followed by albino, maculata and chlorina. the least frequency of auria type of chl mutant was recorded 28.14% (in gamma) and 11.11% (in combined treatment). albina (fig. 4 e & g) mutants were completely lack of chl and could survive only a few days. chlorina (fig. 4 a,b,c) and yellow viridis ( fig. 4 h & i) had green and yellow green leaves, respectively, are lethal mutations. aurea (fig. 3d) had golden yellow coloured leaves and xantha (fig. 3b) had pale yellow coloured seedlings, tigrina (fig. 4d) had yellow colour at edges of the leaves furthermore viridis (fig. 3c ) had dark green colour, these type of mutants not only survive but they complete its full lifecycle. chl content in chl deficient mutants the concentration of green pigmentation in the leaves differed among the different types of chl deficient mutants, ranging from chlorina to xantha type of mutants. the chl content (fig. 5) of various mutants and along with control tissues were examined, the mutants contained significantly less chl than normal plants accept viridis mutant. among the mutants, albina type was totally devoid of chl while xantha (0.83µg g-1fw) and aurea (0.96µg g-1fw) contained the least figure 1. effect of gamma ray and combination treatment on germination and survival in artemisia annua l. (m1 generation). table 1. frequency of chlorophyll mutants induced through gamma and combination treatment (gamma+ems) in m2 generation of artemisia annua l. treatments no. of m1 plant progeny m1 segregates for mutation m2 plant scored m2 mutants mutation frequency (%) m1 plants frequency m2 plant frequency gamma rays (gy) control 500 1090 100 500 27 1097 12 5.40 1.09 200 500 21 1070 23 4.20 2.15 300 500 18 921 9 3.60 0.98 combination treatment (gy+ %) control 500 1073 100+0.1% 500 30 1038 28 6.00 2.82 200+0.1% 500 24 994 13 4.80 1.31 300+0.1% 500 13 882 7 2.60 0.79 19analyzing frequency and spectrum of chlorophyll mutation induced through gamma ray and combination treatment chl and those of viridis (3.86µg g-1fw) the most. th e other chl mutants maculata (1.24µg g-1fw), tigrina (1.06µg g-1fw), chlorina (2.46 µg g-1fw) and yellow viridis (3.01µg g-1fw) contained signifi cantly less chl than those of control (3.24 µg g-1fw). meiotic study meiotic study was done in chl mutant plants which were identifi ed in m2 generation to screen out genetic disturbance caused by both the mutagen. in control plants 9:9 standard configuration was found. different types of anomalies were exhibited by treated sets i.e. precocious movement, stickiness, scattering, laggards, bridges, disturb polarity etc. (fig. 6d–l). stickiness and multivalent was found to be predominant abnormality in treated sets. table 3 represented various abnormality frequency and total abnormality(tab) percentage in treated sets. at lower dose of both the treatment tab% was found to be 6.24±0.15% (gamma) and 8.93±0.17% (combined treatment) while at higher dose of both the treatments tab% was shoot up to 19.71±0.22% (gamma) and 26.63±0.48% (combined treatment) . it shows that abnormality percentage is dose dependent. the results shown in table 4 indicated that the growth habit of all mutant plants (plant height, intertable 2. spectrum and frequency of chlorophyll mutant in diff erent mutagenic treatments. treatment total chl mutants in m2 generation th e relative percentage of chlorophyll mutants(%) albina xantha chlorina tigrina maculata viridis yellow auria gamma ray (gy) 100 12 16.67 33.33 8.33 8.33 0 8.33 16.67 8.33 200 23 13.04 26.09 8.70 13.04 8.70 13.04 8.70 8.70 300 9 11.11 33.33 0 0 0 22.22 11.11 22.22 gamma+ems 100+0.1% 18 11.11 44.44 16.67 0 0 0 16.66 11.11 200+0.1% 13 15.38 30.78 15.38 15.38 0 15.38 15.38 0 3000+0.1% 7 14.29 28.57 0 14.29 28.57 14.29 0 0 0 20 40 60 80 100 120 alb ina xa nth a ch lor ina tig rin a m ac ula ta vir idi s ye llo w vir idi s au ria gamma gamma+ems figure 2. chlorophyll mutations frequency on mutagen basis in artemisia annua l. figure 3. chlorophyll mutants in artemisia annua l.: acontrol, b-semi-xantha mutant at seedling stage, cviridis mutant, daurea mutant 20 girjesh kumar, rajani singh nodal length, leaf area, no. of primary branches, days to 50% f lowering and days to maturity) were different than those of the control plants. as height (cm) of the control plant was observed 103.20±0.88 although at 100gy and 100±0.1% it was increased i.e. 112.30±1.01and 110.30±1.09 which decreased as the figure 4. different types of chlorophyll mutants in artemisia annua l. a. b. c. chlorina mutant, d. tigrina mutant, e. g.. albina mutant, f. maculate mutant, h. i. yellow viridis mutant 21analyzing frequency and spectrum of chlorophyll mutation induced through gamma ray and combination treatment doses of mutagen increases. internodal length, leaf area and primary number of branches were improved at the lower dose of gamma rays but as the doses increases all these character significantly minimally decreased. at lower dose of gamma and gamma+ems plant flowers earlier and matures faster as compared to control. control plants were flower in 56 days and mature in about 155-156 days. while at 100gy and 100+0.1%, days to 50% flowering was observed 50.00±0.57 and 52.00±1.03 furthermore days to maturity was 145.00±4.50 and 143.00±1.00. fig. 7 shows the dwarf variant and tall variant. dwarf variant was found at 300+0.1% while tall variant screen out at 100gy dose and also some plant become prostrate as shown in fig. 7a. 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 control chlorina maculata tigrina viridis yellow viridis albina auria xantha figure 5. chlorophyll content in normal type and chlorophyll deficient mutants of artemisia annua l. figure 6. different types of abnormalities in treated plants of artemisia annua l.: a. stickiness at metaphase i, b. two precocious chromosome at sticky metaphase i, c. stickiness at unoriented anaphase i, d. one laggard chromosome at anaphase i, e. two precocious chromosome at metaphase ii, f. one laggard chromosome at anaphase ii, g. mononucleate condiotion, h. binucleate condition, i. multipolarity(scale bar= 10.13µm) figure 7. some phenotypic variants in artemisia annua l. a-prostrate variant, bdwarf variant ctall variant. 22 girjesh kumar, rajani singh ta bl e 3. a c om pa ra tiv e ac co un t o f c hr om os om al a no m al ie s in in du ce d th ro ug h g am m a ra y an d co m bi na tio n (g am m a+ em s) tr ea tm en t i n a rt em is ia a nn ua l . d os es (g y) n o. o f pm c ’s ob se rv ed m et ap ha si c a bn or m al iti es ( % ) (m ea n± s. e. ) a na ph as ic a bn or m al iti es ( % ) (m ea n± s. e. ) o th . (% ) t. a b. ( % ) po lle n fe rt ili ty (% ) sc pm st u n m v sa br lg u n st a sy d p c on tr ol 33 0 94 .4 1± 1. 46 a 10 0 31 0 0. 43 ±0 .1 0 1. 08 ±0 .1 0 0. 65 ±0 .1 9 0. 43 ±0 .1 1 0. 43 ±0 .1 1 0. 32 ±0 .1 9 0. 54 ±0 .1 0 0 0 0. 89 ±0 .1 0 0. 65 ±0 .0 1 0. 32 ±0 .0 4 0. 53 ±0 .1 0 6. 24 ±0 .1 5 92 .3 7± 1. 85 a 20 0 29 0 0. 80 ±0 .3 0 1. 25 ±0 .2 8 0. 92 ±0 .1 3 1. 14 ±0 .2 1 0. 93 ±0 .2 5 0. 56 ±0 .1 2 0. 81 ±0 .1 3 0. 80 ±0 .1 9 1. 04 ±0 .0 2 0. 69 ±0 .0 1 0. 93 ±0 .2 5 0. 69 ±0 .0 1 0. 58 ±0 .1 2 11 .2 5± 0. 14 87 .1 9± 1. 87 b 30 0 29 1 1. 84 ±0 .1 2 2. 19 ±0 .2 7 1. 26 ±0 .0 9 1. 95 ±0 .0 9 1. 37 ±0 .1 8 1. 27 ±0 .1 4 1. 38 ±0 .2 1 1. 37 ±0 .1 0 1. 62 ±0 .3 4 1. 72 ±0 .1 8 1. 14 ±0 .0 8 1. 37 ±0 .1 6 1. 25 ±0 .2 9 19 .7 1± 0. 22 70 .2 5± 1. 59 c c on tr ol 35 8 95 .2 2± 0. 58 a 10 0+ 0. 1% 38 5 0. 61 ±0 .1 0 0. 86 ±0 .0 7 1. 31 ±0 .1 9 0. 69 ±0 .0 7 0. 51 ±0 .1 4 0. 51 ±0 .1 4 0. 61 ±0 .1 0 0. 43 ±0 .2 2 0. 60 ±0 .0 7 0. 79 ±0 .1 7 0. 78 ±0 .1 6 0. 61 ±0 .0 9 0. 61 ±0 .0 9 8. 93 ±0 .1 7 85 .6 7± 1. 15 a 20 0+ 0. 1% 37 0 1. 44 ±0 .0 9 1. 44 ±0 .2 3 1. 29 ±0 .6 6 1. 53 ±0 .2 3 1. 08 ±0 .1 4 1. 08 ±0 .0 4 1. 17 ±0 .1 0 0. 90 ±0 .0 6 1. 28 ±0 .2 8 1. 35 ±0 .1 4 1. 46 ±0 .2 9 1. 07 ±0 .1 2 1. 43 ±0 .1 5 16 .5 4± 0. 85 77 .1 2± 1. 73 b 30 0+ 0. 1% 36 2 1. 39 ±0 .3 2 1. 66 ±0 .1 9 3. 96 ±0 .3 8 1. 66 ±0 .0 4 2. 67 ±0 .1 9 1. 57 ±0 .1 0 1. 56 ±0 .3 3 2. 41 ±0 .3 8 1. 95 ±0 .3 2 1. 11 ±0 .1 8 2. 94 ±0 .1 8 1. 74 ±0 .2 0 2. 01 ±0 .4 7 26 .6 3± 0. 48 61 .2 9± 0. 85 c a bb re vi at io ns : s .e . s ta nd ar d er ro r; sc sc at te ri ng ; p m pr ec oc io us m ov em en t; st st ic ki ne ss ; u n u no ri en ta io n; m v m ul tiv al en t; sa se co nd ar y as so ci at io ns ; a sy a sy nc hr on ou s; b r b ri dg e; l g l ag ga rd ; d p d is tu rb ed p ol ar ity ; o th o th er s; t .a b. – to ta l a bn or m al iti es , m ea ns a re fo llo w ed b y lo w er ca se le tt er is s ta tis tic al ly s ig ni fic an t a t p < 0 .0 5. ta bl e 4. g am m a ra y an d c om bi na tio n tr ea tm en t ( g am m a+ em s) o n so m e qu al ita tiv e an d qu an tit at iv e tr ai ts o f c hl or op hy ll m ut an ts s ee dl in g in a rt em is ia a nn ua l . ( m 2 ge ne ra tio n) . m or ph ol og ic al tr ai ts c on tr ol m ea n± s. e. c hl or op hy ll m ut an t l in es 10 0 g y m ea n± s. e. 20 0g y m ea n± s. e. 30 0g y m ea n± s. e. 10 0± 0. 1% m ea n± s. e. 20 0± 0. 1% m ea n± s. e. 30 0± 0. 1% m ea n± s. e. pl an t h ei gh t ( cm ) 10 3. 20 ±0 .8 8b 11 2. 30 ±1 .0 1a 10 8. 22 ±1 .6 0b 86 .3 0± 1. 48 c 11 0. 30 ±1 .0 9a 88 .3 0± 1. 44 b 52 .5 0± 1. 30 c in te rn od al le ng th ( cm ) 7. 50 ±0 .0 9a b 8. 60 ±0 .1 3a 7. 40 ±0 .1 5b 6. 90 ±0 .1 3c 8. 31 ±0 .1 1a 6. 30 ±0 .1 5b 5. 60 ±0 .1 4c le af a re a (p er m 2 ) 35 .6 0± 0. 48 b 38 .2 0± 0. 47 a 28 .5 0± 0. 46 c 27 .8 0± 0. 42 c 34 .2 0± 0. 42 b 26 .6 0± 0. 49 b 22 .3 0± 0. 43 c n o. o f p ri m ar y br an ch es 25 .0 0± 0. 86 b 30 .0 0± 1. 08 a 27 .0 0± 0. 86 b 26 ±1 .0 6a 21 ±0 .8 3a b 19 ±0 .7 6b 17 ±0 .6 3c d ay s to 5 0% fl ow er in g 56 .0 0± 0. 43 b 50 .0 0± 0. 57 c 55 .0 0± 0. 60 b 66 .0 0± 2. 16 a 52 .0 0± 1. 03 b 65 .0 0± 2. 60 a 79 .0 0± 2. 79 a d ay s to m at ur ity 15 5. 00 ±1 .0 6b 14 5. 00 ±4 .5 0c 15 0. 00 ±4 .0 1a b 17 0. 00 ±3 .5 5a 14 3. 00 ±1 .0 0c 17 2. 00 ±3 .0 5a 15 9. 00 ±3 .6 8b a bb re vi at io ns : s .e . s ta nd ar d er ro r, m ea ns a re fo llo w ed b y lo w er ca se le tt er is s ta tis tic al ly s ig ni fic an t a t p < 0 .0 5 23analyzing frequency and spectrum of chlorophyll mutation induced through gamma ray and combination treatment discussion in mutation breeding programs, the selection of an effective and efficient mutagen concentration and growth condition is essential to produce a high frequency of desirable mutations(arisha et al. 2014). chl mutation frequency in m2 generation is one of the most dependable measures for evaluating the mutagen-induced genetic alternations. the spectrum of chl mutations was found to be dependent on the genetic background of the genotype. moreover chl mutation frequency increased with the increase in dose of gamma rays both individually as well as in combination with ems in all the varieties. in the present investigation the germination percentage and plant survival were reduced significantly. the reduction in germination may be due to the seeds engrossing the mutagen, which subsequently reaches the meristematic regionof seeds and affects the germ cell (serrat et al. 2014). also, a reduction in germination may be because of the damage of cell constituents (kumar et al. 2013), alteration of enzyme activity or delay or inhibition of physiological and biological processes (talebi et al. 2012).reduction in plant survival in treated population may occur due to various factors such as cytogenetic damage and physiological disturbances (sato and gaul 1967)and disturbances in balance between inhibitors of growth regulators and promoters (meherchandani 1975). ionizing radiation singly not produces much chl mutation as combination treatment produces. among the chemical mutagens, ems is now being widely accepted as the most efficient and influential mutagen which induces highfrequency and wide spectrum of mutation. when ems combined with radiation it not only causes synergistic effect but affect mutation in a specific ways. singh (et al. 1999) reported that combined treatments of gamma rays and emswere most effective in producing chl mutation frequency than their individual treatments in vigna chl mutationsinduced by ems, gamma rays and other mutagens applied individually or in combination were reported by a kumar (et al. 2009) and gandhi (et al. 2014) in vigna radiate, bolbhat and dhumal (2009) and kulkarni and mogle (2013) in macrotyloma uniflorum (lam.)  verdc., sharma et al. (2010) in pisum sativum l. gaur et al. (2013) in capsicum annuum. lower doses of gamma and gamma+ems mutation frequency increases but it significantly decreased at the higher doses. sharma (1970) reported that chl mutation frequency decreased at higher doses when calculated on segregating m1 familiesbasis. for both the treatment higher frequency of chl mutation with moderate doses of mutagens was observed. it seems that the strong mutagens reach their saturation point even at lower or moderate doses in the highly mutable genotype. with increase in dose further than a limit, the strong mutagens become more toxic than the higher doses of relatively weaker mutagens and do not increase mutation frequency (kolar et al. 2011). moreover mutation frequency observed maximum in gamma+ems treatment both in m1 and m2 generation in comparison to gamma. it may be due to ems, a chemical mutagen which causes formation of new sites for mutation. so use of gamma followed by ems suggests that the chemical mutagen is more efficient in inducing mutations of genes needed for chl development (shah et al. 2006). in both the treatment, highest frequency of xantha mutant was observed, the highest frequency of xantha mutants ismay be due to the genes for xanthophylls development that are readily accessible for mutagenic action ( similar reports were already given by lal (et al. 2009), khan (et al. 2005), haq (1990) . these mutants could not survive more due to block in chl synthesis (blixt 1961). in gamma, after xantha viridis was the second highest mutant, these mutant survived tillmaturity. viridis attributed may be due involvement of polygene genes for the formation of chl. in combinedtreatment second highest mutant recorded was albino, these type of mutant formed may be due to deficiency ordegradation of chl formation enzyme. in chickpea all chl mutants including albina type were in general morefrequent in ems treatments than in gamma rays (singh 1988). chl is a vital biomolecule which plays a critical role in the life processes of allplants. plants photosynthesis by absorbing light and transferring light energy to the reaction centers (chl molecule)of the photosynthetic system. thus, chl is essential for plant development and agricultural production (eckhardt et al. 2004; flood et al. 2011). chl development seems to be controlled by many genes that are located on different chromosomal sites (wang et al. 2013). it derivesby the formation of a long chain of biochemical process in which lots of loci were involved. the phenotypes of leaf color mutations are varied and are affected by different genetic and environment factors. mutant plants leaf shows lower or higher chl content than normal leaf. this revealed that rate of change in the content of chl a and chl b was not the same among the mutants, possibly was due to the impair of chl b synthesis during chloroplast development (kolar et al. 2011). ionizing radiation and chemical mutagen at higher concentration affectschloroplast thylakoid membrane which causes disability in chl manufacturing. this chl deficiency reduced the rateof plant growth. mutant plants with a higher (nielsen et al. 1979) like viridis or a lower like chlorine, xantha (vaughn et al. 1978; wu et al. 2007) chl a/b ratiothan that of their 24 girjesh kumar, rajani singh respective normal plants have been reported to be able to survive photoautotrophically. mutations affecting the production of chl are important for identifying gene function and the elucidation of chl metabolism and its regulation (wu et al. 2007). cy tologica l investigation def ines t he specif ic responses of different genotypes to a specific mutagen and it is also provides significant evidences for the selection of desirable traits (kirchhoff et al. 1989).in the present appraisal chl mutant depicts various anomalies such as , stickiness, multivalent, precocious movement of chromosome, laggard and bridges. stickiness could arise due to depolvmerization of ’ nucleic acid caused by mutagenic treatment (avijeet et al. 2011). the formation of multivalent (fig. 6 g) may also be attributed to the abnormal pairing and non-disjunction of bivalents (jabee et al. 2008). jafri (et al.2011) suggested that precocious movement (fig. 6 d&i) of chromosomes was probably caused by spindle dysfunction. laggard formation (fig. 6f,k &l) is due to delayed terminalisation, chromosomal stickiness or failure of chromosomal movement (reddy and munirajappa 2012). due to direct action of mutagen target proteins gets defective and creates the disturbance during chromosome separation and it forms bridges (kumar and gupta 2009). the increment in the chromosomal aberrations might perhaps be due to the interactions of ionizing particles with the protoplasm, mediated through the excitation introduced by radiation that ultimately has increased the aberration frequency (shukla nee tripathi and kumar 2010). when mutagens affects plant tissues internally it get accommodated and damage the cell or genes whichphenotypically can be seen. genetically, during the m1 generation the probability of the occurrence of phenotypicmutation is extremely low and only dominant mutations can be identified (roychowdhury and tah 2013). during the m2 generation, the chancefor identifying visible changes or phenotypic mutations should be higher and mostly due to genetics. thereforeobserved mutations in the m2 generation are considered more stable (parry et al. 2009). the plant height at lower doses of gamma and gamma+ems was significantly increased but higher doses of both the treatment causes inhibition /depression in plant growth. as according to van harten (1998) said, at high amount irradiation could cause the physiological damages such as inhibit cell division, death of cell and growth rate and genetic changes on the plants by producing free electrons radical. internodal length, leaf area and primary number of branches were increased at 100gy dose of gamma. these characters were significantly higher at lower concentration but at higher concentration it shows a reduction pattern. treatment of gamma rays and ems exhibited increase in mean values of number of primary branches in lathyrus sativus (waghmare and mehra (2000). in mutation breeding programme, yield and its attributed traits are very important parameters because ultimately breeders want to improve yield and related characters (shahwar et al. 2020). similar result was givenby hanafiah (et al. 2010) in irradiated glycine max (l.) merr., var. argomulto seeds with gamma rays showsphenotypic variations that occur on m1 plants which affects plant growth development and production and also was given by sinuraya (et al.2017) in allium cepa assay. thilagavathi and mullainathan (2011)concluded that the decrease in quantitative traits have been attributed to the physiological disturbance or chromosomal damage caused to the cellsof the plant by the mutagen as williams (et al.1990) observed that due to nucleotide substitutions and insertion or deletions polymorphism occurred between individuals. in comparison to control treated plant at lower dose (100gy,200gy, 100+0.1%) flowers early and matures more rapidly. at 200gy+0.1% dwarf mutant with early maturing plant was identified. panigrahi (et al.2015) suggested that significant variations in quantitative parameters may showstable gene mutations in the next generations. konzak (et al.1969) in wheat and shakoor (et al. 1978) in triticale reported that polygenes are responsible for semi dwarf character. qin (et al.2008) reported dwarf rice mutants caused by single gene. increased height and number of branches were due to loss of apical dominance which leads to lateral transport of growth hormone which results increased number of branches and bushy appearance as also observed earlier in vicia faba (shahwar et al. 2017), lentil (solanki et al. 2004). physical mutagen causes random change in the growth regulatory genes of plants but chemical mutagen exactly targets their mutagenic site through point mutation. this is the reason combination treatment proved to be more mutagenic and produces good amount of mutants. the selection of effective and efficient mutagens is most important to recover the spectrum while high frequency of desirable mutations and efficiency of a mutagen indicates relatively less biological damage in relation to induced mutation (solanki and sharma 1994). conclusion this investigation revealed the potency of gamma and in combination with ems ,on increasing genetic diversity and demonstrated the successful program of induced mutagenesis in the artemisia annual. in this breeding programme a total of 44 mutant in gamma and 25analyzing frequency and spectrum of chlorophyll mutation induced through gamma ray and combination treatment 67 mutants in combination treatment were segregated. various chl variants were identified in treated sets. xantha was predominant among all the variants as most of the leaves found pale yellow colour. it is noticed that these changes are differentially sensitive to gamma and gamma+ems and the appearance of new mutants would very helpful in maintaining the genetic purity of plant variety. so it should be important to identify desirable mutant plants through isolation and selection method. the cytological analysis of these mutants showed that these changes were induced due to changes in chromosome number, structure, base substitution and deletion. for artemisia ld50 recorded as 200gy. 100gy and 100+0.1%ems was noticed good for plants growth and development as plant height, internodal length, leaf area and primary number of branches improved. so gamma and gamma+ems induced reasonable chl mutations, hence all these treatments could be used in mutation breeding programs for inducing viable mutations only threshold dose of mutagen should be identified. acknowledgements authors are obliged to nbpgr, nainital for providing certified seeds of artemisia annua l. and also of nbri, lucknow for 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in chlorophyll biosynthesis. plant physiol. 145:29–40. caryologia. international journal of cytology, cytosystematics and cytogenetics 75(3): 135-144, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1780 caryologia international journal of cytology, cytosystematics and cytogenetics citation: zahra noormohammadi, masoud sheidai, seyyed-samih marashi, somayeh saboori, neda moradi, samaneh naftchi, faezeh rostami (2022). identification of genetic regions associated with sex determination in date palm: a computational approach. caryologia 75(3): 135-144. doi: 10.36253/ caryologia-1780 received: august 12, 2022 accepted: november 24, 2022 published: april 5, 2023 copyright: © 2022 zahra noormohammadi, masoud sheidai, seyyed-samih marashi, somayeh saboori, neda moradi, samaneh naftchi, faezeh rostami. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid zn: 0000-0003-3890-9001 identification of genetic regions associated with sex determination in date palm: a computational approach zahra noormohammadi1,*, masoud sheidai2, seyyed-samih marashi3, somayeh saboori1, neda moradi1, samaneh naftchi1, faezeh rostami1 1 department of biology, science and research branch, islamic azad university, tehran, iran 2 faculty of biological sciences and biotechnology, shahid beheshti university, tehran, iran 3 date palm & tropical fruits research center, horticultural sciences research institute, agricultural research, education and extension organization (areeo), ahwaz, iran *corresponding authors. e-mail: marjannm@yahoo.com, z-nouri@srbiau.ac.ir abstract. sex determination of date palm seedlings is the challengeable effort for breeders. different studies based on molecular markers and genome sequencing have provided some insight in to the genetic regions related to sex determination in date palms in general. but due to differences in cultivar population structure and also cost of whole genome sequencing, we may need a more suitable approach in developing countries for this task. therefore, we suggest using a combination of different available molecular markers and a computational approach to identify the genetic regions involved in sex differentiation in date palm cultivars. in this study we used twenty-three cultivars including 7 male and 16 female cultivars that were examined by 30 different dominant and co-dominant molecular markers which deal with different genomic regions. grouping of the tree samples based on 178 loci resulted in genetic differentiation of the studied male and female palm trees. multiple correspondence analysis (mca) bi-plot also showed genetic difference within male and female trees. heatmap plot specified those markers which differentiate date palm trees. ssr (simple sequence repeats) and irap (inter retrotransposon amplified polymorphism) markers provided sex linked markers for male cultivars. in present study, we introduced sex specific alleles for iranian male date palm cultivars as a fast track in seedlings. different association studies performed identified the candidate genetic regions which are significantly associated with sex differentiation in date palm cultivars. keywords: dna based markers, dapc, lfmm, mca, phoenix dactylifera, sex determination. 1 introduction date palm (phoenix dactylifera, arecaceae) is the one of the most important fruit products (more than 1.3 m tonnes, faostat, 2019) in 136 zahra noormohammadi et al. iran. date palm trees are propagated by both offshoots and seeds. each female tree produces 0-3 offshoots per year. this kind of propagation is not in a way of classical breeding along with low diversity (adawy et al. 2014). seed germination is supposed to be easiest way for propagation with high heterozygousity while, it is a time consuming method and may take more than 5 years before flowering. date palm is dioecious and the only species of the palmae with sex chromosomes with 2n =36. in fact, this phenomenon makes difficulty for identification of female trees at early stages of growth (maryam et al. 2016). using cytological and biochemical methods may not be used to discriminate date palm male trees from the female trees. however, recently, researchers reported the presence of the occurrence of sex-specific loci in date palm trees (elmeer and mattat 2012, adawy et al. 2014, al-ameri et al. 2016, hassanzadeh and bagheri 2019). these authors used different molecular markers like random amplified polymorphic dna (rapd), inter simple sequence repeat (issr), simple sequence repeat (ssr), sequence characterized amplified region (scar) and start codon targeted polymorphism (scot). different genome sequencing approaches have been carried out on limited number of palm trees of different palm species (torres et al. 2018) to date plan cultivars (al-dous et al. 2011, hazzouri et. al. 2019). these studies have identified the genomic regions associated with sex determination and also a conserved two-locus system present in all palm species. however, due to possible interference of population genetic structure in different date palm cultivars cultivated throughout the world and the high expenses of whole genome sequencing, we suggest to use available molecular markers and analyze the results by using computational approaches to locate the genetic regions associated with dates palm sex determination. therefore, the present study was performed with following objectives:1-to identify sex-specific alleles for iranian male date palm cultivars by using a combination of seven dominant and co-dominant molecular makers like, ssr, issr, scot, irap (inter retrotransposon amplified polymorphism), remap (retrotransposon microsatellite amplified polymorphismand srap (sequence related amplified polymorphism) for date palm sex determination and, 2identify the candidate genetic regions involved in sex determination by applying different computational methods like, fisher exact test, dapc (discriminant analysis of principal components analysis), and lfmm (latent factor mixed model, collins and jombbart 2015, zhang et al. 2019). 2. materials and methods 2.1. plant materials and genetic analysis in total we studied 23 date palm cultivars, of which 7 were male (including: sabzparak, ghannamisabz, wardi, ghannamisorkh 1, ghannamisorkh 2, foreign male 1 and foreign male 2) and 16 female cultivars. two to three trees from each cultivar were randomly selected for molecular studies (totally 63 trees). details of the cultivars with their accession numbers are provided in our previous study (saboori et al. 2021). all cultivars are located omol-tomair station of date palm & tropical fruits research center, ahwaz, iran. for genetic analysis, genomic dna was extracted based on saboori et al. (2021). seven different molecular markers as ssr (6 loci), est-ssr (3 loci), scot (4 loci), issr (5 loci), srap (5 loci), irap (3 loci) and remap (4 loci) were examined for sexual determination (table s1). the touch-up pcr program was used for ssr markers; 94°c for 5 min, initial 10 cycles at 95°c for 30 sec, annealing step for 1 min at 51°c, 47.5 °c, 62 °c, 47.5°c, 46.9 °c and 45 °c for mpdcir078, mpdcir085, pdcuc3-ssr2, mpdcir090, mpdcir048 and mpdcir025 loci respectively, 72°c for 1 min and 30 sec. then 30 cycles were set at 95°c for 30 sec, annealing step (mpdcir078 52°c, mpdcir085 49.9 °c, pdcuc3-ssr2 65 °c, mpdcir090 49.9 °c, mpdcir048 48.8 °c and mpdcir025 48 °c) for 1 min. the extension segment was at 72°c for 1 min and 30 sec following final extension at 72 °c for 15 min. for est-ssr the following procedure was used; 5 min at 94 ºc, 30 sec at 95 ºc, 30 sec at 50 ºc, 52 ºcand 60 ºc for est-pdg3119-rubisco, est-gte and estdpg0633laccase loci respectively and 1 min and 30 sec at 72 ºc. these segments repeated for 35 cycles. final extension was for 5 min at 72 ºc. the scot loci were amplified based on our previous study (saboori et al. 2020) and its data used for sexual determination. the pcr reaction for the issr markers were performed according to the following thermal program: 5 minutes at 94°c, 40 cycles of 30 seconds at 94°c, 60 seconds at 50-52.5°c (50°c for (ga)9t, (ca)7at, and (ga) 9a, 52/5°c for (gt)8ya and (ag) 8yt) and 90 seconds at 72°c and final extension for 7 minutes at 72°c. molecular marker reactions were carried out in 25 µl volume with 20 ng genomic dna and 5 u of taq dna polymerase (bioron, germany), 2x pcr buffer (50 mm kcl; 10 mm tris-hcl, ph;8), 1.5-2 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 µm of each primer. for irap and remap markers, we used thermal program: 3 min of initial denaturation at 94°c, followed 137identification of genetic regions associated with sex determination in date palm: a computational approach by 40 cycles of 94°c for 3 min and annealing temperature 51-54 °c for iraps and 51-56 °c for remaps for 30 s, 72°c for 3 min, and 10 min at 72°c for final extension. all reactions were set up at biorad thermocycler, usa. the band profile of each locus was visualized by using 2.5 % agarose gel electrophoresis. staining of gels were performed by the sybergreen dye. we used the 100 base pair (bps) molecular size ladder for estimation of fragment size (fermentas, germany). 2.2. data analysis all obtained bands of 30 loci were scored as a binary data. multiple correspondence analysis (mca) of molecular data was performed to group the studied palm trees based on sex differentiation as performed in r-package 4.1(abdi and williams 2010). amova test was performed for differentiation of two male and female groups by using genalex ver. 6.5. for association studies, different statistical and bioinformatic approaches are available which have different assumptions. we used dapc (discriminant analysis of principal components), and bayesian based method of lfmm (latent factor mixed model), as suggested by frichot et al (2013), and collins and jombbart (2015). these analyses were followed by bonferroni correction as well as false discovery rate (fdr) tests, to avoid both type i and type ii errors of rejecting true association results. these were done in r package 4.1. similarly, for accuracy of the model presented by. dapc we used cross validation method as implemented in r. 4.1. 3. results in total we obtained 178 loci/ bands by different molecular markers studied. ward dendrogram (fig. 1), differentiated between the studied male and female palm trees as the samples of either group were placed in a separate cluster. therefore, it shows that the molecular markers used in present study can differentiate palm trees based on different sexes. multiple correspondence analysis (mca) of molecular data obtained revealed that about 60% of total genetic variation of the studied samples may be explained by about 10 pca axis (fig. 2). the grouping of the studied palm trees by mca biplot by using the first two pca axes (fig. 3), not only separated male and female trees from each other but also revealed some degree of genetic difference within male and female samples studied. in general, two different genetic groups can be seen in either of these sexes. a heat-map was constructed figure 1. ward dendrogram of male (m) and female (f) date palm trees based on combined molecular data. 138 zahra noormohammadi et al. (fig. 4) to group those molecular markers which show similarity in differentiating palm tree samples. a mova test a lso con f i r med d i f ferent iat ion between two male and female groups (r = 0.129, p = 0.001, table s2). the primers with highest degree of contribution to genetic differentiation of male and female date palm trees have been shown in fig. 5. primer bands irapnikita_2000, 3’ltr_100, issr-(ca)7at-400, ssr-mpdcir048-120, are among the most contributing primers of the first mca axis. similarly, primer bands irapnikita_200, ssr-mpdcir048-200, nikita+ssr2 _100, are among the primers which highly contributed in mca axis 2. a much more detailed information can be obtained from mca-biplot (fig. 6), which shows the presence (y), and absence (n), of the particular primer bands contributed in separating female (numbers 1-20 in fig. 5), and male (numbers 21-40, in fig. 5) palm trees. it is evident from this biplot that both presence and absence of different primer bands of the utilized molecular markers can together differentiate male palm trees from females. we found some loci which a re specif ic in male date palm cultivars (table 1, fig. s1). the mpdcir048(ga)32-ssr locus, showed specific allele between 100-130 bps. the 110 bps band was unique for all male trees including sabzparak, ghannamisabz, wardi, ghannamisorkh, ghannamisorkh2, foreign male1 and foreign male2 cultivars. the 130 bps allele was observed only in ghannamisorkh1, ghannamisorkh2, foreign male1 and foreign male 2 while, allele in size of 120 bps was unique in two male cultivars (sabzparak and wardi). in est-ssr data, one allele in est-pdg3119-rubisco with 200 base pairs in size showed specificity in all male cultivars except sabzparak male cultivar. simi larly, ir ap-3ltr locus produced a l leles between 400 to 1600 bps which were specific at some of the male trees (table 1). for instance, the band in 300 bps in length was amplified in all male trees except sabzeparak male cultivar and were absent in all female date palm trees. figure 2. multiple correspondence analysis (mca) based on 30 loci studied on date palm trees. figure 3. mca biplot: grouping date palm trees based on first two components. numbers are individuals. color gradient shows amount of variance based on mcafrom high to low. m; male trees and f: female trees studied. figue 4. heatmap cluster based on 30 molecular loci and male (m) and female (f) date palm trees studied. 139identification of genetic regions associated with sex determination in date palm: a computational approach 3.1. association studies molecular markers and sex differentiation all association approaches produced almost similar results and identified the same loci as the genetic regions which are associated with sex differentiation. for example, fisher exact test and dapc identified three following marker loci after bonferroni correction at p = 0.01: x18 (srap loci), x50 x51 x53 x64 (irap loci), x91 x98 x108 (remap loci), and x171 x174 x176 (ssr loci). these loci can efficiently differentiate male versus female date palm trees studied (fig. 7). similarly, lfmm analysis of both lasso and ridge methods produced almost similar results after bonferroni correction and false discovery rate (fdr) test (fig. 8). the loci which identified are in agreement with the results of fisher exact test and dapc presented before. therefore, a combination of molecular markers may be used in early-stages sex determination in date palm cultivars with focus on highly associated marker loci. 4. discussion sex determination is one of the main concerns of date palm breeders. different studies have been reported to introduce proper biomarkers but they are restricted to few cultivars. recent gwas study revealed 112 snps related to sex determination in a region with ~6 mb at lg 12 while other 43 snps dispersed on other date palm figure 5. mca axes. 1 and 2, showing primer bands with highest degree of contribution in date palm male and female differentiation. figure 6. mca-biplot based on alleles studied. red numbers : alleles (y=presence and n=absence), blue number: date palm trees numbers. 140 zahra noormohammadi et al. chromosomes (hazzouri et al. 2019). torres et al. (2021) also reported four conserved genes in phoenix males. some mutations in putative genes involved in sex determination (cyp703 and gpat3), is supposed to repress recombination in these regions, leading to gynodioecy and therefore result in establishing male sex in palm tree (torres et al. 2021). however, genetic variations have been observed in both sex linked and autosomal regions. it may call more effort to find proper sex biomarker for date palm cultivars. in present study, we used 30 different loci may be located in different genome regions. base on the allele data, 60% of alleles covered the most variable alleles. the first two mca components showed the highest level of variation and enabled to differentiate male and female trees. most of the identified alleles in these two components belong to ssr and irap loci (fig. 5). we used multiple correspondence analysis (mca) as a statistical precise method for categorical data (binary data, abdi and williams, 2010) mca bi-plot provides a picture which illustrates the variable that can discriminates the studied individual mca bi-plots (fig. 6) depicted all 250 alleles of 30 loci studied. it clearly shows the contribution of each allele to discrimination of male and female trees. moreover, different association studies in present paper identified genomic regions which are associated with sex differentiation in date palm cultivars. in details, we introduced male specific alleles for six important iranian male date palm cultivars. the mpdcir048 ssr locus has two alleles (100, 130 bps) which could discriminate all male cultivars studied (table 2). this ssr locus located in cytosine-s-methyltransferase drm2-like (drm2). jskani et al. (2016) also reported figure 7. dapc plot showing differentiation of female (1) versus male (2). date palm trees based on associated marker loci. figure 8. lfmm results showing associated marker loci with sex differentiation in date palm cultivars studied. 141identification of genetic regions associated with sex determination in date palm: a computational approach male specific alleles of this locus but in different size (250bps) while we detected that allele in both male and female trees. the 190/160 allele pattern for this ssr locus was reported by elmeer and mattat (2012) in qatar male date palm trees. maryam et al. (2016) also reported male specific alleles in this locus (250/250) for pakestani cultivars. it clearly shows the pivotal role of this locus in sex determination although different allelic patterns may be obtained from different male cultivars due to genetic variations of the studied cultivars. on the other hand, wang et al. 2020 reported that mpdirdp52 was sex linked for cultivars collected from china. issr markers as a dominant marker showed less discrimination power for sex discrimination. it is in agreement with the results of the study performed by hassanzadeh khankahdani and bagheri (2019). in other plants like trichosantes dioica roxbi. and hippophae rhamnoides ssp. turkestanica issr markers could show sex discrimination (nanda et al. 2013, adhikari et al. 2014,das et al. 2017) retrotansposone based markers with their abundance, mode of amplification and insertion into the genome, have characteristics suitable for discriminate between species or genotypes (biswas et al. 2010). about 21% of whole genome of p.dactlylifera defined for retrotransposons, mostly including ty1/copia and ty3/gypsy (al-dous et al. 2011, al-mssallem et al. 2013, nouroz and mukaramin 2019). we used irap and remaps’ profiles between male and female date palm trees. irap loci are among loci with the high degree of contribution to total variance in our mca analysis. however, only one of irap loci (3´ltr) showed sex discrimination in 5 male cultivars. this a first report on malelink irap markers in date palm trees. adawy et al. (2014) introduced two scot alleles (850 bps and 1150 bps) in scot36 and scot4 in egyptian female date palm trees as sex-linked markers while in present study we observed these alleles in both gender. cdna-scot allele based on flowering stage was also used for development of scar maker by al-ameri et al. (2016) for determination of male trees of saudi arabia date palm cultivars. they believed that the different gene expression patterns in flowering stage in male and female trees may provide a simple and inexpensive tool for sex determination. however, our literature reviews revealed that sex linked markers in date pale cultivars are specified in local orchards and restricted to the countries. with regard to multilocus nature of ssr markers, it may make different size of alleles for one locus in different cultivars with different geographical locations probably due to newmutations and allele adaptation. 5. conclusion the urgent method for sex determination in date palm nursery orchards is pivotal for breeding industry. our data could provide the highest variant alleles among 30 loci studied by mca for sex determination. we also introduced sex specific alleles for male cultivars as a fast track in seedlings. acknowledgment we acknowledge science and research branch, islamic azad university for providing laboratory. author contribution statement z.n.: conceptualization of the project; m.sh.: analyses of data; s.s, n.m., s.n., f.r.: data collection and lab table 1. specific alleles in male date palm trees. m1 (sabzparak), m2 (ghannamisabz ), m3 (wardi), m4 (ghannamisorkh 1), m5 (ghannamisorkh 2), m6 (foreign male1), m7 (foreign male 2) locus/male m1 m2 m3 m4 m5 m6 m7 ssr (bp) mpdcir048 110/120 100/120 100/120 110/110 110/110 110/110 110/110 110/110 110/120 110/130 110/110 110/130 110/130 110/130 110/130 110/130 110/130 110/130 110/130 110/130 110/130 estpdg3119-rubisco 200 200 200 200 200 200 irap-3’ltr(bp) 400/500 300/500/600/ 300/500/600/ 100/200/300 400/500/600 700/800/900 1500 100/200/300 400/500/600 700/750/1500 100/200/300 400/600/700 900/1000/1500 1600 100/200/300 400/600/800 1500/1600 142 zahra noormohammadi et al. work; s.m.: providing samples; z.n, m.sh. s.m. and s.s. project design data archiving statement all tree samples used in this research are being archived in herbarium of shahidbeheshti university, tehran, iran. the accession numbers will be supplied once available. references abdi h, williams lj (2010) principal component analysis wiley interdisciplinary reviews: comput stat 2: 433459. adawy ss, jiang j, atia ma (2014) identification of novel sex-specific pcr-based markers to distinguish the genders in egyptian date palm trees. int j agric sci res 4: 45-54. adhikari s, saha s, bandyopadhyay tk, ghosh p (2014) identification and validation of a new male sexspecific issr marker in pointed gourd (trichosanthes dioica roxb.). sci world j;2014:216896. doi: 10.1155/2014/216896. al-ameri aa, al-qurainy f, gaafar arz, khan s, nadeem m (2016) molecular identification of sex in phoenix dactylifera using inter simple sequence repeat markers. biomed res intl 2016: 1-5. al-dous ek, george b, al-mahmoud m e, al-jaber my, wang h, salameh ym, malek j a (2011) de novo genome sequencing and comparative genomics of date palm (phoenix dactylifera). nature biotechnol 29: 521. al-mssallem i s, hu s, zhang x, lin q, liu w, tan j, yu j (2013) genome sequence of the date palm phoenix dactylifera l. nature commun 4: 1-9. biswas mk, xu q, deng xx (2010) utility of rapd, issr, irap and remap markers for the genetic analysis of citrus spp. sci hort 124: 254-26. collins c, jombbart t (2015) genome-wide association studies. impeerial college london. mrc center. for outbreak analysis and modeling: 1-61. das k, ganie sh, mangla y. et al. 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(2019) osca: a tool for omic-data-based complex trait analysis. genome biol 20: 107 . https://doi.org/10.1186/s13059-019-1718-z wang y, ihase lo, htwe ym, shi p, zhang d, li d, et al. (2020) development of sex-linked ssr marker in the genus phoenix and validation in p. dactylifera. crop sci 60: 2452-2466. 143identification of genetic regions associated with sex determination in date palm: a computational approach supplementary data table s1. molecular marker primers, their names and sequences used in this study. marker locus name sequence (5´-3´) ssr (bodian et al. 2014) mpdcir025(ga)22-f gcacgagaaggcttatagt mpdcir025(ga)22-r cccctcattaggattctac mpdcir048(ga)32-f cgagacctaccttcaacaaa mpdcir048(ga)32-r ccaccaaccaaatcaaacac mpdcir078(ga)13-f tggatttccattgtgag mpdcir078(ga)13-r cccgaagagacgctatt mpdcir085(ga)29-f gagagagggtggtgttatt mpdcir085(ga)29-r ttcatccagaaccacagta mpdcir090(ga)26-f gcagtcagtccctcata mpdcir090(ga)26-r tgcttgtagcccttcag pdcuc3-ssr2(ga)22-f acattgctcttttgccatgggct pdcuc3-ssr2(ga)22-r cgagcaggtggggttcgggt est-ssr (zhao et al. 2012) est-pdg3119-rubisco-f catactgattattggcacacc est-pdg3119-rubisco-r gtaccataccgtaccagttca (zhao et al. 2012) est-dpg0633laccase -f agactggttaagttggtggag est-dpg0633-laccase-r ctacaaaactgatgtggtggt est-gte-f gcttggccatctatgaaac est-gte-r actctgagcatccatatcg scot (collard and mackill 2009) scot1 caacaatggctaccacca scot2 caacaatggctaccaccc scot36 gcaacaatggctaccacc scot41 caatggctaccactgaca issr (ga)9t gagagagagagagagagat (ca)7at cacacacacacacaat ubc849 gtgtgtgtgtgtgtgtya (ga)9a gagagagagagagagagaa ubc834 agagagagagagagagyt srap (feng et al. 2014) me1 tgagtccaaaccggata me4 tgagtccaaaccggacc em3 gactgcgtacgaattgac em4 gactgcgtacgaatttga irap nikita cgcatttgttcaagcctaaacc 3’ltr tgtttcccatgcgacgttccccaaca 5’ltr1 ttgcctctagggcatatttccaaca remap ssr_pdcir025_r + nikita ssr_pdcir025_r + 3’ltr ssr_pdcir048_r + nikita ssr_pdcir048_r + 5’ltr1 144 zahra noormohammadi et al. table s2. amova test based on date palm trees in two groups: male and female trees. df; degree of freedom, ss: sum of square, ma: mean of square, var: variance. source df ss ms est. var. var% among pops 1 135.730 135.730 3.491 13% within pops 68 1598.284 23.504 23.504 87% total 69 1734.014 26.996 100% stat value p value phipt 0.129 0.001 figure s1. allele pattern of some femal and male date palm trees. left to right: first 5 samples are female and 6-9 male trees. caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 3 2022 firenze university press chromosome mapping of repetitive dnas in the picasso triggerfish (rhinecanthus aculeatus (linnaeus, 1758)) in family balistidae by classical and molecular cytogenetic techniques kamika sribenja1, alongklod tanomtong1, nuntaporn getlekha2,* chromosome number of some satureja species from turkey esra kavcı1, esra martin1, halil erhan eroğlu2,*, fatih serdar yıldırım3 l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations selma tabur1,*, nai̇me büyükkaya bayraktar2, serkan özmen1 characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques kristel ramírez-matadamas1, elva irene cortés-gutiérrez2, sergio moreno-limón2, catalina garcía-vielma1,* contributions of species rineloricaria pentamaculata (loricariidae:loricariinae) in a karyoevolutionary context a cius¹, ca lorscheider2, lm barbosa¹, ac prizon¹, ch zawadzki3, la borin-carvalho¹, fe porto4, alb portela-castro1,4 cadmium induced genotoxicity and antioxidative defense system in lentil (lens culinaris medik.) genotype durre shahwar1,2,*, zeba khan3, mohammad yunus khalil ansari1 biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts and evaluation of their biosafety potential denisa manolescu1,2, georgiana uță1,2,*, anca șuțan3, cătălin ducu1, alin din1, sorin moga1, denis negrea1, andrei biță4, ludovic bejenaru4, cornelia bejenaru5, speranța avram2 polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel (foeniculum vulgare mill.) egizia falistocco methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. sazada siddiqui*, sulaiman a. alrumman comparative study and genetic diversity in malva using srap molecular markers syamand ahmed qadir1, chnar hama noori meerza2, aryan mahmood faraj3, kawa khwarahm hamafaraj4, sherzad rasul abdalla tobakari5, sahar hussein hamarashid6,* nuclear dna 2c-values for 16 species from timor-leste increases taxonomical representation in tropical ferns and lycophytes inês da fonseca simão1, hermenegildo ribeiro da costa1,2,3, helena cristina correia de oliveira1,2, maria helena abreu silva1,2, paulo cardoso da silveira1,2,* nuclear dna content and comparative fish mapping of the 5s and 45s rdna in wild and cultivated populations of physalis peruviana l. marlon garcia paitan*, maricielo postillos-flores, luis rojas vasquez, maria siles vallejos, alberto lópez sotomayor identification of genetic regions associated with sex determination in date palm: a computational approach zahra noormohammadi1,*, masoud sheidai2, seyyed-samih marashi3, somayeh saboori1, neda moradi1, samaneh naftchi1, faezeh rostami1 comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(4): 103-109, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1875 caryologia international journal of cytology, cytosystematics and cytogenetics citation: weera thongnetr, suphat prasopsin, surachest aiumsumang, sukhonthip ditcharoen, alongklod tanomtong, prayoon wongchantra, wutthisak bunnaen, sumalee phimphan (2022). first report of chromosome and karyological analysis of gekko nutaphandi (gekkonidae, squamata) from thailand: neo-diploid chromosome number in genus gekko. caryologia 75(4): 103-109. doi: 10.36253/caryologia-1875 received: september 11, 2022 accepted: december 13, 2022 published: april 28, 2023 copyright: © 2022 weera thongnetr, suphat prasopsin, surachest aiumsumang, sukhonthip ditcharoen, alongklod tanomtong, prayoon wongchantra, wutthisak bunnaen, sumalee phimphan. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. first report of chromosome and karyological analysis of gekko nutaphandi (gekkonidae, squamata) from thailand: neo-diploid chromosome number in genus gekko weera thongnetr1, suphat prasopsin2, surachest aiumsumang3,*, sukhonthip ditcharoen4, alongklod tanomtong5, prayo on wongchantra6, wutthisak bunnaen7, sumalee phimphan3 1 division of biology, department of science, faculty of science and technology, rajamangala university of technology krungthep, bangkok 10120, thailand 2 research academic supports division, mahidol university, kanchanaburi campus, saiyok, kanchanaburi 71150, thailand 3 biology program, faculty of science and technology, phetchabun rajabhat university, phetchabun 67000, thailand 4 division of biology, faculty of science and technology, rajamangala university of technology thanyaburi, khlong luang, pathum thani 12120, thailand 5 program of biology, faculty of science, khon kaen university, muang, khon kaen 40002, thailand 6 center of environmental education research and training, faculty of environment and resource studies,mahasarakham university 44150, thailand 7 mahasarakham university demonstration school (secondary) kham riang sub-district, kantharawichai district, maha sarakham, 44150, thailand *corresponding author. e-mail: topthesun1978@gmail.com, surachest.aiu@pcru.ac.th abstract. the karyotypes of red-eyed gecko are not reported yet. herein, we describe the karyotypes of red-eyed gecko (gekko nutaphandi bauer, sumontha & pauwels, 2008) from thailand. gecko chromosome preparations were directly conducted from bone marrow and testis. chromosomal characteristics were analyzed by giemsa staining, ag-nor banding as well as fluorescence in situ hybridization (fish) using microsatellites d(gc)15 probe. the results showed that the number of diploid chromosomes is 2n=34, while the fundamental number (nf) is 46 in both males and females. the types of chromosomes were 4 large metacentric, 6 large submetacentric, 2 medium telocentric, 2 small metacentric and 20 small telocentric chromosomes. the results of conventional giemsa staining presented the diploid chromosome number differentiation even in the same genus. nors are located at the secondary constriction to the telomere on the long arm of chromosome pair 5. there are no sex differences in karyotypes between males and females. fish with d(gc)15 sequences were also displayed at the telomeres of most other chromosomes. we found that during metaphase i the homologous chromosomes showed synapsis, which can be defined as 19 ring bivalents and 17 haploid chromosomes (n=17) at metaphase ii as a diploid species. the karyotype formula is as follows: 2n (34) = l4m+l6sm+m2t+s2m+s20t. keywords: chromosome, gekko nutaphandi, karyotype, red-eyed gecko. 104 thongnetr weera et al. introduction geckos of the genus gekko laurenti, 1768 are members of a relatively large putatively monophyletic group of lizards that also includes lepidodactylus fitzinger, 1843, luperosaurus gray, 1845, pseudogekko taylor, 1922, and ptychozoon kuhl & van hasselt, 1822 (kluge 1968; russell 1972; bauer et al. 2008). the genus gekko itself is species rich (84 species) and is generally characterized by regional endemism across its broad range in tropical asia. recently, g. nutaphandi, is described from kanchanaburi province in central western thailand (bauer et al. 2008). g. nutaphandi is most similar to g. gecko, g. smithii, g. siamensis, g. verreauxi, and g. albofasciolatus günther, 1872, which have previously been considered to be closely related based on their shared possession of a suite of features. information about karyotypes in gekko is scarce and fragmented, and usually based on conventional staining technique; only a few studies have been published (19 species of 84 species), all of them recent in genus gekko, namely, g. gecko: 2n=38 (singh 1974; solleder and schmid 1984; wu and zhao 1984; trifonov et al. 2011; qin et al. 2012; patawang et al. 2014), g. hokouensis: 2n=38 (chen et al. 1986; kawai et al. 2009; shibaike et al. 2009), g. japonicus: 2n=38 (yoshida and itoh 1974; shibaike et al. 2009; trifonov et al. 2011), g. shibatai and g. vertebralis: 2n=38 (shibaike et al. 2009), g. vittatus and g. ulikovskii: 2n=38 (trifonov et al. 2011), g. tawaensis: 2n=38 (ota 1989a; shibaike et al. 2009), g. taylori: 2n=42 (ota and nabhitabhata 1991), g. monarchus: 2n=44 (ota et al. 1990), g. yakuensis, g. petricolus and g. smithii: 2n=38-42 (ota 1989a), g. kikuchii: 2n=44 (ota 1989a), g. chinensis: 2n=40 (lau et al. 1997), g. subpalmatus: 2n=38 (wu and zhao 1984), g. swinhonis: 2n=38 (chen et al. 1986), g. petricolus: 2n=34 (thongnetr et al. 2022), dixonius hangseesom: 2n=40, d. siamensis: 2n=40, d. melanostictus: 2n=42 (patawang et al. 2022) and cyrtodactylus inthanon: 2n=40 (prasopsin et al. 2022) and shown in table 1. this fact is probably due to the difficulty in obtaining samples for cytogenetic analysis in some species or to problems in obtaining metaphase cells by cell culture induction. moreover, cytogenetic studies using conventional staining techniques provide valuable information on the excellent karyotype diversity shown by these animals. analyses of cytogenetic markers, including the number and karyotype formula, sex determination, b chromosomes, number and location of nucleolar organizer regions (nors), heterochromatin distribution, g-banding, and r-banding, treatments with base-specific fluorochromes and fluorescence in situ hybridization techniques allowed the cytogenetic characterization of populations, species, and supra-specific groups (affonso and galetti jr. 2005). the present study is the first report on the chromosomal characteristics of g. nutaphandi determined using conventional staining, ag-nor banding, and fluorescence in situ hybridization techniques. matherial and methods sample collection, chromosome preparation and chromosome staining we obtained specimens of g. nutaphandi that were collected from kanchanaburi province, western thailand. chromosomes were directly prepared in vivo (ota 1989a; qin et al. 2012) using the following method. with 20% of giemsa solution, the slides were conventionally stained for 30 minutes (patawang et al. 2014). after that, the slides were rinsed thoroughly with running tap water to remove excess stain and placed in air-dry at room temperature. ag-nor banding was analysed according to the method of howell and black (1980). two drops each of 50% silver nitrate and 2% gelatine solutions were added to the slides, respectively. then, they were sealed with cover glasses and incubated at 60°c for 5-10 minutes. they were also soaked in distilled water until the cover glasses were separated. finally, the slides were placed in air-dry at room temperature. they were observed under the microscope. the use of microsatellite investigations which were described by kubat et al. (2008), was followed here with slight modifications. these sequences were directly labeled with cy3 at the 5 -́terminal during synthesis by sigma (st. louis, mo, usa.) fluorescence in situ hybridization (fish) was performed under highly rigorous conditions on mitotic chromosome spreads (pinkel et al. 1986). chromosomal checks, karyotyping and idiograming chromosome counting was carried out on mitotic metaphase cells under the light microscope for 30 cells per specimen to determine the diploid number (2n). twenty clearly observable and well-spread metaphase cells were selected and photographed from each male and female. the short arm length (ls) and the long arm length (ll) of each chromosome were measured to calculate the total length of the chromosome for 20 wellspread metaphase cells. the chromosome types were classified from the method of turpin and lejeune (1965) as metacentric, submetacentric, acrocentric, and telocentric chromosomes. 105first report of chromosome and karyological analysis of gekko nutaphandi (gekkonidae, squamata) from thailand results and discussion diploid chromosome number, fundamental number and karyotype the gecko is a large genus (84 species) of gekkonidae family and until now, no study has investigated the karyotype of g. nutaphandi. furthermore, this is the first report on cytogenetic characterization to use conventional giemsa staining, nor-banding, and fish techniques for this species. for g. nutaphandi, the results indicated a diploid chromosome number (2n) of 34 in all studied samples (figure 1). this result diftable 1. the karyotype reviews among the genes gekko (gekkonidae, squamata). species 2n nf karyotype formulas nors fish locations references gekko gekko 38 50 6m+4sm+2a+26t p4 thailand patawang et al. (2014) 38 44 6bi-arms+32uni-arms singh (1974) 38 wu and zhao (1984) 38 46 8bi-arms+30uni-arms solleder and schmid (1984) 38 trifonov et al. (2011) 38 50 8 m+2sm+2st(a)+26t laos qin et al. (2012) 38 48 8 m+2sm+28t china qin et al. (2012) g. hokouensis 38 56 4 m+6sm+20t+8bi-arms* p19 china, chen et al. (1986) 38 58/59 2 m+8sm+10a+16t+z(t)+w(a) japan kawai et al. (2009) 38 58 4 m+8sm+18t+8bi-arms* p19 taiwan shibaike et al. (2009) 38 58/59 4 m+8sm+16t+8bi-arms*+z(t)w(sm) p19 japan shibaike et al. (2009) 38 42 2m+18sm+16a+zw p19 fish mapping thailand srikulnath (2015) g. japonicus 38 yoshida and itoh (1974) 38 58 4m+8sm+8st+18t p17 japan shibaike et al. (2010) 38 trifonov et al. (2011) g. shibatai 38 58 4m+8sm+18t+8bi-armed p19 japan shibaike et al. (2009) g. vertebralis 38 62 4m+14sm+14t+6bi-arme* p19 japan shibaike et al. (2009) g. vittatus 38 trifonov et al. (2011) g. ulikovskii 38 trifonov et al. (2011) g. tawaensis 38 58 4m+8sm+18t+8bi-armed p19 japan shibaike et al. (2009) 38 56 p19 japan ota (1989a) g. taylori 42 thailand ota and nabhitabhata (1991) g. monarchus 44 46 malaysia ota et al. (1990) g. yakuensis 38 56 p19 japan ota (1989a) g. petricolus 38 54 thailand ota (1989a) g. kikuchii 44 50 taiwan ota (1989a) g. chinensis 40 46 6bi-armed+34uni-armed china lau et al. (1997) g. smithii 38 48 ota (1989a) g. subpalmatus 38 58 wu and zhao (1984) g. swinhonis 38 66 chen et al. (1986) g. petricolus 38 54 4m+2sm+10a+22t p17 (ca)15, (gaa)10 thailand thongnetr et al. (2022) g. nutaphandi 34 46 6m+6sm+22t p5 (gc)15 thailand present study dixonius hangseesom 40 42 2m+38t p13 thailand patawang et al. (2022) d. siamensis 40 42 2m+38t p13 thailand patawang et al. (2022) d. melanostictus 42 44 2a+40t p8 thailand patawang et al. (2022) cyrtodactylus inthanon 40 58 12m+4sm+2a+22t p12 (ca)15 (gc) 15 (cag)10 (gaa)10 thailand prasopsin et al. (2022) note: 2n = diploid chromosome number, nors = nucleolar organizer regions, nf = fundamental number (number of chromosome arms), bi-arm = bi-armed chromosome, m = metacentric, sm = submetacentric, a = acrocentric, t = telocentric chromosome, *=small bi-arms chromosome, and = not available. 106 thongnetr weera et al. fers from most species of the genus gecko. the 2n of this genus range from 38 to 44. (singh 1974; yoshida and itoh 1974; solleder and schmid 1984; wu and zhao 1984; chen et al. 1986; ota 1989a; ota et al. 1990; ota and nabhitabhata 1991; lau et al. 1997; kawai et al. 2009; shibaike et al. 2009; trifonov et al. 2011; qin et al. 2012; patawang et al. 2014; patawang et al. 2022 and prasopsin et al. 2022. the fundamental number (nf) of g. nutaphandi was 46 in both males and females. the karyotype consisted of 4 large metacentric, 6 large submetacentric, 2 medium telocentric, 2 small metacentric, and 20 small telocentric chromosomes (table 1). these results of nf are agreeable with the previous reports of g. gecko (solleder and schmid 1984), g. monarchus (ota et al. 1990), and g. chinensis (lau et al. 1997). the nfs of the genus gekko range from 44 to 62, and karyotypes are composed of both monoand bi-arms chromosomes. nirchio et al. (2002) proposed that species with high nf is an advanced state or apomorphic character, whereas one with low nf is a primitive state or plesiomorphic character. thus, the g. nutaphandi seems to be a more primitive karyotype than other species. the karyotype formula for this species is 2n (34) = l4m+l6sm+m2t+s2m+s20t. there is no evidence of differentiated sex chromosomes in this species which accord with all species of this genus (singh 1974; yoshida and itoh 1974; solleder and schmid 1984; wu and zhao 1984; chen et al. 1986; ota 1989a; ota et al. 1990; ota and nabhitabhata 1991; lau et al. 1997; kawai et al. 2009; shibaike et al. 2009; trifonov et al. 2011; qin et al. 2012; patawang et al. 2014; patawang et al. 2022 and prasopsin et al. 2022. the present study on the meiotic cell division of g. nutaphandi found that during metaphase i (meiosis i), the homologous chromosomes showed synapsis, which can be defined as the 17 bivalent (figure 2a), and 17 haploid chromosomes at metaphase ii (figure 2b) as diploid species. the largest metacentric chromosome pair 1 is the largest bivalent. no diakinesis and metaphase i cell with partially paired bivalents are speculated to be heteromorphic sex chromosomes, and no metaphase ii cells with condensed chromosomes are speculated to be the sex chromosome. chromosome markers from ag-nor banding the development of ag-nor staining technique (howell and black 1980) to detect metaphase chromosome sites of nors has greatly facilitated comparative studies of nors variation. silver staining of nors is considered as one of the standard banding methods and has assumed considerable importance in characterizing a species’ karyotype. the present study was firstly accomplished by using ag-nor staining in g. nutaphandi. the ag-nor positions were shown on the long arm near the centromere of the telocentric chromosome pair 5 (subtelomeric nor) (figure 3). the single pair of nor is the same as in gecko species. compared with other geckos, the nor regions showed two nors appearing near telomeric region of small bi-armed or mono-armed chromosome. an example of the previous reports of the genus gekko had two nors on one pair of small bi-arms chromosomes. g. gecko had two nors on the near telomere of mono-arms chromosome pair 4 (patawang et al. 2014), and g. petricolus had two nors on the long arm near telomere of bi-arms chromosome pair 5 (thongnetr et al. 2022), while there were g. shibatai, g. yakuensis, figure 1. metaphase chromosome plates and karyotypes of g. nutaphandi (a) male (b) female by conventional technique. scale bars = 10 µm. figure 2. metaphase chromosome plates and karyotypes of g. nutaphandi (a) metaphase i (b) metaphase ii by conventional technique. scale bars = 10 µm. 107first report of chromosome and karyological analysis of gekko nutaphandi (gekkonidae, squamata) from thailand g. hokouensis, g. tawaensis, g. vertebralis and g. yakuensis which had two nors on the long arm near the telomere of small bi-arms chromosome pair 19 (ota 1989b; chen et al. 1986; shibaike et al. 2009). the use of nors in explaining kinships depends to a considerable extent on the uniformity of this characteristic and the degree of variety within a taxon (yüksel and gaffaroğlu 2008). the idiogram shows a continuous length gradation of chromosomes. the size differences between the largest and smallest chromosomes exhibit approximately two-fold. the data of the chromosome measurement on mitotic metaphase cells (from all specimens) are shown in table 2. idiograms by conventional giemsa staining and ag-nor banding are shown in figure 4. microsatellite pattern microsatellites or simple sequence repeats (ssrs) are oligonucleotides of 1–6 base pairs in length, forming excessive tandem repeats of usually 4 to 40 units (tautz and renz 1984; ellegren 2004; chistiakov et al. 2006). they show ample distribution throughout eukaryotic genomes, scattered or clustered in euchromatin and heterochromatin. they are highly polymorphic regarding figure 3. metaphase chromosome plates and karyotypes of g. nutaphandi (a) male (b) female by ag-nor banding technique. scale bars = 10 µm. table 2. mean length of short arm chromosome (ls), length of long arm chromosome (ll), length of total chromosomes (lt), relative length (rl), centromeric index (ci), and standard deviation (sd) from 20 metaphases of male and female red-eyed gecko (gekko nutaphandi), 2n (diploid)=34. chro.pairs ls ll lt ci±sd rl±sd chro. size chro. type 1 0.932 1.131 2.035 0.548±0.012 0.132±0.007 large metacentric 2 0.835 1.160 1.961 0.602±0.015 0.120±0.005 large submetacentric 3 0.769 1.303 2.001 0.626±0.023 0.115±0.007 large submetacentric 4 0.784 1.113 1.833 0.586±0.020 0.113±0.007 large metacentric 5* 0.410 1.003 1.372 0.673±0.019 0.084±0.004 large submetacentric 6 0.000 1.216 1.216 1.000±0.000 0.066±0.003 medium telocentric 7 0.000 1.016 1.016 1.000±0.000 0.060±0.003 small telocentric 8 0.000 1.049 1.049 1.000±0.000 0.055±0.002 small telocentric 9 0.000 0.720 0.720 1.000±0.000 0.046±0.003 small telocentric 10 0.000 0.800 0.800 1.000±0.000 0.037±0.004 small telocentric 11 0.000 0.629 0.629 1.000±0.000 0.032±0.003 small telocentric 12 0.000 0.595 0.595 1.000±0.000 0.029±0.003 small telocentric 13 0.000 0.615 0.615 1.000±0.000 0.026±0.003 small telocentric 14 0.000 0.596 0.596 1.000±0.000 0.023±0.004 small telocentric 15 0.000 0.464 0.464 1.000±0.000 0.021±0.003 small telocentric 16 0.195 0.220 0.374 0.520±0.038 0.024±0.004 small metacentric 17 0.000 0.326 0.326 1.000±0.000 0.016±0.003 small telocentric note: chro.=chromosome, * nors bearing chromosomes (satellite chromosome). figure 4. idiogram represents chromosome type, size, and ag-nor banding of g. nutaphandi. 108 thongnetr weera et al. copy number deviation (ellegren 2004). fluorescence hybridization indicates the (gc)15 repeat showing abundance at the telomeric ends of most chromosomes (figure 5), verifying the findings from other gekko groups investigated to date (srikulnath 2015). the distribution of microsatellites was not only restricted to heterochromatin but also dispersed in euchromatic regions of the chromosomes (getlekha et al. 2016). nonetheless, closely related fish species involved in recent speciation events could present a differential pattern in the distribution and quantity of microsatellite sequences on chromosome. in conclusion, we present the first karyotype, nor phenotype, and microsatellite patterns (gc)15 on the chromosomes are specific to species in the g. nutaphandi. more species and techniques should be further studied for more information about the chromosomal diversity and chromosomal evolution in this genus. acknowledgements this research project was financially supported by thailand science research and innovation (tsri). khon kaen university and phetchabun rajabhat university. references affonso pram, galetti jr pm. 2005. chromosomal diversification of reef fishes from genus centropyge (perciformes, 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regions in chalcalburnus mossulensis (pisces: cyprinidae). journal of fisheriessciences.com 2: 587–591. caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 3 2022 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 75(2): 129-141, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1562 caryologia international journal of cytology, cytosystematics and cytogenetics citation: mikail açar, neslihan taşar (2022) a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey). caryologia 75(2): 129-141. doi: 10.36253/caryologia-1562 received: february 01, 2022 accepted: july 06, 2022 published: september 21, 2022 copyright: © 2022 mikail açar, neslihan taşar. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid ma: 0000-0003-3848-5798 nt: 0000-0002-0417-4660 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 department of plant and animal production, tunceli vocational school of higher education, munzur university, tunceli, 62000, turkey *corresponding author. e-mail: mikailacar@munzur.edu.tr abstract. this study performed statistical analyses based on chromosome micromorphology of 18 centaurea taxa, two of which are endemic. anova, correlation analysis, discriminant analysis and cluster analysis were performed to determine the relationships between taxa based on chromosomal features. in addition, according to the data obtained from these analyses, the relationships between taxa and sections were tried to be interpreted. as a result of the analyses, the taxa c. drabifolia sm. floccosa (boiss.) wagenitz & greuter, c. kotschyi (boiss. & heldr.) hayek var. floccosa (boiss.) wagenitz and c. behen l. and c. polypodiifolia boiss pseudobehen (boiss.) wagenitz were located close to each other. these taxa are located in the same sections in the morphological classification. besides, in the discriminant analysis, the taxa of acrocentron, microlophus, cheirolepis sections were closely located compared to all other taxa. however, results were not seen to cover all taxa of the sections. this study revealed various chromosomal characteristics of some centaurea taxa distributed in the eastern anatolia. it also performed statistical analyses on these data. while determining the relationships between taxa according to chromosomal characteristics, comparing chromosomal formulas and indices collectively and evaluating the relationships was found to be relatively consistent with morphological classification. keywords: centaurea, chromosome, endemic, statistical analysis, turkey. introduction members of the asteraceae family occupy a wide range of habitat types and are found in almost every region except antarctica. there are 129 genera and 1156 species of the asteraceae family in turkey. centaurea l. genus, which is one of the important genera of the asteraceae family, is accepted as a systematically problematic genus and spreads globally with approximately 700 species in asia, north africa, america, and europe (güner et al. 2000). in turkey, the genus centaurea is represented by 238 species. 125 of these species are endemic (güner et al. 2012). the endemism rate of this genus, which has many endemic species, is approximately 52%. the systematics of the genus centaurea has changed, especially with the development of molec130 mikail açar, neslihan taşar ular techniques, and some problems have been solved. in the light of these studies, it is known that turkey is the essential gene center of the genus centaurea with many rare endemic species. species of the centaurea in turkey can generally grow in very different habitats such as stony calcareous cliffs, vineyards, roadsides, coastlines, steppe, scrub, fallow areas, sandy beaches, forests, dry meadows, rocky slopes. in addition, although many species of this genus have medicinal properties, especially the flowering above-ground parts or only the flower is used to cure many diseases and relieve pain (yeşilada et al. 2004; gürbüz & yeşilada 2007). many taxonomic, cytotaxonomic, morphological, anatomical and karyological studies have been carried out on centaurea taxa, which are used medically (haratym et al. 2020; fattaheiandehkordi et al. 2021; khammar & djeddi 2012; ) this study was carried out on a statistical evaluation of data obtained from our previous cytological studies (hayta et al. 2017; tasar et al. 2018a; 2018b; 2018c). in the current study, it is aimed to investigate the relationship between taxa, whose karyotype characteristics were revealed using micromorphological chromosome data, and investigate the compatibility and usefulness of this relationship with morphological classification by performing various statistical analyses. material and methods material plant samples belonging to the genus centaurea were collected and numbered between 2011 and 2012 from their natural habitats in different localities in elazığ province (turkey) and its surroundings by field studies. the localities of the taxa are given in table 1. methods chromosome measurements the seeds of the plant samples were sown in petri dishes and germinated in an oven at 20-22 ºc. roots reaching 1–2 cm in length from the germinated seeds were cut and kept in colchicine for 2 hours at room temperature and subjected to pretreatment. afterward, the root tips were placed in carnoy fixative (3:1) and kept in the refrigerator at +4 ºc for 24 hours and fixed. at the end of the period, root tips were hydrolyzed in 1n hcl in an oven at 60 ºc for 5-18 minutes. root tips removed from hydrolysis were stained with feulgen stain for 1 hour in a dark environment at room temperature. then it was washed 2-3 times with tap water. for preparation, the growth meristem part was cut off with a sharp razor blade in a drop of 45% acetic acid on the slide, and the coverslip was closed (elçi 1982). the photographs of each species’ three best somatic cells were taken with a canon digital camera and an olympus bx53 microscope with a 100-lens. the naming system of levan et al. (1964) was used to locate the centromere. the intra-chromosomal asymmetry index (a1) was calculated according to the formula proposed by zarco (1986). the interchromosomal asymmetry index (a2) and karyotype symmetry nomenclature were made according to stebbins (1971). chromosomes measurements of centaurea taxa are given in table 3. data analyses various formulas and indexes were used for analyses based on chromosome characteristics. the measurements were built on haploid datasets. the calculations and abbreviations used in the analysis are as follows. tlc (total length of chromosomes), mtlc (mean of total length of chromosomes), max (maximum length of chromosome), min (minimum length of chromosomes), mla (mean of long arms), msa (mean of short arms), mrv (mean of r-value), mdv (mean of d value), mar (mean of arm ratio), mci (mean of chromosome index), mrlc (mean of relative length of chromosomes), drl (difference of range of relative length), tf% (total form percentage), s% (relative length of shortest chromosome), a1 (intrachromosomal asymmetry index), a2 (interchromosomal asymmetry index), and a (degree of asymmetry). both arm ratios were assumed to be equally affected (adhikary 1974). all karyotype formulas and indexes were determined based on huziwara (1962) (tf%), levan et al. (1964) (r and d values), zarco (1986) (a1 and a2), watanabe (1999) (a), peruzzi & eroğlu (2013) (ci) as well. the abbreviations were taken from rezeai et al. (2014) (rlc%, drl, s%). the formulas are as follows. formulas and indexes r value = d value = length of the long arm of chromosome-length of the short arm of chromosome arm ratio = ci = 131a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia rlc% = x 100 drl = (maximum relative length) (minimum relative length) tf% = x 100 s% = x 100 , (li = lengths of a long arm, si = lengths of a short arm, n = haploid chromosome number). (n = number of homologous chromosome pairs, bi = the average length of short arms in every homologous chromosome pair, bi = the average length of long arms in every homologous chromosome pair). (s = standard deviation of chromosome lengths, = mean of chromosome lengths). a data matrix was constructed according to 17 chromosomal traits in table 4. the discriminant analysis (lda) was used based on the data matrix. next, the table 1. the localities of studied taxa. taxa localities voucher specimen c. aggregata fisch. & c.a.mey. ex dc. subsp. aggregata b7 elazığ; sivrice, gözeli village, kuşakcı mountain slopes, 1550 m. 28.06.2012, based on grids (turkey): a6, a7, a8, a9, b6, b7, b8, b9, c5, c6, c10 taşar 1001 c. virgata lam. b7 elazığ; koçkale village on the elazig-bingöl road, mountainous area, 1380 m. 21.06.2012, based on grids (turkey): a2, a3, a4, a5, a7, a8, a9, b2, b3, b4, b5, b6, b7, b8, b9, b10, c2, c3, c4, c5, c6, c10 taşar 1003 c. balsamita lam. b7 elazığ; sürsürü district, roadside, 1050 m. 02.07.2012, based on grids (turkey): a2, b7, b8, b9, c4, c6, c8 taşar 1006 c. behen l. b7 elazığ; keban road, beşik village entrance, roadside, 1090 m. 15. 07. 2012, based on grids (turkey): b6, b7, b8, b9, c6, c7, c8, c9, c10 taşar 1009 c. polypodiifolia boiss. var. pseudobehen (boiss.) wagenitz b7 elazığ; çemişgezek, danbüken, in the village of avşan, 1090 m. 16.07.2012 , based on grids (turkey): b6, b7 taşar 1010 c. polypodiifolia boiss. var. polypodiifolia b7 elazığ; baskil, kumtarla village, hills, 1090 m. 14.07.2012, based on grids (turkey): a8, a9, b6, b7, b8, b9, c9, c10 taşar 1012 c. carduiformis dc. subsp. carduiformis var. carduiformis b7 elazığ; keban, pınarlar village on arapgir road, field edges, 1430 m. 20-07-2012, based on grids (turkey): a3, a4, a5, a6, a8, b3, b4, b5, b6, b7, c4, c5 taşar 1013 c. urvillei dc. subsp. armata wagenitz b7 elazığ; baskil, radiolink station surroundings, 1350 m. 16.06.2011, based on grids (turkey): a2, a4, a5, b1, b2, b6, b7, c5, c6 taşar 1015 c. urvillei dc. subsp. hayekiana wagenitz b7 elazığ; baskil, kayabeyli village, slopes, 1460 m. 13.06.2011, based on grids (turkey): b6, b7, c3 taşar 1017 c. urvillei dc. subsp. urvillei b7 elazığ; harput, rocky areas around anguzlu baba tomb, 1400 m. 13.06.2011, based on grids (turkey):a1, a2, a3, a4, a5, b1, b2, b6, b7, c1, c2, c3, c4, c5, c6,c7 taşar 1014 c. cynarocephala wagenitz b7-elazığ; sivrice, gözeli village, kuşakçı mountain, 1750 m. 23.06.2012, based on grids (turkey): c8 taşar 1020 c. kurdica reichardt b7 elazığ; baskil roadway, 23. km. roadsides, 1280 m. 13.07.2011, based on grids (turkey): b7, b8, c8 taşar 1022 c. derderiifolia wagenitz b7 elazığ; baskil, haroğlu mountain lower slopes, 1350 m. 22.07.2011, based on grids (turkey): b6, b7 taşar 1024 c. drabifolia sm. floccosa (boiss.) wagenitz & greuter b7 elazığ; baskil, haroğlu mountain, behind the tv station, rocky area, 1950 m. 22.07.2011, based on grids (turkey): a4, a5, a6, b2, b3, b4, b6, b7, c3, c4 taşar 1025 c. kotschyi (boiss. & heldr.) hayek var. floccosa (boiss.) wagenitz b7 elazığ; baskil, yukarı kuluşağı village kuzucuk hamlet mountainous region, 1350 m. 26.08.2012, based on grids (turkey): b6, c6 taşar 1026 c. saligna (k.koch) wagenitz b7 elazığ; palu, baltasi village, the hills behind the military post, 1450 m. 17.07.2012, based on grids (turkey): b6, b7, b8, b9, c9, c10 taşar 1027 c. iberica trev. ex sprengel b7 elazığ; sürsürü district, 1067 m. 22.06.2011, based on grids (turkey): a1, a2, a3, a4, a5, a6, a7, a8, b1, b2, b3, b4, b8, b9, c2, c3, c4, c5, c6, c7, c8, c9, c10 taşar 1028 c. solstitialis l. subsp. solstitialis b7 elazığ; sürsürü district, 1067 m. 22.06.2011, based on grids (turkey): a1, a2, a3, a4, a5, a7, a8, b1, b3, b4, b5, b6, b7, b8, b9, c1, c2, c3, c4, c5, c6, c8, c9 taşar 1029 132 mikail açar, neslihan taşar table 2. the sections of the studied centaurea taxa. section taxa acrolophus c. aggregata subsp. aggregata, c. virgata acrocentron c. urvillei subsp. hayekiana, c. urvillei subsp. urvillei, c. carduiformis subsp. carduiformis var. carduiformis, c. urvillei subsp. armata stizolophus c. balsamita microlophus c. behen, c. polypodiifolia var. pseudobehen, c. polypodiifolia var. polypodiifolia cynaroides c. cynarocephala, c. kurdica cheirolepis c. derderiifolia, c. drabifolia subsp. floccosa, c. kotschyi var. floccosa, c. saligna calcitrapa c. iberica mesocentron c. solstitialis subsp. solstitialis table 3. chromosomes measurements of centaurea taxa (ch. no: chromosome no, c: total length of the chromosome, l: length of the long arm, s: length of the short arm, cp: centromeric position). ch. no c l s cp ch. no c l s cp c. aggregata subsp. aggregata c. urvillei subsp. hayekiana 1 4.74 2.37 2.37 m 1 6.07 3.04 3.04 m 2 4.74 2.47 2.26 m 2 4.54 2.54 2 m 3 4.68 2.53 2.16 m 3 3.86 2.43 1.43 sm 4 4.32 2.95 1.37 sm 4 3.75 1.93 1.82 m 5 4.32 2.95 1.37 sm 5 3.68 2.18 1.5 m 6 4.21 2.95 1.26 sm 6 3.68 2.32 1.36 sm 7 3.89 2.37 1.53 m 7 3 1.64 1.36 m 8 3.68 2.32 1.37 m 8 2.89 1.64 1.25 m 9 3.37 1.68 1.68 m 9 2.57 1.29 1.29 m 10 3.05 1.58 1.47 m 10 2.29 1.14 1.14 m c. urvillei subsp. urvillei c. behen 1 5.28 2.64 2.64 m 1 4.04 2.57 1.46 sm 2 4.66 2.56 2.1 m 2 3.96 2.14 1.82 m 3 4.24 2.8 1.44 sm 3 3.5 1.89 1.61 m 4 4.08 2.04 2.04 m 4 3.46 1.93 1.54 m 5 4 2.28 1.72 m 5 3.43 1.71 1.71 m 6 3.58 2.12 1.46 m 6 3.21 1.61 1.61 m 7 3.4 2.12 1.28 m 7 3.14 1.57 1.57 m 8 2.92 1.66 1.26 m 8 3.07 1.75 1.32 m 9 2.88 2.16 0.72 sm 10 2.8 2.08 0.72 sm c. polypodiifolia var. pseudobehen c. polypodiifolia var. polypodiifolia 1 4.4 2.32 2.08 m 1 4.66 2.61 2.05 m 2 4.04 2.38 1.66 m 2 3.8 1.9 1.9 m 3 4.12 2.92 1.2 sm 3 3.07 1.83 1.24 m 4 3.84 2.56 1.28 sm 4 2.59 1.59 1 m 5 3.6 2.04 1.56 m 5 2.37 1.32 1.05 m 6 3.44 2.16 1.28 m 6 2.37 1.66 0.71 sm 7 3.44 1.72 1.72 m 7 2.12 1.22 0.9 m 8 3.26 1.63 1.63 m 8 1.93 1.24 0.68 sm c. carduiformis subsp. carduiformis c. urvillei subsp. armata 1 6.11 3.63 2.49 m 1 4.74 2.37 2.37 m 2 5.18 3.15 2.03 m 2 4.74 2.47 2.26 m 133a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia ch. no c l s cp ch. no c l s cp 3 4.55 2.93 1.63 sm 3 4.68 2.53 2.16 m 4 4.31 2.49 1.82 m 4 4.32 2.95 1.37 sm 5 4.25 2.39 1.86 m 5 4.32 2.95 1.37 sm 6 4.22 2.65 1.57 m 6 4.21 2.95 1.26 sm 7 4.01 2.8 1.2 sm 7 3.89 2.37 1.53 m 8 3.52 2.09 1.43 m 8 3.68 2.32 1.37 m 9 3.3 2.15 1.15 sm 9 3.37 1.68 1.68 m 10 2.88 1.57 1.31 m 10 3.05 1.58 1.47 m c. cynarocephala c. kurdica 1 6.41 3.62 2.79 sm 1 5.81 3.36 2.44 m 2 5.12 3.5 1.62 m 2 5.17 3.01 2.16 m 3 4.99 3.09 1.9 m 3 4.71 2.36 2.36 m 4 4.35 3.21 1.15 sm 4 4.76 2.86 1.91 m 5 4.29 2.15 2.15 m 5 4.4 2.7 1.69 m 6 4.22 2.62 1.6 m 6 4.18 2.58 1.6 m 7 3.91 2.38 1.53 m 7 4.09 2.7 1.4 sm 8 3.68 2.26 1.41 m 8 4.06 3.03 1.2 sm 9 3.18 1.79 1.38 m 9 3.91 2.41 1.5 m c. derderiifolia c. drabifolia subsp. floccosa 1 2.48 1.46 1.02 m 1 4.42 2.21 2.21 m 2 2.4 1.4 1 m 2 3.84 2.53 1.32 sm 3 2.3 1.39 0.9 m 3 3.84 2.53 1.32 sm 4 2.1 1.31 0.79 m 4 3.37 2.37 1 sm 5 2.06 1.03 1.03 m 5 3.28 2.21 1.08 sm 6 2.07 1.16 0.9 sm 6 3.06 1.85 1.21 m 7 1.98 1.21 0.77 m 7 3 1.79 1.21 m 8 1.9 1.08 0.82 m 8 2.89 1.79 1.11 m 9 1.85 1.1 0.76 m 9 2.76 1.61 1.16 m 10 1.81 1.27 0.53 sm 10 2.84 2 0.84 sm 11 1.67 1.03 0.64 m 11 2.54 1.49 1.05 m 12 1.54 0.84 0.7 m 12 2.21 1.11 1.11 m 13 1.52 1.03 0.48 sm 13 2.13 1.18 0.95 m 14 1.47 0.74 0.73 m 14 2.03 1.13 0.89 m 15 1.35 0.77 0.58 m 15 1.95 1.16 0.79 m 16 1.35 0.77 0.58 m 16 1.89 1.05 0.84 m 17 1.29 0.65 0.65 m 17 1.79 0.95 0.84 m 18 1.13 0.52 0.61 m 18 1.79 1 0.79 m c. kotschyi var. floccosa c. saligna 1 4.93 3.48 1.44 sm 1 6.08 3.44 2.65 m 2 3.81 2.56 1.26 sm 2 5.26 2.95 2.31 m 3 3.81 2.26 1.56 m 3 5.1 2.55 2.55 m 4 3.26 1.63 1.63 m 4 4.66 3.03 1.63 sm 5 3.1 1.8 1.3 m 5 4.52 2.52 2 m 6 3 1.8 1.2 m 6 4.02 2.18 1.84 m 7 2.9 1.8 1.1 m 7 3.9 2.52 1.38 sm 8 2.85 1.74 1.11 m 8 3.47 2.02 1.45 m 9 2.74 1.7 1.04 m 9 3.18 1.98 1.2 m 10 2.56 1.59 0.96 m 11 2.41 1.33 1.07 m 12 2.37 1.19 1.19 m 134 mikail açar, neslihan taşar cluster analysis was made using the manhattan distance index to determine the relationships between centaurea taxa’s chromosome properties (romesburg 2004). in addition, the pearson correlation coefficient (r) analysis was performed to see strong and weak relationships between chromosome traits. at the same time, shapiro wilk normality test was performed. then, the one-way analysis of variance (anova) was performed to determine whether the difference between the data was statistically significant. all the analyses were carried out with paleontostatistics (past) (hammer et al. 2001). results chromosome micromorphological features of 18 centaurea taxa were specified, and statistical analyses were performed on them using formulas created using various chromosome features. mitotic metaphase chromosome images of centaurea taxa are given in figure 1, and karyotype features are given in table 4. one way anova test, which is one of the analyses made according to the chromosome characteristics of the taxa, is given in table 5. according to the values obtained with the formulas using the micromorphological chromosome features of taxa, the data show a normal distribution according to the shapiro-wilk test (p>0.05) and the residual plot graph is shown in figure 2 accordingly. then, according to the one-way anova test p-value, the difference between taxa was statistically significant (p<0.05) (table 5). correlation analysis according to the correlation analysis, there are relations between the r-values of chromosomal data according to the significance level less than p <0.05. particularly a high positive relationship among mtlc-mla-msa, mrv-a1-a, mar-a1-a, mrlc-a2, and a strong negative relations among tlc-mrlc, tlc-a2, mcl-a1, mcla, mci-mar-mrv and mrvtf% values (figure 3). discriminant analysis (lda) according to lda (table 6-7, figure 4), the first two components explained most of the variation according to chromosome data between the taxa. while the first two components explain 93.56% and 4,34% of the variance, respectively, these characters explained 97.9% of the total variation. the variation most affected were tlc, mrlc, mci, and drl%. similarly, since some variables (such as a, a1) have lower values than calculations, the effects on variation in lda have been low. in addition, with the results obtained with the chromosomal characters determined by the formulas, taxa grouped according to sections (given groups) were regrouped (predicted group) by discriminant analysis and distributed into groups with an accuracy of around 5.5%. in other words, when the sections determined according to the morphology of the taxa were regrouped with the characters determined according to the chromosomal formulas in the analysis, the overlap was around 5.5%. ch. no c l s cp ch. no c l s cp 13 2.37 1.19 1.19 m 14 2.3 1,26 1.04 m 15 2,22 1.19 1.04 m 16 2.22 1.56 0.67 sm 17 2.07 1.04 1.04 m 18 1.44 0.89 0.56 m c. iberica c. solstitialis subsp. solstitialis 1 2.95 1.84 1.11 m 1 3.53 2.39 1.14 sm 2 2.76 1.68 1.08 m 2 2.81 1.58 1.22 m 3 2.42 1.49 0.93 m 3 2.72 1.47 1.25 m 4 2.29 1.31 0.98 m 4 2.28 1.22 1.06 m 5 2.25 1.26 0.99 m 5 2.25 1.36 0.89 m 6 2.01 1.2 0.82 m 6 2.23 1.12 1.12 m 7 1.97 0.99 0.99 m 7 2.17 1.42 0.75 sm 8 1.84 1.06 0.78 m 8 1.81 1.17 0.64 sm 9 1.84 1.24 0.61 sm 10 1.57 0.84 0.73 m 135a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia cluster analysis according to the cluster analysis results made according to the upgma algorithm and manhattan distance index, the taxa are divided into 3 main groups (figure 5). these groups are also divided into subgroups among themselves. the stizolophus and cynaroides sections were found together in group 1, the microlophus section in group 2, and the cheirolepis section in group 3. especially c. drabifolia subsp. f loccosa and c. kotschyi var. floccosa; c. behen and c. polypodifolia var. pseudobehen; c. urvillei subsp. armata and c. aggregata subsp. aggregata stand out as closely related taxa. within these relationships, c. urvillei subsp. armata and c. aggregata subsp. aggregata taxa were found to be close to each other in terms of chromosomal characteristics, although they were in different sections. discussion this study investigated 18 taxa belonging to 8 sections of genus centaurea (table 2). among the investigated taxa, c. derderifolia, c. saligna are endemic to turkey. no statistical study of this scale has been encountered based on chromosome characteristics on the genus. in some studies, the cluster analysis data can yield similar trees with the morphological classification of the taxa (açar & satıl 2019; arabaci et al., 2021; dirmenci et al. 2019; genç et al. 2021). figure 1. mitotic metaphase chromosomes of centaurea taxa (1. c. aggregata subsp. aggregata, 2. c. virgata, 3. c. balsamita, 4. c. behen, 5. c. polypodiifolia var. pseudobehen, 6. c. polypodiifolia var. polypodiifolia, 7. c. carduiformis subsp. carduiformis var. carduiformis, 8. c. urvillei subsp. armata, 9. c. urvillei subsp. hayekiana, 10. c. urvillei subsp. urvillei, 11. c. cynarocephala, 12. c. kurdica, 13. c. derderiifolia, 14. c. drabifolia subsp. floccosa, 15. c. kotschyi var. floccosa, 16. c. saligna, 17. c. iberica, 18. c. solstitialis subsp. solstitialis, scale bars: 10 µm). 136 mikail açar, neslihan taşar ta bl e 4. k ar yo ty pe c ha ra ct er is tic s of c en ta ur ea t ax a (t lc : t ot al l en gh t of c hr om os om es , m t lc ( m ea n of t ot al l en gt h of c hr om os om es , m a x : m ax im um l en gt h of c hr om os om e, m in : m in im um l en gt h of c hr om os om e, m la : m ea n of l on g a rm s, m sa : m ea n of s ho rt a rm s, m rv : m ea n of r v al ue , m dv : m ea n of d v al ue , m a r : m ea n of a rm r at io , m c i: m ea n of c hr om os om e in de x, m r lc : m ea n of r el at iv e le ng th o f c hr om os om es , d r l: d iff er en ce o f r an ge o f r el at iv e le ng th , t f% : t ot al f or m p er ce nt ag e, s % : r el at iv e le ng th o f sh or te st c hr om os om e, a 1: in tr ac hr om os om al a sy m m et ry i nd ex , a 2: in te rc hr om os om al a sy m m et ry i nd ex ). c en ta ur ea t ax a t lc m t lc m a x m in m la m sa m rv m dv m a r m c i m r lc d r l t f% s% a 1 a 2 a c . a gg re ga ta s ub sp . a gg re ga ta 41 .0 0 4. 10 2. 95 1. 26 2. 42 1. 68 1. 44 0. 74 1. 52 41 .1 8 10 .0 1 4. 10 0. 41 0. 43 0. 27 0 0. 10 0. 17 9 c . u rv ill ei s ub sp . h ay ek ia na 36 .3 3 3. 63 3. 03 1. 14 2. 02 1. 62 1. 24 0. 40 1. 27 44 .5 8 10 .0 1 10 .4 3 0. 45 0. 38 0. 18 2 0. 10 0. 10 9 c . u rv ill ei s ub sp . u rv ill ei 37 .8 4 3. 78 2. 64 0. 72 2. 25 1. 54 1. 46 0. 71 1. 68 39 .4 3 10 .0 1 6. 55 0. 41 0. 27 0. 36 8 0. 10 0. 18 7 c . v ir ga ta 63 .5 2 3. 53 4. 63 0. 63 2. 23 1. 29 1. 73 0. 94 1. 83 38 .0 6 5. 56 6. 88 0. 37 0. 14 0. 34 8 0. 06 0. 26 6 c . b al sa m ita 33 .9 5 2. 61 2. 49 0. 64 1. 62 0. 99 1. 62 0. 63 1. 68 38 .4 2 7. 69 7. 72 0. 38 0. 26 0. 35 6 0. 08 0. 23 8 c . b eh en 27 .8 1 3. 48 2. 57 1. 32 1. 89 1. 58 1. 20 0. 31 1. 21 45 .6 8 12 .5 0 3. 47 0. 45 0. 51 0. 14 7 0. 13 0. 09 1 c . p ol yp od iif ol ia v ar . p se ud ob eh en 30 .1 4 3. 77 2. 32 1. 20 2. 22 1. 55 1. 43 0. 67 1. 49 41 .4 2 12 .5 0 3. 77 0. 41 0. 52 0. 26 7 0. 13 0. 17 7 c . p ol yp od iif ol ia v ar . p ol yp od iif ol ia 22 .9 1 2. 86 2. 61 0. 68 1. 67 1. 19 1. 40 0. 48 1. 51 40 .6 6 12 .5 0 11 .9 3 0. 42 0. 26 0. 29 9 0. 12 0. 16 8 c . c ar du ifo rm is s ub sp . c ar du ifo rm is v ar . c ar du ifo rm is 42 .3 3 4. 23 3. 63 1. 15 2. 58 1. 65 1. 57 0. 93 1. 60 38 .9 6 10 .0 0 7. 63 0. 39 0. 32 0. 35 2 0. 10 0. 22 1 c . u rv ill ei s ub sp . a rm at a 41 .0 0 4. 10 2. 95 1. 37 2. 42 1. 68 1. 44 0. 74 1. 52 41 .1 8 10 .0 0 4. 10 0. 41 0. 46 0. 27 1 0. 10 0. 17 9 c . c yn ar oc ep ha la 40 .1 5 4. 46 3. 62 1. 38 2. 74 1. 73 1. 59 1. 01 1. 66 38 .7 3 11 .1 1 8. 06 0. 39 0. 38 0. 34 9 0. 11 0. 18 4 c . k ur di ca 41 .0 9 4. 57 3. 36 1. 20 2. 78 1. 80 1. 54 0. 98 1. 63 38 .7 2 11 .1 1 4. 63 0. 40 0. 36 0. 34 5 0. 11 0. 22 6 c . d er de ri ifo lia 32 .2 7 1. 79 1. 46 0. 58 1. 04 0. 75 1. 39 0. 29 1. 42 42 .2 8 5. 55 4. 17 0. 42 0. 40 0. 24 8 0. 06 0. 21 2 c . d ra bi fo lia s ub sp . fl oc co sa 49 .6 3 2. 75 2. 53 0. 79 1. 66 1. 09 1. 52 0. 57 1. 54 40 .3 8 5. 55 5. 31 0. 40 0. 31 0. 30 5 0. 06 0. 20 6 c . k ot sc hy i v ar . fl oc co sa 50 .3 6 2. 79 3. 48 0. 56 1. 66 1. 13 1. 47 0. 53 1. 48 41 .2 4 5. 56 6. 91 0. 41 0. 16 0. 27 7 0. 06 0. 19 1 c . s al ig na 40 .1 9 4. 46 3. 44 1. 20 2. 58 1. 89 1. 36 0. 69 1. 42 41 .9 1 11 .1 1 7. 21 0. 42 0. 35 0. 26 6 0. 11 0. 15 4 c . i be ri ca 21 .9 0 2. 19 1. 84 0. 61 1. 29 0. 90 1. 43 0. 39 1. 45 41 .3 8 10 .0 0 6. 29 0. 41 0. 33 0. 28 1 0. 10 0. 17 7 c . s ol st iti al is s ub sp . s ol st iti al is 19 .8 0 2. 47 2. 39 0. 64 1. 46 1. 01 1. 45 0. 45 1. 49 40 .9 5 12 .5 8. 71 0. 41 0. 27 0. 28 7 0. 12 0. 18 5 ta bl e 5. o ne w ay a n o va te st r es ul ts . te st fo r eq ua l m ea ns su m o f s qr s df m ea n sq ua re f p (s am e) b et w ee n gr ou ps : 45 34 3. 3 16 28 33 .9 6 35 1. 6 8. 14 9e -1 79 w ith in g ro up s: 23 29 .6 4 28 9 8. 06 10 4 pe rm ut at io n p (n =9 99 99 ) to ta l: 47 67 2. 9 30 5 1e -0 5 om eg a2 : 0. 94 83 137a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia wagenitz (1975) divided the genus centaurea into 34 sections in flora of turkey. wagenitz (1975) also stated that the genus in flora of turkey is taxonomically difficult and noted that much more studies are needed. in addition, he emphasized that it is especially important to obtain cytological data. there are many taxonomic diffigure 2. shapiro wilk normality test(p=0.4809>0.05)-residual plot. figure 3. correlation analysis between karyotype characteristics ((tlc: total lenght of chromosomes, mtlc (mean of total length of chromosomes, max: maximum length of chromosome, min: minimum length of chromosome, mla: mean of long arms, msa: mean of short arms, mrv: mean of r value, mdv: mean of d value, mar: mean of arm ratio, mci: mean of chromosome index, mrlc: mean of relative length of chromosomes, drl: difference of range of relative length, tf%: total form percentage, s%: relative length of shortest chromosome, a1: intrachromosomal asymmetry index, a2: interchromosomal asymmetry index). 138 mikail açar, neslihan taşar ficulties in the genus, which has a large number of taxa according to the flora of turkey. therefore, studies on the genus containing many such species will provide important data. in this study, taxa from 8 sections were discussed. relationships between taxa were tried to be revealed based on cytological data. in the cluster analysis, which is one of the analyses, although the grouping to cover the whole section was not fully formed, some taxa could be located close to each other in the same section. however, as seen in the discriminant analysis, individuals belonging to the same section in a certain way could not be found in the same spot. in addition, some taxa were close in both discriminant analysis and cluster analysis, and this result was also found to be consistent with morphological classification. this result shows us a strong relationship between these taxa, such as c. drabifolia subsp. floccosa and c. kotschyi var. floccosa and c. behen and c. polytable 6. discriminant analysis (lda) of centaurea taxa showing the eigenvalues of the total variance. pc eigenvalue % variance 1 185.98 93.56 2 8.6347 4.344 3 2.3673 1.191 figure 4. discriminant analysis scatter plot diagram (different colors and numbers refer to different sections, black(1): acrolophus, aqua (2): acrocentron, blue(3): stizolophus, green(4): microlophus, yellow(5): cynaroides, red(6): cheirolepis, purple(7): calcitrapa, pink(8): mesocentron). table 7. discriminant analysis (lda) of centaurea taxa showing the given (morphological sections) and predicted groups (after the analyses). taxa given group classification c. aggregata subsp. aggregata 1 5 c. urvillei subsp. hayekiana 2 5 c. urvillei subsp. urvillei 2 5 c. virgata 1 5 c. balsamita 3 4 c. behen 4 7 c. polypodiifolia var. pseudobehen 4 7 c. polypodiifolia var. polypodiifolia 4 7 c. carduiformis subsp. carduiformis var. carduiformis 2 5 c. urvillei subsp. armata 2 5 c. cynarocephala 5 6 c. kurdica 5 6 c. derderiifolia 6 5 c. drabifolia subsp. floccosa 6 5 c. kotschyi var. floccosa 6 5 c. saligna 6 5 c. iberica 7 4 c. solstitialis subsp. solstitialis 8 8 139a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia podifolia var. pseudobehen. also these taxa are located in the same sections in the morphological classification. in addition, in the discriminant analysis, the taxa of acrocentron; microlophus; cheirolepis sections were located close to each other according to the diagram compared to all other taxa (figure 4). on the discriminant analysis, there was a 5.5% overlapping classification between the sections in which they were classified according to the taxa morphological classification and the new groups that emerged according to the relations between them as a result of the analyses made according to the chromosomal formulas (table 7). accordingly, most of the taxa in the cherloides section were found in the cynaroides section. again, according to the analysis, sections 1 and 3, acrolophus and stizolophus sections were not included in the new grouping. as a result, other sections are sufficient to classify these taxa. however, the most important result is that the morphological classification and the classification made according to the data series obtained from the chromosomal formulas show similarity at a rate of 5.5%. moreover, it has no linear relationship in taxonomic terms. genç et al. (2021) reached the same conclusion in their study. accordingly, chromosomal formulas are not suitable for evaluating together with each other to draw a meaningful conclusion. however, it would be more appropriate to compare taxa one by one. cluster analysis can be a helpful tool in classifying taxa. accordingly, sections of acrocentron, microlophus, cynaroides were grouped with each other. this result was also relatively similar in the discriminant analysis. in general, when this analysis is performed according to morphological data, while there is a more directly proportional grouping, according to the data using the formulas obtained with chromosomal micromorphological data, although there is consistency in some sections, there may be a possibility that the classification to be made using these data in classifying taxa in general terms may be incorrect. in conclusion, there is no general overlap between morphological classification and chromosomal micromorphological-based classification. in addition, there were taxa located close to each other in both morphological data and chromosomal micromorphological data. undoubtedly, these taxa are estimated to be closely related to each other. however, the results obtained from the formulas were seen as characters while creating data sets, and analyses were made in that way. the validity of this needs to be investigated better with more data and evaluation of taxa from different perspectives. this study also provides important data for this situation. this study revealed various chromosomal characteristics of centaurea taxa distributed in eastern anatolia. it also performed statistical analyses on these data 1 2 3 figure 5. cluster analysis according to karyotype characteristics show that 3 main groups (same colored taxa are located in the same section). 140 mikail açar, neslihan taşar and revealed that comparing chromosomal calculations separately in taxa would be more beneficial in morphological classification than classifying them together into analysis structures. references açar m, satıl f. 2019. distantes r. bhattacharjee (stachys l. /lamiaceae) altseksiyonu taksonları üzerinde karşılaştırmalı anatomik ve mikromorfolojik çalışmalar [comparative micromorphological and anatomical investigations on the subsection distantes r.bhattacharjee (stachys l./lamiaceae)]. kahramanmaraş sütçü i̇mam üniversitesi tarım ve doğa dergisi. 22(ek sayı 2): 282-295. adhikary ak. 1974. precise determination of centromere location. cytologia. 39: 11-16. arabaci, t., çelenk, s., özcan, t., martin, e., yazici, t., açar, m., ... & dirmenci, t. 2021. homoploid hybrids of origanum (lamiaceae) in turkey: morphological and molecular evidence for a new hybrid. plant biosystems-an international journal dealing with all aspects of plant biology, 155(3): 470-482. dirmenci t, özcan t, açar m, arabacı t, yazıcı t, martin e. 2019. a rearranged homoploid hybrid species of origanum (lamiaceae): o. × munzurense kit tan & sorger. botany letters. 166(2): 153-162. elçi ş. 1982. sitogenetikte gözlemler ve araştırma yöntemleri [observations and research methods in cytogenetics]. elazığ: fırat üniversitesi fen edebiyat fakültesi yayınları, uğurel matbaası, no:3; 166 s. genç h, yildirim b, açar m, çetin t. 2021. statistical evaluation of chromosomes of some lathyrus l. taxa growing in turkey. caryologia. 74(3): 107-117. doi: 10.36253/caryologia1124 gurbuz i, yesilada e. 2007. evaluation of the antiulcerogenic effect of the sesquiterpene lactones from centaurea solstitialis ssp. solstitialis by using various in vivo and biochemical techniques. j. ethnopharmacol. 112: 284–295. güner a, özhatay n, ekim t, başer khc. 2000. flora of turkey and the east aegean islands, vol. 11 (supplement 2). edinburgh: edinburgh university press; 656 p. güner a, aslan s, ekim t, vural m, babaç mt. 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(poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(2): 89-99, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1571 caryologia international journal of cytology, cytosystematics and cytogenetics citation: made pharmawati, ni nyoman wirasiti, luh putu wrasiati (2022) genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate. caryologia 75(2): 89-99. doi: 10.36253/caryologia-1571 received: february 08, 2022 accepted: march 24, 2022 published: september 21, 2022 copyright: © 2022 made pharmawati, ni nyoman wirasiti, luh putu wrasiati. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid mp: 0000-0002-3064-4582 genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 1 biology study program, faculty of mathematics and natural sciences, universitas udayana, jalan raya kampus unud, jimbaran, kecamatan kuta selatan, kabupaten badung, bali 80361, indonesia 2 agroindustrial technology study program, faculty of agricultural technology, universitas udayana, jalan raya kampus unud, jimbaran, kecamatan kuta selatan, kabupaten badung, bali 80361, indonesia *corresponding author. e-mail: made_pharmawati@unud.ac.id abstract. several environmental pollutants can cause damage to chromosomes, one of which is the heavy metal nino3. some plant extracts have antigenotoxic properties that result in a decrease in chromosomal damage. member of flowering plants that need to be tested is seagrass. one seagrass species is enhalus acoroides which was found to contain phytochemical compounds. this study aimed to analyse the genotoxic effect and the potential of encapsulated e. acoroides leaf extract as antigenotoxic against nickel nitrate nino3. the extraction was conducted using a mixture of chloroform and ethanol, and crude extract encapsulated using maltodextrin and tween 80. chromosomal aberrations were evaluated using the squash technique of allium cepa var. aggregatum root tips. triphenyltetrazolium chloride and evans blue staining were used to observe mitochondrial and apoptotic activities. the results showed that at higher concentrations (250 ppm and 500 ppm), the encapsulated e. acoroides extract decreased mitotic indices; however, no chromosome aberration observed. nino3 itself induced a genotoxic effect as observed by low mitotic index and a high percentage of chromosome aberration. the modulation of nino3 effect by adding the encapsulated e. acoroides extract at low concentration (100 ppm) increased mitotic index compared to treatment with ni alone, but did not reduce chromosome aberration. simultaneous encapsulated e. acoroides extract and ni treatment, significantly reduced nuclear fragmentation and nuclear lesion. the encapsulated e. acoroides extract can repair several types of nuclear damage but cannot minimise chromosomal damage. keywords: chromosome aberration, enhalus acoroides, heavy metal, nuclear abnormality, seagrass. introduction heavy metals are hazardous inorganic environmental pollutants due to their toxicity. however, when present in low amounts, several heavy metals such as cu, fe, mn, co, zn, and ni are required for plants and animals as 90 made pharmawati, ni nyoman wirasiti, luh putu wrasiati micronutrients (singh et al. 2020). recently, due to high industrial activities and the extensive use of fertilizer and pesticides, heavy metals are present in enormous amounts in the environment, posing a serious global environmental threat. one of the most common heavy metal contaminants found in the environment is nickel (ni), along with arsenic (as), cadmium (cd), chromium (cr), copper (cu), mercury (hg), lead (pb), and zinc (zn) (huang et al. 2020). nickel is widely distributed in the environment including air, water, soil, and biological materials. it is mainly derived from natural sources such as windblown dust, resulting from the weathering of rocks and soils, forest fires, and volcanic activity. nickel is also present in the environment due to the combustion of coal, diesel oil, and fuel oil, as well as the incineration of trash and sewage (cempel and nikel 2006). in plants, high nickel concentrations can inhibit growth by causing oxidative damage and disrupting nutrient uptake and translocation (amjad et al. 2020). nickel has also been reported to have cytotoxic and mutagenic effects in plants (gantayat et al. 2018). natural ingredients with bioactive compounds that can fight mutagenic and carcinogenic effects are now getting more and more attention. compounds capable of reducing the mutagenicity of physical and chemical mutagens are referred to as antimutagens. however, considering that all mutagens are genotoxic, then compounds that reduce dna damage caused by genotoxic agents are also called antigenotoxic agents (lópez-romero et al. 2018). for example, aqueous extracts of medicinal plants, spondias mombin, nymphea lotus and luffa cylindrica reduced chromosomal and nuclear aberration induced by pbno3 in a. cepa root tip cells (oyeyemi and bakare 2013). butanol and ethyl acetate fractions of parkinsonia aculeata l. leaf extract demonstrated the most significant reduction in chromosomal abnormalities in a. cepa cells treated with maleic hydrazide, indicating that they had chemo-preventive efficacy (sharma et al. 2018). previously, sharma et al. (2012) reported that using the a. cepa root chromosomal aberration assay, the chloroform extract of brasicca juncea seeds possesses antigenotoxic potential against mercury-induced genotoxicity. exploration of antitoxic properties from natural products has also been done at marine organisms such as ulva fasciata (rodeiro et al. 2015), sargassum sp. (kilawati and islamy 2019). however, limited studies were conducted in seagrass. seagrass are flowering plants that grow in a marine environment. one of the seagrass species is enhalus acoroides (l.f.) royle. enhalus acoroides is tropical seagrass, a member of the family of hydrocharitaceae, found throughout the indo-pacific region, including southern japan, southeast asia, northern australia, southern india, and sri lanka (short and waycott 2010). in indonesia, this species is distributed widely in papua, north maluku, ambon, sulawesi, bali, java, borneo, and sumatra in indonesia (kiswara and hutomo 1985). extract of e. acoroides leaves contains phytochemical compounds such as phenols, flavonoids, and tannins as well as several pigments including chlorophyll, lutein, pheophytin, and b-carotene (pharmawati and wrasiati 2020). it has been known that flavonoids, phenolic compounds and pigments have antioxidant activity. to extend self-life and protect oxidative stability of plant extract, microencapsulation is often applied (yusop et al. 2017) using colloidal particles such as maltodextrin, arabic gum, or chitosan (özkan and bilek 2014, šturm et al, 2019). the aim of this study was to analysed the genotoxic effect of encapsulated e. acoroides leaves extracts and its antigenotoxic potential against heavy metal nickel nitrate ni(no3)2.6h2o using allium cepa var. aggregatum root tips assay. materials and methods sample collection, extraction and encapsulation leaves of e . acoroides were col lected f rom semawang beach, denpasar, bali, indonesia. the methods of den hartog and kuo (2006) and mckenzie and yoshida (2009) were used to identify e. acoroides based on morphological traits. the voucher specimen was deposited in the herbarium biology udayana (hbump10), biology study program, universitas udayana. leaves were washed in running water, cut into 10 cm long, and air-dried for three days. the leaves were then further dried for one day using an oven at 50°c. using a blender, the dried leaves were mashed and sieved through a 60-mesh sieve. using 200 ml of chloroform: ethanol at a 9:1 (v/v) ratio, up to 20 g of dried leaves powder was extracted. the extraction was done using a soxhlet extractor. the solvent was filtered using whatman filter paper, and the filtrate was vacuum evaporated using an ika® rv10 rotary evaporator at 40°c and 100 mbar (pharmawati and wrasiati 2020) the encapsulation of crude extract was conducted using a 20% maltodextrin solution. as much as 10% extract of e. acoroides and 2% tween 80 were mixed with the encapsulated solution and homogenized at 6000 rpm for 30 minutes. after that, the mixture was dried up to 8% moisture content, mashed with a blender and then sieved through a 60 mesh sieve (sulistyadewi et al. 2014). 91genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate treatment of allium cepa var. aggregatum root the base of a. cepa var. aggregatum bulbs were soaked in water to induce roots. when the length of the root was approximately 1 cm, bulbs were transferred to a glass jar containing treatment solutions. the treatments were 30 ppm ni in the form of ni(no3)2.6h2o, encapsulated e. acoroides extract at 100 ppm, 250 ppm, and 500 ppm, and combined 30 ppm ni with each of 100 ppm, 250 ppm and 500 ppm of encapsulated e. acoroides extract. the treatments were given for 72 hrs. as controls, bulbs were soaked in h2o. three bulbs were used in each treatment. chromosome preparation modified procedures were used to prepare mitotic squash (sharma and sharma 1994). following treatments, roots were rinsed in distilled water and cut in the morning. roots were then soaked in farmer’s fixative containing of ethanol and acetic acid (3:1) for 24 hrs in the refrigerator. for hydrolysis, root tips were cut 2 mm long and treated with 1n hcl for 15 minutes. the root tips were cleaned in distilled water before being coloured with 2% acetoorcein for 20 min. excess stain was absorbed using filter paper. stained root tips were covered with cover glass and then squashed. slides were inspected for mitotic chromosomes and aberration using microscope binocular xsz 107bn (nanjing bw optics and instrument co.) with 400x total magnification. the photographs were taken using the top mount camera optilab advance (miconos). the data was collected from a total of six roots of three bulbs for each treatment and six fields chosen at random from each root. metabolic activity following the treatment and control procedures, 5 root tips were removed and soaked in 0.5% 2,3,5-triphenyl tetrazolium chloride (ttc) for 15 minutes in the dark at 35°c. the root tips were then analysed qualitatively after being rinsed with distilled water. furthermore, the roots were soaked in 95% ethanol to extract the colourful triphenyl formazan complex. the absorbance was measured at 490 nm (vazhangat and thoppil 2017). three replications were conducted in this experiment. apoptotic activity the evans blue staining method was used to investigate the loss of cell viability. after treatments, five roots with identical lengths were cut and dyed with a 0.25% (w/v) aqueous evans blue solution for 15 minutes before being rinsed with distilled water for 30 minutes. the experiment was conducted with three replications. the roots were then macro-imaged to determine cell death qualitatively. roots then were soaked in 3 ml of n,ndimethylformamide for 1 hour at room temperature for a quantitative estimation by measuring the absorbance of evans blue at 600 nm (vazhangat and thoppil 2017). data analyses the mitotic index (%) was computed as the number of dividing cells divided by the total number of cells ´ 100. the chromosomal aberrations were calculated by dividing the number of abnormal cells by the total number of cells counted × 100 (sarac et al. 2019). phase index (%) was determined by calculating the number of dividing cells in phases by the total number of dividing cells ´ 100 (kumar and thonger 2016). the antigenotoxicity of encapsulated e. acoroides extract was determined by calculating the inhibitory activity of chromosomal aberration induced by ni. the formula used was following prajitha and thoppil (2016). inhibitory activity (%)= a−b: a−c × 100, where a: number of aberrant cells induced by ni, b: number of aberrant cells induced by the mixture of ni and encapsulated e. acoroides extract, c: number of aberrant cells induced in the control statistical analyses were performed using minitab 20, with randomised experimental design. the differences between treatments were analysed using the tukey test with a 95% confidence level. the data were presented as mean ± standard deviation, except for the data of the types of aberration. results mitotic index using allium cepa var. aggregatum root tips, the antigenotoxic potential of encapsulated extract of e. acoroides leaves was investigated. one of the metrics used to assess antigenotoxicity was mitotic activity as measured by the mitotic index. the mitotic indices were significantly affected by the treatments (p<0.01). treatment of a. cepa root with ni reduced mitotic index significantly. there are no differences between the mitotic index of control and treatment with 100 ppm encapsulated extract of e. acoroides leaves. the concentration of 250 ppm and 500 ppm encapsulated extract had a sig92 made pharmawati, ni nyoman wirasiti, luh putu wrasiati nificantly lower mitotic index than control, but significantly higher than nickel (table 1). treatment with nickel resulted in the lowest mitotic index indicating genotoxic activity of nickel. when nickel and encapsulated extract of e. acoroides leaves were given simultaneously, the mitotic indices were higher than the mitotic index of ni alone; however, statistical analysis showed that only the addition of 100 ppm encapsulated extract had a significant increase of the mitotic index. table 1 shows the mitotic indices of control, treatment with ni, encapsulated e. acoroides extract, and combined treatment of ni and encapsulated extract. phase index the distribution of mitotic phases was shown in table 2. the treatments significantly affected prophase, metaphase and telophase indices (p<0.05), while anaphase index was not affected by treatments. the majority of chromosomes in all treatments and control were in metaphase. treatment of nickel resulted in the highest percentage of metaphase chromosomes, and ni inhibited telophase as indicated by the significantly lowest index of telophase in ni treatment. the addition of encapsulated e. acoroides extract to the ni treatment increased the percentage of telophase. chromosomal aberration and nuclear abnormality statistical analysis shows that the treatments affected chromosomal aberration (p<0.01) and nuclear abnormality (p<0.01). mitotic chromosomal aberrations were detected in all treatments including control (table 1) and control has a very low percentage of aberration. treatment with ni resulted in 1,677% of aberrant chromosomes. the percentage of the aberrant chromosome at root tips treated with encapsulated e. acoroides extract at the concentration of 100 ppm, 250 ppm and 500 ppm had no significant difference to control, suggesting that the encapsulated extract had no or very low genotoxic effect. simultaneous treatments of ni and encapsulated e. acoroides extract at concentrations 100 ppm, 250 ppm, and 500 ppm showed a similar percentage of chromosomal aberration to treatment with ni alone. modulation of ni-induced genotoxicity with encapsulated e. acoroides extract showed no significant reduction of chromosomal aberration. the inhibitory activities of encapsulated extract to the genotoxic activity of ni were only 4.9%, 6.5%, and 14.4% with simultaneous addition of 500 ppm, 250 ppm, and 100 ppm encapsulated extract. the types of chromosomal aberration at mitotic phases included prolonged prophase, stickiness, fragment, chromosoma l brea k /fragmentation at metaphase, diagonal metaphase, diagonal telophase, chromosome bridge, star anaphase, fragment at anaphase, and vagrant telophase. figure 1 shows normal mitotic phases, while figure 2 shows types of aberrant chromosomes. table 3 shows the percentage of each type of chromosomal aberration. nuclear abnormalities were observed in a different set of fields of view than that of chromosomal aberration. the nuclear abnormalities observed were micronuclei, nuclear fragments, and nuclear lesions (figure table 1. the mitotic index and percentage of chromosomal aberration of a. cepa root tip cells induced by ni, encapsulated e. acoroides leaves extract and mixture of ni and encapsulated extract. treatment (ppm) mitotic index chromosome aberration (%) control 5.036± 0.497a 0.091±0.1b 30ni 2.248±0.497c 1.677±0.487a 100ea 5.048±0.864a 0.553±0.462b 250ea 3.612±0.444b 0.542±0.227b 500ea 3.383±0.418b 0.551±0.2097b 30ni+100ea 3.262±0.29b 1.453±0.343a 30ni+250ea 2.905±0.2176bc 1.575±0.1974a 30ni+500ea 2.779±0.489bc 1.601±0.289a ni=nickel in the form of ni(no3)2.6h2o; ea=encapsulated e. acoroides leaves extract. means with same letters at the same column are not significantly different. table 2. phase index of mitosis of a. cepa root tip cells after treatment with ni, encapsulated e. acoroides leaves extract and mixture of ni and encapsulated extract. treatment (ppm) prophase index metaphase index anaphase index telophase index control 21.68±8.02a 27.8±5.38b 27.08±7.57a 23.44±5.08a 30 ni 19.22±8.82a 59.17±18.87a 19.14±13.58a 2.74±3.83b 100 ea 26.6±8.27a 32.96±4.89b 20.13±7.21a 18.31±7.08a 250 ea 22.98±6.02a 37.88±5.54b 16.61±4.96a 24.22±3.21a 500 ea 24.11±9.85a 37.72±13.86b 14.91±4.54a 21.57±3.49a 30 ni+100 ea 35.1±12.19a 29.42±11.86b 19.3±6.22a 16.17±5.75a 30 ni+250 ea 33.86±7.03a 36.21±4.82b 14.77±10.07a 15.16±5.46a 30 ni+500 ea 22.69±8.88a 43.34±15.44ab 17.97±2.83a 16±9.7a ni=nickel in the form of ni(no3)2.6h2o; ea=encapsulated e. acoroides leaves extract. means with same letters at the same column are not significantly different. 93genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate 3, table 4). micronuclei were not detected in control and in treatment using 100 ppm, 250 ppm of encapsulated e. acoroides extract, while it was detected at 500 ppm encapsulated e. acoroides extract but not significantly different than control. treatments with ni alone and combined ni and encapsulated e. acoroides extract resulted in the formation of micronuclei and statistically, they were not significantly different, although the percentage of combined treatments was much lower than ni alone. fragmented nuclei were not observed in control and in treatment with 100 ppm, 250 ppm, and 500 ppm of encapsulated e. acoroides extract. the highest percentage of nuclear fragmentation was induced by ni treatment only. the encapsulated e. acoroides extract also induced nuclear fragmentation in a significantly lower percentage than ni treatment. the encapsulated e. acoroides extract given simultaneously with ni, significantly reduced the percentage of fragmented nuclear (table 4). another nuclear abnormality observed was nuclear lesion and ni treatment showed the highest percentage. the addition of encapsulated extract to ni treatments significantly reduced the percentages of nuclear lesions compared to ni treatment (table 4). metabolic activity the triphenyl tetrazolium chloride (ttc) staining was used to examine the influence of ni and encapsulated e. acoroides extract on mitochondrial function. treatment of roots with ni revealed a substantial decrease in mitochondrial activity. visually, the encapsulated e. acoroides extract as well as combined encapsulated extract and ni shows an increase in mitochondrial activity (figure 4). based on the absorbance value of 490 nm, the encapsulated extract at concentrations of 100 ppm and 250 ppm has no effect, while 500 ppm extract reduced mitochondrial activity; however, the reduction was significantly less than treatment with ni (table 5). simultaneous treatment of encapsulated extract at 100 ppm and ni showed improvement of mitochondrial activity compared to ni alone. in comparison, the addition of 250 ppm and 500 ppm encapsulated extract to ni treatment did not show improvement of mitochondrial activity. apoptotic activity evans blue stain was used to analyse in situ cell death by assessing the cell membrane’s integrity. living cells keep the dye out due to the semipermeable nature of cell membranes. on the other hand, damaged cells are unable to remove the dye and are thus stained blue (roy et al. 2019). figure 5 shows the visualization of cell death using evan’s blue staining. evan’s blue staining method for in situ cell death revealed that the encapsulated e. acoroides extract at concentrations 100 ppm, 250 ppm, and 500 ppm showed less colour than treatment with ni only. simultaneous treatment of ni and encapsulated e. acoroides extract also showed a reduction of blue colour indicating a reduction of cell death (figure 5). quantitative analysis using a spectrophotometer is shown in table 5. statistical analysis revealed that 100 ppm and 250 ppm of encapsulated extract had similar effect as control. the 500ppm extract showed higher absorbance than control, but significantly lower than ni alone. the data of in situ cell death is similar to that of mitochondrial activity. the 100 ppm encapfigure 1. normal mitosis of a. cepa root tip cells. a. prophase; b. metaphase; c. anaphase; d. telophase. scale bar=10μm. figure 2. types of chromosomal aberrations of a. cepa root tip cells after treatment with ni, encapsulated e. acoroides leaves extract and combined ni and encapsulated extract. a=prophase abnormality with fragments; b= sticky metaphase; c= fragment at metaphase; d=chain metaphase; e=vagrant telophase (circle); f=diagonal anaphase; g=chromosome bridge; h=diagonal metaphase; i=fragment at anaphase; j=star anaphase; k=sticky anaphase. scale bar=10μm. arrows indicate abnormalities. 94 made pharmawati, ni nyoman wirasiti, luh putu wrasiati sulated extract when given simultaneously with ni demonstrated significantly less cell death than treatment with ni. discussion as a result of increased urbanization and industrialisation, toxic metal poisoning has become a global issue. moreover, accumulated heavy metal in plants has been known to induced chromosome abnormalities as shown by sabeen et al. (2020). this present study confirmed that nickel has a genotoxic effect by significantly decreasing mitotic index and inducing chromosomal aberration. at the concentration of 30 ppm ni(no3)2.6h2o the reduction of the mitotic index was 44.53% in comparison to control. the decline of the mitotic index below 50% has sub-lethal effects and is known as the limit value of cytotoxicity (madike et al. 2019). the genotoxic effect of nickel has been studied using nickel chloride (nicl2) (ganesan and panneerselvam 2013), nickel sulfate (niso4.6h2o) (pavlova 2017) and nickel nitrate ni(no3)2 (sarac et al. 2019). the concentration of 30 ppm ni was used in this study, to evaluate a lower concentration than that used by sarac et al. (2019) which was 50 ppm. nickel at 30 ppm induced chromosomal aberration where chromosome stickiness and chromosome break table 3. the percentage of each type of chromosomal aberration of a. cepa root tip cells after treatment with ni, encapsulated e. acoroides leaves extract and combined ni and encapsulated extract. treatment (ppm) frag.pro (%) sty.meta (%) frag.meta (%) ch.meta (%) diag.meta (%) diag.ana (%) star.ana (%) sty.ana (%) frag.ana (%) bridge (%) vr.telo (%) control 0c 0.091b 0c 0a 0a 0a 0b 0a 0a 0a 0a 30ni 0.256ab 0.509a 0.656ab 0.062a 0.062a 0.042a 0.1ab 0a 0.081a 0.023a 0a 100ea 0.069b 0.180ab 0.071c 0.071a 0.032a 0a 0.039ab 0.026a 0.027a 0.039a 0a 250ea 0.034bc 0.156ab 0.226c 0.026a 0.033a 0a 0b 0.033a 0.034a 0a 0a 500ea 0.029bc 0.151ab 0.233c 0.032a 0a 0.037a 0.034ab 0a 0.036a 0a 0a 30ni+100ea 0.394ab 0.221ab 0.357bc 0.034a 0.069a 0.029a 0.225a 0.059a 0.064a 0a 0a 30ni+250ea 0.224ab 0.292ab 0.73ab 0.027a 0a 0a 0.086ab 0.1a 0.027a 0.027a 0.065a 30ni+500ea 0.229ab 0.181ab 0.758a 0.034a 0.029a 0.029a 0.13ab 0.068a 0.073a 0.073a 0a ni=nickel in the form of ni(no3)2.6h2o; ea=encapsulated e. acoroides leaves extract. frag.pro=prophase abnormality with fragments; sty.meta= sticky metaphase; frag.meta=fragment at metaphase; ch.meta=chain metaphase; diag.meta=diagonal metaphase; diag.ana=diagonal anaphase; star.ana=star anaphase; sty.ana=sticky anaphase, frag.ana=fragment at anaphase; bridge=chromosome bridge; vr.telo=vagrant telophase. means with same letters at the same column are not significantly different. table 4. the percentages of nuclear abnormalities of a. cepa root tip cells after treatment with ni, encapsulated e. acoroides leaves extract and combined ni and encapsulated extract. treatment (ppm) micronuclei (%) nuclear lesion (%) nuclear fragmentation (%) control 0±0b 4.57±5.75d 0±0b 30 ni 0.222±0.192a 90.67±3.58a 0.73±0.609a 100 ea 0±0b 20,11±2.84c 0±0b 250 ea 0±0b 20.03±3.78c 0±0b 500 ea 0.020±0.063b 21.76±3.84c 0±0b 30 ni+100 ea 0.083±0.092ab 60.43±8.2b 0.083±0.091b 30 ni+250 ea 0.082±0.092ab 62.73±6.29b 0.079±0.087b 30 ni+500 ea 0.091±0.1ab 67.48±6.14b 0.076±0.084b ni=nickel in the form of ni(no3)2.6h2o; ea=encapsulated e. acoroides leaves extract. means with same letters at the same column are not significantly different. table 5. metabolic and apoptotic activities of a. cepa root tips after treatment with ni, encapsulated e. acoroides leaves extract and combined ni and encapsulated extract. treatment (ppm) metabolic activity apoptotic activity control 0.482±0.034a 0.166±0.03c 30 ni 0.157±0.006d 0.448±0.053a 100 ea 0.486±0.05a 0.268±0.07bc 250 ea 0.479±0.024a 0.286±0.071bc 500 ea 0.334±0.026b 0.301±0.035b 30 ni+100 ea 0.277±0.022bc 0.304±0.015b 30 ni+250 ea 0.242±0.057bcd 0.320±0.011ab 30 ni+500 ea 0.222±0.024cd 0.344±0.053ab ni=nickel in the form of ni(no3)2.6h2o; ea=encapsulated e. acoroides leaves extract. means with same letters at the same column are not significantly different. 95genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate were at a high percentage. this agrees with sarac et al. (2019) and kaur et al. (2019) who observed that chromosome break and chromosome stickiness were the major types of chromosomal aberration found due to heavy metal treatments. nickel promotes the generation of a large quantity of reactive oxygen species (ros) which is a factor of nickel toxicity. reactive oxygen species harm all cellular components in plants, including cell membranes, lipids, pigments, enzymes, chloroplasts, and nucleic acids (gopal and nautiyal 2012). excessive ros promotes dna break which can be observed through the chromosomal break (ganesan and panneerselvam 2013). chromosome stickiness is caused by chromosome loss of physical identity due to physical attachment of chromatin material or inter-chromosomal connections (asita et al. 2017). heavy metal complexes are very reactive, and their complexes interact directly or indirectly with dna, histone, or non-histone proteins, causing chromosomal surface properties to change, making them sticky (kumar and srivastava 2015). the cytotoxicity of encapsulated e. acoroides extract was tested by calculating the mitotic index. the mitotic index of encapsulated e. acoroides extract at a concentration of 100 ppm was similar to control. higher concentrations of encapsulated extract decreased mitotic index but were still higher than the mitotic index of ni treatment. this suggests that at 250 ppm and 500 ppm, the encapsulated e. acoroides extract was less toxic than ni. the reduction in the mitotic index shows that the encapsulated e. acoroides extract inhibits mitotic activity in a. cepa. the reduction in mitotic index is attributable to compounds in the aqueous extracts that have cytotoxic effects, as the mitotic index is a quantitative measure of mitotic activity in an organism or a particular organ (sreeranjini and siril 2011). when ni and encapsulated extract were given simultaneously, only 100 ppm encapsulated extract resulted in a significant increase of the mitotic index. this indicates that the encapsulated e. acoroides extract had the potential in modulated ni-inhibited mitotic activity by increasing the proliferative activity of cells. the antigenotoxic activity of low concentrations of plant extract was also reported by prajitha and thoppil (2016). the lower concentration (5 ppm) of amaranthus spinosus employed in the antigenotoxicity experiment was beneficial in reversing the genotoxicity. treatment with higher concentrations of encapsulated e. acoroides extract combined with ni did not significantly increase mitotic index. a study using chenopodium album extract found that at low concentration, the extract reduced the genotoxic effect induced by ems (ethylmethane sulfonate). at higher concentrations, c. album extract showed synergistic action with ems, resulting in an increased genotoxic effect (asita et al. 2015). in the present study, encapsulated e. acoroides extract did not have a synergetic effect with ni since the addition of encapsulated extract together with ni, had no significantly different with ni alone on the chromosomal aberration. based on analysis of phase index, ni treatment had a significantly higher metaphase index, while the anaphase indices were not significantly different between treatments. this means that in ni treatment the anaphase index was low. the telophase index of ni treatment was significantly lower than other treatments. figure 5. analysis of apoptotic activity using evans blue staining of root of a. cepa. a=control, b=30 ppm ni, c=100 ppm ea, d=100 ppm ea, d= 250 ppm ea, e=500 ppm ea, f=30ppmni+100 ppm ea, g=30 ppm ni+250 ppm ea, h=30 ppm ni+500 ppm ea. ea=encapsulated e. acoroides leaves extract. figure 4. analysis of metabolic activity using ttc staining of root of a. cepa. a=control, b=30 ppm ni, c=100 ppm ea, d=100 ppm ea, d= 250 ppm ea, e=500 ppm ea, f=30ppmni+100 ppm ea, g=30 ppm ni+250 ppm ea, h=30 ppm ni+500 ppm ea. ea=encapsulated e. acoroides leaves extract. figure 3. types of nuclear abnormalities of a. cepa root tip cells after treatment with ni, encapsulated e. acoroides leaves extract and combined ni and encapsulated extract. a. nuclear fragmentation; b. micronucleus; c. nuclear lesion. scale bar=10μm. 96 made pharmawati, ni nyoman wirasiti, luh putu wrasiati according to asita et al. (2017), a decrease in the proportion of dividing cells in a + t indicates that the chromosome spindles were poisoned, resulting in metaphase arrest. low anaphase and telophase indices can cause daughter cells to be damaged, limiting plant growth. the simultaneous addition of ni and encapsulated e. acoroides extracts at all concentrations significantly increased the telophase indices. the percentage of chromosome abnormality between control and treatment with all concentrations of encapsulated e. acoroides extract were not significantly different, indicating the possibility of the non-toxic effect of encapsulated e. acoroides extract. this result is important since the encapsulated e. acoroides extract had an antiproliferative effect by reducing the mitotic index. therefore, it can be further explored in anticancer research. chromosome aberrations were detected in the combined treatment of ni and encapsulated e. acoroides extract at all concentrations tested. although there were decreases in percentages of chromosome aberration in combined ni and encapsulated extract treatments, the percentages were not significantly different from that of ni treatment alone. this result indicates that the concentrations of encapsulated e. acoroides extract used unable to effectively suppress chromosome aberration induced by ni. however, it is worth noting that there was 14.4% inhibitory activity of ni when 100 ppm encapsulates e. acoroides extract was given simultaneously with 30 ppm ni. this suggests that the mixtures were less genotoxic than ni alone. nickel induced the formation of micronuclei and nuclear fragmentation at low levels but formed nuclear lesions in extremely high percentages. nuclear lesions provide cytological evidence of dna biosynthesis inhibition (sajitha and thoppil 2018). at all concentrations tested, the encapsulated e. acoroides extract did not induce micronuclei and chromosome fragmentation. however, it induced nuclear lesions at low percentages, significantly lower than induced by ni. the control group had a low level of nuclear lesion, which could be due to unintentional dna changes. according to nefic et al. (2013), root tip cells show a very low frequency of spontaneous abnormalities. in ttc analysis, the roots treated with ni were unable to convert ttc to red coloured tf, indicating a significantly lower activity of the mitochondrial respiratory chain compared to control. the roots treated with encapsulated e. acoroides extract at 250 ppm and 500 ppm demonstrated no effect on mitochondrial activity. the addition of lower concentration of encapsulated e. acoroides extract (100 ppm) to 30 ppm ni increased mitochondrial activity compared to treatment with ni alone. apoptotic activity was highly induced in ni treated root, while encapsulated e. acoroides extract showed lower apoptotic activity than ni, but higher activity than control. this suggests that encapsulated e. acoroides extract was less toxic than ni. supplementation of encapsulated e. acoroides extracts to ni, visually resulted in lower apoptotic activity than ni alone as observed as less blue colour. however, when measured using a spectrophotometer, there were no differences between the apoptotic activities at ni treatment and ni supplemented with 250 ppm and 500 ppm encapsulated e. acoroides extract. lower concentration of encapsulated e. acoroides extract (100 ppm) when given together with 30 ppm ni, induced reduction of apoptotic activity. the effects of simultaneous addition of ni and encapsulated e. acoroides extract at a lower concentration to metabolic activity and apoptotic activity agreed with their effect on the mitotic index. according to prajitha and thoppil (2016), a higher concentration of an extract can have mutagenic effect and a lower concentration can have an antimutagenic effect or vice versa. in mice, lower levels of b-carotene increased the anticlastogenic activity of cyclophosphamide-induced clastogenicity, but there was no protective impact at higher concentrations. this finding implies distinct processes of b-carotene modulation and a probable shift in the balance of the promutagen activation/detoxification mechanism (salvadori et al. 1992). similar reasoning may apply to the effect of simultaneous addition of ni and low concentration encapsulated e. acoroides extract on increasing mitotic index and metabolic activity and reducing apoptotic activity. enhalus acoroides leaves extract contained phytochemical compounds, including phenols, tannins, and flavonoids. the ftir analysis confirmed the presence of flavonoid and polyphenols as a high c-h out-of-plane bending (oop bend) vibration for the substituted benzene ring was identified in the extract (pharmawati and wrasiati 2020). these phytochemical components in the plant extracts may be responsible for the reduced mitotic index in a. cepa root meristematic cells when roots were treated with encapsulated e. acoroides leaves extract. on the other hand, these phytochemical compounds may contribute to the increasing mitotic index, lowering nuclear abnormalities when the encapsulated extract is present together in the ni treatment. this kind of result where plant extract showed the opposite effect was also observed by prajitha and thoppil (2016) in amaranthus spinosus extract. the encapsulated e. acoroides leaves extract also contained pigments such as chlorophyll b, 97genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate ethyl-chlorophyllide a, mg-free chlorophyll b, lutein, mg free chlorophyll a, pheophytin, and β-carotene (pharmawati and wrasiati 2020). it is well known that phenolic compounds, tannins, flavonoids, chlorophyll, and carotenoids have antioxidant properties (aryal et al. 2019). antioxidants containing phenolics can prevent the generation of free radicals and/or stop the spread of autoxidation. at the same plant pigments can chelate metals and transfer hydrogen to oxygen radicals, delaying oxidation (brewer 2011). in the present investigation, the encapsulated e. acoroides extract was found to have preventive activity, as evidenced by the reduction and reversion of nuclear damages (nuclear lesions and nuclear fragmentations) caused by ni. however, the encapsulated extract cannot reduce chromosomal aberration. preincubation with the encapsulation extract before ni treatment needs to be evaluated to test the ability of encapsulated extract to suppress chromosomal abnormalities. further study is also needed to test the protective activity of the encapsulated extract on animal cells. acknowledgment the authors thank universitas udayana for supporting this study through the study program flagship research scheme no. b/773/un14.2.8.ii/pt.01.03/2021 funding universitas udayana through the study program flagship research scheme no. b/773/un14.2.8.ii/ pt.01.03/2021 references amjad m, raza h, murtaza b, abbas g, imran m, shahid m, naeem ma, zakir a, iqbal mm. 2020. nickel toxicity induced changes in nutrient dynamics and antioxidant profiling in two maize (zea mays l.) hybrids. plants. 9(1):5. 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thailand piyaporn saensouk1, surapon saensouk2,*, rattanavalee senavongse2 effect of ag nanoparticles on morphological and physio-biochemical traits of the medicinal plant stevia rebaudiana sherzad r. abdull, sahar h. rashid*, bakhtiar s. ghafoor, barzan s. khdhir morphometric analysis and genetic diversity in hypericum l. using sequence related amplified polymorphism wei cao1, xiao chen2,*, zhiwei cao3 population differentiation and gene flow of salicornia persica akhani (chenopodiaceae) xiaoju zhang1, li bai2,*, somayeh esfandani-bozchaloyi3 scot molecular markers are efficient in genetic fingerprinting of pomegranate (punica granatum l.) cultivars shiva shahsavari1, zahra noormohammadi1,*, masoud sheidai2,*, farah farahani3, mohammad reza vazifeshenas4 first record of nucleus migration in premeiotic antherial cells of saccharum spontaneum l. (poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 145-154, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-174 citation: h. nemat farahzadi, s. arbabian, a. majd, g. tajadod (2020) long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder. caryologia 73(1): 145-154. doi: 10.13128/caryologia-174 received: february 26, 2019 accepted: february 23, 2020 published: may 8, 2020 copyright: © 2020 h. nemat farahzadi, s. arbabian, a. majd, g. tajadod. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod department of biology, faculty of sciences, islamic azad university, north-tehran branch, tehran, iran *corresponding author. e-mail: helalfarahzadi@yahoo.com, arbabias@gmail.com, ahmad_majd2005@yahoo.com, tajadodg@gmail.com abstract. pepper is one of the most important crop plants. recently, the global need for this plant has been widely increased due to its use in the food and pharmaceutical industry. we invested the effects of different concentrations of zinc on the development of male and female gametophytes of bell pepper (capsicum annuum l. var california wonder). the plants were cultivated with different concentrations of zinc nitrate (0 (control), 2.5, 5, 7.5, 10 and 15 mm) in a greenhouse under experimental conditions. buds and flowers are harvested at different stage of development (in 6 sizes) from may to jun. they were fixed in faa and maintained in 70% alcohol, then embedded in paraffin, sliced with a microtome and analyzed using a light microscope. microscopic studies showed that developmental process of ovule, gynoecium and pollen grain in bell pepper plants was taking to ordinary process in dicotyledonous plants. according to the results, increased zinc concentration resulted in a disorder in the reproductive phase, which caused the treatment 1 to enter the reproductive phase with a 4-week delay. in addition, the other of the treatments did not enter the reproductive phase and were wilt during the growth period. the developmental stages of gynoecium and anther in treatment 1 were similar to the control. however, a number of abnormalities and irregularities wereo bserved including signs of nuclei disintegration, deformation of embryo sac, accumulation of dark materials and deformation of pollen grains. keywords. bell pepper, male gametophyte, female gametophyte, zinc. introduction heavy metals refer to a group of elements with a density of more than 5 gr cm-3. a few of them (co, fe, mn, mo, ni, zn and cu) are essential micronutrients, which are necessary for normal growth, oxidation and reduction reactions, electron transferring, and other important metabolic processes in plants (rai et al., 2004). increase in zinc occurs mainly due to the environ146 helal nemat farahzadi et al. mental pollution following industrial and agricultural activities such as  smelter and incinerator emissions, spreading from mine wastes, excessive use of chemical fertilizers and zinc-containing insecticides, using sewage (waste water), sludge or other industrial and mineral fertilizers contaminated with zinc (pedler et al., 2004; giuffré et al., 2012). as it is rapidly absorbed by plants, it can be very toxic. growth inhibition is a common phenomenon attributable to poisoning with zinc in plants. the more poisoning occurs, the less the product will become, and it eventually overcomes the growth and inhibits it (broadley et al., 2007; marschner, 2012), which is mainly because of the degradation of the photosynthesis activity. this affects photochemical reactions (assche and clijsters, 1986), carbonic anhydrase activity (ló pezmillán et al., 2005) biosynthesis of chlorophyll (assche and clijsters, 1986) and the integrity of the cell membrane (wissemeier and horst, 1987). if the concentration of zinc becomes higher than the critical level, it will lead to a decrease in growth and no flower production (rout and das, 2003). pepper is one of the most important crop plants. the need for pepper cultivation has doubled over the past 20 years (fao, 2017). given the excessive increase in chemical fertilizers in agriculture, the increase in the amount of heavy metals in the environment, and the economic and nutritional importance of the pepper in recent decades in the world, the study aimed to examine the effect of zinc nitrate on anther and gynoecium development in this plant. materials and methods seeds from capsicum annuum l. var california wonder were provided from the plant gene bank of seed and plant improvement institute of karaj, iran. after sterilizing the seeds, they were cultured in a sterilized soil (obtained from behkam company). six treatment groups with different concentrations of zinc (0 (control), 2.5, 5, 7.5, 10 and 15 mm) were selected and used for irrigation from the first irrigation until the end of the growth period. during the propagation stage, temperature was 25 ± 2 ° c, humidity 75-80%, and 16 h day light. buds and flowers in all stages of development (in 6 sizes) were collected every week from may to jun, 2017. the collected plant materials were fixed in a faa (37% formalinglacial acetic acid-70%alcohol, 2:1:17 v/v) for 12 hours, then were stored in 70% alcohol and dehydration in an ethanol series and embedding in paraffin, specimens were sliced by shandon as325 rotary microtome. staining of serial sections of 6-7 µm was carried out hematoxylineosine. specimens were viewed with a olympus model bx43 light microscope connected to olympus digital camera. at least 15 flowers were observed for each developmental stage and the best samples were chosen for photographs. results generative meristem and flowering from the fifteenth to sixteenth weeks from the first day of cultivating, the generative meristems started their activity in the control plants and the buds emerged. in order to study the stages of flower development, the buds were considered in six stages: developmental stage 1: 1 to 2 mm diameter; developmental stage 2: 3 to 4 mm diameter; developmental stage 3: 3 to 5 mm diameter with the corolla hidden in calyx; developmental stage 4: 9 to 10 mm diameter corolla is in calyx; developmental stage 5: semiopen corolla with a 10-15 mm diameter; and development stage 6: approximately 20 mm in diameter the mature flower was considered with distinct corolla (figure 1). after the plant reaching the stage of f lowering, the vegetative meristem is transformed into a generative meristem. figure (2a-e) shows the developmental stages of generative meristem and the formation of different part of the flower. the generative meristem has a greater volume and densely stained compared to the vegetative meristem, which is the result of increased of mitosis activity in the apical meristem, tonica and corpus regions. microscopic studies of generative meristem show that this meristem is enlarged and expanded and its stainability is almost homogeneous in different parts. their cells are homogeneous, dense and more stainable compared to vegetative meristem. the terminal part of this meristem is sporogenous meristem (sp.m) and its figure 1. flower bud sizes: stage 1: 1-2 mm diameter (st1). stage 2: 3-4 mm diameter (st2). stage 3: 3-5 mm diameter (st3). stage 4: 9-10 mm diameter (st4). stage 5: 10-15 mm diameter (st5). stage 6: 20 mm diameter (st6). 147long-term effect of different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. lower part, which is less stainable, is called receptacle meristem (re.m). with the formation of generative meristem, the structural components of the flower such as sepals (se), stamen primordia (st.p), and petal primordia (pe.p) gradually become distinct (figure 2a). simultaneous to the formation of stamen, petals (pe) are formed, as a result of the activity of the peripheral part of sporogenous meristem against the stamen, and in the final stage, the ovary primordium (ov.p) is formed (figure 2b). sepals (se) appear with the activity and the remaining divisions of the initial ring (figures 2a-b). in the next steps, from the outside to inside, sepals (se), petals (pe), stamen primordia (st.p) and ovary primordium (ov.p), can be distinguished, respectively. the proliferated and deformed mass of the ovary primordium indicate the beginning of the ovary (ov) and style (st) organization (figure 2 b-c). style, ovary, and ovules primordia were formed in the gynoecium. following the mentioned changes and with gradual development of flower, sepals, petals, stamens, gynaecium, and ovules primordia can be detected (figure 2d), and the full flower structure with mature stamen and carpel is shown in figure 2e. microsporogenesis and pollen grains development bell pepper (capsicum annuum l.) flowers are bisexual (figure 2c-e). the flower is hexamerous (figure 2f). all the stamens are equal in size, and they surround the style and the stigma. in addition, the anthers are tetrasporagiate and contain four pollen sacs (figure 3 a-d). first, the stamens primordia are formed, then in the middle part, the pistil primordium is formed. as seen in figure 2b, the development of the stamens is faster than the gynoecium and ovule. when the pistil primordium is still seen in form of cell masses without any differentiation, the stamens (filament, anther, and various layers of the anther wall and the cells of the sporogenous tissue) (figure 2a-b) are observed and recognized clearly. the young anther cell wall contains an epidermis, endothecium, two middle layers and tapetum from the outside to the inside (figure 4a). young anther layers wall cells have the same size and their large nuclei occupy most of the cell volume (figure 4a). a series of rectangular cells form a row of single nucleus epidermis (figure 4a). before maturation, endothecium forms a row of single-nucleus rectangular cells (figure 4b). figure 2. longitudinal section of floral bud in different stages of flower development: (a): stage 1. (b): stage 2. (c): stage 3. (d): stage 4. (e): stage 5; (f) transverse section of floral bud (ib – idioblast; m.an – mature anther; ov.p – ovary primordium; p – petal; pe.p – petal primordium; p.o – ovule primordium; se – sepal; st.p – stamen primordium; st stigma; st: stamen; sty – style; y.an – young anther). 148 helal nemat farahzadi et al. during maturity, these cells are radially divided, and the singledouble outer endostium form up to 3 layers of cells towards the connective tissue (figure 4c-d). at this time, these cells form thick fibrous and the middle layers are decomposed (figure 4c-d). first, the tapetal cells are secretive single-cellular (figure 4a). the nuclear division in these cells usually occurs before the onset of meiosis in the pollen mother cells (pmcs) and they become bi-nuclei (figure 4c-e). during development of the anther, these cells develop radially and they are filled with dense materials (figure 4c). tapetal cells differ in the morphology characteristics: on the outer surface of anther wall, they are stretched, rectangular and relatively uniform, whereas they become longer radially towards the connective tissue and are hypertrophied (figure 4f). sporogenous cells of anther have a dense stainable cytoplasm their large nuclei occupy most of the cell volume (figure 5a). first, they are placed next to each other, then separated from each other and differentiated into pmcs (figure 4a-b). first, to create pollen grains, these cells have to undergo a meiosis, but before initiation of the division process of pmcs, secrete a spefigure 3. transverse section of anther in different stages of development. (a) young anther; (b) second meiotic division in anther; (c) anther at the binucleate pollen grain stage; (d) longitudinally dehiscent anther in cross section (fi – filament; p – pollen; pc – placentoids; sp – primary sporogenous layer; st – stamen; sto – stomium). figure 4. (a) the young anther wall layers and bilayer sporogenous tissue; (b) anther sporogenous tissue is forming; (c) degenerating middle layers and dividing endothecium cells at a single locule of anther. (d) transverse section of a single locule of mature anther before dehiscence showing degenerating intersporangial septum and anther wall with only epidermis and endothecium; (e) two-nucleated glandular tapetal cells on the outer side of anther wall; (f) transverse section of microsporangium showing outer and inner binucleate tapetal cells, disintegrating callose in microspores tetrads and anther wall layers (et – endothecium; ep – epidermis; it – inner tapetum; ml – middle layer; mt – microspore tetrads; md – microspore diad; ot – outer tapetum; pmc – pollen mother cell; p – pollen grain; pc – placentoids; t – tapetum). 149long-term effect of different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. cial callose wall around them, surrounding the whole cell (figure 5a). after the meiosis and the formation of the dyad (figure 4c) and then the tetrad (figure 4c, 5b), when the callose is decomposed, all microspores are released from tetrad into the lucule (figure 5c). they have dense cytoplasm and thin cell walls (figure 5c-e). at the end of the single nucleus stage, the microspore nucleus is pushed to one side. following the expansion of microspore, the nucleus is divided and two dissimilar nuclei (generative and vegetative) are produced (figure 5d). mature pollen grains normally have the large vegetative cell and a small generative cell (figure 5d). at this stage, only the epidermis and endostium exist in the anther wall (figure 5f). anthers dehiscence occurs through a longitudinal gap in the stomium (figure 5g). it is formed by a layer of at most 20 cells under the epidermis, which is highly stainable and contains calcium crystals (shown with an arrow on figure 5g). gynoecium, ovule and embryo development in bell pepper (capsicum annuum l.) the gynoecium is syncarpous, and the ovary is superior and figure 5. (a) pollen mother cell; (b) transverse section of anther with microspore tetrahedral tetrads; (c) microspore tetrads and diads stages. (d) binucleate pollen grains. (e) tricolporate pollen grains. (f) mature anther before dehiscence showing anther wall with only epidermis and endothecium; (g) stomium degenerating epidermis cells and anther dehiscence (ep – epidermis; et – endothecium; gc – generative cell; md – microspore diad; mt – microspore tetrads; ot – outer tapetum; pmc pollen mother cell; sc – sand crystal; vc – vegetative cell). 150 helal nemat farahzadi et al. ring shaped. the ovary is trilocular and consists of two carpels. each carpel has a large number of ovules on the placenta axis (figure 6a). the ovule is unitegmic, hemianatropous and tenuinucellate (figure 6b). the embryo sac in the ovule is of polygonum type. the uninucleate archeospore is developed under the apical epidermis of the ovules and acts directly as a primary sporogenous cell. thus, the meiosis in the megasporcyte initially forms as dyad and eventually as a linear tetrad (figure 6b). in the tetrads, the sister megaspores are gradually decomposed, and the development of the embryo sac begins with the chalazal megaspore (figure 6b). three mitosis divisions continuously produce functional megaspore in the embryonic sac of two, four, and eight nuclei (figure 6c). thus, the development of the embryonic sac confirms the polygonum type (figure 6e). for the formation of an embryo sac, the megaspore volume increases and its nucleus is divided into two nuclei, migrating to each pole of the cell (chalaza, micropyle) (figure 6d). then large vacuole is created between the nuclei. the nuclei are divided two times, and four nuclei of haploid are formed on each pole of the embryo sac (figure c-d). a nucleus migrates to the middle part of the embryo sac from each pole, polar nuclei are formed, and the remaining three nuclei evolve into three cells at each pole (chalaza, micropyle). there are three adjacent cells to the micropyle which are formed, the middle is called egg cell (oosphere), and the two symmetric lateral cells which are called synergid opposite to the chalaza, as well as three cells opposite to the micropyle which are called antipodal (figure 6f). these cells have a short life and die before fertilization in the embryo sac. the giant cells underneath the inner epifigure 6. (a)section of a single locule of ovary; (b) megaspore mother cell (mmc) beneath the apical epidermis of the nucellus; (c) end of the mmc second meiosis and linear arrangement of the megaspores; (d) eight-nucleate immature embryo sac; (e) eight-nucleate immature embryo sac; (f) eight-nucleate and seven-celled mature embryo sac (ant – antipodal; eg – egg cell; ent – endothelium; es – embryo sac; fc – functional megaspore; mmc – megaspore mother cell; nu – nucellus; ov – ovule; sy – synergid). figure 7. (a) transverse sections of ovary wall with giant cell; (b) transverse sections of ovary tissue with idioblast (gc – giant cell; ib – idioblast; ovovule). 151long-term effect of different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. dermis of the ovary wall are observed in developmental stages. during the developmental stages of the ovary, they enlarge (figure 7a). idioblasts containing calcium crystals are found in the ovule and ovary parenchyma cells (figure 7b). changes pollen grain and embryo sac in treatments in treatment 1 (2.5 mm), the plants entered the reproductive phase in the 19th to the 20th week with a 4-week delay. the plants in other treatment groups did not enter the generative phase (the number of culture periods was repeat three times, and no generative phase was observed). the process of male gametophyte development in this treatment was similar to the control. pollen grains from zinc-treated plants showed that the shape of pollen grains was changed from the normal spherical state to a smaller shape, and a large number of pollen grains had irregular shapes. a large number of pollen grains showed the accumulation of dark materials (figure 8a-b). in the development of female gametophyte, the examination of microscopic slides in the plants under treatment showed signs of nuclear degradation and embryo sac changes (figure 8c-d). figure 8. (a-b) transverse sections of anther from treatment 1. abnormalities in pollen grains and collected black materials in pollen grain; (c-d) transverse sections of ovule from treatment 1. change of the shape of embryo sac and also degradation of egg apparatus in plants (et – endothecium; ep – epidermis; es – embryo sac; ot – outer tapetum; p – pollen grain). 152 helal nemat farahzadi et al. discussion the studies on the structural characteristics of male and female gametophytes and the details of microsporogenesis and macrosporogenesis in various genera of solanaceace showed that bisexual are common in this family and dioecy are relatively rare (davis, 1966), hunzikera, 2001, talebi et al., 2016, ramezani et al., 2018). microscopic studies showed that developmental process of ovule, gynoecium and pollen grain in bell pepper plants was taking to ordinary process in dicotyledonous plants. the differentiation of the various parts of the floral from the vegetative meristem was consistent with the results of munting (1974) on c.annuum. the stamens in c.annuum are composed of relatively large anthers equal throughout the filaments. these stamens are tetrasporangiate and contain four pollen sacs (dharmadhaj and prakash, 1978), a distinct state in potato family (endress, 1996). the taptum is glandular and its cells are binuclear. secretive tapetum is observed in some solanacease, such as a. belladonna (yurukova-grancharova et al., 2011), w. somnifera (ghimire and heo, 2012), p. hybrida (chehregani and ramezani, 2016), s.tuberosum (talebi et al., 2016). in our observations, the structural characteristics of the tapetum cell layer were different from that of the outer surface of the anther wall and to the connective tissue in the bell pepper (c.annuum), that was consistent with the results of yurukova-grancharova et al. (2011) on a. belladonna, chehregani and ramezani (2016) ) on p. hybrid and ramezani et al. (2018) on c.annuum. although in these plants, the tapetal cells are different not only in structural but also in the number of nuclei, the number of nuclei in the tapetal cells is inconsistent in all families of solanaceace (tobe, 1989). as stated, for a.belladonna (yurukovagrancharova et al., 2011) it has four nuclei, for w. somnifera (ghimire and heo, 2012) two nuclei, for p. hybrid (chehregani and ramezani, 2016) four nuclei, and for s.tuberosum (talebi et al., 2016) it has several nuclei. our results showed that tapetal cells of c.annuum had two nuclei, similar to w. somnifera (ghimire and heo, 2012) and c.annuum (ramezani et al., 2018), whereas dharmadhaj and prakash (1978) reported three nuclei for c.annuum. at the time of maturation, each pollen grain of pepper has two nuclei; the presence of two-nucleus pollen grains is commonly found in solanaceace (davis, 1966, dharamadhaj and prakash, 1978, poddubnaya-arnoldi, 1976, talebi et al., 2016, ramezani et al. 2018). the sandy crystals accumulate in the anther cells of c.annuum. crystals appear in the early stages of flower development in most solanaceace anthers (d’arcy and averett, 1996). when the c.annuum pollen grains are matured, the anther wall breaks down, and these calcium oxalate crystals are released (ramezani et al., 2018). our results confirmed the previous studies by the authors for different speices of solanaceace (d’arcy and averett, 1996; rezanejad, 2006; chehregani and ramezani, 2016, ramezani et al., 2018). the released calcium crystals stick to the pollen and dissolve in the style mucous, and calcium ions can generate pollen for germination and pollen tube growth (iwano et al., 2004). the differentiation of male gametophyte in bell pepper (c.annuum) happens early from the female gametophytes, which is consistent with the results of munting (1974) and ramezani et al. (2018). the development of the embryo sac is done as a common pattern in all solanaceace (davis, 1966). in some c.annuum samples, we observed bilocular or trilocular ovary. indeed, the bilocular ovary is a characteristic feature of solanaceace (ghimire and heo, 2012, ramezani et al., 2018). c. annuum ovule is hemiantropous that is consistent with the results of some varieties of c. annuum (munting, 1974) and ramezani et al. 2018 on the same variety. the present study showed that the development of the female gametophyte c. annuum l. var. california wonder is of polygonum type. polygonum type is typically known as solanaceace (mohan and kamini, 1964, mohan, 1966, munting, 1974, dharmadhaj and prakash, 1978, yurukova-grancharova et al., 2011, ghimire and heo, 2012, brito et al. 2015, ramezani et al., 2018). according to van assche (1986), high doses of zinc inhibit many metabolic activities in the plant by damaging the mitochondrial structure in cells (rout and das, 2003). the high accumulation of zinc in the cytosol of the plant cell also results in impaired cellular function and inhibition of respiration and energy responses associated with cell growth that might reduce the growth and development of the whole plant (bonnet et al., 2000). this element affects the process of cell division by interrupting the interphase, prolonging the stage of prophase, g2, and stopping the synthesis of the synthesized proteins required by the cell cycle and nucleic acid synthesis (el-ghamery et al., 2003). the continuity, integrity and permeability of the membrane are impaired due to toxicity of zinc. at the molecular level, it also affects the expression of many genes (wang et al., 2009). nutritional conditions play an important role in flower formation (taiz & zeiger, 2010). the zinc stress and increase in its concentration disrupt the plant’s balance nutrition. on the other hand, the concentration of zinc affects hormonal balance and causes a disruption in hormonal balance and because hormones act as genetic regulators in inducing expression of the involved genes (rout and das, 2003). according to aloni reports in 2006, delayed 153long-term effect of different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. flowering is due to a hormonal disorder such as auxin, which is due to gene disruption. in treatment 1, the flowering began with delay and the rest of the treatments did not enter the reproductive phase, although stresses such as heavy metals typically cause premature aging of plants, and from the developmental perspective one of the symptoms of aging is flowering. however, in the interpretation of latency and lack of flowering in c.annuum seen under the influence of zinc stress in our study, one can state that the nutritional conditions of the plant plays an important role in the formation of the flower. studying the c/n ratio in different plants has shown that each time a plant prepares for flower formation, this ratio goes higher (taiz & zaiger, 2010). increase in the concentration of zinc causes disruption of absorption in other elements, including iron (zeng et al., 2011), which causes a delay or lack of flowering. studies on microscopic sections of control and treatment samples showed that the general trend of ovule and pollen formation in plant treatment 1 and control was the same. however, for the plants in treatment 1, a large number of pollen grains smaller than the normal size were found. the results were consistent with mohsenzadeh (2011) on reseda lutea l., yousefi (2011) about the effect of heavy metals pollution on chenopodium botrys l., albooghobaish et al. 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(ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. and mathematical analysis cihangir alaca1, ali özdemir1, bahattın bozdağ2, canan özdemir2,* clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, 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partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 73(3): 89-96, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-191 caryologia international journal of cytology, cytosystematics and cytogenetics citation: e. karlik (2020) display of sukkula distributions on barley roots via in situ hybridization. caryologia 73(3): 89-96. doi: 10.13128/caryologia-191 received: march 17, 2019 accepted: june 19, 2020 published: december 31, 2020 copyright: © 2020 e. karlik. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. display of sukkula distributions on barley roots via in situ hybridization elif karlik the university of istinye, faculty of arts and sciences, department of molecular biology and genetics, istanbul/turkey e-mail: elif.karlik@istinye.edu.tr abstract. retrotransposon are an abundant and ancient parts of the plant genomes that especially ltr retrotransposons influence the genome size and evolution. sukkula is a non-autonomous and active, relatively high copy-number retroelement. in this study, we performed fluorescence in situ hybridization (fish) to observe the distributions of sukkula elements (ltrs and internal-domain) by using labelled-pcr products. the localizations of sukkula elements (ltrs and internal-domain) were observed under confocal microscope on hordeum vulgare l. cv. hasat root preparations. our results revealed that sukkula elements is still active and spread through the whole barley chromosomes. additionally, the re-sequencing analysis of sukkula ltrs demonstrated that ltrs sequences had ~65 bp gain. these analyses represent a valuable resource to reveal genome organization of barley and large sized plants. keywords: fluorescence in situ hybridization, retrotransposon, sukkula, barley, ltrs, internal-domain introduction beginning with the pioneering work of barbara mcclinton, transposable elements (tes) have become to take part a central position in the plant genome studies. tes consist of dna fractions capable of chromosomal movement, either via replicative or conservative (cut-and-paste) mechanisms (doolittle and sapienza 1980; orgel and crick 1980; finnegan 1989). eukaryotic tes contain two main classes; class i elements and class ii elements. class i elements are also known as retrotransposons move through using an rna intermediate, while class ii elements move through the genome using a dna intermediate (finnegan 1989). in plants, the vast majority of repetitive dna in the nuclear genomes is derived from the proliferation of mostly class i elements called as retrotransposons (sanmiguel et al. 1996; vicient et al. 1999; hawkins et al. 2006; neumann et al. 2006; vitte and bennetzen 2006) which are subdivided as two major subclasses; long terminal repeat (ltr) retrotransposons and non-ltr retrotransposons. ltr retrotransposons, which typically comprise gag and pol protein coding orfs encoding several enzymes (reverse transcriptase 90 elif karlik – rt; protease – pr; rnaseh – rh; integrase – int) responsible for reverse transcription and integration of daughter sequences into new chromosomal locations, constitute the largest fraction of the tes (eickbush and malik 2002; havecker et al. 2004; hawkins et al. 2006; neumann et al. 2006; vitte and bennetzen 2006). moreover, ltr retrotransposons are found in plants are subdivided in two main superfamilies, gypsy-like and copia-like (also known as metaviridae and pseudoviridae, respectively), which include the same protein coding domains. however, these domains are rearranged in different order in both ltr retrotransposon types (eickbush and malik 2002; havecker et al. 2004). sukkula elements were first identified in barley genome at the barley mlo locus and to an insertion sequence present in the 3’ ltr of one bare1element (manninen and schulman 1993). shirasu et al. (2000) later determined two ~5 kb sequence similar this insertion in a 66 kb stretch of barley genome that were also found to be flanked by 5 kb direct repeats. therefore, these sequences were named as sukkula elements means “shuttle” in finnish. however, sukkula ltr copies are found to be gypsylike retrotransposons, but non-autonomous elements belonging to a novel group of retroelements, large retrotransposon derivatives or lards (kemekawa et al. 1999; kalendar et al. 2004). moreover, sukkula elements consist of reverse transcriptase in appx. 3.5 kb central domain which is found to be conserved as in primary sequence and secondary structure, including no open reading frames (orfs) encodes typical retroelement proteins. according to these features of sukkula elements, they are trims (terminal-repeat retrotranposons in miniature) in their lack of a protein-coding domain (kalendar et al. 2004). active retrotransposons are important for genome diversification in plants, because of their transposition and accumulation potentials in the genome, thus it can change the overall genome structure (wessler et al. 1995; vicient et al. 1999; schulman and kalendar 2005). fluorescence in situ hybridization (fish) using target-specific dna probes have become important tool in modern biology and cell research (hausmann and cremer 2003). in plants, introducing fish probes is more difficult, because of the cell wall and the cytoplasm of the plants that they hinder chromosome spreading and low metaphase indices (salvo-garrido et al. 2001). by using fish technique, the distribution of retrotransposon families has been reported in various plants such as hordeum vulgare, allium cepa, aegilops speltoides, brachypodium distachyon and glycine max (vicient et al. 1999; lin et al. 2005; kiseleva et al. 2014; shams and raskina 2018; li et al. 2018). bare1 distributions on barley chromosomes have been demonstrated by using bac clones a probe via fish (vicient et al. 1999). in barley, fish technique was also used to reveal gene organization and to integrate the genetic linkage map with a physical map (stephens et al. 2004). the aim of this study was to present the distributions of sukkula elements (ltrs and internal-domain) in hordeum vulgare l. cv. hasat chromosomes using labelled-pcr products via fish. sukkula localization patterns were observed under confocal microscope on barley root preparations. we also performed sequencing studies and the sequence analysis of sukkula elements (ltrs and internal-domain) to elucidate sukkula sequence alterations in barley. our results indicate that sukkula elements (ltrs and internal-domain) are still active and under genome evolution. materials and methods plant materials barley (hordeum vulgare l.) cv. hasat was provided from directorate of trakya agricultural research institute. the seeds were grown at growth chamber for germination period under controlled conditions (16 h light/8 h dark, 25°c ± 2°c) and relative humidity was kept at 60–75%. the plants were harvested after 72 hours, directly treated with liquid nitrogen and then stored at –80°c until dna extraction. gdna isolation gdna were isolated from 200 mg of the samples by using the cetyltrimethylammonium bromide (ctab) precipitation method was modified as previously described in mafra et al. (2008). specifically, 200 mg homogenized sample was incubated with 1 ml edward’s buffer (0.5% (w/v) sds, 250 mm nacl, 25 mm edta, 200 mm tris ph 8.0) at 95oc for 5 min (cold spring harbor laboratory 2005). they were then spun down at relative centrifugal force of 16,000 g in a microcentrifuge for 15 min, and the supernatant was isolated twice with chloroform. then, the aqueous phase was incubated with 2 volumes of ctab precipitation solution, after which the ctab protocol was followed as previously described (mafra et al. 2008). dna yield and purity were measured by uv spectrophotometry at 230, 260 and 280 nm using a nanodrop 2000c instrument (thermo scientific usa). dna integrity was evaluated by agarose gel electrophoresis, samples were separated on 1% agarose gels containing ethidium bromide nucleic acid stain in 1x tae buffer. 91display of sukkula distributions on barley roots via in situ hybridization chromosome preparation for fish analysis grains were placed randomly in petri dishes containing filter paper soaked in only water to germinate in an incubator at 18-25°c in the dark for 3 days. then, root tips of barley cv. hasat were harvested, then directly fixed in carnoy fixative (3:1 ethanol:acetic acid solution) without any chemical pre-treatment, stored roots at 4ºc. chromosome preparations and fish analysis were performed according to jenkins and hasterok (2001, 2007) with modifications. the slides were checked under the light microscope (olympus u-tvo.5xc-3) and kept in a freezer at -20 ºc. development of probes and labelling the fish probes used in this study were generated from two set of data which is the sukkula (internaldomain) gene and ltr sequences. to investigate the distribution of sukkula, we amplified internal-domain and ltr sequences of sukkula using designed specific primer sets table 1. the probes for internal-domain and ltr sequences designated by using idt’s primerquest© tool (2012). gc% and tm values of probes were around 50 and between 50°c and 55°c, respectively. the sequences of sukkula ltr and internal-domain were obtained from barley (ay054376 for ltr and intern-domain). probe synthesis was carried out individually by using sukkula ltr and internal-domain primers. the reactions were carried out in total volume of 50 μl including 18.25 μl nuclease-free water, 25 μl of hotstart pcr master mix (bio-rad), 1.5 μl of each primer (10 μm/μl), 1.75 μl of tetramethylrhodamine-dutp (tritc) (1 mm), and 2 μl template dna (40 ng/μl). pcr conditions were as follows: 94°c for 5 min followed by 40 cycles of 94°c for 25 s, annealing 50°c for 25 s and 72°c for 30 s. the reaction was completed by a final extension step at 72°c for 5 min. fluorescence in situ hybridization (fish) analysis the fish analysis procedure was performed based on jenkins and hasterok protocol (2001, 2007) with modif ications. chromosome spreads were scanned under ×40 objective light microscopes to define the number and quality of well-spread metaphase plates, and they were treated with 100 μg/ml of rnase at 37°c for 1 h. the hybridization mixture consist of 20 μl of deionised formamide (50%), 8 μl of dextran sulphate (10%), 4 μl of 20x ssc (2x ssc), 2 μl of 10% sds (0.5%), 10 μl of probe (75-200ng/slide), 1 μl of blocking dna (sonicated salmon sperm dna) (25100x probe) and added sterile dh2o to bring final volume 40 μl. final concentrations were indicated in parenthesis. the mixture was denatured at 85°c for 10 min and kept on ice for 10 min. a 38 μl aliquot of the hybridization mixture was applied onto each slide, covered with a coverslip, and sealed with paper bond. both chromosomal dna and probe dna on the slides were denatured together in a thermal cycler at 70°c for 6 min and hybridized with each other at 37°c overnight in a humid dark box. afterwards, hybridization the chromosome spreads were washed three times in 2x ssc: once 2x ssc to float coverslips off; once in 15% formamide/0.1x ssc, and again once in 15% formamide/0.1x ssc, each for 10 min at 42°c. then, slides were washed in 2x ssc for 3 min at 42°. this step was repeated twice with fresh 2x ssc at 42°. ultimately, slides were washed three times in 2x ssc for 3 min at rt. after, slides were dehydrated in alcohol series (70, 90 and 100%), each for 1 min at rt and waited in the dark for 15-20 min. vectashield-dapi mounting-staining medium (7-10 μl) was dropped onto the chromosome spreads, which were then stored at 4°c until used. image acquisition for imaging the slides, the following wavelengths were utilized for fluorescence detection: 551-575 nm for probes labelled with tritc and 420-480 nm for dapi in leica dm5500 confocal microscope. the different fluorescent images were acquired separately. afterwards, they were merged into single composite images. the signal images were analysed by adobe photoshop cc 2014. table 1. primers used in this study. no primer name sequence (5’→3’) 1 sukkula ltr f ccctccttccctcttctctaat 2 sukkula ltr r ccatactctgaacctgatcctaaac 3 sukkula ltr sequencing f aaccagtcaaccagcatagg 4 sukkula ltr sequencing r ggagagggagagataagaggaa 5 sukkula internaldomain f ccttgcacttgatggctact 6 sukkula internaldomain r cggatgagacacggaagaaa 92 elif karlik sequence analysis for sequence analysis of sukkula ltrs, we performed pcr reaction. the pcr products were resequenced. th e sequence homology search was conducted in barley genome by using blastn in the ensembl website (http://plants.ensembl.org/barley). however, the re-sequencing results of sukkula ltrs were compared the original sukkula ltr sequences using clustalomega (altschul et al. 1990). results and discussion total copy number of tes in plant genomes expands from as little as a few hundred in those with smaller genome sizes, including arabidopsis, to hundreds of thousands in their larger genome counterparts (e.g. maize, triticum, hordeum) (bennett and leitch 2005). comparison studies suggest that the same general te types are found in all plant species, however the relative proportions of diverse classes and subclasses can diff er dramatifigure 1. display of sukkula internal-domain distributions in barley root preparations via fish. figure 2. display of sukkula ltrs distributions in barley root preparations via fish. 93display of sukkula distributions on barley roots via in situ hybridization cally (kejnovsky et al. 2012). moreover, ltr retrotransposon turnover in monocots have been demonstrated to be extremely rapid with both gains and losses of tes comparing with eudicots (ma et al. 2004; vitte and bennetzen 2006). in the present study, distribution of sukkula ltrs and intern-domain were observed on hordeum vulgare l. cv. hasat root tips preparations using fish analysis (see figure 1 and 2). because of the large genome size of barley, single-copy probes are mostly designed from bac or yac contigs (vicient et al. 1999; acevedo-garcia et al. 2013; bustamante et al. 2017). in the current study, we used direct pcr products derived from barley genomik dna to generate single-stranded probes were short, ~ 421 bp for ltrs and 451 bp for internal-domain which were highly specifi c and stable for hybridization, therefore it leads to very good amplifi cation of the signals. in addition to display of sukkula elements in barley chromosomes, our group were able to observe the distributions of sire1 env and gag by using barley root tips via fish (karlik and gozukirmizi unpublished data). nowadays, it is figure 3. a) gel fi guration of sequencing primers results. b) demonstration of sequencing analysis results. original genbank sequence ay054376 compared with the sequences recovered from bottom gel band and upper gel band displayed in figure 3a. 94 elif karlik also possible to use short direct labelled-pcr products to observe lncrnas on barley chromosomes by using fish (karlik et al. 2018). the impact of retrotransposons especially ltr-retrotransposon proliferations and loss on genome structure and evolution of plant species have been studied in species with smallor medium sized genomes. however, large sized genomes have been reported in monocotyledonous species, including maize (2.3 gigabase pairs) and barley (5.1 gigabase pairs) (schnable et al. 2009; mascher et al. 2017), thus we studied with barley in the present study. sukkula element is known as non-autonomous ltr retrotransposons which use other retrotransposons proteins for transposition, thus we observed that sukkula elements (ltrs and internal-domain) distributed on whole barley genome (see figure 1 and 2). however, some studies demonstrated the prevalence or eventual decay of tes in the different genomic regions depends on the process of selection and “host control” in a very long evolutionary time (rebollo et al. 2012; vitte et al. 2014). kartal-alacam et al. (2014) has investigated sukkula polymorphism rates in non-cultured mature embryos, 40and 80-days old callus materials by using irap and ipbs techniques that sukkula is the second most active retrotransposon in barley genome. moreover, our study indicated that transposition of sukkula elements does not depend on the process of selection and “host control”. kalendar et al. (2004) suggested that sukkula ltrs are rarer than bare1 and not distributed in the barley genome. however, their fish results demonstrated sukkula ltrs with a high copy number. their labelledltr probes were hybridized with the chromosome arms except the telomeres, nucleolar organizing regions, and centromeres, where the signals are blocking. interestingly, we also observed a high copy number, in addition with our ltr and internal-domain probes labelled the regions in the whole chromosomes, including telomeres, nucleolar organizing regions, and centromeres (see figure 1 and 2). however, our fish results are consistent with the chromosome hybridization data were confirmed by pcr reaction that both sukkula segments (ltrs and internal-domain) were found to be present on all barley chromosomal segments. the abundance of repetitive dna is mostly responsible for genome size variations in species or interspecies that especially in ltr-retrotransposons, these differences in abundance may be originated from extreme amplification through retro-transposition or from dna loss by unequal homologous recombination, which produced solo-ltrs (flavell 1986; lisch 2013). during the probe synthesis, we noticed the two bands in agarose gel electrophoresis analysis (see figure 3a). then, we performed sequencing analysis to reveal the difference between two bands. therefore, re-sequencing analysis of sukkula ltrs has revealed that sukkula ltrs had some gains, especially ~ 65 bp during the evolutionary time, indicating that this event may depend on dna gain by unequal homologous recombination (see figure 3b). additionally, sukkula ltrs sequences (ay054376) demonstrated 95.20% sequence identity to bottom gel band and 58.81% identity to upper gel band. in conclusion, we were able to observe the distributions of the sukkula ltrs and internal-domain elements via fish by using labelled-pcr products in barley root preparations. sukkula is a non-autonomous ltr retrotransposon which is still 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angiosperm genome evolution. proc natl acad sci usa. 103:17638–17643. wessler sr, bureau te, white se. 1995. ltrretrotransposons and mites: important players in the evolution of plant genomes. curr opin genet dev. 5(6):814-821. caryologia. international journal of cytology, cytosystematics and cytogenetics 75(4): 111-121, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1721 caryologia international journal of cytology, cytosystematics and cytogenetics citation: narges firoozi, ghasem karimzadeh, mohammad sadegh sabet, vahid sayadi (2022). intraspecific karyomorphological and genome size variations of in vitro embryo derived iranian endemic asafoetida (ferula assa-foetida l., apiaceae). caryologia 75(4): 111-121. doi: 10.36253/caryologia-1721 received: june 29, 2022 accepted: december 06, 2022 published: april 28, 2023 copyright: © 2022 narges firoozi, ghasem karimzadeh, mohammad sadegh sabet, vahid sayadi. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. intraspecific karyomorphological and genome size variations of in vitro embryo derived iranian endemic asafoetida (ferula assa-foetida l., apiaceae) narges firoozi, ghasem karimzadeh*, mohammad sadegh sabet, vahid sayadi department of plant genetics and breeding, college of agriculture, tarbiat modares university, tehran p. o. box 14115-336, iran *corresponding author. e-mail: karimzadeh_g@modares.ac.ir abstract. asafoetida (ferula assa-foetida l.) is one of the endemic medicinal plants in iran. analysis of karyomorphology and 2cx dna measurements (monoploid genome size) of 18 iranian endemic ferula assa-foetida populations were performed. the in vitro embryo-derived root tips were examined for karyological studies, via technique of squash and stain with 2% (w/v) aceto-orcein. seeds of the ferula samples and leaves of solanum lycopersicum as standard reference (2c dna = 1.96 pg) were stained with propidium iodide (pi), using flow cytometric (fcm) technique. all the studied populations were diploids (2n = 2x = 22) with mean chromosome length (cl) of 3.95 μm, varied from 3.05 μm (p7) to 4.94 μm (p18). the mean total chromosome volume (tcv) was 0.98 μm3, ranged from 0.47 μm3 (p7) to 1.57 μm3 (p3). two-typed chromosomes (“m”, “sm”) formed three classes of karyotype formula. karyotypes were mostly symmetrical and fell in 1a and 2a stebbins category. the monoploid genome size of iranian endemic ferula assa-foetida populations is being stated for the first time; its mean value was 4.51 pg, ranged from 4.09 (p4) to 4.69 pg (p16). intraspecific karyomorphological and genome size variations were clearly confirmed in studied ferula assa-foetida. keywords: chromosome, ferula assa-foetida, flow cytometry, karyotype, 2cx dna. 1. introduction ferula assa-foetida belongs to apiaceae family that grows in iran, kashmir in pakistan, and afghanistan. asafoetida production from ferula assafoetida as a source is confined to southern iran (farhadi et al. 2019; barzegar et al. 2020). iranian flora consists of 30 species of ferula, most of which are endemic (khajeh et al. 2005; farhadi et al. 2019). it is herbal and permanent and enlarges to 2 cm high (khajeh et al. 2005). apiaceae family had very low germination owing to seed dormancy (nadjafi et al. 2006). hence, the germination of ferula’s seeds was complicated. to accelerate the breakage of 112 narges firoozi et al. its seed dormancy, various methods were applied such as soaking with running water and treating with either chilling temperature or ga3 (keshtkar et al. 2008; hassani et al. 2009; zare et al. 2011). the oleo-gum-resin has got from taking away of the stems or cut off the roots that have a sulfurous smell and bitter taste. ferula species, due to its chemical compounds, play a useful role in the treatment of various diseases. the oleo-gum-resin is antiseptic, antifungal, antibiotic, laxative, indigestion, antiviral, antidiabetic whooping cough, cramp, infertilitypain, and cancer chemopreventive (aruna and sivaramakrishnan 1992; dehpour et al. 2009; lee et al. 2009). the attribute properties of these plants have sesquiterpene coumarins and e few monoterpenes (elrazek et al. 2001). terpene coumarins have anti-hiv activity (zhou et al. 2000). in plant sciences, for a large number of plants in order to dna content’s screening is used of flow cytometry (fcm) as a powerful and relifigure 1. geographic distribution of sampled ferula assa-feotida l. on the map of iran, using arcgis 113intraspecific karyomorphological and genome size variations of in vitro embryo derived iranian endemic asafoetida able technique (e.g., loureiro et al. 2005; mahdavi and karimzadeh 2010; karimzadeh et al. 2010, 2011; abedi et al. 2015; tarkesh esfahani et al. 2020; zarabizadeh et al., 2022). it mainly focused on cell cycle analysis, measurement of nuclear dna content, and determination of ploidy level. it can be used to determine monoploid and holoploid genome size (doležel et al. 2003, 2007; greilhuber et al. 2005(. furthermore, in plant systematics and plant breeding, karyotypes can make available evidence and data for species identification and the study of populations resulting from a cross between individuals (anjali and srivastava 2012). in a study on ferula assa-foetida, it was found that this plant is diploid with a chromosome number 22, grouping in 2a class according to stebbins classification (zhao et al. 2006). likewise, the same chromosome number of 22 was reported by el-alaoui-faris et al. (2006) in five different species of ferula; f. gouliminensis, f. cossoniana, f. tingitana, f. sauvagei, and f. atlantica. furthermore, in a study conducted in china on two species of f. liacentiana and f. bungeana, the 22-chromosome number was also reported (qixin and menglan, 1993). it is significant to note that some studies and reports on f. assa-foetida in outside of its native range have mistakenly identified the species. in other words, some species that produce asafoetida are often misidentified as f. assa-foetida (chamberlain, 1977). awareness of genetic diversity and the management of genetic resources are considered as the main parts of plant breeding programs. the first step in plant breeding is to understand the genome structure and the germplasm collection (lee et al. 2021). taken together, these situations indicate the need for basic investigations especially cytogenetic studies. the key aim of the current survey was to study intraspecific genome size and karyo-morphological variations among 18 f. assa-foetida populations of iran. 2. materials and methods seeds of 18 iranian endemic populations of ferula assa-foetida l. were used for this study. the germplasm collection of the iranian biological and resource center (ibrc), tehran, iran from where the seeds were obtained. geographical description, climatic information, the population codes used in this study and the gene bank codes are present in table 1. since previously reported methods, including chilling temperature and treating ga3, was not satisfactory to achieve good germination, hence, in vitro embryo culture was the best, and the most suitable technique (zare et al. 2011; suran et al. 2016). 2.1. in vitro embryo culture at first, seeds were soaked in running water for 24 h sterilized as follows: table 1. local information of the collected iranian endemic ferula assa-foetida l. mean rainfall (mm) mean temp (ºc) altitude (m) longitude (e) latitude (n) local collection locations population code 13.66 20.20 1817 52º 33’ 00.0’’ 33º 27’ 54.0’’ esfahan, iran p1 60.52 15.25 2750 51º 26’ 55.3’’ 30º 55’ 49.9’’ kohkiloyeh boyerahmad, iran p2 15.87 20.82 1795 54º 20’ 00.0’’ 29º 12’ 00.0’’ fars, iran p3 5.54 22.91 669 56º 55’ 50.6’’ 33º 35’ 39.1’’ khorasan, iran p4 4.32 20.42 2158 54º 38’ 23.8’’ 32º 06’ 43.4’’ yazd, iran p5 5.08 20.54 1720 54º 09’ 53.6’’ 31º 38’ 13.4’’ yazd, iran p6 5.08 20.54 831 55º 38’ 20.4” 33º 06’ 43.2” yazd, iran p7 4.32 20.42 2158 54º 38’ 23.6’’ 32º 06’ 43.4’’ yazd, iran p8 5.08 20.54 1950 54º 14’ 42.5’’ 31º 38’ 41.7’’ yazd, iran p9 5.08 20.54 2160 54º 09’ 53.6’’ 31º 38’ 13.5’’ yazd, iran p10 5.08 20.54 3279 54º 05’ 27.0’’ 31º 37’ 41.2’’ yazd, iran p11 9.04 20.00 2164 57º 54’ 50.4” 29º 18’ 10.8” kerman, iran p12 12.05 17.07 2000 56º 45’ 00.0’’ 30º 48’ 00.0’’ kerman, iran p13 9.04 20.00 2200 56º 25’ 00.0’’ 31º 08’ 00.0’’ kerman, iran p14 12.05 17.07 1900 57º 07’ 00.0’’ 30º 17’ 00.0’’ kerman, iran p15 4.05 21.05 2600 57º 18’ 00.0’’ 30º 30’ 00.0’’ kerman, iran p16 11.49 16.67 1850 55º 07’ 57.0’’ 30º 17’ 40.0’’ kerman, iran p17 7.04 19.23 2335 56º 60’ 00.0’’ 30º 20’ 20.4’’ kerman, iran p18 114 narges firoozi et al. · wash the seeds with five drops of washing liquid for 2 min. · seeds placement under running water for 5 min. · seeds placement in ethanol 70% for 2 min. · wash seeds with distilled water for 5 min. · seeds placement in sodium hypochlorite 5.25 (v/v) for 20 min. · wash seeds with dsh2o three times and each time for 2 min in laminar airflow. after this step, the embryos were excised by push the bottom of seeds and were transferred to murashige and skoog medium (murashige and skoog 1962). the embryos in petri dishes were placed in an incubator at photoperiod with a 16 h light/8 h dark and 25 °c. it was germinated after three to four days. 2.2. karyomorphological analysis for the preparations of karyology, actively around one cm-long in vitro growing roots were removed and pre-treated in colchicine (0.05% (w/v)) for 4 h in the dark at 4 °c to impel metaphase arrest. after that, the roots were subsequently fixed in freshly carnoy’s fixative (3 absolute ethanol: 1 glacial acetic acid (v/v) ratio) at 4 °c for 24 h (karimzadeh et al. 2010, 2011). using distilled water, the fixed roots were washed then in a water bath its hydrolyzed in 1m hydrochloric acid (hcl) for 10 min at 60 °c, after these steps, staining was performed for 3 h with aceto-orcein (2% (w/v)) at 25 °c (karimzadeh et al. 2011). the five well-expand monolayer metaphase plates from various individuals were examined per ferula assa-foetida populations. photographs were taken in high resolution via a light microscope (bx50 model, olympus optical co., ltd., tokyo, japan) armed with an digital camera (dp12, olympus optical co., tokyo, japan). eight chromosomal parameters were either investigated as short arm (s) and long arm (l) lengths, or measured as chromosome length (cl = l + s), r-value (s/l), arm ratio (ar; l/s), total chromosome volume (tcv = πr2 cl, where r = average chromosome radius), form percentage (f% = s/scl), and centromeric index (ci = s/cl). chromosome types were determined, via levan et al. (1964) formula and idiograms were drawn from the mean values. the following parameters were also assessed for analysis karyotype asymmetry: the difference of range relative length (rl%max rl%min), karyotype total form percentage (tf% = σs/σcl × 100), dispersion index (di = (mean ci × cvcl) /100), mean centromeric asymmetry (mca), coefficient of variation of chromosome length (cvcl) (paszko 2006; peruzzi et al. 2009), romero-zarco (1986) intrachromosomal (a1) and interchromosomal (a2) asymmetry indices; and stebbins asymmetry categories for the investigation of karyotype asymmetry were assessed (stebbins 1971). 2.3. flow cytometric analysis for each asafetida population, the monoploid 2cxvalue was calculated through flow cytometric analysis. hence, to prepare nuclear suspensions, four seeds (jedrzejczyk and sliwinska, 2010) of each asafetida sample along with the healthy fresh young leaves of solanum lycopersicum cv. stupicke (2c dna = 1.96 pg; doležel et al., 2007) as the internal reference standard plant were chopped with a sharp razor blade in ice-cold woody plant buffer (wpb, loureiro et al., 2007). flow cytometric analysis was carried out via pi (propidium iodide) staining method. the nuclei suspension was examined via the f low cytometer (bd facscanto ii, bd biosciences, bedford, ma, usa), through bd facsdivatm software. the gained data were transferred to flowing software (ver. 2.5.0, cell imaging core, turku centre for biotechnology) to be editable in partec flomax software (ver. 2.4e, partec, münster, germany). the range of gating zone was calculated on obtained histograms from fcm, via the flomax. healthy fresh young leaves of the asafetida sample’s seeds and the standard reference were chopped, using a sharp razor blade. this action performed in ice-cold nuclear extraction buffer wpb (woody plant buffer, loureiro et al. 2007). the chopped seeds and leaves were filtered via a 30 μm green nylon mesh (partec, münster, germany). one ml of staining buffer, 50 µg ml-1 of pi (fluka) solution and 50 µg ml-1 of rnase (sigma-aldrich corporation, mo, usa) stock solution were added to each sample. for the stained nuclei, the relative fluorescence intensity was calculated via fcm on a linear scale. for each sample, flow cytometrically minimum 5000 nuclei were evaluated. the values of the means of g1 peak were used to estimate the absolute dna amount of the sample (doležel et al. 2003, 2007; greilhuber et al. 2005; doležel and bartoš 2005; mahdavi and karimzadeh 2010; karimzadeh et al. 2011) as follows: sample 2cx dna (pg) = (sample g1 peak mean/standard g1 peak mean) × standard 2c dna (pg). monoploid genome size was estimated based on above converting formula in the form of base pair (doležel et al. 2003). it should be noted that one pg of dna equivalent to 978 mega base pairs (mbp) were considered (doležel et al. 2003). 115intraspecifi c karyomorphological and genome size variations of in vitro embryo derived iranian endemic asafoetida 2.4. statistical analysis in first normality test was carried out for the obtained data from karyotypic and f low cytometric studies and then were investigated for karyological data in fi ve replications and for nuclear dna content three replications based on a completely randomized design (crd). differences between means were measured through the least signifi cant diff erence (lsd). minitab 17 soft ware package was used for multivariate statistical analysis. principal components analysis (pca) was then carried out based on data matrix to evaluate the contribution of each karyotypic parameter to the ordination of populations. a cluster analysis on chromosomal parameters was carried out, through the ward’s method and the euclidean distance to assess similarities and variations amid the populations. 3. results all 18 iranian endemic populations of ferula assafoetida were diploid (2n = 2x = 22). obtained karyotypes from somatic complement and the haploid complement’s idiograms of studies f. assa-foetida populations are demonstrated in fi gures 2 and 3, respectively. based on the anova results, among populations for either all chromosomal parameters, or monoploid genome size were signifi cant diff erences (p < 0.01; 2cx dna; table 2). th e mean chromosome length (cl) was determined as 3.95 μm, varied from 3.05 μm (p7) to 4.94 μm (p18). th e mean ci of the complement, varied from 41.15% (p18) to 45.57% (p1). th e mean tcv was 0.98 μm3, ranged from 0.47 μm3 (p7) to 1.57 μm3 (p3). according to numerous karyotypic symmetrical indices tested, f. assa-foetida populations indicated various symmetrical clusters. th e maximum value of tf% was recognized in p1 (45.3%) and the lowest value was identifi ed in p14 (41.3%). th e uppermost and the lowermost values of the various range of relative length (drl) were detected in p8 (4.79%; the most asymmetric) and p1 (2.25%; the most symmetric), respectively. th e highest and lowest values of cvcl %were identifi ed in p8 (15.53%; the maximum asymmetric) and p1 (7.17%; the maximum symmetric), respectively. similar to the results of drl and p1 p2 p3 p4 p5 p6 p7 p8 p9 p10 p11 p12 p13 p14 p15 p16 p17 p18 figure 2. karyotypes of somatic chromosome complement of 18 iranian endemic ferula assa-foetida l. (n = 2x = 22) populations. scale bars = 5 µm. ch r omosome leng th (µ chr oosome leng th (µ p1 p2 p3 p4 p5 p6 p7 p8 p9 p10 p11 p12 p13 p14 p15 p16 p17 om p18 figure 3. idiograms of haploid chromosome complement of 18 iranian endemic ferula assa-foetida l. (2n = 2x = 22) populations. 116 narges firoozi et al. cvcl%, the highest value of dispersion index (di) was detected in p8 (0.07; the most asymmetric), while p1 displayed the lowest (0.03; the most symmetric). in conclusion, three (drl, cvcl% and di) among five karyotypic symmetrical indices examined, confirmed that all 18 f. assa-foetida populations examined p8 and p1 performed to have the most asymmetrical and symmetrical karyotypes, respectively. the highest value of mca was identified in p14 (18.75 %) while p1 demonstrated the lowest value (9.1 %). two-typed chromosomes were recognized in all populations by using levan et al. (1964) chromosome nomenclature: “m” (centromere at medium region) and “sm” (centromere at sub medium region); formed 3 different karyotypic formulas as follows: 22m (nine populations), 20m+2sm (six populations) and 18m+4sm (three populations; table 3). karyotypes of all populations were ordered in the 1a and 2a classes of stebbins classification (stebbins 1971). for further detailed studies of asymmetry, a1 and a2 indices were also calculated (romero-zarco, 1986). the highest a1 = 0.30 was found in p14, showing the highest inter chromosomal difference, resulted in asymmetric karyotype and the lowest a1 = 0.16 is related to the p1 which demonstrations the highest symmetry. the highest a2 = 0.16 was related to table 2. anova of chromosomal parameters (a) and monoploid genome size (2cx dna; b) of ferula assa-foetida l. s.o.v. df ms s l cl ar r-value f% tcv ci a) chromosomal parameters population 17 12.58** 0.19** 0.18** 2.35** 2.42** 1.85** 20.12** 2.32** error 972 0.79 0.005 0.005 0.97 0.97 0.98 0.66 0.97 b) monoploid genome size (2cx dna) s.o.v. df 2cx dna population 17 0.066** error 36 0.004 ** significant difference (p < 0.01). table 3. karyotypic parameters of ferula assa-foetida l. (2n = 2x = 22). populations stebbins’ category karyotype formula cvcl% di drl% tf% mca asymmetry indices (romero-zarco, 1986) a2 a1 p1 1a 22m 7.17 0.03 2.25 45.30 9.00 0.07 0.16 p2 1a 20m+2sm 13.78 0.06 4.21 43.54 14.11 0.14 0.23 p3 1a 22m 14.45 0.06 4.40 43.34 14.26 0.14 0.24 p4 1a 20m+2sm 10.88 0.05 3.27 43.26 14.24 0.11 0.24 p5 1a 22m 13.16 0.06 3.89 43.21 13.48 0.13 0.23 p6 1a 22m 12.79 0.05 4.03 42.61 15.58 0.13 0.26 p7 1a 22m 14.01 0.06 4.29 42.97 14.92 0.14 0.24 p8 1a 22m 15.53 0.07 4.79 42.95 14.34 0.16 0.24 p9 1a 22m 13.71 0.06 4.22 43.05 14.05 0.14 0.23 p10 1a 22m 11.65 0.05 3.65 42.67 14.89 0.12 0.25 p11 1a 20m+2sm 12.86 0.05 3.99 43.06 15.31 0.13 0.25 p12 1a 20m+2sm 14.94 0.06 4.48 41.78 17.14 0.15 0.27 p13 2a 18m+4sm 13.78 0.06 4.24 41.50 17.59 0.14 0.28 p14 1a 18m+4sm 13.73 0.06 4.07 41.31 18.75 0.14 0.30 p15 1a 22m 14.94 0.06 4.62 42.67 13.03 0.15 0.22 p16 2a 20m+2sm 11.67 0.05 3.43 42.58 16.06 0.12 0.26 p17 1a 20m+2sm 13.37 0.06 4.12 41.39 17.25 0.13 0.28 p18 1a 18m+4sm 13.92 0.06 4.10 41.67 17.69 0.14 0.29 117intraspecifi c karyomorphological and genome size variations of in vitro embryo derived iranian endemic asafoetida p8; they have the highest chromosomal asymmetry and the lowest a2 = 0.07 was related to p1, having the highest intra chromosomal symmetry (table 3). the scatter diagram of these indices (figure 4) shows fi ve groups of populations. th e principal component analysis of the karyotypic parameters was carried out to estimate total variation and its parameters quota in populations. th e pca representing that the fi rst three principal components account for 99% of the cumulative variation, and they were plotted in a 2-dimensional graphic (figure 5). th e ward (khodadadi et al. 2014) phenogram constructed based on karyotype similarities (figure 6) shows six major clusters. th e principal component analysis resulting populations arrangement from this exam entirely fi ts with that obtained with the ward grouping analysis. th us, the obtained results suggested that populations within one cluster, having the high homology in chromosomal diff erences. the result of fcm data was confirmed for normality and analyzed based on completely randomized design (crd) with three replicate cells. anova showed amongpopulations high signifi cant diff erence (p < 0.01) for monoploid genome size (2cx dna content; table 2). th e mean value was 4.51 pg (table 4), varied from 4.09 pg in p4 to 4.69 pg in p16. th e histograms obtained for 27 a 2 a1 figure 4. scatter plot of intrachromosomal (a1) and interchromosomal (a2) asymmetries of 18 iranian ferula assa-foetida l. populations 28 first component s ec on d c om p on en t figure 5. diagram resulting from principal components analysis (pca) of ferula assa-foetida l. populations. 29 observations d is ta n ce figure 6. dendrogram showing the phenetic relationships among the studied populations of ferula assa-foetida l. populations. table 4. mean (± se) comparisons of monoploid genome size (2cx dna) of iranian endemic ferula assa-foetida l. (2n = 2x = 22). 1cx dna (mbp) 1cx dna (pg) mean 2cx dna (pg) ± se population 2166.27 2.215 4.43 ± 0.075efg p1 2146.71 2.195 4.39 ± 0.047fg p2 2122.26 2.170 4.34 ± 0.006g p3 2000.01 2.045 4.09 ± 0.042h p4 2210.28 2.260 4.52 ± 0.036bcde p5 2185.83 2.235 4.47 ± 0.010def p6 2259.18 2.310 4.62 ± 0.035ab p7 2176.05 2.225 4.45 ± 0.049defg p8 2244.51 2.295 4.59 ± 0.012abc p9 2273.85 2.325 4.65 ± 0.015a p10 2190.72 2.240 4.48 ± 0.060cdef p11 2176.05 2.225 4.45 ± 0.025defg p12 2234.73 2.285 4.57 ± 0.019abcd p13 2229.40 2.280 4.60 ± 0.038abc p14 2244.51 2.295 4.59 ± 0.026abc p15 2293.41 2.345 4.69 ± 0.030a p16 2283.63 2.335 4.67 ± 0.019a p17 2268.96 2.320 4.64 ± 0.056ab p18 2000.01-2293.41 2.045-2.345 4.09-4.69 range means with the same symbol letter in a “mean 2cx dna (pg)” column are not signifi cantly diff erent (p > 0.01), using lsd test. 118 narges firoozi et al. analyzing nuclear dna amount included two peaks (figure 7): the left peaks refer to the g1 of solanum lycopersicum cv. stupicke as reference standard plant (doležel et al. 2007) and the right peaks to the g1 of f. assa-foetida samples. in other words, using 1cx dna monoploid genome size in mbp, the mean value of all populations was 2205.39 mbp (table 4). 4. discussion ferula assa-foetida belongs to apiaceae family includes about 170 identifi ed taxa, of which 30 species have been detected in different phytogeographical regions in iran (zomorodian et al., 2018). it is an important perennial herb with medicinal benefi ts which is native to central asia and iran (farhadi et al. 2019). many pharmaceutical properties and medical benefi ts had been reported for ferula (dehpour et al. 2009; lee et al. 2009). for increasing the potential applicability of this genus, this perennial plant still needs more investigation on the genetic variability, expanding the range of research on its genetic characteristics as well as develop breeding methods. in the current study, we studied the iranian endemic ferula assa-foetida populations. eighteen iranian endemic populations of asafoetida (f. assa-foetida l.) were cytogenetically assessed on the basis of karyomorphology and genome size in the current study; they were all diploids (2n = 2x = 22). mostly, in ferula genera the diploids are more frequent and obtained results in the present study are in agreement with those reported on ferula assa-foetida (zhao et al. 2006) and on other species of ferula (wanscher 1931; qixin and menglan 1993; ghaff ari et al. 2005; el-alaoui-faris et al. 2006; sağiroğlu and duman 2006; bernard et al. 2007). th e obtained results of anova show a signifi cant diff erence in terms of all chromosomal traits. (table 2), confi rming intraspecifi c karyomorphological diversity in the studied iranian endemic asafoetida populations, which would be benefi cial for the success of the breeding programs. the mean chromosome length (cl) was 3.95 μm, varied from 3.05 μm (p7) to 4.94 μm (p18). in other words, a remarkable 1.89 μm variation in chromosome size was detectable among such a number of examined iranian endemic asafoetida. in the present investigation two-typed chromosomes (“m”, “sm”) formed three classes of karyotype formula, comprised: 22m for nine populations (p1, p3, p5p10, p15), 20m+2sm for six populations (p2, p4, p11, p12, p16, p17(, and 18m+4sm for three populations (p13, p14, p18). zhao et al. (2006), studying on ferula fukanensis, similarly reported the two-typed chromosomes of “m” and “sm”. th e studied karyotypes were grouped in the 1a and 2a classes based on classifi cation of stebbins. in a study on ferula fukanensis, karyotypes were classifi ed as 2a (zhao et al. 2006), which was consistent with the results of the current study in the p13 and p16 populations. it has been alleged that symmetric karyotypes have a lower grade of evolution in comparison with asymmetric karyotypes (stebbins 1971). understanding taxon evolution, and interrelations are facilitated through the information obtained from karyotype, and chromosome morphology (furo et al. 2020; sayadi et al. 2021). in the current investigation, the scatter diagram shown the populations in fi ve groups that exactly fi ts with the principal component analysis resulting species arrangement. furthermore, the results noted that populations inside a cluster have the maximum homology of chromosomes. according to number of n uclei p1 p2 p3 p4 p5 p6 p7 p8 p9 p10 p11 p12 p13 p14 p15 p16 p17 p18 relative nuclear dna content (a. u.) figure 7. flow cytometric histograms of monoploid genome size (2cx dna (of 18 iranian endemic ferula assa-foetida l. populations. th e left peaks refer to g1 of the solanum lycopersicum cv. stupicke (2c dna = 1.96 pg) reference standard and the right peaks refer to g1 of ferula assa-foetida l. samples. 119intraspecific karyomorphological and genome size variations of in vitro embryo derived iranian endemic asafoetida karyotypes studies, the most fertile offspring can be produced by crossing the populations having the maximum chromosomal homology. the results of principal component analysis, and cluster analysis for the chromosomal traits, it is possible to introduce populations in groups 2 and 3 due to the shortest distance, to intersect and produce maximum fertile offspring. accordingly, crossing between populations in a cluster is recommended, for instance between p13 with p14, p16, or p17, and also p12 with p18. the karyotypes in the primitive species are usually highly symmetrical, but that is not necessarily the case. in other words, a distinct and more evidence is ever required to evaluate the direction in changes of karyotype (peruzzi and eroǧlu 2013). it has been noted that the evolutionary relationships via asymmetry indices usage for the establishment may not be straightforward. it can be stated that the genus diversity may have resulted from the structural changes. some differences in asymmetry indices and karyotype formula between species may have contributed to this diversity (seijo and fernandez 2003). as stated in the stebbins (1971) classification, the karyotypes were mostly symmetric (table 3). in the present study, to achieve greater measurement accuracy, additional parameters were also assessed in addition to stebbins asymmetry categories, including tf%, di, cvcl, mca, a1, and a2 asymmetry indices for karyotype asymmetry analysis (table 3). some have argued and suggested that stebbins’ (1971) classification as a qualitative method is not so strong and lower flexible regarding the types of conclusions it can provide (paszko, 2006). the average of di% was 5.6% (from 3% in p1 to 7% in p7). all of the populations were symmetric based on tf% parameter, that the mean value was 42.71%, ranging from 41.3% (p14) to 45.3% (p1). some chromosomal disorders are probably a factor of gradual changes in the amounts of tf%. the appearance changes in the chromosomes morphology were happens due to the various causes such as translocated or duplicated chromosomes (das et al. 1998). when the populations are similar in stebbins classification, estimates a1 and a2 parameters of romero-zarco (1986) to determine the further asymmetric karyotype are necessary. as a1 index gets lower in p1 represents karyotype symmetry and a higher value in p14 assessed greater asymmetry. the a2 parameter was 13.22%, ranging from 7% (p1) to 16% (p8). preventing interspecific cross accomplishment and offspring infertility may have ensued from the difference in resulting karyotype symmetry. obvious intraspecific diversity was also detected in terms of monoploid genome size (2cx dna) among examined iranian endemic asafoetida populations. hence, on the other hand, the 2cx dna amounts of 18 iranian endemic f. assa-foetida l. populations are being reported for the first time, having the mean value of 4.51 pg, varied from 4.09 (p4) to 4.69 pg (p16). in other words, in our knowledge, few reports were found in the literature for the genome size estimation even in other ferula species. for example, 2.90 pg was reported for 2c dna amount of f. communis by olmedilla et al. )1985) and 4.92 pg in f. heuffelii by siljak-yakovlev et al. (2010). changes in the genome size (increases or decreases) may have participated in the genus diversity and evolution (seijo and fernandez 2003). cytogenetical investigations as a valuable method have been significantly performed in phylogenetic relationships amongst plants; and its obtained information has been of appreciable value in understanding taxon evolution and interrelations. these results may provide relevant information for f. assa-foetida l. breeding studies. acknowledgement authors gratefully thank tarbiat modares university, tehran, iran, for their support to the study. author contribution n. firoozi carried out the experiments under the supervision of prof. g. karimzadeh and the advisory of assist. prof. m. s. sabet. the manuscript prepared and revised by v. sayadi. all authors approved the final manuscript. this research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors. references abedi r, babaei a, and 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(asafetida), a threatened medicinal herb. trakia j. sci. 9(2): 57-61. zhao x, ma x.j, kaisar s, fu c.l, and chen r.y. 2006. karyotypes analysis of ferula fukanensis. chin. med. j. 31(2): 114-116. zhou p, takaishi y, duan h, chen b, honda g et al. 2000. coumarins and bicoumarin from ferula sumbul: anti-hiv activity and inhibition of cytokine release. phytochemistry. 53(6): 689-697. zomorodian k, saharkhiz j, pakshir k, immeripour z and sadatsharifi a. 2018). the composition, antibiofilm and antimicrobial activities of essential oil of ferula assa-foetida oleo-gum-resin. biocatal. agric. biotechn. 14: 300-304. caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 4 2022 firenze university press avicennia genus molecular phylogeny and barcoding: a multiple approach laleh malekmohammadi1, masoud sheidai1,*, farrokh ghahremaninejad2, afshin danehkar3, fahimeh koohdar1 studying some morphological responses of stevia (stevia rebaudiana bertoni) to some elicitors under water deficiency basoz sadiq muhealdin1, sahar hussein hamarashid1,*, fairuz ibrahim ali1, nakhshin omer abdulla1, syamand ahmad qadir2 morphological and cytogenetic characterization in experimental hybrid aloe jucunda reyn. x aloe vera (l.) burm. f. (asphodelaceae) wendy ozols-narbona*, josé imery-buiza assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions seyedeh mahsa hosseini1, sepideh kalatejari1, mohsen kafi2,*, babak motesharezadeh3 assessment of protein and dna polymorphisms in corn (zea mays) under the effect of non-ionizing electromagnetic radiation ekram m. abdelhaliem1,*, hanan m.abdalla1, ahmed a. bolbol1, rania s. shehata1,2 chromosome counts of some species of wetland plants from northwest iran saeedeh sadat mirzadeh vaghefi*, adel jalili delimiting species using dna and morphological variation in some alcea (malvaceae) species based on srap markers chnar hama noori meerza1, basoz sadiq muhealdin2, sahar hussein hamarashid2,*, syamand ahmad qadir3, yusef juan4 mapping cap-a satellite dnas by fish in sapajus cay paraguay and s. macrocephalus (platyrrhini, primates) simona ceraulo, francesca dumas* determination of genome size variation among varieties of ilex cornuta (aquifoliaceae) by fow cytometry peng zhou1, jiao li2, jing huang1, fei li1, qiang zhang2,*, min zhang1,* first report of chromosome and karyological analysis of gekko nutaphandi (gekkonidae, squamata) from thailand: neo-diploid chromosome number in genus gekko weera thongnetr1, suphat prasopsin2, surachest aiumsumang3,*, sukhonthip ditcharoen4, alongklod tanomtong5, prayoon wongchantra6, wutthisak bunnaen7, sumalee phimphan3 intraspecific karyomorphological and genome size variations of in vitro embryo derived iranian endemic asafoetida (ferula assa-foetida l., apiaceae) narges firoozi, ghasem karimzadeh*, mohammad sadegh sabet, vahid sayadi cytogenetic studies in the centaurea aucheri group (sect. phaeopappus) seyed mahmood ghaffari¹,*, seyed mohsen hesamzadeh hejazi² karyomorphology, genome size, and variation of antioxidant in twelve berry species from iran saeed mohammadpour1, ghasem karimzadeh1,*, seyed mahmood ghaffari2 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(3): 21-32, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-167 caryologia international journal of cytology, cytosystematics and cytogenetics citation: a. acar, z. türkmen, k. çavuşoğlu, e. yalçin (2020) investigation of benzyl benzoate toxicity with anatomical, physiological, cytogenetic and biochemical parameters in in vivo. caryologia 73(3): 21-32. doi: 10.13128/ caryologia-167 received: february 12, 2019 accepted: april 13, 2020 published: december 31, 2020 copyright: © 2020 a. acar, z. türkmen, k. çavuşoğlu, e. yalçin. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. investigation of benzyl benzoate toxicity with anatomical, physiological, cytogenetic and biochemical parameters in in vivo ali acar1,*, zafer türkmen2, kültiğin çavuşoğlu2, emine yalçin2 1 vocational school of health services, department of medical services and techniques, giresun university, giresun, turkey 2 department of biology, faculty of science and art; giresun university, giresun, turkey *corresponding author. e-mail: aliacar@outlook.com abstract. in this study, the toxic effects of benzyl benzoate, which is widely used in the food, cosmetics, agriculture and pharmaceutical sectors, have been investigated using allium cepa l. test material. in the determination of toxicity, physiological parameters with determination of root lengths, weight gains and germination percentages; cytogenetic changes with determination of chromosomal abnormalities formation, micronucleus (mn) frequency and mitotic index ratio (mi); anatomical changes with determination of anatomical differentiations in root tip cells; biochemical changes with lipid peroxidation and antioxidant enzyme analysis were determined and the obtained data were evaluated statistically. the bulbs were divided into four groups consisting of one control and three application groups, bulbs of the control group were treated with tap water and the bulbs of the application groups were treated with benzyl benzoate at doses of 10,000, 25,000 and 50,000 mg/l for 72 hours. at the end of the study, it was determined that germination percentage, weight gain and root length and mi ratio decreased, chromosomal abnormalities, mn formation, mda, sod and cat levels increased dose-dependent in the application groups when compared with the control group. depending on the application, it has been determined that root cells have chromosomal abnormalities such as fragments, sticky chromosomes, chromosome bridges, unequal distribution of chromatins and c-mitosis. furthermore, when compared with the control group, it was determined that benzyl benzoate administration caused anatomical changes in root tip cells. it was determined that these changes were in the form of necrosis, cell deformation, flattened cell nuclei, cortex cell deformation, accumulation of certain substances in cortex cells, wall thickening in cortex cells and unclear vascular tissue. in conclusion, it was determined by physiological, anatomical, cytogenetic and biochemical parameters that benzyl benzoate showed a dose-dependent toxic effect in allium cepa l. root cells. also, the parameters used in the study were determined to be useful biomarkers for the determination of toxicity. keywords: benzyl benzoate, allium cepa l., toxicity, chromosomal abnormalities, lipid peroxidation, antioxidant enzymes. 22 ali acar, zafer türkmen, kültiğin çavuşoğlu, emine yalçin introduction with the increase in the world population, it is rapidly increasing in consumption in many areas such as food, medicine, clothing and cosmetics. due to this increase, production quantities of foodstuffs, medicines and cosmetic products, that have an important place in human life, are increasing day by day. these increases in production and consumption cause competition among the manufacturers, that encourages the use of various chemicals to companies who want to gain more profit and find faster solutions to some problems occurring in production, marketing and storage. the negative effects caused by the chemicals used are not investigated sufficiently and the damages that can be given to the ecosystem, especially the human health and environment, are ignored (searle, 1995). organisms are exposed to many foreign substances and are commonly referred to as xenobiotics. exposure of people to xenobiotics can be caused by food additives, cosmetics and medicines that are directly consumed by people and used in the substances, while indirect exposure may be due to pesticides and agricultural drugs used in agricultural control (akay, 2004; alam and jones, 2014). the increasing use of chemical substances increases the importance of toxicological studies. it is possible to examine the possible effects of chemical substances that can be absorbed by respiration, nutrition or skin through toxicological researches, to develop strategies for control purposes and to prohibit use (vural, 2005). benzyl benzoate is a chemical compound which is widely used in food, cosmetics, agriculture and pharmaceutical sectors. it is also widely used in combating mites and insects. the aim of this study was to investigate the physiological, cytogenetic, anatomical and biochemical effects of benzyl benzoate that is one of the most frequently used chemical substances, on allium cepa l. benzyl benzoate is an ester of benzyl alcohol and benzoic acid, also known as benzoic acid phenylmethyl ester, benzoic acid benzyl ester, benzyl benzene carboxylate, benzyl phenyl formate and phenylmethyl benzoate. it is a chemical compound with the closed formula c14h12o2, open formula c6h5cooch2c6h5, molecular weight 212.25 g/mol, density 1.118 g/cm3, melting point 18-20 °c and boiling point 323 °c (hassan and mossa, 1981; ash and ash, 2004). it has a sharp burning taste and is colorless, oily and liquid. benzyl benzoate is rapidly hydrolyzed to benzyl alcohol and benzoic acid in vivo, and benzyl alcohol is then oxidized to glycine-conjugated benzoic acid to form hippuric acid (hassan and mossa, 1981). benzyl benzoate can be formed as a result of condensation of benzyl alcohol and benzoic acid and also from benzaldehyde by tishchenko reaction (kamm and kamm, 1941). it is naturally found in various essential oils with peru and tolu balsams. in addition, it has been determined that benzyl benzoate was found to be 89.5% in the oils obtained from the leaves of the cinnamomum sulphuratum nees plant and 98.2% in the root crusts oils (kar, 2003; rameshkumar and george, 2006). benzyl benzoate has a wide variety of application areas. used as a diluent and solvent in solid aromatics, as a stabilizer in perfume compositions due to its low volatility, as solvent for substances such as cellulose acetate and nitrocellulose, instead of camphor in cellulose and plastic pyroxylin compounds and also in many different sectors and products such as confectionery and chewing gum products (kar, 2003). benzyl benzoate can also be used as an additive in foods. benzyl benzoate use has been approved by the eu food processing agents as a sweetening food additive (eu, 2012), and carrier solvent by the fao/who expert committee on food additives (jecfa, 1996). it is also known that benzoic acid and its compounds are used in foods such as chocolates, beverages, oils, sauces, milk powders, fats, ketchup, mayonnaise, bakery products, dry yeasts, sugars, gums, salads and cookies (erkmen and bozoğlu, 2008). benzyl benzoate is also known to be used in the cosmetic industry. in a study conducted in england, it was reported that benzyl benzoate was found in 23% of the cosmetics examined in research (buckley, 2007). benzoic acid and its species are generally used as ph adjuster and preservative in cosmetic products (wenninger et al., 2000). benzyl benzoate is also used to increase agricultural yield by combating pests in agricultural production. benzyl benzoate has been reported to be acaricidal (mcdonald and tovey, 1993) and insecticidal (jantan et al., 2005). one of the most common uses of benzyl benzoate is the health field. it is widely used to combat lice and to treat scabies. benzyl benzoate is topically applied for the treatment of scabies. it should be applied to all skin surfaces from the scalp to the soles of the skin during the treatment process and should be avoided from contact with the eye and its use in inflamed or cracked skin. if used in children may show an irritant effect (stuart et al., 2009). benzyl benzoate has been in use in the treatment of scabies since 1937. it is formulated as emulsions in concentrations from 20% to 35%. as a complaint after application, there is burning, irritation and tenderness in the case of intense contact. it may cause irritation dermatitis, especially in the genital area and on the face (campbell and rew, 1986; habif, 2016). 23investigation of benzyl benzoate toxicity with anatomical, physiological, cytogenetic and biochemical parameters in in vivo the mechanism of action of benzyl benzoate against the disease agent sarcoptes scabiei l. is not yet known (micali and lacarrubba, 2016). when used topically, it is a relatively toxic compound. although its chronic effects are not known, it may cause mild allergic reactions which may be lost after the end of the exposure. when used as an acaricide, it may cause diarrhea, peristalsis, enterospasm, intestinal colic, spastic constipation, pylorospasm, hypertension, contraction of seminal vesicles and bronchospasm in the intestines (wexler et al., 2005). a 7-year-old male patient was reported to have died after marrow transplantation for aplastic bone marrow disease. the death cause could not be determined precisely, but the month before the diagnosis, his body was washed with ethyl alcohol, water, polysorbate and ascabiol (containing 10% benzyl benzoate and 2% disulfiram) and death was probably caused by the chronic overdose of the scabicide (hayes and laws, 1991). it was reported that oral ld 50 values of benzyl benzoate varied from 1700 mg/kg in rats to 22440 mg/ kg in dogs. when applied to animals too often or over a large area of the skin, saliva can trigger signs of systemic toxicity such as pylorization, incoordination of muscles, tremors, the progression of hind limbs, severe convulsions, shortness of breath and death. when administered in high doses to laboratory animals, it may cause incoordination, hyperexcitation, convulsions, ataxia and respiratory paralysis (wexler et al., 2005). in addition, the joint fao/who expert committee on food additives (jecfa) has determined the adi (average daily intake) value of benzyl benzoate as 5 mg/kg, indicating the amount that a person can consume without a health risk throughout his or her life, based on the body weight of the individual (who, 2001). in this study, allium cepa l. test was used to investigate the effects of benzyl benzoate. allium cepa l. test was considered to be suitable for evaluating chromosome damage and changes in the mitotic cycle because of the small number of chromosomes (2n = 16) and the large structure. analysis of chromosomal changes allows the determination of structural changes, however, it is achievable to observe the numerical changes occurring in chromosomes. in addition, it has many advantages such as low cost, multiple roots, short test duration, storage and ease of use, ease of observing nucleus and abnormal events (fiskesjö, 1985; liu et al., 1995). the data obtained as a result of the allium cepa l. test provide accurate estimates of the effects of the agent investigated on other living biodiversity. cytotoxicity tests using in vivo plant testing systems, such as allium cepa l., have been approved by several researchers performing in vitro and in vivo animal testing and the results obtained are similar (vicentini et al., 2001; teixeira et al., 2003). in the evaluations, 76% of 148 chemicals evaluated with allium cepa l. test gave positive results, this test was accepted as a standard test to determine the chromosomal damage caused by chemicals (grant, 1982). applying chemicals to allium cepa l. root tips, cell progression can be blocked at one of the stages of the cell cycle or cell division. the applied chemical has a toxic effect by causing chromosomal abnormalities, bridge and mn formation in the root tip cells (bonciu et al., 2018). materials and methods preparation of research materials and application groups this study was carried out using benzyl benzoate at doses of 10,000 mg/l, 25,000 mg/l and 50,000 mg/l. approximately equally large and healthy allium cepa l. bulbs are used as research material. the bulbs were divided into four groups of one (1) control, three (3) administration groups and placed in glass beakers 85x100 mm in diameter and allowed to germinate at room temperature for 72 hours. during the application period, the bulbs in the control group were treated with tap water and the bulbs in the application groups were treated with benzyl benzoate at doses of 10,000 mg/l, 25,000 mg/l and 50,000 mg/l. at the end of the period, the root ends were washed with distilled water and prepared for cytogenetic analysis using standard crushing preparation techniques (qian, 2004). measurement of physiological parameters at the end of the application period, the root lengths were determined with the millimetric ruler based on the radicular formation of the bulbs germinating and the weight gains were determined by the precision scales by taking into consideration the weight differences obtained before and after the application. germination percentages were determined using equation 1. germination = number of germinated bulbs x 100 (1) percentage total number of bulbs chromosomal abnormalities, micronucleus (mn) test and mitotic index (mi) determination the root tips cut about 0.5 cm in length were fixed for two hours at the ‘clarke’ fixator, washed for 15 min24 ali acar, zafer türkmen, kültiğin çavuşoğlu, emine yalçin utes in 96% ethanol and stored in +4° c at 70% ethanol. in the next step, the root tips were hydrolyzed in 1n hcl for 17 minutes at 60 ° c, incubated in 45% acetic acid for 30 minutes and stained with acetocarmine for 24 hours, then crushed with 45% acetic acid and photographed at x500 magnification in a binocular research microscope (qian, 2004; staykova et al., 2005). the criteria determined by fenech et al. (2003; 2010) were taken into consideration in the determination of the existence of mn. prepared preparations were examined in a binocular research microscope to determine mitotic index (mi) ratio and the percentage of mi was determined by using equality 2. mitotic = number of divided cells x 100 (2) index (%) total number of analyzed cells anatomical damages in order to determine the anatomical damages in the root tip meristematic cells, the allium cepa l. root ends were washed with distilled water and the cross-sections were taken and stained with methylene blue. biochemical analysis determination of lipid peroxidation lipid peroxidation measured according to the method specified by ünyayar et al. (2006) measuring the amount of malondialdehyde (mda). the values of the mda content are taken from the measurements of three independent samples and are expressed as mean + standard error (se) µmol/g fresh weight (fw). antioxidant enzyme analysis approximately 0.5 g of the tissue sample taken from the control and administration group root tips were cut into small pieces by washing with deionized water and homogenized by trituration with 5 ml chilled sodium phosphate buffer. the homogenates were transferred to new tubes and centrifuged at 10,500 rpm for 20 minutes at room temperature, and the supernatant was stored at +4 ° c for antioxidant enzyme analysis. superoxide dismutase (sod) determination superoxide dismutase (sod) activity was determined by making some modifications to the method of beauchamp and fridovich (1971). one unit sod enzyme activity was determined as the amount of sod enzyme required for 50% inhibition of nbt reduction under application conditions. the values of the sod content were taken from the measurements of three independent samples and are expressed as mean + standard error (se) u/mg fresh weight (fw). catalase (cat) determination catalase activity (cat) was determined according to the method determined by beers and sizer (1952). the cat activity unit was defined as 0.1 unit change at 240 nm absorbance. the values of the cat content were taken from the measurements of three independent samples and expressed as mean ± standard error (se) od240nm/ min.g fresh weight (fw). statistical analysis spss statistics v 23.0 (ibm corp., usa, 2015) package program was used for the analysis of statistical data. the statistical differences between the groups were evaluated by using one-way anova and duncan tests. data were given as mean ± sd values and p value was considered as statistically significant when less than 0.05. results and discussion the effects of benzyl benzoate on germination percentage, root length and weight gain are shown in table 1. treatment of the a. cepa test material with different doses of benzyl benzoate showed a germination inhibitory effect. the germination percentage was 100% in group i, 83% in group ii, 63% in group iii and 47% in group iv. it was determined that the germination pertable 1. effects of different doses of benzyl benzoate on germination percentage, root length and weight gain. groups germination percentage (%) average root length ±sd weight gain (g) group i 100 8.25±0.81a +7.71 group ii 83 5.58±0.75b +4.88 group iii 63 3.13±0.60c +2.96 group iv 47 1.30±0.37d +0.44 *group i: control, grup ii: 10,000 mg/l benzyl benzoate, grup iii: 25,000 mg/l benzyl benzoate, grup iv: 50,000 mg/l benzyl benzoate. means with the different letters are statistically significant (p<0.05). 25investigation of benzyl benzoate toxicity with anatomical, physiological, cytogenetic and biochemical parameters in in vivo centage decreased with the increase in the benzyl benzoate doses. although there is no comprehensive study investigating the effects of benzyl benzoate on root length in the scientific literature, there are similar studies conducted with other food additives and chemicals that support the results. for example, in a study conducted by singh (2017), tartrazine, which is used as a coloring additive in food, medicine and cosmetic fields, was applied to the vigna radiata (l.) r. wilczek seeds at 0.05 ppm and 0.1 ppm doses, consequently 90% germination occurred in the 0,05 ppm tartrazine treated group, 0.1 ppm tartrazine treated group is not germinated and decrease in germination percentage due to application doses. bidlan et al. (2004) investigated the effects of different doses of hexachlorocyclohexane on germination in radish and mung bean. as a result, the percentage of germination for both species was reported to decrease due to the application dose. benzyl benzoate also showed an inhibitory effect on another physiological parameter, root length. with the increase in the dose of benzyl benzoate, it was determined that the root length decreased. the average root lengths of the groups were measured as 8.25±0.81 cm in group i, 5.58±0.75 cm in group ii, 3.13±0.60 in group iii and 1.30±0.37 in group vi. the maximum root length was determined in the control group and the lowest root length was determined in group iv treated with a 50,000 mg/l dose of benzyl benzoate. there are similar studies conducted with other food additives and preservatives by other researchers that support our findings. in a study conducted by adeyemo and farinmade (2013), monosodium glutamate (msg), which is used as a flavor-enhancing additive in foods, is given to a. cepa test material with doses of 1.0 g/l, 3.0 g/l, 5.0 g/l and 7.0 g/l, changes in the root length were measured for five days. as a result, it was reported that msg application inhibits root growth at all test concentrations and this is statistically significant at higher doses. in another study by koç and pandir (2018), a. cepa test material was treated with different doses of sunset yellow (e-110) and brilliant blue (e-133) additives, which were used as a coloring agent in foodstuffs. it was reported that root lengths decreased due to increasing application dose. in the study, it was determined that benzyl benzoate application had an inhibitory effect on weight gain which is another physiological parameter. at the end of the 72 hours application period, the mean weight gains were measured as 7.71 g in group i, 4.88 g in group ii, 2.96 g in group iii and 0.44 g in group iv. it was determined that there was an inverse ratio between the dose of benzyl benzoate administered and weight gain. although there is no comprehensive study investigating the effects of benzyl benzoate on weight gain, there are similar studies in the treatment of some diseases and chemical substances used in the fight against insects. in a study conducted by arslanoglu (2011), a. cepa test material was administered basudin 60 em insecticide at doses of 600, 1200 and 1800 ppm, resulting in a reduction in weight gain due to increased dose. in another study, çavuşoğlu et al. (2012b) applied 100 mg/kg, 250 mg/kg and 500 mg/kg doses of thiamethoxam insecticide to the a. cepa test material and reported that the weight gain was reduced depending on the application doses. in our study, cytogenetic effects of benzyl benzoate application were investigated by determining chromosomal damages, mn formation and mi ratio. the effects of benzyl benzoate on mn formation are shown in table 2 and figure 1. benzyl benzoate was found to promote the formation of mn in root tip cells of a. cepa depending on the dose of administration. as a result of microscopic examinations, there was an average of 0.30 ± 0.48 mn formation in the control group. in group i, which is the control group, only a few mn formation was observed and it was determined that mn formation increased with increasing dose of benzyl benzoate. the mean mn formation was determined as 0.30±0.48 in group i, 11.80±2.74 in group ii, 24.70±3.34 in group iii and 47.60±5.34 in group iv. similar studies have been carried out with other food additives and chemicals that support our findings. for example, dönbak et al. (2002) investigated the cytogenetic effects of boric acid, which is used as a preservative additive in foods, on the a. cepa test material, resulting in boric acid causing the formatable 2. effects of different doses of benzyl benzoate on mn formation and mitotic index (mi). groups mn frequency average±sd mitotic index (mi) percent mi (%) group i 0.30±0.48d 901.50±32.35a 9.02 group ii 11.80±2.74c 808.40±30.84b 8.08 group iii 24.70±3.34b 699.50±32.86c 7.00 group iv 47.60±5.34a 534.70±16.67d 5.35 *group i: control, grup ii: 10,000 mg/l benzyl benzoate, grup iii: 25,000 mg/l benzyl benzoate, grup iv: 50,000 mg/l benzyl benzoate. for the determination of the mitotic index, 1,000 cells at each root tip in each group and 10,000 cells in total were analyzed. for the formation of mn, 100 cells each root tip in each group and were 1,000 cells in total were analyzed. data were shown as mean ± standard deviation (sd) (n = 10). the statistical significance between the means was determined by using “one-way” anova analysis of variance following the duncan’s test. the averages indicated by different letters in the same column were statistically significant (p<0.05). 26 ali acar, zafer türkmen, kültiğin çavuşoğlu, emine yalçin tion of mn. in another study, gomes et al. (2013) investigated the cytogenetic effects of bordeaux red (e-123), tartrazine yellow (e-102) and sunset yellow (e-110), which are used as colorant additives in foods, in a. cepa root tip cells. as a result, it has been reported that the application of these food additives causes the formation of mn. the effects of benzyl benzoate on mn formation are shown in table 3 and figure 1. as a result of microscopic observations, it was determined that the frequency of chromosomal damages induced by benzyl benzoate application was fragment> sticky chromosome> chromosome bridge> unequal distribution of chromatin> c-mitosis. the highest effect of benzyl benzoate on chromosomes was determined as fragment formation. while no statistically significant chromosomal damages were observed in the control group (except several sticky chromosomes, chromosome bridge and c-mitosis), all chromosomal damages in the application groups increased due to the application dose and these increases were statistically significant (p <0.05). similar studies have been performed with other food additives and insecticides that support our findings. in a study conducted by türkoğlu (2007), boric acid, sodium benzoate, figure 1. chromosomal damages and formation of mn induced by benzyl benzoate (a: fragment, b: sticky chromosome, c: chromosome bridge, d: unequal distribution of chromatin dağılımı, e: c-mitosis, f: mn). table 3. effects of different doses of benzyl benzoate on the frequency of chromosomal abnormalities groups number of root tips number of mitotic cells frg sc cb udc cm group i 10 100 0.00±0.00d 0.30±0.48d 0.20±0.42d 0.00±0.00d 0.10±0.32d group ii 10 100 17.10±3.81c 13.40±2.91c 10.80±2.53c 8.20±2.20c 3.60±1.07c group iii 10 100 33.10±4.77b 27.60±4.48b 20.90±5.15b 14.80±4.08b 9.50±2.07b group iv 10 100 65.20±7.36a 46.70±8.10a 39.90±8.12a 30.20±6.70a 23.40±5.62a *group i: control, grup ii: 10,000 mg/l benzyl benzoate, grup iii: 25,000 mg/l benzyl benzoate, grup iv: 50,000 mg/l benzyl benzoate. data were shown as mean ± standard deviation (sd) (n = 10). for chromosomal abnormalities, 100 cells at each root tip in each group and 1000 cells in total were analyzed. the statistical significance between the means was determined by using “one-way” anova analysis of variance following the duncan’s test. the averages indicated by different letters in the same column were statistically significant (p<0.05). (frg: fragment, sc: sticky chromosome, cb: chromosome bridge, udc: unequal distribution of chromatin, cm: c-mitosis). 27investigation of benzyl benzoate toxicity with anatomical, physiological, cytogenetic and biochemical parameters in in vivo potassium citrate and citric acid used as preservatives in foods were applied to a. cepa bulbs in 20, 40, 60, 80 and 100 ppm doses, as a result, it was reported that chromosome damage occurred in anaphase bridge, c-mitosis, laggard chromosome and sticky chromosome, chromosome damages occurred due to the increase in application time and doses. in another study, singh et al. (2007) treated hordeum vulgare l. seeds with 0.01%, 0.1% and 0.5% concentrations of prophenophos insecticide and mancozeb fungicide. as a result, it was reported that chromosomal abnormalities occurred due to the application doses and chromosomal damages were reported as disturb metaphase, bridge, laggards, chromosomal breakage and diagonal anaphase. in this study, it was determined that benzyl benzoate had an inhibitory effect on mitotic index (mi) in a. cepa root tip cells. the effect of benzyl benzoate application on mitotic index (mi) in root cells of a. cepa is shown in table 2. when the data were analyzed, mi was 9.02% in the control group, 10.85% in group ii, 7.00% in group iii and in group iv it was determined as 5.35%. depending on these findings, it was determined that mi was decreased with increasing doses of benzyl benzoate, in other words, the mi and benzyl benzoate administration dose showed inverse proportions. in addition, the differences between the groups were determined to be statistically significant (p <0.05). similar studies have been carried out with other food additives and insecticides that support our findings. njagi and gopalan (1982) applied different doses of sodium benzoate and sodium sulfite used as preservative additives in foods to the root tip cells of vicia faba l., as a result, a reduction in mi was reported due to the administration dose. in another study, rencüzoğulları et al. (2001), treated a. cepa test material with sodium metabisulphite used as a coloring additive in foods at doses of 7.5 mg/l, 15 mg/l and 30 mg/l for 10 and 20 hours, consequently, it was determined that mi decreased in both applications duration due to application doses. as a result of microscopic observations, it was observed that the application of benzyl benzoate caused significant anatomic damages in a. cepa root tip cells. anatomical examinations of root tips of the control and application groups application group are shown in figures 2 and 3. these damages are in the form of necrosis, cell deformation, flattened cell nucleus, accumulation of some substances in cortex cells, cortex cell deformation, cortex cell wall thickening and unclear transmission tissue (figure 2). as a result of the investigations, it was found that there was no anatomical change in the root figure 2. anatomical changes induced by benzyl benzoate in root cell meristematic cells of a.cepa (a: necrosis, b: cell deformation, c: flattened cell nucleus, d: cortex cell deformation, e: accumulation of some substances in cortex cells, f: cortex cell wall thickening, g: unclear transmission tissue). figure 3. the appearance of the control group root tip meristematic cells (a: normal appearance of cortex cells, b: the usual shape of the cell nucleus, c: the usual appearance of the transmission tissue, d: normal appearance of epithelial cells). 28 ali acar, zafer türkmen, kültiğin çavuşoğlu, emine yalçin tip cells and tissues of the control group (figure 3), but an increase in anatomic damage rates was also observed in the application groups due to the increasing doses of benzyl benzoate. since there is no comprehensive study on the anatomic damage caused by benzyl benzoate in plant root tip cells, the results are discussed with data from other chemical agents. bıçakcı et al. (2017) reported that the application of diazinon cause anatomic damages in the root tip cells of a. cepa as unclear conduction tissue, flattened cell nucleus, cell deformation, thickening of cortex cell walls, necrosis and accumulation of some substances in the tissue of transmission, and the frequency of these damages increased due to application dose. çavuşoğlu et al. (2011) reported that the application of glyphosate causes anatomic changes in the cell-root cell of a. cepa, unclear vascular tissue, cell deformation, unclear epidermis layer, binuclear cell and abnormal cell nucleus. in our study, the effects of benzyl benzoate on lipid peroxidation in a. cepa root tip cells were also investigated. lipid peroxidation is a metabolic process that causes oxidative degradation of reactive oxygen species (ros) and lipids, especially polyunsaturated fatty acids and mda production (odjegba and adeniran, 2015). mda is an oxidative product of membrane lipids and a biological marker that indicates the level of oxidative stress (janero, 1990). ros damage the peroxidation of biological molecules including lipids, proteins, rna and dna (shah et al., 2001; dinakar et al., 2010). free oxygen groups can act on dna and cause mutations in nucleic acids and changes in chromosomes (yarsan, 2014). the effects of benzyl benzoate application on root mda levels are shown in figure 4. when mda levels were examined, the lowest mda level was 10.00 µmol/g fw in the control group. sod levels were found to be 1.45 times higher in group ii, 1.83 times higher in group iii and 2.21 times higher in group iv compared to the control group. it was determined that the level of mda increased with the increase in the benzyl benzoate doses. in addition, it was observed that the differences in the mda level between the groups were statistically significant (p <0.05). in the literature, because of the lack of a comprehensive study investigating the effect of benzyl benzoate on lipid peroxidation in plant test materials, our findings are discussed with similar studies examining the effects of other chemicals on lipid peroxidation in plants test materials. in the investigations chromium (iv) application of a. cepa root tip tissues (patnaik et al. 2013), high-dose lead application on allium sativum l. (liu et al. 2009), bentazone herbicide application in rice (wang et al. 2008) reported an increase in mda levels in tissues according to applications. various defense mechanisms have been developed by aerobic organisms against free radicals. one of the so-called protective antioxidants cat decomposes hydroperoxides or hydrogen peroxide and sod reduces the formation of free radicals and active oxygen by quenching and modifying active oxygen (comporti, 1993). the effects of benzyl benzoate application on root sod and cat activity are shown in figure 5 and 6 respectively. the lowest sod levels were measured as 75.00 u/mg fw in the control group. sod levels in benzyl benzoate application groups were 94.00 u/ mg in group ii, 157.00 u/mg in group iii and 180.70 u/ mg in group iv. it was determined that the level of sod increased with the increase in benzyl benzoate dose. in addition, it was observed that the sod level differences between the groups were statistically significant (p <0.05). the level of sod is thought to be increased as a result of ros formation in the form of superoxide radifigure 4. effects of benzyl benzoate application on mda levels. (group i: control, grup ii: 10,000 mg/l benzyl benzoate, grup iii: 25,000 mg/l benzyl benzoate, grup iv: 50,000 mg/l benzyl benzoate). vertical bars denote standard error (se). figure 5. effects of benzyl benzoate application on sod activity (group i: control, grup ii: 10,000 mg/l benzyl benzoate, grup iii: 25,000 mg/l benzyl benzoate, grup iv: 50,000 mg/l benzyl benzoate). vertical bars denote standard error (se). 29investigation of benzyl benzoate toxicity with anatomical, physiological, cytogenetic and biochemical parameters in in vivo cals by exposure to benzyl benzoate. superoxide is a key component of signal transduction triggers genes responsible for antioxidant enzymes, including sod (alavarez and lamb, 1997). the lowest cat levels were measured as 1.17 od240nm/min.g fw in the control group. cat levels in benzyl benzoate application groups were 1.42 od240nm/min.g fw in group ii, 1.81 od240nm/min.g fw in group iii and 2.24 od240nm/min.g fw in group iv. increased levels of sod and cat were determined by increasing benzyl benzoate dose. similarly, in many studies, antioxidant enzyme activities have been reported to increase in allium species due to stress-induced by other chemicals. application of different doses of chlorpyrifos and mancozeb insecticides caused an increase in cat and sod levels in a. cepa leaves (fatma et al. 2018), the increase in sod and cat levels due to the application doses and duration of cypermethrin insecticide on a. cepa root tips (çavuşoğlu et al. 2012a), the application of cadmium inhibited sod and cat levels in allium sativum l. leaves but when the application was continued the increase in sod and cat levels was reported (zhang et al. 2005). conclusions when all the data obtained in the study were examined; benzyl benzoate application showed inhibitory effects on physiological parameters investigated in a. cepa test material. this is thought to be due to the inhibitory effect of benzyl benzoate on the cell cycle. inhibition in physiological parameters is considered as an indicator of benzoate toxicity. benzyl benzoate increases the occurrence of chromosomal damage with the frequency of mn and decreases in the rate of mi suggests that it may be caused by the production of more ros that can be detoxified by the cellular defense mechanisms and by causing the damage to dna by being tolerated. increases in mda, sod and cat levels also support this situation. in addition, due to the application of benzyl benzoate, the anatomical changes occurring in the transmission tissue and root tip cells may be due to the defense mechanisms developed to inhibit the cellular uptake of the benzyl benzoate developed by the plant. despite these mechanisms, high benzyl benzoate doses may be caused the penetration of the substance into the plant. as a result; benzyl benzoate, which is used in many different fields, such as food, health, cosmetics and agricultural production, has been identified using a. cepa test material, which can show toxic effects if it reaches certain concentrations. for this reason, the use of benzyl benzoate exposure should be avoided considering the damages that can be caused to living things. in cases such as disease treatments where the use is essential, the appropriate dosage and duration range should be determined and the areas of use should be limited. in this study conducted using a. cepa test material, it was found that physiological parameters such as germination percentage, root length and weight gain are important precursors in the rapid detection of toxicity. cytogenetic parameters such as chromosomal abnormalities, mn formation and mi ratio are sensitive biomarkers in the biological monitoring of toxicity. in addition, it was determined that the determination of mda, sod and cat levels contributed to explaining the causes and effect mechanisms of toxicity, the anatomical changes occurring in the root tip meristematic cells were found to contribute to the understanding of the cellular responses of the plant during the incorporation of the chemical agent into the plant. acknowledgments this study was supported by the giresun university scientific research projects unit (grübap) under fenbap-c-200515-14 coded project. references [eu] european commission. 2012. commission implementing regulation (eu) no 872/2012 of 1 october 2012 adopting the list of flavouring substances provided for by regulation (ec) no 2232/96 of the european parliament and of the council, introducing figure 6. effects of benzyl benzoate application on cat activity (group i: control, grup ii: 10,000 mg/l benzyl benzoate, grup iii: 25,000 mg/l benzyl benzoate, grup iv: 50,000 mg/l benzyl benzoate). vertical bars denote standard error (se). 30 ali acar, zafer türkmen, kültiğin çavuşoğlu, emine yalçin it in annex i to regulation (ec) no 1334/2008 of the european parliament and of the council and repealing commission regulation (ec) no 1565/2000 and commission decision 1999/217/ec: official journal of the european union. 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[accessed : 2018 may 10] http://apps.who.int/food-additives-contaminants-jecfa-database/chemical.aspx?chemid=1171 yarsan e. 2014. lipid peroksidasyon olayı ve önlenmesine yönelik uygulamalar [lipid peroxidation event and its applications for prevention] van vet j. 9(1):89-95. turkish. zhang h, jiang y, he z, ma m. 2005. cadmium accumulation and oxidative burst in garlic (allium sativum). j plant physiol. 162(9):977-984. caryologia. international journal of cytology, cytosystematics and cytogenetics 72(4): 29-39, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-172 citation: m.n. moura, d.c. cardoso, b.c.l. baldez, m.p. cristiano (2019) genome size in ants: retrospect and prospect. caryologia 72(4): 29-39. doi: 10.13128/cayologia-172 published: december 23, 2019 copyright: © 2019 m.n. moura, d.c. cardoso, b.c.l. baldez, m.p. cristiano. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. genome size in ants: retrospect and prospect mariana neves moura1,2, danon clemes cardoso2,*, brenda carla lima baldez3, maykon passos cristiano2,* 1 programa de pósgraduação em ecologia, , universidade federal de viçosa, cep: 36.570–000, viçosa, minas gerais, brazil 2 departamento de biodiversidade, evolução e meio ambiente, universidade federal ouro preiato – ufop, cep: 35.400–000, ouro preto, minas gerais, brazil 3 programa de pósgraduação em ecologia de biomas tropicais, universidade federal de ouro preto, cep: 35.400–000, ouro preto, minas gerais, brazil *corresponding authors: danon@ufop.edu.br; maykon@ufop.edu.br abstract. genome size is very useful in studies regarding taxonomy, evolution, and reproductive biology in many animal groups, including insects. herein, we assembled the information about genome size in ants, compiling the dna content estimated so far, in order to evaluate the methods, the tissues and the internal standard applied to estimate the genomes size. all values were placed in a phylogenetic tree to put it in an evolutionary context and the means of the subfamilies were further compared statistically to investigate changes and trends in the variation across taxa. the compiled data resulted in 86 specimens of ants, comprising 69 different species. this number represents 0.52% of the total number of 13,369 ant species described, covering only 40 from 333 valid extant genera. the average formicidae genome size was 0.36 pg (± 0.13). most of the estimates were obtained through flow cytometry (83.5%), commonly using brain tissues, with drosophila melanogaster as internal standard (76%). differences in dna content of ant species may be related to differences in the amount of heterochromatin and is not related with chromosome number. the evaluation of the genome size estimations currently available for ants has highlighted their scarcity. such information would be valuable as independent data for the study of ant diversity and evolutionary biology. further, we conclude that the standardization of the techniques used and a large–scale study on ant genome size are urgently required, given the importance of this insect group and the needs for the improvement in our knowledge on ant genome. keywords. c-value, dna content, genetic diversity, genome, evolution, phylogeny. introduction ants comprise a monophyletic group with approximately 13,369 valid species distributed throughout the planet, with exception of extreme northern and southern latitudes (bolton, 2018). they are one of the largest groups among insects in species diversity and biomass and together with some wasps and bees, are known as eusocial insects and comprise the order hymenoptera (hölldobler and wilson, 1990; ardila–garcia et al., 2010). 30 mariana neves moura et al. they represent an important insect group to investigate the relationship between the genealogical lineages and the distribution patterns of species, due to their occurrence in different habitats of the most diverse ecosystems (goodisman et al., 2008). currently, the family formicidae is divided into 17 extant and 3 extinct subfamilies, spanning 333 valid extant genera and 154 extinct genera (bolton, 2018). the subfamily myrmicinae is the largest and most diverse subfamily worldwide, covering about 47% of all ant species (françoso and brandão, 1993; brandão, 1999). genome size, also named dna content, dna amount, or dna c–value, has been described as a trait that ‘uniquely lies at the intersection of phenotype and genotype’, and the genome size of eukaryotes varies over five orders of magnitude, with a distribution skewed toward small values, around 2 picograms (pg) (oliver et al., 2007). this variation does not seem to be correlated with the complexity of the organism or with the number of genes in eukaryotes, leading to what is called the “c–value paradox” (moore, 1984; gregory, 2001, 2005a; eddy, 2012). it has been questioned, for example, why similar organisms with similar amounts of coding sequence have different amounts of dna. while changes in gene sequences are often slow and gradual, changes in genome size can be rapid and abrupt as a consequence of chromosomal rearrangements or duplications (alberts et al., 2007). the main methods used to estimate the total nuclear genome size are image cytometry, flow cytometry (fcm), and complete genome sequencing (gregory, 2005b). image cytometry was the first method used to determine genome size estimates. basically, it operates by statically imaging a large number of cells stained with specific chemicals or fluorochromes, using optical microscopy (torresan et al., 1994; basiji et al., 2007). in contrast, flow cytometry evaluates the relative fluorescence intensity of suspended nuclei, also stained with specific fluorochromes, and presents the data in a typical histogram with a higher peak relative to the nuclei in the g0/g1 phase of cell cycle, and a lower peak, relative to the nuclei in g2 phase (price et al., 2000; dolezel and bartos, 2005). the complete genome sequencing method, on the other hand, provides the complete dna sequence of the genome of an organism at a single time with the precise order of the nucleotides and an estimate of the genome size after its assembly (klug et al., 2014). a fourth less common technique known as biochemical analysis (bca) was used during the early studies of genome size. it includes ‘the chemical extraction and quantification of dna combined with cell counts to give an average dna amount per nucleus or the reassociation kinetics, in which the dna molecule was denatured and then the time taken for the strands to renature is used to calculate the amount of dna (gregory, 2005b). among the methods, flow cytometry has been shown to be the least cost and time expensive technique when compared to other molecular tools and provides rapid generation of accurate results (merkel et al., 1987; doležel et al., 2007). according to gregory (2018), haploid dna contents (c–values, in picograms pg) are currently available for 6,222 species of animals (3,793 vertebrates and 2,429 invertebrates), with insects representing 21.6% of this total. li and heinz (2000) performed the first dna content estimation of an ant by mean of biochemical analysis (bca), to quantify the genome of solenopsis invicta buren, 1972. subsequently, johnston et al. (2004) also estimated the genome size of s. invicta but now using flow cytometry. yet, in 2008, tsutsui et al. (2008) carried out the first comprehensive study regarding the evolution of the genome size in ants, reporting genome size estimates for 40 species from nine subfamilies. this was the last inclusion of a large number of ant species estimates to the genome size database that was followed by the study of ardila–garcia et al. (2010), which added a further 29 species. these two studies raised different questions about genome size, being the first a study of genome size evolution in formicidae and the second a study of correlation between genome size with parasitism and eusociality in the order hymenoptera as a whole. it is important to note that they applied different methodologies in genome size estimation: in tsutsui et al. (2008) the dna content was estimated by using only flow cytometry, while ardila–garcia et al. (2010) also performed the fiad method (feulgen image analysis densitometry) to estimate the dna content, and then compared the results from both techniques. later, others studies explored the dna content of ants, however in some cases covering only one species through complete genome sequencing (e.g. nygaard et al., 2011) or, in other cases, considering specifically an ant genus through flow cytometry. the genome size of the genus mycetophylax emery, 1913 (sensu klingenberg and brandão, 2009) was estimated by cardoso et al. (2012) that explored the data placing them in a phylogenetic context, also correlating it with chromosome number of fungus–growing ants; and aguiar et al. (2016) that evaluated three camponotus mayr, 1861 species, exploring their correlation with the karyotype of the studied species. despite the importance of genome size, little is known about the ecological and evolutionary consequences of dna amount in ants. yet, the biological sig31genome size in ants nificance and evolution of the genome size diversity in other groups has received much more attention over the last decades (dufresne and jeffery, 2011; alfsnes et al., 2017; pellicer et al., 2018). the diversity of genome size in plants has been shown to correlate with several phenotypic features of cells and ultimately the organisms. for instance, plant species with larger genomes are adapted to xeric and higher elevation environments (e.g. bottini et al., 2000). here, we evaluate the available information about the genome size of ants, assembling the dna content estimated so far, in order to provide insights into the distribution, evolution and possible consequences of ant genome size diversity. we have also investigated and verified the needs of a re–evaluation in the genome size data (dna c–value) for ants, as well the technique used in the estimation of the dna content in respect of methodological issues such as: the internal standard and tissues used in the analysis. the basic information about ant genomes analyzed here may improve our knowledge about the evolution and diversification regarding this diverse group of insects and may help as a baseline and guidance for future studies about ant genome biology. materials and methods to evaluate the knowledge about nuclear dna content on ants, we compiled the haploid genome size estimates for ants and other insect groups from the animal genome size database (gregory, 2018) and from the literature by searching in the publication databases scopus® and isi web science knowledgetm, by using the terms: “genome size”, “dna amount”, “c–value” and “ants”. based on the seven manuscripts found on ant genome size, we evaluated the method used to measure genome size, the type of tissue and the internal standard used to obtain the total content of dna. to examine the genome size variation over formicidae subfamilies we compiled the estimates in a table of all the values available in the literature, expressed in picograms of dna (pg) and mega base pairs (mbp). then we manually placed them in the phylogenetic tree proposed by moreau and bell (2013) by collapsing branches with equal names (same operational units otus) and separating the subfamilies by color. general linear models were built to check for differences between the averaged genome sizes of the sampled subfamilies. the differences in genome size average for each subfamily were assessed by variance analysis of the glm. when the p-value of anova was significant (p < 0.05), a contrast analysis at 5% level was then performed to determine which mean was different. all the statistical analysis was performed in r v2.15.1 software (r core team, 2013) and glm was submitted to residual analysis to evaluate adequacy of normal error distribution (crawley, 2013). results and discussion overview: number of estimates, methods, tissues and internal standards used the compiled data resulted in 86 specimens of ants whose genome size had been estimated, comprising 69 different species (table 1). this number represents 0.52% of the total number of 13,369 ant species accepted until now, covering only 40 genera from 333 accepted (bolton, 2018). from 17 existing subfamilies, we only found estimates for nine, with myrmicinae having the largest number of species evaluated (32 spp.) (figure 1). the number of estimates may reflect the richness of this subfamily that is the most diverse within formicidae. yet, formicinae and dolichoderinae together bear 20 spp. with dna content estimates available. these three subfamilies represent 65% of dna content estimates on ants. the two main methods used to estimate dna content in ants were fcm and fiad. a third method, biochemical analysis (bca), was used in a pioneering work from li and heinz (2000) in order to estimate the genome size sole for solenopsis invicta. it is important to mention that s. invicta has the genome size estimates by all three methods listed above and different values were obtained in each estimate: 0.60 pg by bca (li and heinz, 2000), 0.47 pg by fiad (ardila-garcia et al., 2010) and 0.77 pg by flow cytometry (johnston et al., 2004). such huge variation in genome sizes may be explained by the occurrence of different ploidy levels in s. invicta or even outcomes due the different techniques employed in the studies. cytogenetical evidence suggests that there may be different levels of ploidy in s. invicta. all genome sizes are estimated by mean of comparison with nuclei of reference standard, whose genome size is known that is called the “internal standard”. in the genome size estimation drosophila melanogaster meigen, 1830 (0.18 pg), scaptotrigona xantotricha moure, 1950 (0.43 pg) and tenebrio molitor linnaeus, 1758 (0.52 pg) are the internal standards most commonly used considering hymenoptera as a whole. most of the estimates were obtained using d. melanogaster as internal standard (76%), while fcm was the most common method used (83.5%). generally, brain tissue is used to estimate nuclear genome size, but cells (hemocytes) obtained through hemolymph smears have also been tested (ardi32 mariana neves moura et al. table 1. overview of the genome size data available in literature for formicidae species. subfamily species 1c-value (pg) 1c-value (mbp) method cell type standard references amplyoponinae amblyopone pallipes (haldeman, 1844)* 0.34 332.52 fcm br dm tsutsui et al., 2008 amblyopone pallipes (haldeman, 1844)* 0.37 361.86 fcm br dm ardila-garcia et al., 2010 dolichoderinae dolichoderus mariae (forel, 1885) 0.18 176.04 fcm br dm ardila-garcia et al., 2010 dolichoderus taschenbergi (mayr, 1866) 0.23 224.94 fcm br dm ardila-garcia et al., 2010 dorymyrmex bicolor wheeler, 1906 0.25 244.5 fcm br dm tsutsui et al., 2008 dorymyrmex bureni (trager, 1988) 0.18 176.04 fiad he tm ardila-garcia et al., 2010 forelius pruinosus (roger, 1863) 0.22 215.16 fiad he tm ardila-garcia et al., 2010 linepithema humile (mayr, 1868) 0.26 254.28 fcm br dm tsutsui et al., 2008 linepithema humile (mayr, 1868) 0.26 250.8 genome sequencing ns ns smith et al., 2011 liometopum occidentale emery, 1895 0.29 283.62 fcm br dm tsutsui et al., 2008 tapinoma sessile (say, 1836) 0.37 361.86 fcm br dm ardila-garcia et al., 2010 tapinoma sessile (say, 1836) a 0.38 371.64 fcm br dm tsutsui et al., 2008 tapinoma sessile (say, 1836) b 0.61 596.58 fcm br dm tsutsui et al., 2008 dorylinae cerapachys edentata 0.22 215.16 fcm br dm tsutsui et al., 2008 eciton burchelli (westwood, 1842) 0.27 264.06 fcm br dm tsutsui et al., 2008 labidus coecus (latreille, 1802) 0.37 361.86 fcm br dm tsutsui et al., 2008 ectatomminae ectatomma tuberculatum (olivier, 1792) 0.71 694.38 fcm br dm tsutsui et al., 2008 formicinae camponotus castaneus (latreille, 1802) 0.31 303.18 fcm br dm tsutsui et al., 2008 camponotus crassus mayr, 1862 0.29 283.62 fcm br sx aguiar et al., 2016 camponotus floridanus (buckley, 1866) 0.23 224.94 fiad he tm ardila-garcia et al., 2010 camponotus floridanus (buckley, 1866) 0.245 240 genome sequencing ns ns bonasio et al., 2010 camponotus pennsylvanicus (de geer, 1773) 0.33 322.74 fcm br dm tsutsui et al., 2008 camponotus renggeri emery, 1894 0.29 283.62 fcm br sx aguiar et al., 2016 camponotus rufipes (fabricius, 1775) 0.29 283.62 fcm br sx aguiar et al., 2016 formica pallidifulva wheeler, 1913 0.39 381.42 fcm br dm tsutsui et al., 2008 lasius (acanthomyops) latipes (walsh, 1863) 0.27 264.06 fcm br dm ardila-garcia et al., 2010 lasius alienus (foerster, 1850) 0.31 303.18 fcm br dm tsutsui et al., 2008 lasius minutus emery, 1893 0.23 224.94 fcm br dm ardila-garcia et al., 2010 paratrechina longicornis (latreille, 1802) 0.18 176.04 fiad he tm ardila-garcia et al., 2010 prenolepis imparis (say, 1836) 0.30 293.4 fcm br dm tsutsui et al., 2008 myrmeciinae myrmecia varians mayr, 1876 0.28 273.84 fcm br dm tsutsui et al., 2008 myrmicinae acromyrmex echinatior (forel, 1899) 0.36 335 fcm br crbc sïrvio et al., 2006 acromyrmex echinatior (forel, 1899) 0.32 313 genome sequencing ns ns nygaard et al., 2011 aphaenogaster rudis (texana group n16) enzmann, 1947 0.43 420.54 fcm br dm ardila-garcia et al., 2010 aphaenogaster rudis (texana group n17) enzmann, 1947 0.46 449.88 fcm br dm ardila-garcia et al., 2010 aphaenogaster rudis (texana group n22b) enzmann, 1947 0.44 430.32 fcm br dm ardila-garcia et al., 2010 aphaenogaster fulva roger, 1863 0.42 410.76 fcm br dm ardila-garcia et al., 2010 aphaenogaster treatae forel, 1886 0.50 489 fcm br dm ardila-garcia et al., 2010 apterostigma dentigerum wheeler, 1925 0.65 635.7 fcm br dm tsutsui et al., 2008 atta cephalotes (linnaeus, 1758) 0.31 303.18 fcm br dm tsutsui et al., 2008 atta cephalotes (linnaeus, 1758) 0.30 290 genome sequencing ns ns suen et al., 2011 atta colombica guérin-méneville, 1844 0.31 303.18 fcm br dm tsutsui et al., 2008 atta texana (buckley, 1860) 0.27 264.06 fcm br dm ardila-garcia et al., 2010 33genome size in ants subfamily species 1c-value (pg) 1c-value (mbp) method cell type standard references crematogaster hespera buren, 1968* 0.28 273.84 fcm br dm tsutsui et al., 2008 eurhopalothrix procera (emery, 1897) 0.39 381.42 fcm br dm tsutsui et al., 2008 messor andrei (mayr, 1886)* 0.26 254.28 fcm br dm tsutsui et al., 2008 monomorium viridum brown, 1943 0.50 489 fiad he tm ardila-garcia et al., 2010 mycetophylax conformis (mayr, 1884) 0.32 312.96 fcm br sx cardoso et al., 2012 mycetophylax morschi (emery, 1888) 0.32 312.96 fcm br sx cardoso et al., 2012 mycetophylax simplex (emery, 1888) 0.39 381.42 fcm br sx cardoso et al., 2012 myrmecina americana emery, 1895 a 0.26 254.28 fcm br dm tsutsui et al., 2008 myrmecina americana emery, 1895 b 0.31 303.18 fcm br dm tsutsui et al., 2008 pheidole dentata mayr, 1886 0.24 234.72 fiad he tm ardila-garcia et al., 2010 pheidole floridana emery, 1895 0.21 205.38 fiad he tm ardila-garcia et al., 2010 pheidole hyatti emery, 1895 0.33 322.74 fcm br dm tsutsui et al., 2008 pogonomyrmex badius (latreille, 1802) 0.27 264.06 fcm br dm tsutsui et al., 2008 pogonomyrmex barbatus (smith, 1858) 0.24 235 genome sequencing ns ns smith et al., 2011 pogonomyrmex californicus (buckley, 1867) 0.25 244.5 fcm br dm tsutsui et al., 2008 pogonomyrmex coarctatus mayr, 1868 0.29 283.62 fcm br dm tsutsui et al., 2008 pyramica rostrata (emery, 1895) 0.28 273.84 fcm br dm tsutsui et al., 2008 sericomyrmex amabilis wheeler, 1925 0.45 440.1 fcm br dm tsutsui et al., 2008 solenopsis invicta buren, 1972 0.62 606.36 bca br ns li and heinz 2000 solenopsis invicta buren, 1972 0.77 753.06 fcm br dm johnston et al., 2004 solenopsis invicta buren, 1972 0.47 459.66 fiad he tm ardila-garcia et al., 2010 solenopsis invicta buren, 1972 0.49 482 genome sequencing ns ns wurm et al., 2011 solenopsis molesta emery, 1895 0.38 371.64 fcm br dm ardila-garcia et al., 2010 solenopsis xyloni mccook, 1880 0.48 469.44 fcm br dm tsutsui et al., 2008 temnothorax ambiguus (emery, 1895) 0.31 303.18 fcm br dm ardila-garcia et al., 2010 temnothorax texanus (wheeler, 1903) 0.32 312.96 fcm br dm ardila-garcia et al., 2010 tetramorium caespitum 0.26 254.28 fcm br dm tsutsui et al., 2008 tetramorium caespitum (linnaeus, 1758) 0.27 264.06 fcm br dm ardila-garcia et al., 2010 trachymyrmex septentrionalis (mccook, 1881) 0.25 244.5 fiad he tm ardila-garcia et al., 2010 ponerinae dinoponera australis emery, 1901 0.57 557.46 fcm br dm tsutsui et al., 2008 harpegnathos saltator jerdon, 1851 0.34 330 genome sequencing ns ns bonasio et al., 2010 odontomachus bauri emery, 1892 0.49 479.22 fcm br dm tsutsui et al., 2008 odontomachus brunneus (patton, 1894) 0.33 322.74 fiad he tm ardila-garcia et al., 2010 odontomachus brunneus (patton, 1894) 0.44 430.32 fcm br dm tsutsui et al., 2008 odontomachus cephalotes smith, 1863 0.43 420.54 fcm br dm tsutsui et al., 2008 odontomachus chelifer (latreille, 1802) 0.54 528.12 fcm br dm tsutsui et al., 2008 odontomachus clarus wheeler, 1915 0.42 410.76 fcm br dm tsutsui et al., 2008 odontomachus haematodus (linnaeus, 1758) 0.51 498.78 fcm br dm tsutsui et al., 2008 ponera pennsylvanica buckley, 1866 0.55 537.9 fcm br dm ardila-garcia et al., 2010 ponera pennsylvanica buckley, 1866 0.60 586.8 fcm br dm tsutsui et al., 2008 pseudomyrmicinae pseudomyrmex ejectus (smith, 1858) 0.29 283.62 fiad he tm ardila-garcia et al., 2010 pseudomyrmex gracilis (fabricius, 1804) 0.35 342.3 fcm, fiad br, he dm, tm ardila-garcia et al., 2010 pseudomyrmex gracilis (fabricius, 1804) 0.40 391.2 fcm br dm tsutsui et al., 2008 method: fcm = flow cytometry, fiad = feulgen image analysis densitometry; cell type: br = brain tissue, he = haemocyte; standard: dm = drosophila melanogaster, crbc = chicken red blood cells, sx = scaptotrigona xantotricha, tm = tenebrio molitor, ns = not specified. *valid names: stigmatomma pallipes (haldeman, 1844); crematogaster laeviuscula mayr, 1870; veromessor andrei (mayr, 1886), respectively. 34 mariana neves moura et al. la-garcia et al., 2010). considering s. xantotricha, this internal standard was started to be used in studies comprised stingless bees, and after with ants by the same research group (tavares et al. 2010, cardoso et al. 2012, aguiar et al. 2016). since no genome size histograms are available in either ardila-garcia et al. (2010) or tsutsui et al. (2008), it is impossible to compare the usefulness of one or another internal standard considering the other two studies (cardoso et al. 2012 and aguiar et al. 2016) used s. xantotricha. in studies with plants, the choice of an appropriate internal standard considers the genome size magnitude of standard and studied group, mainly to avoid superposition of picks. concerning the methods employed in genome size estimation, the study from ardila-garcia et al. (2010) is the only one that multiple species in the same work had the genome measured by two methods. they evaluated by fiad and fcm the genome size on odontomachus brunneus, pseudomyrmex gracilis, and solenopsis invicta and showed that the estimates using the first method tended to be smaller. the authors argue that the values from both techniques do not differ statistically. however, it is difficult to say that this difference is solely due to the technique itself, since both the tissue and the internal standard used during the analysis were different. the nuclear dna content of some ants has also been measured using a fourth method, which utilized complete genome sequencing techniques in species such as acromyrmex echinatior (forel, 1899) (nygaard et al., 2011), atta cephalotes (linnaeus, 1758) (suen et al., 2011), camponotus floridanus (buckley, 1866) (bonasio et al., 2010), harpegnathos saltator jerdon, 1851  (bonasio et al., 2010), linepithema humile (mayr, 1868) (smith et al., 2011), pogonomyrmex barbatus (smith, 1858) (smith et al., 2011) and solenopsis invicta (wurm et al., 2011) (table 1). the genome size of ac. echinatior was 313 mbp (or 0.32 pg considering 1 pg = 978 mbp; (doležel et al., 2003)) obtained with complete genome sequencing (nygaard et al., 2011) and 335 mbp (0.36 pg) by fcm (sïrvio et al., 2006). this difference can be attributed to the loss of repetitive regions and some chromosomal regions, such as telomeres, through genome sequencing techniques (gregory, 2005b). the same was observed in a. cephalotes, whose genome size estimated by complete genome sequencing was 290 mbp (approximately 0.30 pg) (suen et al., 2011) and by fcm was 303.18 mbp (approximately 0.31 pg) (tsutsui et al., 2008). the differences were greater in s. invicta, whose genome size was obtained with all four different techniques (bca, fiad, fcm, and genome sequencing): 606 mbp (0.62 pg) (li and heinz, 2000) by bca, 459 mbp (0.47 pg) (ardila-garcia et al., 2010) by fiad, 753 mbp (0.77 pg) (johnston et al., 2004) by fcm and 482 mbp (0.49 pg) (wurm et al., 2011) by genome sequencing. values obtained with fiad and genome sequencing are more similar. so, considering the loss of certain repetitive regions of dna by the complete genome sequencing and the difficulties in using other techniques such as bca and fiad (mainly due to the low number of repetitions available to estimate de dna amount) the use of fcm has proven to be the most efficient methodology to obtain accurately the total dna content. genome size evolution the reported dna c–value of insects range from 0.07 pg (clunio tsushimensis tokunaga, 1933 – diptera) to 16.93 pg (podisma pedestris linnaeus, 1758 – orthoptera) and out of 1344 estimates found, 1224 (91%) were comprised of values between 0.07 to 2.00 pg (gregory, 2018). from 27 orders of insects, 24 currently have estimates of genome size, with diptera accounting for the largest number of measurements (386 specimens, 29% of the total), followed by coleoptera (278 specimens, 21% of the total) and hymenoptera (240 specimens, 18% of the total). the average genome size for the formicidae (hymenoptera) was 0.36 pg (± 0.13), with values ranging from 0.18 pg (the smallest value, found in dolichoderinae and in formicinae) to 0.77 pg in s. invicta (myrmicinae) (table 1; figure 2), being always less than 1 pg. this is in accordance with the pattern already observed for others eukaryotes that most of the distribution of genome size is skewed towards smaller values (oliver et al., 2007), since it is evident that the number of species declines as the genome doubles in size. as can be seen in figure 2 the variation of genome size among species of a subfamily is similar to the variafigure 1. distribution of the number of species across formicidae subfamilies with published genome size estimates. the list of species is presented in table 1. 35genome size in ants figure 2. phylogeny of the extant formicidae. phylogenetic tree redrawn from moreau and bell (2013). the figure highlights the subfamilies containing species with estimated genome size. aside of each terminal on the tree the genome size is shown in picograms (pg) of dna and also the mean genome size per formicidae subfamilies. 36 mariana neves moura et al. tion found between subfamilies. significant differences in genome size were observed between the subfamilies sampled (anova, p-value < 0.01). through contrast analysis, most of the subfamilies grouped statistically (group average = 0.34 pg, p-value > 0.05) except for ponerinae, whose average was different from the others (average = 0.47, p-value < 0.01). the subfamilies ectatomminae (ectatomma tuberculatum (olivier, 1792), 0.71 pg) and myrmeciinae (myrmecia varians mayr, 1876, 0.28 pg) were not considered in the analysis because only one value for each was available, so it was not possible to calculate a mean for the comparison test (figure 2). differences in the genome size were also observed between genera within the sampled subfamilies and mainly between species of the same genus, as observed in atta fabricius, 1804 spp. (e.g. atta cephalotes = 0.31 pg and atta texana (buckley, 1860) = 0.27 pg), camponotus spp. (e.g. camponotus floridanus = 0.23 pg and camponotus pennsylvanicus (de geer, 1773) = 0.33 pg) and odontomachus spp. (e.g. odontomachus brunneus (patton, 1894) = 0.33 pg and odontomachus chelifer (latreille, 1802) = 0.54 pg) (table 1, figure 2). these differences in genome size among closely related species have been associated in several studies with the amount of heterochromatin in the chromosomes (lopes et al., 2009; tavares et al., 2010; cardoso et al., 2012), transposable elements (kidwell, 2002; vieira et al., 2002) and other repetitive genome sequences (gregory and hebert, 1999; petrov, 2001). in some species, as ectatomma tuberculatum and apterostigma dentigerum wheeler, 1925 the differences in genome size was correlated with whole genome duplication events given the large genome size of this both species when compared with the others of formicidae (0.71 pg and 0.65 pg, respectively) (tsutsui et al., 2008). the correlation between genome size and chromosome number has been reported in some studies for ants, for example, cardoso et al. (2012) within fungus– growing ants. in their study, they found a relationship between these two characteristics being sericomyrmex amabilis wheeler, 1925 the species with the highest number of chromosomes and also the largest genome size and other two species with the lowest number of chromosomes also had the smallest genome size. correlation between chromosome and genome size has been reported for some insects. for instance, ardila–garcia and gregory (2009) also found this positive correlation among species of damselflies, but not in dragonf lies (insecta: odonata). lack of correlation between genome size and chromosome number has been shown in the highly eusocial stingless bees of meliponini tribe (hymenoptera: apidae) (tavares et al., 2012). yet, body size was correlated with genome size among dragonflies and damselflies (ardila–garcia and gregory, 2009), but not among stingless bees (tavares et al., 2010) or ants (tsutsui et al., 2008). these contradictory observations remain the issue whether genome size is shaped by neutral or natural selection. it has been proven that changes in genome size are related to the addition and deletion of heterochromatin and that species with low amounts of heterochromatin also have lower dna content per haploid nucleus, likewise the reverse is also true (tavares et al., 2017). although conclusion remarks still unlike due the limited availability of data and sampling representing more genera and species, important question could be addressed when more data became available. considering the assembled data e evidences from other social insects, as bees, we propose that the differences in dna content among ant species may also be related to the different amount of heterochromatin in the chromosomes. nevertheless, we emphasize that this can only be confirmed after a detailed study of chromosomal structure and chromosome counts across genera and subfamilies. conclusions and perspectives the compilation of the genome size data currently available in the literature for ants has highlighted the scarcity of estimates for this hyper–diverse family (with only 0.52% of known species having been estimated). little is known about the methodologies employed and the lack of standardization of the works makes it problematic to compare the different estimates (ardila–garcia et al., 2010; doležel and greilhuber, 2010), especially regarding the buffer to isolate the nuclei, tissue and internal standard used. also, the mechanisms involved in the evolution of the genome in ants are still unknown, especially those related to the total amount of heterochromatin in chromosomes and their relationship with genome size; the whole–genome duplication events, which could explain the large variation of the genome of some species, such as ectatoma tuberculatum and apterostigma dentigerum (tsutsui et al., 2008); and polyploidy events as in solenopsis invicta males (glancey et al., 1976; lorite and palomeque, 2010). our analysis highlight the importance and accuracy of the use of fcm to estimate the genome size of species and the possibility of obtaining robust results, since a large number of nuclei (10.000 or more per sample) are analyzed to determine the dna content. therefore, the standardization of the techniques used and a large–scale study of the ant genome size are urgently required, given the ecological and economic importance of this group contributing to 37genome size in ants our knowledge on ant evolution by using another genetic diversity and independent dataset. conflict of interest the authors declare that they have no conflict of interest. acknowledgments this study was carried out as part of the phd thesis of the first author. author is grateful to capes for the scholarship. the authors thanks fapemig (fundação de amparo à pesquisa de minas gerais), cnpq (conselho nacional de desenvolvimento científico e tecnológico) and ufop (universidade federal de ouro preto) for their financial support. author received a research productivity fellowship from fapemig (ppm–00126–15). references aguiar hjac barros lac soares faf carvalho cr pompolo sg (2016) estimation of nuclear genome size of three species of camponotus (mayr 1861) (hymenoptera: formicidae: formicinae) and their cytogenetic relationship. sociobiology 63: 777–782. http:// dxdoiorg/1013102/sociobiologyv63i2948 alberts b, johnson a, lewis j, raff m, roberts k, walter p (2007) molecular biology of the cell fifth ed. garland science new york. alfsnes k, leinaas hp, hessen do (2017) genome size in arthropods, different roles of phylogeny habitat and life history in insects and crustaceans. ecology and evolution 7: 5939–5947. https://doi.org/10.1002/ ece3.3163 ardila–garcia am, gregory tr (2009) an exploration of genome size diversity in dragonflies and damselflies (insecta: odonata). journal of zoology 278: 163–173. https://doi.org/10.1111/j.1469-7998.2009.00557.x ardila–garcia am, umphrey gj, gregory tr (2010) an expansion of the genome size dataset for the insect order hymenoptera with a first test of parasitism and eusociality as possible constraints. insect molecular biology 19: 337–346. https://doi.org/10.1111/j.13652583.2010.00992.x basiji da, ortyn we, liang l, venkatachalam v, morrissey p (2007) cellular image analysis and imaging by flow cytometry. clinics in laboratory medicine 27: 653–70. https://doi.org/10.1016/j.cll.2007.05.008 bolton, b. 2018. an online catalog of the ants of the world. available from http://antcat.org. 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(2011) the genome of the fire ant solenopsis invicta. proceedings of the national academy of sciences of the united states of america 108: 5679–5684. substantia an international journal of the history of chemistry vol. 2, n. 1 march 2018 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 72(4): 93-104, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-164 citation: z. bouziane, r. issolah, a. tahar (2019) analysis of the chromosome variation within some natural populations of subterranean clover (trifolium subterraneum l., fabaceae) in algeria. caryologia 72(4): 93-104. doi: 10.13128/caryologia-164 published: december 23, 2019 copyright: © 2019 z. bouziane, r. issolah, a. tahar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. analysis of the chromosome variation within some natural populations of subterranean clover (trifolium subterraneum l., fabaceae) in algeria zahira bouziane1,3, rachida issolah2,*, ali tahar3 1 université abbès laghrour, khenchela, algérie 2 inraa, crp mehdi boualem, division de recherche sur les ressources phytogénétiques, bp 37, baraki, alger, algérie 3 université badji mokhtar, laboratoire de biologie végétale et de l’environnement, annaba, algérie *corresponding author: rachida.issolah@yahoo.com abstract. nine natural populations of subterranean clover (trifolium subterraneum l.) coming from different eco-geographical sites of the north-east algeria, have been studied for their chromosome number and karyotype features. the study is part of the evaluation and valorization of plant genetic resources of fodder and pastoral interest in algeria. the results of mitosis detect two groups of populations, and reveal diversity in the number among and within populations. the algerian populations of t. subterraneum are characterized by two chromosomic formulas. the first formula (2n=2x=16m) (median), more common in most of the studied populations, is in conformity with previous reports in this species. the karyotype of these populations is symmetrical for size and form. the second (2n=2x= 18m), is detected for the first time and described as a new chromosomal formula in t. subterraneum. the latter is relatively more frequent than the first one and characterizes the populations coming from high altitude areas. the karyotype (2n=2x=18m) is relatively symmetrical. at the level of the two established karyotypes, satellites are highlighted at the first pair. a variation in the size and frequency of these satellites is observed. the species exhibits regular meiotic behaviour, confirming the presence of two basic chromosome numbers (x=8 and 9). the study also highlights the role of ecological factors (altitude and rainfall) of the originating environment of algerian populations in the variation and evolution of chromosome numbers in t. subterraneum. the new cytogenetic data can be exploited in the taxonomy of the species in algeria in order to select and develop this plant genetic resource in the agricultural field. keywords. chromosomes, intraspecific variability, karyotype, subterranean clover, trifolium subterraneum l. introduction the genus trifolium is one of the largest genera of the fabaceae family (sub-family, papilionoideae). it has more than 255 annual and perennial spe94 zahira bouziane, rachida issolah, ali tahar cies (zohary and heller 1984; gillet and taylor 2001). most of them are of great agricultural importance and widely grown as fodder and green manure (ellison et al. 2006). the genus trifolium is originating from mediterranean, because of the greatest diversity of numbers and chromosome forms have been found in this region (taylor 1985). it has been subdivised into eight sections: lotoidea, paramesus, mystillus, vesicaria, chronosemium, trifolium, trichocephalum and involucrarium (zohary and heller 1984). the principal geographical centers of diversity of trifolium are the mediterranean basin, the west of north america, and the highlands of eastern africa (ellison et al. 2006). the cytotaxonomic studies carried out on trifolium have shown that it presents a surprising variety of chromosome numbers, and the changes in the number of chromosomes have played a large part in its evolution (falistocco et al. 2013). britten (1963) and pritchard (1969) have shown that an aneuploid series of basic numbers x=5, 6, 7 and 8 are found in this genus. the presence of x=8 in about 80% of the species suggests that x=8 is the ancestral number of the genus (senn 1938; pritchard 1969; zohary and heller 1984; ellison et al. 2006), from which the numbers x = 7, 6 and 5 are derived. polyploidy is more common in perennial species (kiran et al. 2010; falistocco et al. 2013). subterranean clover (trifolium subterraneum l., sect trichocephalum), commonly known as the burrowing clover or sower, is a winter annual species, native to the mediterranean basin, west asia and the atlantic coast of western europe (gladstones and collins 1983  ; zohary and heller 1984). the plant of subterranean clover is autogamous, characterized by mechanisms of burial of reproductive structures, ensuring thus, its own self-regenerating (masson 1997). the species constitutes an heterogenous complexesis, divided into three subspecies:  subterraneum, brachycalycinum and yanninicum (katznelson 1984), identifiable enough by their morphophysiology, karyotypes, isozymes and polymorphisms for molecular markers (piluzza et al. 2005). in algeria, the subterranean clover is very common in the tell and the mountain meadows (quezel and santa 1962), adapted to different ecological conditions (issolah et al. 2015). this species is represented by three varieties belonging to the subterraneum subspecies (subsp. subterraneum var. subterraneum, var. brachycladum, var. flagelliforme) on the eight varieties described in algeria (zohary and heller 1984). despite the agronomic importance of the species in the world, as cattle feed and soil improvement, its cytological characterization remains very restricted. this is because of the small size of chromosomes like all the other species of trifolium (zohary and heller 1984). the first investigations on t. subterraneum focused only on the determination of the chromosome number (2n=16), but without establishing the karyotype (weselxen 1928; yates and brittan  1952; brock 1953; hutton and peak 1954; zohary and katznelson 1958; kliphuis 1962; britten 1963; katznelson and morley 1965a). later, some karyotype studies were performed in spain (angelo et al. 1975, 1977, 1983), iran (hezamzadeh hijazi and ziaeinasab 2006) and italy (falistocco et al. 1987; falistocco et al. 2013). the present study is interested in the evaluation and the valorization of the phytogenetic resources of fodder and ¦pastoral¦ interest in algeria. its aim is the analysis of the chromosomal diversity presents in the natural populations of trifolium subterraneum l., and the establishment of its karyotype. it follows the different studies carried out on natural fodder legumes (issolah and abdelguerfi 1999a; issolah and khalfallah 2007;  issolah et al. 2006,  2012,  2015, 2016). material and methods plant materials the trifolium subterraneum specimens were collected by inr aa (national institute of agronomic research of algeria), in july 2010. nine natural populations sampled from north-east algeria (issolah et al. 2015), were the subject of a karyological study (table 1). chromosome counting the seeds belonging to the nine studied populations, were scarified to remove in tegumentary hardness, and then germinated on wet filter paper in petri dishes at room temperature. the root tips meristems (1 to 1.5 cm in length) were excised in the morning between 8 am8.30 am and pretreated with α-bromonaphthalene (1%) at room temperature for 2h45mn. the use of this pretreatment increases the number of metaphase mitotic cells, allows the chromosomes to be well spread in the cell, straightens the chromatids, and contracts the chromosomes, which makes primary and secondary constrictions very noticeable (singh 2018). for chromosomes analysis, root tips were hydrolyzed in 1n hcl and stained in lactopropionic orcein (dyer 1963). the chromosomic observations were repeated several times. for each population, five plates of chromosomes were selected from at least 30 individuals (seeds). then, they were observed and photographed using a primo star zeiss 95analysis of the chromosome variation of subterranean clover (trifolium subterraneum) in algeria microscope. chromosome counts were performed on metaphase plates with well individualized chromosomes. karyotype analysis the karyomorphological analysis was carried out according to the following parameters: the lenght of long arm (l), short arm (s), the total length of the chromosome (lt = l + s), the difference between arms (d = l-s), and the relative length (lr (‰) = 1000 x tl/ σtl). centromere position and chromosome types were determined from the two parameters: arm ratio (r= l/s), and centromeric index (ci % = s/lt x100) according to the nomenclature of levan et al. (1964). for determining the asymmetry of the karyotype, three parameters were estimed: [(ias. k% = (σ l/σ lt) x100 (aran and saito 1980)], the ratio between the longest and the shortest chromosome pairs (r), and the inter-chromosomal asymmetry coefficient (a2) (standard deviation of chromosome length / mean chromosome length) (romeo zarco 1986). chromosome measurements, based on five plates per population, were performed using the axiovision software (1999-2009). the different karyotype calculations were made thanks to excel (2007). meiosis to confirm the results corresponding to the numbers found by mitosis (presence of supernumerary chromosome pair for certain populations), the meiotic behaviour of the nine populations was also analysed. for this purpose, a trial has been conducted at the experimental station of inraa (november 2014). each population was represented by twenty individuals (seeds) and sowed in total randomization (field) for identifying the different phases of meiosis (laboratory). the flower buds collect period was spread over a month before flowering (recovering flower buds of variable size). for each plant, at least five flower buds were collected (april 2015) in the early morning (from 8h), then fixed in carnoy solution (ethanol-acetic acid 3  : 1, v/v) for at least 48 h at 4c°. after dissection of the anthers, the pollen mother cells (pmc) were crushed in an acetic carmine drop 1% (jahier et al. 1992). observations and photographs at different phases were performed using a primo star zeiss microscope. results chromosome counting all mitotic metaphase plates of investigated populations of the species trifolium subterraneum l. showed a diploid number of chromosomes (2n = 16) (figure 1). this number is frequently observed in individuals of the populations 12/10; 13/10; 19/10; 20/10 and 33/10. however, the somatic metaphases of the four populations 22/10; 23/10; 25/10; 26/10, have presented along with the characteristic number of the species (2n = 16), a second and new number of chromosomes (2n = 18), often encountered during this study in these later populations (figure 1). the two chromosome numbers (2n = 16 and 18) are observed within the cells of the same individual, and also in different individuals of the same population. this indicates a chromosomical variation within and between the populations of trifolium subterraneum. the analysis of 15 individuals per population, indicated that the variation of the chromosome numbers (2n = 16 and 18) was not in the same frequency in these figure 1. mitotic metaphases of algerian natural populations of trifolium subterraneum l. with two chromosomes numbers 2n=16 and 2n=18 respectively: (a) population 12/10; (b) population 13/10; (c) population 19/10; (d) population 20/10; (e) population 22/10; (f ) population 23/10; (g) population 25/10; (h) population 26/10; (i) population 33/10; (j) population 22/10 (2n=18); (k) population 25/10 (2n=18); (l) population 26/10(2n=18) . arrows: satellites. bar: 2.5µm. 96 zahira bouziane, rachida issolah, ali tahar latter populations (figure 2). indeed, within the populations (22/10 and 23/10), the frequency of the number (2n = 16) represents twice the frequency of the number (2n=18) (0.67 and 0.33; 0.64 and 0.36, respectively). however, very similar frequency values are shown in the other two populations (25/10 and 26/10) (0.56 and 0.44; 0.5 and 0.46 respectively) (figure 2). karyotype analysis in all investigated populations, the morphology and chromosome structure are almost identical (table 2-5). our results showed that the chromosomes of the algerian population of the species trifolium subterraneum l. are small. the size of the chromosomes varies from 1.02 μm (table 3) to 3.01 μm (table 2). the total lengths of diploid chromosome set are comprised between 12.82 μm (table 4) and 18.87 μm (table 2). the mean value of the total length (tlg) of all studied populations is 1.92μm. the results of this study indicate also that the population 22/10 (2n =16) is characterized by the highest values for the selected parameters, like the mean value of chromosome length, which gives an estimated size of the genome (18.87 μm) and the largest first pair and eighth pair (3.01μm-1.72μm) (table 2). thus, we note that the two additional chromosomes present in the populations (2n = 18), have the same form, with a mean size of 1.09 μm (figure 1, table 3 and 5). the results of this study indicated also that satellites are located at the first chromosome pair within all investigated populations. a variation of the size and an abundance of these satellites are noticed. thus, the metaphase plates of the populations characterized by (2n= 16), present a considerable size of these satellites compared to that noted on the plates of the populations characterized by (2n=18) with 0.25 μm ± 0.022; 0.18 μm ± 0.025, respectively. these satellites are more abundant in the metaphases of populations with 2n = 18 compared to those with 2n = 16. their frequencies are 0.70 and 0.44, respectively (figure 1). otherwise, the results of the centromeric index (ic) and the ratio between the long arm and the short arm (r) allowed us to determine the homologous chromosomes and to classify the different chromosomal types. therefore, all the studied populations are characterized by the karyograms, presenting median chromosomes (figure 3b). figure 2. frequencies of the two chromosomes numbers (2n=16 and 18) in the four populations of trifolium subterraneum l. (15 individuals / population). table 1. geographical origin and ecological characteristics of the sampling sites of nine populations of trifolium subterraneum l. in algeria nº of populations origin altitude (m) rainfall (mm) 12/10 guelma 170 600 13/10 guelma 200 558 19/10 tarf 665 661 20/10 tarf 555 661 22/10 souk ahras 950 800 23/10 souk ahras 1040 700 25/10 souk ahras 800 900 26/10 souk ahras 1110 700 33/10 skikda 110 562 source (issolah et al. 2015) table 2. morphometric data within the population 22/10 (2n=16) of trifolium subterraneum l. in algeria. ch p l (µm) (±sd) s (µm) (±sd) tl (µm) rl ‰ d r ci % ct 1 1.69 (0.41) 1.32 (0.26) 3.01 159.36 0.37 1.28 43.90 m-sat 2 1.42 (0.31) 1.26 (0.43) 2.68 141.96 0.17 1.13 46.86 m 3 1.53 (0.38) 1.08 (0.35) 2.61 138.52 0.45 1.42 41.33 m 4 1.26 (0.50) 1.09 (0.40) 2.35 124.38 0.17 1.15 46.45 m 5 1.23 (0.50) 1.05 (0.43) 2.28 120.85 0.18 1.17 45.98 m 6 1.18 (0.40) 1.05 (0.31) 2.23 118.11 0.13 1.12 47.12 m 7 1.05 (0.29) 0.94 (0.32) 1.99 105.65 0.11 1.12 47.16 m 8 0.92 (0.31) 0.80 (0.19) 1.72 91. 17 0.12 1.15 46.61 m i1as% =54.48 ∑tl=18.87 tlg=2.36 r1=1.75 a2(1)=0.14 97analysis of the chromosome variation of subterranean clover (trifolium subterraneum) in algeria the values of the asymmetry index ias% (arano and saito, 1980), the ratio between the largest and the smallest chromosome pairs (r), and the interchromosomal index a2 (romero zarko 2006) gives indications on the evolution of chromosomes in plants. the results of the three parameters [(r1: 1.75, r3 =1.78), (i1as% = 54.48, i3as% = 55.81), and (a2 (1) = 0.14, a2(3) = 0.19)] (table 2 and 4) are weak and indicate that the karyotype (2n=16m) is very symmetrical for the size and the form. it is therefore primitive. nevertheless, although the asymmetry indices are low (i2as%: 56.69, i4as% 55.07) in the populations (2n = 18), they showed a karyotype with more or less uniform sizes except for the ninth pair. this is reflected by relatively high values of the ratio (r) and interchromosomal asymmetry a2, compared to those found for the karyotype (2n = 16) (table 3 and 5). meiosis analysis the study of meiotic behaviour showed that the nine natural populations of the species trifolium subterraneum exhibit normal and regular meiosis, with dominance of bivalents at the diakinesis, metaphases i and figure 3. karyotype of trifolium subterraneum l. in algeria. (a) somatic metaphasis (2n=18, population 23/10); (b) karyogram; (c) idiogram; arrow (satellites). bar: 2µm. table 3. morphometric data within the population 23/10 (2n=18) of trifolium subterraneum l. in algeria. ch p l (µm) (±sd) s (µm) (±sd) tl (µm) rl ‰ d r ci % ct 1 1.45 (0.17) 1.05 (0.14) 2.50 146.23 0.40 1,38 42.06 m-sat 2 1.39 (0.13) 1.02 (0.16) 2.41 140.86 0.37 1,37 42.27 m 3 1.33 (0.11) 0.93 (0.08) 2.26 132.46 0.39 1,42 41.31 m 4 1.09 (0.32) 0.89 (0.08) 1.98 116.14 0.20 1,23 44.84 m 5 1.07 (0.25) 0.80 (0.22) 1.87 109.50 0.27 1,33 42.97 m 6 1,01 (0.24) 0.81 (0.19) 1.82 106.51 0.21 1,26 44.30 m 7 0.92 (0.25) 0.81 (0.19) 1.73 101.41 0.11 1,13 46.86 m 8 0.85 (0.30) 0.63 (0.04) 1.48 86.93 0,21 1,34 42.77 m 9 0.57 (0.24) 0.45 (0.15) 1.02 59.96 0.11 1,25 44.42 m i2as%=56.69 ∑tl=17.08 tlg=1.90 r2=2.45 a2(2)=0.25 table 4. morphometric data within the population 25/10 (2n=16) of trifolium subterraneum l. in algeria. ch p l (µm) (±sd) s (µm) (±sd) tl (µm) rl ‰ d r ci % ct 1 1.14 (0.23) 0.91 (0.02) 2.05 159.96 0.22 1.24 44.57 m-sat 2 1.12 (0.10) 0.82 (0.01) 1.94 151.56 0.31 1.37 42.14 m 3 0.96 (0.09) 0.80 (0.01) 1.76 137.50 0.16 1.20 45.60 m 4 0.99 (0.01) 0.73 (0.02) 1.72 134.38 0.26 1.35 42.30 m 5 0.83 (0.06) 0.67 (0.02) 1.50 117.00 0.16 1.24 44.50 m 6 0.80 (0.02) 0.61 (0.15) 1.41 110.16 0.19 1.31 43.09 m 7 0.73 (0.06) 0.56 (0.20) 1.29 100.78 0.17 1.30 43.41 m 8 0.60 (0.17) 0.56 (0.18) 1.15 90.04 0.04 1.08 48.16 m i3as%=55.81 ∑tl=12.82 tlg=1.6 r3=1.78 a2(3)=0.19 98 zahira bouziane, rachida issolah, ali tahar table 5. morphometric data within the population 26/10 (2n=18) of trifolium subterraneum l. in algeria. ch p l (µm) (±sd) s (µm) (±sd) tl (µm) rl‰ d r ci % ct 1 1.36 (0.49) 1.25 (0.36) 2.61 160.10 0.11 1.09 47.94 m-sat 2 1.22 (0.43) 1.04 (0.41) 2.26 138.20 0.18 1.17 46.01 m 3 1.25 (0.58) 0.90 (0.33) 2.15 131.45 0.35 1.38 41.96 m 4 1.14 (0.50) 0.85 (0.39) 1.99 121.50 0.29 1.34 42.75 m 5 1.00 (0.52) 0.75 (0.43) 1.75 107.40 0.25 1.33 42.94 m 6 0.91 (0.38) 0.70 (0.28) 1.61 98.97 0.21 1.30 43.50 m 7 0.82 (0.16) 0.68 (0.23) 1.50 92.08 0.14 1.20 45.42 m 8 0.77 (0.30) 0.71 (0.25) 1.48 90.55 0.06 1.09 47.88 m 9 0.56 (0.05) 0.47 (0.02) 1.03 62.48 0.09 1.20 45.15 m i4as%=55.07 ∑tl=16.38 tlg=1.82 r4=2.54 a2 (4)=0.26 ch p: chromosome pair, l: long arm, s: short arm, lt: total length of chromosome, lr (‰)  : relative length, d  : long arm short arm  ; r  : long arm / short arm, ic : centromeric index, ct : chromosome type, ias% : asymmetry index, r  : longest / shortest pair, σtl  : total lenght of diploid set, tlg : average of total length, a2: interchromosomal asymmetry index, (sd) : standard deviation, sat: satellites. figure 4. pollen meiosis in some natural populations of trifolium subterraneum l. in algeria l. (a) pollen cell  ; (b) diakinesis (population 22/10, n=x=8)  ;  (c) diakinesis (22/10, n=x=9)  ;  (d) diakinesis (population 23/10, n=x=8)  ; (e) metaphase i (population 23/10, n=x=8)  ; (f ) metaphase i (population 23/10 n=x=9) ; (g) anaphase i ; (h) telophase i ; (i) tetrade. bar: 2 µm. 99analysis of the chromosome variation of subterranean clover (trifolium subterraneum) in algeria anaphases i (figure 4). this allowed us to authenticate the basic haploid number (x = 8) for the populations (12/10; 13/10; 19/10; 20/10; 33/10). likewise, it confirms the presence of the two chromosome numbers (2n = 16 and 18) detected in mitosis, within the four populations (22/10; 23/10; 25/10; 26/10), through the appearance of two basic haploid numbers (x = 8) and (x = 9). discussion in this study, the chromosome numbers, karyogram, idiogram and karyotype asymmetry of naturel populations of trifolium subterraneum, were determined. mitotic metaphases showed both the same chromosome number (2n=16) in all studied populations. this number was previously reported by several authors within different ecotypes and varieties from several areas (senn 1938; angelo 1975, 1977, 1983; zohary and heller 1984; hezamzadeh hijazi and ziaeinasab 2006; vizintin et al. 2006; falistocco et al. 1987; falistocco et al. 2013), considering x=8, as being the ancestral basic chromosome number of the species. meanwhile, four populations presented two numbers of chromosome (2n=16 and 18) within the cells of the same individual, and also in different individuals of the same population. the number of chromosomes, as one of the genetic variations, is extremely variable ranging from low numbers to relatively high numbers (eroğlu and per 2016). a change in the basic chromosome number of a species represents dysploidy (yakovlev 1996). according to the same author, this change can occur either in the direction of an increase (ascending dysploidy) or a decrease (downward dysploidy). in plants, this last case seems to be the most frequent, it results from the simultaneous or successive action of several cytogenetic mechanisms (robertsonian translocation, deletion …) (yakovlev 1996). contandriopoulos (1978) reports 2n = 30, 32 and 34 for sideritis libanotica labill. this author notes that dysploidy still seems anarchic and has not succeeded to form populations with stable karyotypes having their own geographical distribution and a particular morphological differentiation. in such case, according to the same author, it would seem more judicious to speak about hyper and hypoaneuploidy. aneuploidy may present the beginning of the mechanism leading to dysploidy, provided that the individuals carrying the aneuploid number are able to multiply then impose itself in the population (contandriopoulos 1978). yakovlev (1996) considers that a variable chromosome number within the same population is both an aneuploidy and dysploidy phenomenon, witch is difficult to draw the line between these two phenomena, especially when it is polyploid taxa. an intra-specific dysploidy represents a transitiona l step towards a def initive change in t he basic chromosome number (yakovlev 1996). the populations in which such change has occurred and fixed represent, well probably, the direct ancestors of future dysploïde species (yakovlev 1996). in the genus trifolium, many variations of the nombre de chromosomes (2n = 16, 14, 12, and 10) characterize different diploid species, and in some instances cytological variants occur within the same species (falistocco et al. 2013). brock (1953) counted two different chromosome numbers (2n = 12 and 16) in the species trifolium subterraneum growing in various regions. this author suggested that the difference could be the result of a chromosomal rearrangement without loss of genetic material. in the same genus, two basic numbers (x = 8 and 9) were highlighted within the populations of two species of trifolium: t. ornithopodiodes from the british isles (rutland 1941; muñoz-rodríguez 1995), and also in t. montanum var. montanum. of iberian peninsula (bleier 1925a; muñoz-rodríguez 1995). issolah and abdelguerfi (1999b), evenly showed the presence of two basic chromosomes numbers (x = 5 and 6) in the algerian populations of trifolium scabrum. according to pritchard (1969) and zohary and heller (1984), the dysploidy is consistently linked to the annual species, and are most common within sections that are at a more advanced stage of evolution, such as trifolium and tricocephalum, in which all the four basic numbers (x =8,7,6 and 5) may be found. uslu (2012) has shown that taxa in the trifolium section, growing in turkey, have three numbers (x = 6, 7 and 8). within the tribe trifolieae, darlington and jamaki (1945) and darlington and wylie (1945) reported three basic numbers (x = 7, 8, and 9). the last basic number (x = 9) was detected in europe in trigonella ornithopodiodes l. (dc) (darlington and wylie 1945). this species was reclassified later, for taxonomic reasons, in the trifolium genus (allen and allen 1981). within the fabaceae family, several cases, observing more than one basic chromosome number, have been reported in different genera including onobrychis, with x = 7 and x= 8 (hejazi et al. 2010, arslan et al. 2012) and genista where the most common number of chromosomes is 2n = 48, with the exception of the aneuploid number (2n = 44) revealed in genista ovina (bacchetta et al. 2012). the same process was detected in species of the genus hedysarum, among which, h. pallidum (2n = 16 and 18) (benhizia et al. 2003); h. coronarium (2n = 100 zahira bouziane, rachida issolah, ali tahar 16 and 2n = 18) (issolah et al. 2006)  and h. perrauderianum (2n = 32 and 18) (benhizia et al. 2013). in the poaceae family, dysploidy was observed in lygeum spartum l., whose cytogenetic study revealed two basic chromosome numbers, in two algerian populations of different origins (2n = 16 and 40) (abddaimboughanmi et al. 2009). according to the same authors, the population (2n = 40), also presented a variability of the chromosome number within the same individual. yakovlev et al. (2017) have shown that constitutive heterochromatin, dna gc rich and rrna are involved in chromosomal rearrangements during the change in basic chromosome numbers in mediterranean species of the genus reichardia roth. (asteraceae). these species are characterized by three basic chromosome numbers (x = 9, 8 and 7), which have contributed to the evolution of the genus in the mediterranean region (yakovlev et al. 2017). concerning chromosome size, our results (1.023.1μm) seem to be relatively inferior to those found by falistocco et al. (2013) on italian accessions of trifolium subterraneum (2.5-3.5μm). but then, this size appears to be very similar to that recorded in t. lappaceum species of iran (3.03 μm), but smaller than the sizes reported in other trifolium species of iran (t. angustifolium: 14.56 μm, t. leucanthum: 12.32 μm, t. tumens: 11.09 μm) (alimardani et al. 2014). our data are also close to those found within some trifolium species in turkey, such as t. echinatum (1.41-2.74 μm)  and t. phleoides (1.73-2.78 μm) (uslu 2012), and appear to be superior to those recorded by kiran et al. (2010) in t. speciosum willd. (0.99-1.64 μm) and t. campestris scherb (1.13-1.73 μm). within the same family (fabacaea), the size of t. subterraneum chromosomes, found during our study, is relatively close to those reported for some species of the genera hedysarum, astragalus and asparagus studied in algeria (benhizia et al. 2003 ; issolah et al. 2006, benhizia et al. 2013; baaziz et al. 2014 and boubetra et al. 2017). our observations highlighted satellites at the first pair of chromosomes. the presence of satellites and their location on the first chromosome pair joins the result found by falistocco et al. (2013) on italian accessions. according to falistocco et al. (1987)  and falistocco et al. (2013), these satellites are present in the three subspecies of t. subterraneum (subterraneum, brachycalycinum, yanniniccum), and their size can be used for discriminating the three subspecies. the satellites are more important in yanninicum and medium in the other two subspecies (falistocco et al. 1987). in all populations, the chromosomes are median. this confirm the results of falistocco et al. (2013) on italian accessions, characterized also by median chromosomes, whereas, angelo et al. (1983) have described two chromosomes types (median and submedian) for spanish ecotypes. moreover, two types of karyotypes were identified for the iranian accessions: the first consists on eight median pairs; the second karyotype is composed by six median pairs and two submedian pairs (hezamzadeh hijazi and ziaeinasab 2006). karyotype asymmetry is an important parameter in karyological studies (eroğlu 2015). in our case, the karyotype (2n=16) of algerian populations of trifolium subterraneum is very symmetrical. this seems to be a common trait with italian populations of t. subterraneum karyotype (falistocco et al. 2013), but differs from the iranian ones. the latter populations of t. subterraneum (2n = 16) are characterized by low intrachromosomal symmetry (hezamzadeh hijazi and ziaeinasab 2006). on the other hand, the karyotype of the population 2n = 18 is considered relatively symmetrical because of the high value of interchomosomal asymmetry. thus, muñoz-rodríguez (1995) does not consider the karyotype of the species trifolium ornithopodioides (2n=18) as asymmetrical, despite the high value of the asymmetry index a2 (0.20). the author noticed this, because of the more or less uniform sizes of the chromosome pairs, except for the first pair, which was larger than the others (muñoz-rodríguez 1995). in the species reichardia picroides (asteraceae), yakovlev (1986) has suggested that this is a case of secondary symmetry due to chromosomal rearrangements. the analysis of pollen meiosis confirmed the results obtained in mitosis. at the end of these results we have found that the algerian populations of t. subterraneum are characterized by two chromosomal formulas. the first, (2n = 2x = 16m) (median) usually reported by previous authors, and the second (2n = 2x = 18m) revealed for the first time in this species throughout our present work. it is important to note that the new formla (2n = 2x = 18m) is observed particularly in populations sampled from high altitude sites (800-1110 m), belonging to the same biogeographic area and characterized by a high rainfall (700-900 mm). consequently, the variation in the chromosome number observed in the populations of this species and the appearance of a new chromosome pair seems to be influenced by these two ecological factors (altitude and rainfall). meanwhile, t he same popu lations considered through our study have been the subject of previous work on the ecological characterization of the natural habitat of t. subterraneum in algeria (issolah et al. 2015). thus, the results of this latest study have shown that the variation of the edaphic, climatic, and topographic characteristics of the origin sites of these popu101analysis of the chromosome variation of subterranean clover (trifolium subterraneum) in algeria lations influences the distribution of this species in the north-est algeria (issolah et al. 2015). significant relationships were found between altitude and rainfall and the physico-chemical parameters of the soils of these populations, and the effect of altitude was relatively more pronounced notably on the nitrogen, clay, ph and c / n ratio (issolah et al. 2015). abdelguerfi et al. (2006) indicate that t. subterraneum is more prevalent in heavily watered and moist regions. rossiter and collins (1988a, 1988b) and cocks (1992) also observed greater variability of subterranean clover populations in high rainfall areas in australia. various studies have shown that differences in the origin’s areas of populations and the variation of the environmental factors of the natural habitat may explain the intra-specific differences. thus, they can affect the variation of chromosome numbers, ploidy level, chromosome structure, and asymmetry of karyotype in certain species belonging to the genera: trifolium (issolah and abdelguerfi, 1999b, issolah 2006); hedysarum (issolah et al. 2006, benhezia et al. 2013); bellevalia and muscari (azizi et al. 2016); asparagus (boubetra et al. 2017). environmental factors also, influenced karyotype parameters in aegilops (poaceae) species (baik et al. 2017). significant relationships were found between altitude, total lengths chromosome set and interchromosomal asymmetry on the one hand and, on the other hand, between rainfall and intrachromosomal asymmetry (baik et al. 2017). according to hayward and breese (1993), natural habitats are rarely, if ever, uniform in space and time and can encompass several distinct micro-niches or go through large seasonal fluctuations. although trifolium subterraneum is a self-pollinating species, allard and adams (1969) and hayward and breese (1993), report that fluctuations and variation in edaphic conditions at the site of origin trigger in self-pollinated species, a disruptive selection that produces and maintains high levels of variability in wild populations. in italy, a relationship between many morphological characteristics and the ecological factors of the environment of origin (altitude and rainfall) has been determined in several populations of t. subterraneum from sicily (piano et al.1993, pecetti and piano 1998). in a large collection of subsp. subterraneum germplasm of sardinia, piano et al. (1996, 2002) found that the level of complexity for various traits varied greatly among populations and was influenced by the climatic characteristics of the collection sites. within the genus trifolium, interesting relationships have been found between many morphological characteristics and some ecological factors (altitude and rainfull) of the environment of origin of several spontaneous algerian populations belonging to various species (t. campestre, t. glomeratum, t. tomontosum, t. resupinatum, t. scrabrum, t. lampaceum, t. spumosum) (issolah and abdelguerfi 1993, 1995, 2003 ; issolah 2006). in addition, medoukali et al. (2015), do not report any significant relationship between the morphological characteristics and the environment of origin of populations belonging to several trifolium species (t. angustifolium, t. lappaceum, t. resupinatum, t. tomentosum, t. scabrum, t. campestre, t. fragiferum, t. pallidum, t. pallescens, t. squarrosum, t. glomeratum, t. cherleri, t. stellatum, t. repens and t. spumosum). nevertheless, a large genetic variation of isoenzymes has been observed (medoukali et al. 2015). although the species is self-pollinated with cleistogamous flowers (katznelson and morley 1965), there is a possibility of occasional cross breeding, and this exceptional rarefaction could be of great importance for the evolution of t. subterraneum. marshall and broué (1973) estimated the cross-pollination rate of the australian clover populations at 0.15%. variation released by occasional hybridization can then be fixed by selfing and made available to natural selective pressures (cocks 1992b). according to piano (1984), natural populations of subterranean clover were formed by clusters of several genetically distinct strains. this would probably explain the chromosomal variation observed in this study within and between populations. as a result, the different populations of t.subterrarenum would have been crossed. meanwhile, four populations from the same region exhibited the same somatic behaviour (2n = 16 and 18) (within the same individual and between different individuals) and meiotic (n = x = 8 and n = x = 9). these populations would probably be evolved in time, since they belong to a species of the “trichocephaleum” section considered, according to zohary and heller (1984), as the most evolved section compared to other sections of the genus trifolium. this section is therefore composed of species, whose interaction, with the various ecological characteristics of the natural habitat, would affect the chromosomal rearrangements and evolutionary trends of the populations within t. subterraneum species. conclusion this study permitted to identify and analyse the intraspecific diversity of the chromosome numbers and karyotypes within nine natural populations of trifolium subterraneum, originating from the different areas of the north eastern algeria. two chromosome numbers 102 zahira bouziane, rachida issolah, ali tahar are distinguished in this species: 2n=16 (x=8) and 2n=18 (x=9). the first number (2n=16), is widely detected by previous authors, while the second one (2n=18) is newly observed in algerian populations of this species. the latter number (2n=18) is frequently met in populations coming from the high altitude areas. the ecological conditions of the origin’s environment of the populations would have an effect on the changes in the genetic and karyological structure, particularly the altitude factor. this karyological approach provides new information that will help researchers to elucidate and complete the systematics and the nature of diversity within trifolium subterraneum species. however, thorough investigations of the morphological and molecular aspects of these natural populations would be necessary, to determine the limits of dysploidy. furthermore, comparative analysis with other populations from different origins would help to understand more about the genome evolution process of t. subterraneum populations in their environment of origin. this would permit to valorize and develop this plant genetic resource in the mediterranean area, especially in algeria. references allen o n, allen e.k.1981. the leguminoseae, macmillon.c.o, london. abdeddaim-boughanmi k and kaid-harche m. 2009. structure, ultrastructure of the anther pollen microsporogenesis and morphology of pollen grains of two populations of lygeum spartum l. in algeria. americ j. agric. and biol sci. 4 (3) 201-205. abdelguerfi a, abdelguerfi–laouar m, m’hammedi bouzina m, guittonneau gg, huguet t, abbas k, mebarkia a, aouani m e and madani t. 2006. distribution et écologie de quelques fabaceae spontanées d’intérêt pastoral et / ou fourrager en algérie. workshop international sur la diversité des fabacées fourragères et de leurs symbiotes : applications biotechnologiques, agronomiques et environnementales. alger, 19-22 février 2006: 27-36. alimardani f, torabi s, naghavi r, ebrahimi a. 2014. study of cytological among some trifolium species of iran. interciencia. 39 (4) 147– 151. allard rw. 1965. genetic systems associated with colonizing ability in predominantly self-pollinated species. in: baker h.g. and stebbins g.l. 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(buddlejaceae) and castilleja arvensis schltdl. & cham. (orobanchaceae) aldo ruben andrada1,*, valeria de los ángeles páez1, m.s. caro1,2, p. kumar3 population genetic studies in ziziphus jujuba mill.: multiple molecular markers (issr, srap, its, cp-dna) farah farahani1, atieh sedighzadegan2, masoud sheidai2, fahimeh koohdar2 karyotypes of danubian lineage brown trout and their hybrids melike alemdag, rafet cagri ozturk, sebnem atasaral sahin, ilhan altinok* chromosome counts and karyotype analysis of species of family apocynaceae from egypt samia heneidak1,*, esra martin2, fahim altinordu2, abdelfattah badr3, halil erhan eroğlu4 geographical distribution and karyotype of nannospalax ehrenbergi (nehring 1898) (rodentia, spalacidae) in iraq zaitoon ahmed hamad1, alaettin kaya2, yüksel coşkun2,* population genetic study of ziziphus jujuba mill.: insight in to wild and cultivated plants genetic structure seyyedeh tahereh nabavi1, farah farahan2, masoud sheidai3,*, katayoun poursakhi1, mohammad reza naeini4 analysis of the chromosome variation within some natural populations of subterranean clover (trifolium subterraneum l., fabaceae) in algeria zahira bouziane1,3, rachida issolah2,*, ali tahar3 megagametophyte differentiation in zephyranthes drummondii d. don and zephyranthes chlorosolen (herb.) d. dietr. (amaryllidaceae) charles f. crane caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 45-55, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-185 citation: b. huri gölge, f. vardar (2020) temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots. caryologia 73(1): 45-55. doi: 10.13128/caryologia-185 received: march 8, 2019 accepted: november 2, 2019 published: may 8, 2020 copyright: © 2020 b. huri gölge, f. vardar. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* marmara university, faculty of arts and sciences, department of biology, göztepe, 34722, i̇stanbul, turkey *corresponding author. e-mail: filiz.vardar@gmail.com abstract. aluminum (al) is the third most elements found in the earth crust and al toxicity is one of the most dangerous toxicants in terms of plants. as soil acidity increases due to a number of environmental factors, al becomes soluble and transforms into toxic forms. in the present study, barley (hordeum vulgare l.) roots were exposed to 100 µm alcl3 solution for short (1/2, 1, 2, 3, 4, 5, 6 and 7 h) and long (24, 48, 72 and 96 h) term to reveal time dependent programmed cell death evidences. at the end of time periods, al+3 accumulations, loss of plasma membrane integrity and lipid peroxidation increased time dependently. on the other hand, increase in caspase-1 like enzyme activities were observed in al toxicity beginning from ½ h. similar to apoptosis seen in animals, cytochrome c release from mitochondria to cytoplasm was also determined quantitatively. as a result of our research, increase of cytochrome c release from mitochondria to cytoplasm was time dependent which is one of the indicators of programmed cell death. finally, under al stress, genomic dna fragmentation was measured by flow cytometry, and it was determined that dna fragmentation was visible at first hours, but it was more significant after long term application in barley roots. in conclusion; the presented study highlights the adverse effects of al on barley roots and importance of clarifying the relationship between al toxicity and time dependent programmed cell death mechanism. keywords. aluminum, caspase-1 like activity, cytochrome c, dna fragmentation, lipid peroxidation, programmed cell death. introduction aluminum (al), as an abiotic stress factor, exists as third most abundant mineral forming 8% in earth’s crust (matsumoto 2000; abate et al. 2013). it has been known that al is the primary limiting factor on plant growth and development in acidic soils. considering the 67% of the world’s potential arable lands are acid soil, most of the agronomic plants are face to al toxicity (kochian et al. 2004; abate et al. 2013; ma et al. 2014). non-toxic al appears as insoluble aluminosilicates or oxides, but when soil ph decreases (ph<5) al solubilized into reactive and phytotoxic forms being in a range of 10–100 μm (matsumoto 2000; ciamporova 2002; vardar and ünal 2007). several 46 büşra huri gölge, filiz vardar researches revealed that al adversely affects the plant within a few minutes even at low micro-molar doses; as a result of this al is considered as major constraint for crop yield (vitorello et al. 2005; abate et al. 2013). considering the whole plant, root apex (root cap, meristem and elongation zone) is the first target organ and accumulates more al (matsumoto 2000; vardar et al. 2006). several studies prove that al induce morphological, biochemical and physiological alterations. besides, it is a genotoxic agent leading adverse effects on dna structure and function. moreover, al toxicity decreases mitotic index and causes chromosome aberrations (frantzios et al. 2000; vardar et al. 2011). recent works also revealed that al also adversely affects dna methylation and polymorphism on ltr retrotransposons suggesting these alterations may be a defense mechanism to al stress (guo et al. 2018; taşpınar et al. 2018). there is a close relation to al toxicity and ros (reactive oxygen species) production triggering oxidative damage in the cell. over accumulation of ros cause damage on lipid, protein, carbohydrate, photosynthetic pigments and dna leading to programmed cell death (pcd) (darko et al. 2004; sharma and dubey 2007; gupta et al. 2013). pcd is considered as an alternative adaptive mechanism in plants to enable the survival of whole organism under extreme environmental stresses (jackson and armstrong 1999; drew et al. 2000; vardar et al. 2018). pcd is a genetically regulated cell suicide process and coordinated by specific proteases and nucleases (wang et al. 2011; vardar and ünal 2012; wituszyńska and karpiński 2013; petrov et al. 2015). it has been identified by common characteristics in eukaryotes such as cell shrinkage, vacuolization, cytochrome c release, specific protease activation, chromatin condensation, dna fragmentation and finally breakdown of the cell (vardar and ünal 2008; papini et al. 2011; wang et al. 2012; poor et al. 2013). there are several researches concerning abiotic stress induced pcd in plants, but al stress-induced pcd is still need to be investigated to clarify its toxicity and tolerance mechanism. even if there are a few research on temporal occurrence of al-induced pcd (vardar et al. 2015; 2016), more detailed time dependent analyses are of the essence. therefore, we designed a detailed analysis addressing al-induced pcd in the course of short and long term exposure in hordeum vulgare roots. material and methods plant material the seeds of barley (hordeum vulgare l. cv çetin 2000) which were obtained from the field crops central research institute (ankara, turkey) were sterilized with 1% sodium hypochloride solution for 10 min. after rinsing, seeds were placed on moistened filter paper in petri dishes for germination. the petri dishes were kept in a plant growth room with fluorescent tubes giving an irradiance of 5000 lux (day/night 16/8 respectively), temperature of 23 ± 2 °c, and relative humidity 45–50% for 48 h. the barley seedlings which reached 0.5-1 cm root elongation were immersed in 100 µm alcl3 (ph 4.5) with different time intervals. in the present study the exposure time designed in two groups: short (½, 1, 2, 3, 4, 5, 6 and 7 h) and long time (24, 48, 72 and 96 h) exposure. distilled water was used for the control group. fifty seeds were used for each experimental group. all of the analyses were assessed with three replicates for statistical validity. analysis of variance of all the experimental data was performed with spss 13.0 computer program. student ttest was used to determine the statistical significance of differences among the means at p < 0.05. determination of al uptake al+3 ion uptake in control and treated roots was assessed by hematoxylin staining method (ownby 1993). the intact barley roots were stained with solution containing 0.2% (w/v) hematoxylin and 0.02% (w/v) kio3 for 15 min in dark. then the roots were washed with distilled water and 10 root tips (1 cm) immersed in 4 ml 1n hcl (v/v) for 1 h. after immersion, hcl solution was measured at 490 nm spectrophotometrically. determination of loss of plasma membrane integrity the loss of plasma membrane integrity in control and treated roots was detected by evans blue staining method (pandey et al. 2013). the intact barley roots were stained with 0.25% (w/v) evans blue solution for 30 min. after rinsing in distilled water for 10 min, 10 root tips (1 cm) were homogenized in 1 ml of 1% (w/v) sodium dodecyl sulphate (sds) solution. after centrifugation at 13500 rpm for 10 min, the supernatant was measured at 600 nm spectrophotometrically. determination of lipid peroxidation lipid peroxidation was measured by the amount of malondialdehyde (mda) produced after reaction with thiobarbituric acid (tba) (cakmak and horst 1991). the 47temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots control and treated roots (0.4 g) were homogenized in 2 ml 0.1% (w/v) trichloroacetic acid (tca). after centrifugation at 12000 g for 20 min, the supernatants (0.5 ml) were added on 0.6% (w/v) tba in 20% (w/v) tca (2 ml) and boiled at 95 °c for 30 min. the mixture was transferred to ice immediately. after color change the samples centrifuged at 12000 g for 10 min. absorbance of the tba-reactive substance was determined as tba-mda complex at 532 and 600 nm. determination of caspase-1 like activities control and alcl3 treated root tips (0.3 g) were ground in ice-cold mortar with 1 ml extraction buffer (50 mm hepes-koh – ph 7, 10% sucrose, 0.1% chaps, 5 mm dtt, and 1 mm edta) according to lombardi et al. (2007). the homogenates were transferred on ice for 10 min and centrifuged at 14000 rpm (+4 ºc) for 10 min. protein concentration was determined by nano photometer. for determination of caspase-1 activity, enzo’s caspase-1/ice colorimetric protease activity assay kit was used. according to manufacturer’s instructions, equal amounts of protein extracts were incubated at 37 °c for 90 min with p-na (p-nitroaniline)-labeled substrates yvad. the caspase-1 like activity was measured at 400 nm and calculated according to the standard curve. comparison of the absorbance of p-na from an apoptotic sample with an un-induced control allows determination of the fold increase in caspase activity. determination of cytochrome c release control and alcl3 treated root tips (0.2 g) were ground in ice-cold mortar with 3 ml 0.1 m phosphate buffer saline (pbs, ph 7.7). the homogenates were centrifuged (+4°c) for 15 min and the supernatant re-centrifuged at 16000 rpm for 15 min. after centrifugation, supernatant was collected as cytoplasmic phase and pellet is collected as mitochondrial phase. the pellet was re-suspended with 200 µl pbs (huang et al. 2014). for determination of cytochrome c (cyt c) release, mybiosource’s plant cyt c elisa kit was used. according to manufacturer’s instructions, both cytoplasmic and mitochondrial phases were incubated with hrp-conjugated reactive at 37°c for 60 min. after rinsing with washing solution, chromogen a and b solution were added and incubated at 37°c for 15 min. along with stop solution, cytochrome c was measured at 450 nm and calculated according to the standard curve. determination of dna fragmentation dna fragmentation was analyzed by flow cytometry in control and al treated roots. for this purpose, cystain® uv precise p kit special for plants was used. the nuclei were isolated from 6 root tips by careful slicing with a razor blade in 0.4 ml nuclei extraction buffer. the extract was collected with micropipette and stained with 1.6 ml dapi in a micro centrifuge tube. the tubes were transferred on ice for 1-2 min and then filtered to test tubes. the dna fragmentation of nuclei was analyzed with flow cytometry (sysmex). results to determine the temporal effects of al, the barley roots were exposed to 100 µm alcl3 (ph 4.5) for short (0, ½, 1, 2, 3, 4, 5, 6 and 7 h) and long (24, 48, 72 and 96 h) time points. after al exposure al+3 ion uptake, loss of plasma membrane integrity, lipid peroxidation, caspase-1 like activities, cytochrome c release and dna fragmentation were analyzed in relation to time dependent programmed cell death (pcd) occurrence. according to hematoxylin analysis al+3 ion uptake increased by 8.7%, 30.4% and 73.9% at ½, 1 and 2 h, respectively. ongoing times al uptake raised by about 1.5 fold up to 5 h, 1.8 fold at 6 h and 2.1 fold at 7 h (fig. 1a). after long term exposure, al uptake increased exponentially by 4.6, 7.5, 11.3 and 12.1 fold at 24, 48, 72 and 96 h, respectively (fig. 1b). evans blue analysis is frequently used to determine the loss of plasma membrane integrity. it has been known that the dye can penetrate through ruptured membranes and stains the damaged cells.  in this respect after al exposure plasma membrane rupture determined in barley root cells time dependently. based on our results the dye uptake increased by 25.3%, 34.6%, 46.7%, 74.7%, 72%, 78.7%, 94.7% and 97.3% from ½ to 7 h respectively (fig. 2a). after long time al exposure evans blue uptake increased progressively. it was increased by 2.4, 3.5, 4 and 4.3 fold at 24, 48, 72 and 96 h, respectively (fig. 2b). under oxidative stress conditions, over production of reactive oxygen species (ros) cause lipid peroxidation. after al exposure mda content one of the end product of lipid peroxidation showed an increment even at ½ h and it raised 6-7 fold up to 7 h (fig. 3a). the increment showed 7.5, 9.7, 8.1 and 6-fold increase at 24, 48, 72 and 96 h, respectively. although the mda content was highest at 48 h, it decreased at 72 and 96 h with regard to 48 h (fig. 3b). 48 büşra huri gölge, filiz vardar figure 1. hematoxylin content of control and 100 µm alcl3 treated barley roots at short (a) and long (b) time points. the data with (*) are significantly different from the control at p < 0.05 level. figure 2. evans blue uptake of control and 100 µm alcl3 treated barley roots at short (a) and long (b) time points. the data with (*) are significantly different from the control at p < 0.05 level. figure 3. lipid peroxidation of control and 100 µm alcl3 treated barley roots at short (a) and long (b) time points. the data with (*) are significantly different from the control at p < 0.05 level. 49temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots although there are no functional homologs of animal caspases, there are true caspase-like activities in plants during pcd. although the results of caspase-1 like activities showed f luctuations between exposure groups, al caused increase both at short and long time points. the increased activities were among 1.33 – 1.78 than that of control (fig. 4a, b). it has been known that cyt c is released from inner mitochondrial membrane to cytoplasm during pcd. in barley roots, after al exposure cyt c release started from ½ h and increased exponentially. it was 33.1% in ½ h, 40.6% in 1 h, 42.8% in 2 h, 55.6% in 3 h, 86.6% in 4 h, 85.2% in 5 h, 80.2% in 6 h and 98.1% in 7 h (fig. 5a). after long term exposure it increased about 2 fold in 24 and 48 h, and about 2.4 fold in 72 and 96 h (fig. 5b). dna fragmentation is one of the significant markers of pcd and flow cytometry is one of the analyses that used. the fragmented dna can be seen as peaks at the left of g1 in the histogram. after the data analysis, the cell rate of having fragmented dna was 23.7%, 17.3%, 31.4%, 37.1%, 15.0%, 37.8%, 25.1% and 30.6% between ½ 7h, respectively (fig. 6). at long term exposure dna fragmentation rate was more significant. it was 63.7%, 49.6%, 62.6% and 64.4% between 24-96 h, respectively (fig. 7). according to the flow cytometry al caused dna fragmentation both in short and long term exposure. discussion al toxicity is one of the major inhibitors of plant growth and development in acidic soils (abate et al. 2013; ma et al. 2014). although multiple studies have been performed in understanding physiological and molecular mechanism of al toxicity and tolerance in the last few decades, there is limited studies concerning time dependent occurrence of al-induced pcd. therefore, we figure 4. % fold caspase-1 like activities of control and 100 µm alcl3 treated barley roots at short (a) and long (b) time points. the data with (*) are significantly different from the control at p < 0.05 level. figure 5. cytochrome c level in mitochondria and cytoplasm of control and 100 µm alcl3 treated barley roots at short (a) and long (b) time points. the data with (*) are significantly different from the control at p < 0.05 level. 50 büşra huri gölge, filiz vardar determined the temporal effects of al correlating with al+3 ion uptake, loss of plasma membrane integrity, lipid peroxidation, caspase-1 like activities, cyt c release and dna fragmentation in relation to pcd. al toxicity adversely affects the cellular processes due to the strong and rapid interactions of al with apoplasmic and symplasmic targets. it has been widely known that al accumulates more in root apex and strongly binds to negative charged materials such as cell walls and membranes (matsumoto 2000). several researches revealed that primer cellular responses to detrimental effects of al could arise within second to minutes along with al penetration (kochian et al. 2005; singh et al. 2017). based on our al+3 ion uptake results, al penetrated into root apex beginning from ½ h and increased on the advancing hours. although the al uptake was very slight (8.7%) at the beginning, its impact was very severe in barley roots. considering the loss of plasma membrane integrity and lipid peroxidation, the toxicity was very significant even at ½ h al exposure. al uptake increased gradually depending on time and at the same time loss of plasma membrane integrity enhanced. considering the sharp increase of mda even at ½ h suggests that al is not detrimental on only plasma membrane. it’s also very injurious on whole cellular membrane system including organelles. although mda content rose sharply up to 48 h, the reduction of mda at 72 and 96 h may be related to increased generation of the other end product aldehydes such as 4-hne. according to general consensus al induces oxidative stress as an abiotic stress factor in plants (matsumoto 2000). whereas al is not a transition element, it catalyzes formation of ros inducing oxidative stress (gupta et al. 2013). over-accumulation of ros induces damage of biological molecules such as proteins, dna and lipids. membrane lipids are the more significant targets of ros and culminate in lipid peroxidation eventually affecting normal cellular functions such as reduction in membrane fluidity, increase in phospholipid exchange, leakage of membrane and membrane rupture. besides lipid peroxidation is considered as one of the hallmarks of pcd (gill and tuteja 2010; woo et al. 2013; nath and lu 2015). moreover, it has been reported that al induces swollen mitochondria with several vacuoles, disrupted plasma membrane, and nuclear deformations following figure 6. dna fragmentation of control and 100 µm alcl3 treated barley roots at short term exposure. (x) axis denotes cell counts and (y) axis denotes fluorescence intensity. 51temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots the mitochondrial pathway of pcd (gupta et al. 2013; singh et al. 2017). there is a close relation to ros triggered cyt c release from the inner mitochondrial membrane to cytoplasm constituting mitochondrial pathway of pcd. ros provokes the cardiolipin oxidation in mitochondrial inner membrane weakening the bonds of cyt c. ros also triggers the ca2+ transition as a secondary signal from er to mitochondria reducing the mitochondrial membrane potential (δψm). after δψm decrease, cyt c released to intra-membrane space translocate to the cytoplasm from the mitochondrial permeability transition pores (mptp) in the outer mitochondrial membrane (ott et al. 2007; williams et al. 2014). in pcd cyt c release stimulates caspase-like activities resulting in execution of pcd (li and xing 2010; petrov et al. 2015; gunawardena and mccabe 2015). huang et al. (2014) indicated that al-induced ros accumulation and pcd is closely related. according to their results 100 µm alcl3 caused increase in mptp opening, reduction of δψm, cyt c translocation to cytoplasm and caspase 3-like activity subsequent to ros accumulation in arachis hypogaea roots. it has been concluded that al induced pcd is mediated by ros figure 7. dna fragmentation of control and 100 µm alcl3 treated barley roots at long term exposure. (x) axis denotes cell counts and (y) axis denotes fluorescence intensity. 52 büşra huri gölge, filiz vardar related cascade of cellular events resulting in mitochondrial alterations and caspase like activities. simultaneously, zhan et al. (2014) established the relation between ros accumulation and mitochondrial alterations during al-induced pcd in arachis hypogaea. the researchers reported that increase in mitochondrial ros induced reduction of mitochondrial ca concentration, opening of mptp, collapse of δψm and cyt c release were more extensively in al sensitive cultivar depending on al concentration. similarly, in the present study al application caused cyt c release to the cytoplasm depending on time. the best of our knowledge all of the researches subjecting cyt c release were qualitative analysis and no report has been published of quantitative analysis of cyt c release during pcd in plants except our study. in addition to alterations of mitochondria several researchers reported caspase like activities induced by ros increase and cyt c release after al application (li and xing 2011; huang et al. 2014; aytürk and vardar 2015; yao et al. 2016). caspases are cysteine proteases initiating and amplifying pcd and cleave their cellular substrates at certain aspartic acid residues (mcilwain et al. 2013). whereas there are no functional homologs of caspases in plants, caspase-like activities have been elucidated. according to recent reports, vacuolar processing enzymes (vpes) belonging to cysteine protease family cleave synthetic caspase-1 substrate yvad (tyrval-ala-asp) in plants (hatsugai et al. 2004; cai et al. 2014; rocha et al. 2017). besides synthetic caspase-1 inhibitor (ac-yvad-cho) blocks vpe activation (sexton et al. 2007; misas-villamil et al. 2013). kariya et al. (2013; 2018) reported al-induced dose dependent vpe activity at transcriptional level in nicotiana tabacum. the researchers also indicated that the presence of vpe inhibitor (ac-yvad-cho) suppressed pcd symptoms. aytürk and vardar (2015) revealed that short term al toxicity also increased caspase-3, -8 and -9 like activities which are responsible for dna fragmentation and initiation of apoptosis in animal systems in rye, barley, oat and triticale roots. similar to the stated studies, al application caused caspase-1 like activity from ½ h to 96 h in barley roots. although it has been reported that al toxicity induces different caspase like activities, using specific caspase-1 substrate is more accurate approach because vpe specific to plants has caspase-1 like activity. dna fragmentation which is one of the important signatures of pcd originates from internucleosomal dna cleavage by specific proteases and nucleases. in light and electron microscopy fragmented dna can be seen as a compact mass at the periphery of the nucleus (vardar and ünal 2012). dna fragmentation can also be analyzed by tunel reaction, comet assay, laddering on the agarose gel and flow cytometry (tripathi et al. 2016). flow cytometry is based on dna staining with a florescence dye and analyze of the cell cycle (shen et al. 2017). in the histogram of flow cytometry, the first peak indicates g0/g1 cycle and the second peak indicates g2/m. during cell death dna was cleaved into oligonucleosomal fragments. as a result of this, dna content decreases in apoptotic cells and fragmented dna accumulates at the left of g0/g1 peak (yamamada et al. 2006). vardar et al. (2015; 2016) revealed dna fragmentation using comet assay and agarose gel electrophoresis under al stress in early hours in wheat, rye, barley, oat and triticale. according to our results the accumulation of fragmented dna was also visible after short term al application. the fluctuation in the rate of cells having fragmented dna may suggest a recovery effort in short term exposure. however the rate of dying cells is up to 50% and dna fragmentation is more severe in long term exposure. as distinct from the previous results, we put forward the long term effect of al on dna fragmentation. although there are several studies concerning dna fragmentation revealed by flow cytometry during senescence (yamada et al. 2003; 2006), there is no study available during abiotic stress as well as al stress. however, jaskowiak et al. (2018) examined the effect of al on the cell cycle of barley roots by flow cytometry. the researchers revealed that the dna replication and mitotic index reduced, but g2/m phase increased. besides in conclusion; al caused loss of plasma membrane integrity, lipid peroxidation, cyt c release, caspase-1 like activity and dna fragmentation which are characteristic features of pcd. although al uptake, plasma membrane integrity, lipid peroxidation, cyt c release increased time dependently, caspase-1 like enzyme begun to activate at ½ h and did not represented very wide difference during short or long term al application. moreover, dna fragmentation was progressive at long term exposure during al-induced pcd. acknowledgement this work was supported by the research foundation of marmara university (bapko) under grant fenc-ylp-091116-0501 and fen-e-090517-0270. conflict of interest the authors declare that they have no conflict interest. 53temporal analysis of al-induced programmed cell death 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activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha tf_template_word_windows_2016 evaluation of the antigenotoxic potential of fresh bovine whey in onion meristematic roots exposed to quizalofop-p-tefuryl florica colăa, elena bonciua*, mugurel colăa, nicoleta anca șuțanb adepartment of agricultural and forestry sciences, university of craiova, faculty of agronomy, libertatii 19, 200421 craiova, romania bdepartment of natural sciences, university of pitesti, târgul din vale 1, 110040 pitesti, romania *corresponding author: elena.agro@gmail.com this article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the version of record. please cite this article as: florica colăa, elena bonciua, mugurel colăa, nicoleta anca șuțanb (2022). evaluation of the antigenotoxic potential of fresh bovine whey in onion meristematic roots exposed to quizalofop-p-tefuryl. caryologia, just accepted mailto:elena.agro@gmail.com evaluation of the antigenotoxic potential of fresh bovine whey in onion meristematic roots exposed to quizalofop-p-tefuryl abstract. whey is a protein complex derived from milk, being a functional food with multiple health benefits. in this paper, the antigenotoxic potential of fresh bovine whey (fbw) in onion (allium cepa) meristematic roots exposed to quizalofop-p-tefuryl (qpt) herbicide was evaluated using the allium assay. firstly, the allium cepa meristematic roots obtained after a short germination of 24 hours in distilled water were subjected to a pre-treatment with fbw in three different concentrations (500, 1000 and 2000 µl/l) for 24 hours. after that, there was a post-treatment with qpt herbicide (100, 500 and 1000 µl/l), for another 24 hours. all variants were tested alongside a negative control (onion root tips in distilled water) and a positive control (onion root tips treated with 500 µl/l qpt). the genotoxic effects of qpt were observed in all treatment variants, through the low rate of mitosis and through the induction of a large number of chromosomal and nuclear abnormalities (bridges, laggards, rings and strap nuclei). on the other hand, the fresh bovine whey improved the mitotic activity and reduced the index of chromosomal aberrations in variable percentages, in all treatment variants. these results suggest the cytoprotective potential of fbw against the cytotoxic and genotoxic effects of the tested herbicide. although the mechanism of antigenotoxicity is unknown, it seems plausible that the whey protein acts as a blocking agent by chemical or physical interaction with the qpt components. nevertheless, additional studies are needed to determine with certainty this potential. keywords: allium assay, herbicide, genotoxic, whey, antigenotoxic introduction genotoxicity is the ability of various agents to cause damage to genetic material and to affects cellular components involved in the functionality and behavior of chromosomes (bhattachar, 2011; nagarathna et al. 2013). agents able of causing genetic toxicity are described as genotoxic and are called genotoxins. one of the categories of genotoxins that cause damage to genetic material is pesticides (anguiano-vega et al. 2020; kim et al. 2017). higher plants are used in many experiments as indicator plants that show the cytogenotoxic effects of chemicals that pollute the environment (yıldız et al. 2009; bonciu et al. 2018; deveci özkan et al. 2019). in this context, allium cepa l. is a widely used indicator plant for testing the genotoxic/antigenotoxic potential of various chemicals (bonciu et al., 2018; datta et al., 2018; khanna and sharma, 2013). pesticides (fungicides, insecticides and herbicides) are chemicals used against pests and weeds in agriculture. certain pesticides cannot be broken down by microorganisms or the human body's enzymatic equipment, and they can accumulate. for this reason, pesticides represent a source of toxic risk, due to their persistence in soil (datta et al. 2018; srimurali et al. 2015), plants (deveci özkan et al. 2019; roșculete et al. 2019) and in the human body (andersson et al. 2021; hernandez et al. 2016; van maele-fabry et al. 2019; yusa et al. 2015). keeping weeds under control is a constant practice of modern agriculture to ensure high yields by suppressing the growth of unwanted wild species that compete for the same resources with agricultural plants (bartucca et al. 2019; loddo et al. 2020). but, the frequent and excessive use of these chemicals has been identified as a serious threat to the environment and human health (parveen et al. 2016). quizalofop-p-tefuryl (qpt) is a post-emergence herbicide used for the control of the grass weeds in agricultural and horticultural crops (potato, sugar beet, sunflower, oilseed rape, peanut, etc.). the active ingredient is rapidly adsorbed by the leaves of grass weeds, producing their well-wishing and consequent death. its mode of action involves the inhibition of acetyl coa carboxylase activity. whey is full of cysteine, a substance necessary for the production of glutathione, a powerful antioxidant that protects the body from infections and toxins (gupta et al. 2017). thus, whey helps strengthen the immune system. glutathione is also quite effective in the treatment of the thyroid gland, cancer, sclerosis and parkinson's disease (marshall et al. 2004). current studies suggest that the fresh bovine whey (fbw) is much more than a protein source with a particularly nutritious composition of essential amino acids (jaladat et al. 2022; park et al. 2021; walzem et al. 2002). thus, fbw is a real complex cocktail, which contains, in addition to proteins, peptides, complex lipids and oligosaccharides; all of these substances act as a growth factors, toxin binding factors, antimicrobial peptides, prebiotics and immune regulatory factors in mammals (pan et al. 2013; teixeira et al. 2019). consumption of whey products can modulate redox biomarkers to reduce oxidative stress (giblin et al. 2019). this bioactivity has been partially attributed to the whey peptides using a series of biochemical or cellular in vitro assays (abdel-wahhab et al. 2013; corrochano et al. 2019; garg et al. 2018; falkowski et al. 2018). a number of studies have demonstrated the antioxidant bioactivity of whey products and increasing glutathione levels (brandelli et al. 2015; yadav et al. 2015; zhang et al. 2012). glutathione (tripeptide with an important antioxidant role in the body of plants, animals, fungi and bacteria) prevents the destruction of some cellular components and detoxifies various endogenous and exogenous toxins (kasperczyk et al. 2013; pizzorno et al. 2014). some cellular lines exposed to various whey products showed increases in glutathione levels, with some exceptions (corrochano et al. 2019). on the other hand, some studies suggest the adverse effects of fbw when it is consumed in excess (amanzadeh et al. 2003; vasconcelos et al. 2021). practically, 20-25 grams of whey protein powder can be consumed every day, depending on the active or sedentary lifestyle. whey is contraindicated for people allergic to dairy products and the high consumption of whey protein can lead to an increase in the percentage of body fat and stress on the kidneys, an increase in cardiovascular and osteoporosis risks, the appearance of nausea, headaches, cramps, reduced appetite, etc. (aparicio et al. 2011; aydın et al. 2018; hattori et al. 2017; vasconcelos et al. 2021). fbw (ph < 5.1) as a by-product from the manufacture of hard, semi-hard or soft cheese and rennet casein is known as sweet whey. the main constituents of fbw are shown in table 1 (dinkci, 2021). the antigenotoxic potential of whey proteins in the field of medicine was suggested by many results (aydın et al., 2018; jaladat et al., 2022; marshall, 2004; park et al., 2021; teixeira et al., 2019). on the other hand, in the specialized literature there is a lack of results regarding the antigenotoxic potential of fbw following plants exposure to various chemical substances, such as pesticides. in this context, we initiated this study for evaluation of the antigenotoxic potential of fbw in onion (a. cepa) meristematic root tips exposed to qpt herbicide. materials and methods plant material in this study, a number of ten onion (a. cepa, 2n=16) bulbs (procured from a local vendor) were used as biological material for each treatment variant. the outer scales were removed, and older dry roots were scrapped off in order to promote the emergence of new roots. qpt is the active substance of pantera herbicide (producer chemtura s.r.l. italy). this was purchased from a local specialty store for phytopharmaceutical products and was used as the test substance. the onion bulbs were immersed in glasses with distilled water for a short germination (24 h) and then were subjected to a pre-treatment with fbw in three different concentrations (500, 1000 and 2000 µl/l) for 24 hours. after that, there was a post-treatment with qpt herbicide (100, 500 and 1000 µl/l), for another 24 hours. the qpt herbicide concentrations were established according to the dose recommended in agricultural practice. the concentrations of fbw were randomly set, because in the literature there is no data related to the testing of fbw in plants, but only some results on animals. the length of the meristematic roots was measured and recorded after each treatment stage as roots length average (rla). likewise, microscopic analyses were performed after each treatment stage and for each sample, in order to determine the mitotic index (mi), the indices of mitosis phases (ip=prophase index; im=metaphase index; ia=anaphase index; it=telophase index) and to identify the chromosomal aberrations. for this study, the variants were tested alongside a negative control (onion root tips in distilled water) and a positive control (onion root tips treated with 500 µl/l qpt herbicide). the experiment was performed in laboratory, at room temperature (24±2°c). microscopic preparations after measuring and recording the root growth following germination, pre-treatment with fbw and respectively post-treatment with qpt, the biological material was prepared for the microscopic stage. thus, a. cepa roots were fixed in ethanol: acetic acid (3:1) and hydrolysed in 1n hydrochloric acid (hcl) at 60ºc for 5 min. roots with a length of approximately 1 cm were stained through immersion in 3-5 ml schiff reagent (30 minutes) and then transferred on clean slide and crushed in drop of 2% acetocarmine. the microscopic preparations were performed by squash technique. all slides were labelled before microscopic analysis. five random microscopic fields from each slide were scored. the viewing microscopic area was divided into three viewing sections and then, in each viewing section, the cells were counted and recorded in prophase, metaphase, anaphase and telophase. the mi and mitosis phase index were calculated using the following formulas: mi (%) = × 100 ip (%) = × 100 im (%) = × 100 ia (%) = × 100 it (%) = × 100 the index of the total abnormalities (ita) was also calculated: ita (%) = × 100 chromosomal aberrations and nuclear anomalies were determined by scoring cells with bridges, laggards, rings and strap nucleus in randomly picked three zones per slide. photomicrographs of cells showing mitosis, chromosomal aberrations and nuclear anomalies were taken using the digital microscope optika b-190tb (optika, italy), 1000x magnification. statistical analyses the experiment was organized according to a randomized complete design with three replications and minimum 1000 cells were analysed. statistical analysis was done using ms excel 2016. the data obtained were analysed by one-way analysis of variance (anova) and duncan’s multiple range test by using statistical software spss version 20 for windows. significance was considered at p < 0.05. data were expressed as mean ± standard error (se) (gomez and gomez, 1984). the experiment was conducted in triplicate and minimum 1000 cells were analysed for each sample. results the length of the meristematic roots was measured and recorded after each treatment stage (figure 1). thus, after 24 hours germination in distilled water, rla recorded values ranged between 0.3 and 0.6 cm, while the value of the negative control was 0.5 cm. after pre-treatment with fbw, the highest rla value was found in sample v2 (1000 µl/l) 1.9 cm, followed by v1 (500 µl/l) 1.7 cm and v3 (2000 µl/l) 1.6 cm. thus, a more intense growth of onion roots is found in all variants, compared to the negative control, in variable percentages between 58.3-33.3%. on the other hand, after post-treatment with qpt herbicide, the highest rla value was found in variant v2 (500 µl/l) – 2.3 cm, followed by v1 (100 µl/l) – 2.1 cm and v3 (1000 µl/l) 1.7 cm. thus, a more intense growth of onion roots is found in v2 and v1 samples, compared to the negative control, in variable percentages between 27.7-16.6%. in the case of v3 sample, the rla value was 5.5% lower than the negative control. table 2 presents the results of the influence of fbw and qpt on the mi and mitosis stages index in a. cepa root tips. it was found that, following pre-treatment with fbw, the mi in two variants, namely v1 (62.6%), respectively v2 (65.3%), was higher compared to the negative control (54.2%). after the treatment with 2000 µl/l fbw (v3), the mi value was lower (51.4%) than the value recorded for the negative control. on the other hand, higher values of mi were found in all variants compared to the positive control. regarding the post-treatment with the qpt herbicide, an increased mi was recorded in the same variants, namely v1 (66.2%) and v2 (71.5%), compared to the negative control. the treatment with 1000 µl/l qpt (v3) induced a decrease of mi compared to the value recorded by the negative control, but higher than the value recorded by the positive control. it can be appreciated that the 2000µl/l fbw and 1000µl/l qpt concentrations induced a mitodepressive effect in meristematic root cells of a. cepa in a time and concentration-dependent manner. the indices of mitosis phases had different values compared to the control variant, between 78.7-88.4% ip, 3.0-5.5% im, 3.1-5.1% ia and 5.5-10.7% it, respectively. the results regarding chromosomal and nuclear abnormalities induced by fbw and qpt in a. cepa root tips are presented in table 3. in all treatment variants, the appearance of a variable number of chromosomal aberrations and nuclear anomalies was observed, the most important being the following: bridges, laggards, rings and strap nuclei (figure 2). thus, the bridges were observed with a frequency ranged between 2.0-3.4% after the treatment with fbw and an increased frequency of 2.4-4.1% after the treatment with qpt, respectively. laggards had values between 4.2-5.1% after the treatment with fbw and respectively 4.0-5.4% in the case of treatment with qpt. also, cells with ring chromosomes had values between 1.5-2.2% in the case of treatment with fbw and respectively 1.9-2.2% in the case of treatment with qpt. on the other hand, the nuclear abnormalities of the strap nucleus type were recorded with a frequency of 2.2-3.9% and 3.0-5.3%, after the treatment with fbw and qpt, respectively. it was found that all these abnormalities recorded higher values than the negative control but much lower than the positive control. a similar evolution of the records was determined for the index of the total abnormalities (ita%), that ranged between 9.9-14.6% (after fbw treatment) and 11.3-16.9% (after qpt treatment). discussion in the last years, many researchers have found novel bioactive phytocompounds able to counteract the effects of some physical and chemical mutagens that can affect the health of humans, animals and the environment (bonciu et al., 2018; dimitrov et al., 2006; franco-ramos et al, 2020). the ability of a substance to cause cytotoxicity is measured by its capability to decrease cell proliferation (franco-ramos, 2020). the challenge of identifying and developing of some therapies through plants help or some dietary strategies represents a major public health challenge. thus, several studies have shown the antigenotoxic potential of different plants (lópez-romero et al., 2018; park et al., 2018; stavric et al., 1996). also, whey protein supplementation is a dietary strategy widely used in the field of oncology (abdel-wahhab et al., 2013; gupta and prakash, 2017; teixeira et al., 2019). this emerging dietary strategy harbouring several benefits translated well into animal models of cancer and in humans. at the molecular level, whey protein subfractions display appealing anti-cancer effects (teixeira et al., 2019). in order to highlight the cytological activity and the occurrence of some cytogenetic abnormalities induced to qpt in plant meristematic roots it was chosen allium test because is one of the simplest, inexpensive and valuable tests for determining the cytotoxicity of chemical substances on plants. as shetty et al. (2017) states, a. cepa root chromosomal aberration assay was chosen as it offers an easily adaptable method for screening mitotic/genotoxic/antigenotoxic activity of any bioactive chemical. in our study, in all qpt herbicide treatment variants, the appearance of a variable number of chromosomal aberrations and nuclear anomalies was observed, the most important being bridges, laggards, rings and strap nuclei. the results obtained showed the strong cytotoxic effect of qpt herbicide, by reducing the mi in onion roots. thus, it can be appreciated that the 1000 µl/l qpt induced a mitodepressive effect in meristematic root cells of a. cepa in a time and concentration-dependent manner. in this context, there are many results regarding the cytogenotoxic effects of herbicides on plants or animals. thus, the results obtained by dimitrov et al. (2006) show that the tested herbicide (stomp) did not induce chromosomal aberrations in plant cells of crepis capillaris l., instead it was increased their incidence in mouse bone marrow cells. in the same study on the other hand, the herbicide increased the frequency of micronuclei in both test systems. the authors suggest that the induction of some nuclear and chromosomal aberrations in plant cells may have been due to the herbicide's disruptive effect on the spindle, as all herbicide concentrations produced c-mitosis. also, the increased frequency of chromosomal aberrations in mouse bone marrow cells may be due to the biosynthesis of genotoxic metabolites (dimitrov et al. 2006). some dairy products (milk and yogurt) supplemented with red ginseng extract have been shown to have antioxidant and antigenotoxic effects (park et al. 2018). also, some studies have shown the potential of lactobacilli and bifidobacteria in dairy products to inhibit the genotoxic activity of chemical compounds (burns et al. 2000; lopitz-otsoa et al. 2006; tavan et al. 2002). our results showed that fbw has improved mitotic activity and reduced chromosomal abnormalities in the meristematic cells of a. cepa induced by qpt, a fact that suggests the antigenotoxic potential of fbw. the fbw reduced the aneugenic and clastogenic effects of qpt in a. cepa cells. although the mechanism of antigenotoxicity is unknown, it seems plausible that the whey protein acts as a blocking agent by chemical or physical interaction with the qpt components. however, the continuation of studies in this direction remains open in order to establish with certainty the antigenotoxic potential of fbw in plant cells. conclusions the experimental results from the present study show that the application of qpt herbicide produced some cytotoxic and genotoxic effects on the meristematic cells of a. cepa. these toxic effects increased significantly at dose dependently, but application of fbw ameliorated these negative 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review. anal chim acta. 891:15-31. zhang qx, ling yf, sun z, zhang l, yu hx, kamau sm, lu rr. 2012. protective effect of whey protein hydrolysates against hydrogen peroxide-induced oxidative stress on pc12 cells. biotechnol lett. 34:2001-2006. table 1. the main constituents of fbw* constituents % total solids 6.00-6.50 water 93.00-94.00 fat 0.05 protein 0.60-0.65 npn (non-protein nitrogen) 0.20 lactose 4.50 ash (minerals) 0.50 calcium 0.03 phosphorus 0.04 sodium 0.04 potassium 0.14 chloride 0.09 lactic acid 0.05 *source: dinkci, 2021 table 2. results of the influence of fbw and qpt on the mi and mitosis stages index in a. cepa meristematic roots* specification variant/conc. (µl/l) mi±se (%) ip±se (%) im±se (%) ia±se (%) it±se (%) nc 54.2 ±1.8a 82.9±0.2a 3.9±0.2a 5.5±0.3a 7.7±0.5a pc 33.2 ±1.8b 85.1±0.4a 2.1±0.1a 3.1±0.3a 9.7±0.6a fbw v1/500/24 62.6 ± 2.1b 84.2±0.5a 3.5±0.4a 4.2±0.5b 8.1±0.5a v2/1000/24 65.3 ±2.9b 83.5±0.9a 3.4±0.2a 3.2±0.1c 9.9±0.7a v3/2000/24 51.4 ± 3.1a 85.6±0.7b 3.2±0.2a 4.1±0.5b 7.1±0.4a qpt v1/100/24 66.2 ± 2.4b 80.3±0.4a 4.7±0.5b 4.4±0.2b 10.6±0.5b v2/500/24 71.5 ± 4.8c 78.7±0.8a 5.5±0.2b 5.1±0.3a 10.7±0.5b v3/1000/24 42.7 ± 1.9d 88.4±0.9c 3.0±0.3a 3.1±0.1c 5.5±0.2c *means with the same letter in the same column for each application time do not differ statistically at the level of 0.05. nc=negative control; pc=positive control; mi=mitotic index; se=standard error; ip=prophase index; im=metaphase index; ia=anaphase index; it=telophase index. data are means ± se of three replicates. table 3. results regarding the chromosomal and nuclear abnormalities induced by fbw and qpt in a. cepa root tips* specification variant/concentration (µl/l)/exposure time (h) b (%) l (%) r (%) sn (%) ita±se (%) nc 0.2 0.8 0.1 1.2 2.3±0.5a pc 4.8 6.2 2.7 7.4 21.1±1.1b fbw v1/500/24 2.0 4.2 1.5 2.2 9.9±0.4a v2/1000/24 2.7 4.5 1.9 3.1 12.2±0.7a v3/2000/24 3.4 5.1 2.2 3.9 14.6±0.6a qpt v1/100/24 2.4 4.0 1.9 3.0 11.3±0.4a v2/500/24 3.5 4.1 2.2 4.3 14.1±0.4b v3/1000/24 4.1 5.4 2.1 5.3 16.9±0.9c *means with the same letter in the same column for each application time do not differ statistically at the level of 0.05. nc=negative control; pc=positive control; b=bridges; l=laggards; r=rings; sn=strap nucleus; ita= index of the total abnormalities; se=standard error. figure 1. a. cepa roots lenght average (rla) (cm) after a short germination of 24 hours in distilled water, followed by pre-treatment with fbw for 24 hours and a post-treatment with qpt herbicide for another 24 hours. (a) (b (c) (d) figure 2. the main chromosomal aberrations and nuclear anomalies induced by pre-treatment with fbw and post-treatment with qpt in a. cepa cells: bridge (a), double bridge and laggard chromosome (b), ring chromosomes in metaphase (c) and strap nuclei (d). arrows indicate abnormalities. caryologia. international journal of cytology, cytosystematics and cytogenetics 75(3): 91-99, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1895 caryologia international journal of cytology, cytosystematics and cytogenetics citation: sazada siddiqui, sulaiman a. alrumman (2022). methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. caryologia 75(3): 91-99. doi: 10.36253/caryologia-1895 received: october 12, 2022 accepted: december 02, 2022 published: april 5, 2023 copyright: © 2022 sazada siddiqui, sulaiman a. alrumman. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid ss: 0000-0001-5448-7617 methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. sazada siddiqui*, sulaiman a. alrumman department of biology, college of science, king khalid university, abha 61413, saudi arabia *corresponding author. e-mail: sasdeky@kku.edu.sa; kalasaz@yahoo.co.in abstract. methomyl is a carbamate pesticide that is frequently applied to crops all over the world. this research aims to evaluate the pisum sativum l mitotic process and potential genotoxicity. the cell proliferation kinetics (cpk) frequencies demonstrated changes in kinetics of mitotic process, and study of mitotic index (mi) demonstrated that methomyl had cytotoxic properties. in fact, the telophases ratio dropped at 0.1% to 0.5% methomyl treatment, while there was an increase in prophases, metaphases, and anaphases from 0.1% to 0.5% in a dose dependent manner. in terms of genotoxicity, methomyl cause an increase in the frequency of clastogenic and aneugenic chromosomal abnormalities at metaphase-anaphase at 0.1% to 0.5%. the effects on the mitotic spindle were further confirmed by an increase in the frequencies of c-mitosis from 0.1 to 0.5% methomyl treatment. the outcome of the current analysis indicates that regularly used insecticides methomyl has a considerable cytotoxic effect on mitotic cells of pisum sativum l. keywords: methomyl, mitotic index, clastogenic, aneugenic, cmitosis pisum sativum l. introduction in public areas, agricultural lands and gardens, pesticides are extensively used to eradicate weeds, undesirable pests, and diseases transmitted by vectors. nevertheless, the prolonged usage of pesticides may leave behind toxic remains that, through surface drains, spray drift, runoff, spray leftovers, and leaching may pollute nearby surface water and ground natural water bodies (mojiri et al. 2020; chandra et al. 2021). the accumulation of residual pesticides in aquatic and marine organisms food chains can pose a risk to human health and have a detrimental effect on ecological systems (lukaszewicz et al. 2019; jing et al. 2022a; abdel-wahab et al. 2021). from many decades, pesticides have been a crucial component in reducing crop loss and increasing output. due to these advantages, farmers are spraying pesticides on crops more frequently and using modern techniques like drones (nie et al. 2020). however, the propensity of pesticides to bio accumulate in edible goods may have an undesirable impact on human 92 sazada siddiqui, sulaiman a. alrumman health (yu et al. 2016; ramadan et al. 2020). beyond their maximum residue limits (mrls), pesticides in water and agricultural products have the potential to cause both acute and chronic illnesses in people (li and jennings 2018; amaç and liman 2021). carbamates are a diverse group of chemicals that are used as insecticides. the acetylcholinesterase (ache) enzyme is selectively affected by carbamates, which results in a buildup of acetylcholine and overstimulation of the nervous system in both target and non-target species, including human beings (eddleston et al. 2004). in the areas where onions, cucumbers, cabbage, and chili peppers are grown, methomyl, a carbamate insecticide, is frequently used (ramadan et al. 2020). acute poisoning may result from methomyl consumption by using contaminated agrifoods and water via occupational or non-occupational ways (jing et al. 2022 b). due to its highly effective biological action in controlling pests and safeguarding the crops, methomyl (c5h10n2o2s), s-methyl-1-n[(methyl carbamoyl)oxy]-thioacetimidate, belongs to carbamate pesticide group that is commonly applied in various countries (laicher et al. 2022; pietrini et al. 2022). several pesticides are designed to strike a particular group of targets, although their noxious constituents will affect the whole organism, both target and non-target (castellanos et al. 2022). according to a study, methomyl causes genotoxic effects in fish (afaf et al. 2022). fish and aquatic creatures including danio rerio, coastal aquatic system and water spinach have also shown toxicity to methomyl (jablonski et al. 2022; camilo-cotrim et al. 2022). dna damage is a preliminary biotic phenomenon which could disrupt biological developments and structures and produce genotoxic disorders associated with carcinogenic complications (acar et al. 2022; siddiqui and sulaiman 2022 a and b; el-houseiny et al. 2022). as per a recent report, numerous species undergo carcinogenic progressions due to various causes, such as dna damage instigated by chemical contaminants (pesavento et al. 2018; velázquez et al. 2022; liman et al. 2022). this study aims to analyze the potential adverse effects of methomyl on mitotic processes and dna integrity in the terrestrial plant pisum sativum l. material and methods purchasing of chemicals and seeds methomyl insecticide were bought from sigma chemicals ltd., united states (cas no. 16752-77-5). pisum sativum l (pea) seeds were procured from a licensed trader at a community market in abha, saudi arabia. exposure conditions even sized p. sativum l seeds were chosen, presoaked for 12 hours in distilled water and then divided into various groups of 30 seeds each. after that, the seeds were exposed to various methomyl concentrations (0.1, 0.2, 0.3, 0.4, and 0.5%) for 1 h by soaking in 250 ml solutions of methomyl. double-distilled water was used to soak the seeds in the control group. throughout the treatment time, the containers were shaken repeatedly to make available ample aeration to the seeds. following treatment, seeds were extensively rinsed with double distilled water (ddw) to eliminate any remaining traces of adhering methomyl and were placed in petri dishes on moisturized whatman filter paper. for the following 72 hours, the petri dishes were kept in dark in a plant growth cabinet at 25±2°c. the experiment was conducted on newly emerging roots that were 1-2 cm long. the complete experiment was conducted thrice in identical conditions. evaluation of mitotic kinetics and genotoxicity one to two cm long roots were collected between 8 to 10 am, soaked for 24 h in a fixation solution (ethanol: glacial acetic acid, 3:1), then transferred to 70% ethanol, maintained at 5°c till microscopic examination. for each sample, 10 roots were hydrolyzed in 1n hcl for 10 minutes, and with 2% acetocarmine solution, root tips were dyed for 10 minutes for preparing each slide. chromosome preparation was done from root tips as stated by qian et al. 1998 with minor modifications (siddiqui and suleiman 2022b). to calculate the mi, which is a proportion of dividing cells, 1000 cells from each sample were evaluated. the no. of cells in each division phase to all mitotic cells was used to compute cpk frequencies. all mitotic cells were studied in a light microscope under oil immersion (100 x). all slides were examined blind and coded. ratio of aberrant cells over 500 metaphase/anaphase cells per root tips were used to calculate the frequency of chromosomal aberrations. chromosomal aberrations were categorized as per their origin in clastogenic (resulting in chromosomal breakage) or aneugenic (disrupting spindle function and leading to asynchronic chromosomal migration). laggards and vagrants chromosomes have been scored with regards to aneugenic abnormalities. single bridges, fragments, double bridges, and sticky chromosomes were taken into considera93methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. tion in clastogenic aberrations. c-mitosis as defined by grant (1978) is an inactivation of spindle ensued by a haphazard scattering of chromosomes over the cell and is quantified and scored by assessing the frequency over 100 metaphases per root tips. all the aberrant and c-mitosis cells were studied in a light microscope under oil immersion (100x). all slides were examined blind and coded. statistical analysis a one-way anova test using gpis 1.13 software (graphpad, california, usa) was applied to find significance of differences in variables. all results were articulated as mean ± standard error. results it is clear from the results that methomyl is toxic to mi, cpk, cmitosis, aneugenic and clastogeneic aberrations. the observed mi, cpk, c-mitosis, aneugenic and clastogeneic aberrations are well represented in (fig. 1, table 1, fig. 2, fig. 3 and fig. 4). the clastogenic abnormalities observed were single bridges, fragments, double bridges, sticky chromosome and aneugenic abnormalities were laggards and vagrants. effect of methomyl treatment on mitotic index of p. sativum l. fig. 1 shows how methomyl affected the mi of root tip cells in p. sativum. in control group, seeds treated with ddw for 1 hour had a mi of 9.3%. from 0.1 to 0.2% methomyl treated seeds, a non-significant decline (p>0.05) in mi was observed and at 0.3% concentration, there was a significant decrease (p< 0.05) in mi and at 0.4 to 0.5%, a very significant decrease (p< 0.01) in mi was reported in comparison to control for 1 hour. overall, mi decreases dose dependently in all concentrations from 0.1 to 0.5%. effect of methomyl in cell proliferation kinetics (cpk) of p. sativum l cell proliferation kinetics (cpk), assessed as the ratio of prophases, metaphases, anaphases and telophases revealed a rise in prophase, metaphase and anaphase from (0.1 to 0.5%) and a decrease in telophase at 0.1 to 0.5% of methomyl treated root tips in comparison to control (table 1). a significant increase (p<0.05) was reported in prophase at 0.4 % (58.34±1.2); metaphase at 0.2% (27.3±3.8), and anaphase at 0.2% (21.3±1.9) but a significant decrease (p<0.05) was observed in telophase at 0.3% (19.12 ±3.6) in comparison to control. prophase at 0.5% (60.12±2.6); metaphase from 0.3 to 0.5% (27.4±3.5; 28.45±3.6; 29.40±1.2 respectively) and anaphase from 0.3 to 0.5% (23.1±1.7; 24.5±1.9; 25.5±1.6 respectively) resulted in a very significant increase (p<0.01) and telophase from 0.4 to 0.5% (17.6±2.5; 16.80±3.6) showed a very significant decrease (p<0.01) in comparison to control. effect of methomyl treatment on c-mitosis of p. sativum l fig. 2 demonstrates how methomyl affects c-mitosis in p. sativum root tips cells. seedlings treated for 1h with figure 1. effect of methomyl on mitotic index of p. sativum for 1 h. *p<0.05; compared to control group. data are mean of three replicates ±se, 0.0 = control group. table 1. effect of methomyl on cell proliferation kinetics in p. sativum l. concentration (%) prophases metaphases anaphases telophases 0.0 52.5±4.8 21.7± 2.7 18.5±3.4 23.12.8±2.3 0.1 50.7±4.6 23.5±1.7 20.4±2.4 21.5.6±3.0 0.2 50.4±2.4 27.4±3.5* 21.3±1.9* 20.9±1.30 0.3 55.7± 2.2 27.3.3±3.8** 23.1±1.7 ** 19.12±3.6* 0.4 58.34±1.2* 28.45±3.6** 24.5±1.9 ** 17.6±2.5** 0.5 60.12±2.6 ** 29.40±1.2** 25.5± 1.6 ** 16.80±3.6** *p<0.05; **p<0.01 compared to control group. data are mean of three replicates ±se, 0.0 = control group. 94 sazada siddiqui, sulaiman a. alrumman ddw in the control group exhibited 0% c-mitosis. a significant increase (p<0.05) in the number of c-mitosis cells were seen in seeds treated with 0.1% methomyl for 1 hour and from 0.2 to 0.5%, there was a very significant increase (p<0.01) in c-mitosis cells in comparison to control for 1 hour. overall, c-mitosis increases dose dependently in all concentrations ranging from 0.1 to 0.5%. effect of methomyl on aneugenic and clastogeneic aberration cells in p. sativum l the incidence of aneugenic aberrations (laggards and vagrants) in metaphase-anaphase plates in the control group was zero. percentage of aneugenic aberrations (laggards and vagrants) in the metaphase-anaphase plate dose dependently increased with methomyl treatment (fig. 3 and fig. 5). seeds treatment with 0.1% methomyl resulted in a 1-fold increase and 0.2% treatment resulted in a 1.37fold increase which was not significant and 0.3% methomyl treated seeds resulted in a 2.4-fold increase which was significant (p<0.05) in comparison to control. further increase in concentration from 0.4 to 0.5% methomyl treated seeds resulted in a rise in incidence of aneugenic aberrations, 5.9-fold, and 9.56-fold respectively, which was very significant (p<0.01) in comparison to control. the incidence of clastogeneic aberrations (single bridges, fragments, double bridges and sticky chromosome) at metaphase-anaphase plates in control group was zero (fig. 4, and fig. 5). percentage of root tip cells with clastogeneic aberrations (single bridges, fragments, double bridges and sticky chromosomes) at metaphaseanaphase plate increased dose dependently with methomyl treatment (fig. 4). treatment of seeds with 0.1% methomyl resulted in 1.81-fold increase which was nonsignificant as compared to control. however, from 0.2 to 0.5 % methomyl treated seeds resulted in 6.25-fold, 12.19-fold, 18.92-fold and 21.28-fold increase in clastogeneic aberrations respectively which was very significant (p<0.01) in comparison to control. 0.0 0.1 0.2 0.3 0.4 0.5 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 ** ** **c -m it o si s (% ) methomyl concentration (%) * ** figure 2. effect of methomyl on c-mitosis in p. sativum for 1 h. *p<0.05; **p<0.01 compared to control group. data are mean of three replicates ±se, 0.0 = control group. 0.0 0.1 0.2 0.3 0.4 0.5 0 2 4 6 8 10 12 ** t o ta l a n eu g en ic a b er ra ti o n s (% ) methomyl concentration (%) * ** figure 3. effect of methomyl on total aneugenic aberrations in p. sativum for 1 h. *p<0.05; **p<0.01 compared to control group. data are mean of three replicates ±se, 0.0 = control group. 0.0 0.1 0.2 0.3 0.4 0.5 0 5 10 15 20 25 30 ** ** * * t o ta l c la st o g en ic a b er ra ti o n s (% ) methomyl concentration (%) * ** figure 4. effect of methomyl on total clastogenic aberrations in p. sativum for 1 h. *p<0.05; **p<0.01 compared to control group. data are mean of three replicates ±se, 0.0 = control group. 95methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. discussion this study shows that reduction in cell division indicates that tested methomyl have a mitodepressive potential. when mitotic activity is reduced, the amount of dna also declines. this could be due to the blockage of cell cycle in the g2 phase or dna synthesis inhibition or stopping the cell from starting mitosis (siddiqui et al. 2007; siddiqui et al. 2012; siddiqui and alrumman 2020 a and b). the significant decrease in mitotic index observed in this study might be the effect of methomyl interfering with the cell cycle by blocking g2 phase of cell cycle or dna synthesis inhibition, or it could be the outcome of a rise in the frequency of chromosomal anomalies with analogous increase in methomyl concentration. these findings are also consistent with the outcomes of several research teams which have stated the cytotoxic effects of ethephon (ayşe and kılıç 2017; bonciu et al. 2022), various synthetic plant growth regulators (singh et al. 2022; asif et al. 2022), and various pesticides (lukaszewicz et al. 2019; siddiqui and alrumman 2022 a and b; omeiri et al. 2022; hafez et al. 2022; bandopadhyay et al. 2022). in this study, methomyl raised the percentage of metaphase, prophase and anaphase and reduced the percentage of telophase in all concentrations in a dose dependent manner, as per the outcomes of proportions of distribution of specific mitotic stages. there is an increase at all concentrations of metaphase, prophase and anaphase phases. the outcomes are consistent with the findings of liman et al. (2010), priya et al. (2014), and ozkul et al. 2016). furthermore, the percentage of telophase stage decreased in comparison to control. these findings suggest that decline in telophase stages and henceforth mi might be due to arrest of one or more mitotic stages or due to a slowdown in the rate of cell development during mitosis (ping et al. 2012). c-mitosis was found in the present study. c-mitosis was created by unstable microtubules (odeigah et al. 1997) or disruptions in the development of spindle fibers (shimoi et al. 2019; haliem 1990). the incidence of c-mitosis in root tip cells of pisum sativum shows that spindle formation was harmfully affected (el-ghamery et al. 2000). considerable numbers of c-mitosis detected in this study implies that methomyl is a strong spindle figure 5. clastogenic and aneugenic aberrations in methomyl treated p. sativum l root tip cells. clastogenic aberrations a to f: a) bridge in anaphase; b) single bridge in telophase; c-d) chromosome fragment in metaphase; e) double bridge at anaphase; f) sticky chromosome at metaphase. aneugenic aberrations g to j: g-h-i) chromosome vagrant at metaphase; j) vagrant chromosome at anaphase; k –l) c-mitosis in metaphase; bar 10 μm. 96 sazada siddiqui, sulaiman a. alrumman inhibitor. c-mitosis is also an indication of spindle poisoning, as per rank (2003). the cause of the generation of c-mitosis might be due to disruptions in spindle formation, affected by methomyl. in relation to genotoxicity, methomyl enhanced the incidence of clastogenic as well as aneugenic anomalies at the metaphase-anaphase plate. single bridges, fragments, double bridges and stickiness, were the clastogenic anomalies whereas vagrants and laggards were the aneugenic anomalies observed in the present study. in treated seeds, a number of bridges were created in anaphases i and ii plate. bridges were most likely formed by breakage and combining of chromosomal bridges, which got enhanced with methomyl treatment. chromosome stickiness and subsequent failure of free anaphase division or irregular translocation or inversion of chromosomal fragments can all lead to the creation of chromosomal bridges (jing et al. 2022a; honles et al. 2022). the fusion of broken chromosomes was the primary cause of the formation of bridges as per rosculet et al (2019; honles et al. 2022). increases in methomyl concentrations were associated with stickiness. stickiness may result from partial detachment of nuclear proteins and alterations in their association design or from partial detachment of nucleoproteins and alterations in their association design or due to nucleic acid depolymerization activated by methomyl treatment. disruptions in cytochemical balance reaction may lead to stickness (dewitte et al. 2010; rosculet et al. 2019). nucleic acid depolymerization because of herbicidal treatment or by partial detachment of nucleoproteins (kaufman et al. 1955) or by incomplete separation of nucleoprotein variation in their organization design (evans 1962) might cause stickness. the fragments formed from chromatid and chromosomal break imply its mutagenic events within the cell. in a previous study, siddiqui et al. (2020 a,b) had reported that pesticides cause various chromosomal anomalies. generation of giant cells having diverse chromosomal anomalies had been reported in a previous study by food colorants (prajitha and thoppil 2016). the laggards observed during the current study may result from failure of chromosome movement or from deferred ending of stickiness of ends of chromosomes. at metaphase i, chromosome lagging could result from disturbances in bivalents motion to equatorial plate. single univalent lagging was the most common incidence (zeyad et al. 2019). laggards and bridges could be created due to deferred ending of stickiness of ends of chromosomes (kaur and grover 1985). laggards are responsible for the formation of micronuclei at telophase i. acentric fragments or laggards are liable for micronuclei generation at telophase ii and hence it leads to the changes in size and number of pollen grains arising from mother cells. the other frequent aneugenic form of anomaly observed in dividing cells was vagrant chromosomes. as per rank (2003), vagrant chromosomes are pointers of spindle poisoning. these aberrations might have developed as a result of the disruption in spindle formation, which was affected by methomyl treatment. genotoxic stress or genomic instability caused by dna damage may result in illnesses, senescence, alterations in gene expression or cellular aging (bonciu et al. 2018; iturburu et al. 2018; shabbir et al. 2021; omeiri et al. 2022; castellanos et al. 2022). in both plants and animals, a rise in genomic instability has been advocated as a basis for the decline in population fitness. genotoxicity biomarkers must be taken into consideration when assessing potential noxious effects in aquatic organisms since genotoxic substances have the potential to cause damage that extends beyond the individual and can be seen over several generations (frenzilli et al. 2009; fioresi et al. 2020; ergin et al. 2020 amac and liman 2021; menzyanova et al. 2022). conclusion according to the findings of the current study, methomyl can alter kinetics of mitotic cell process in root tip cells and can have genotoxic effects on p. sativum l through aneugenic and clastogenic processes. these findings raise concern about noxious effects of pesticides on non-target organisms. for the benefit of human welfare, additional genotoxicological and risk assessment studies needs to be conducted on various eukaryotic test systems. funding we are grateful to king abdul aziz city for science and technology (kacst), riyadh, saudi arabia for funding this project under grant number: 13-agr211907. acknowledgments we are grateful to king abdul aziz city for science and technology (kacst), riyadh, saudi arabia for funding this project under grant number: 13-agr211907. we also express our gratitude to king khalid univer97methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. sity, saudi arabia for providing administrative and technical support. references abdel-wahab si, aioub aa, salem re, el-sobki ae. 2021. do the herbicides pinoxaden, tribenuronmethyl, and pyroxsulam influence wheat (triticum aestivum l.) physiological parameters? 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chromosome mapping of repetitive dnas in the picasso triggerfish (rhinecanthus aculeatus (linnaeus, 1758)) in family balistidae by classical and molecular cytogenetic techniques kamika sribenja1, alongklod tanomtong1, nuntaporn getlekha2,* chromosome number of some satureja species from turkey esra kavcı1, esra martin1, halil erhan eroğlu2,*, fatih serdar yıldırım3 l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations selma tabur1,*, nai̇me büyükkaya bayraktar2, serkan özmen1 characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques kristel ramírez-matadamas1, elva irene cortés-gutiérrez2, sergio moreno-limón2, catalina garcía-vielma1,* contributions of species rineloricaria pentamaculata (loricariidae:loricariinae) in a karyoevolutionary context a cius¹, ca lorscheider2, lm barbosa¹, ac prizon¹, ch zawadzki3, la borin-carvalho¹, fe porto4, alb portela-castro1,4 cadmium induced genotoxicity and antioxidative defense system in lentil (lens culinaris medik.) genotype durre shahwar1,2,*, zeba khan3, mohammad yunus khalil ansari1 biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts and evaluation of their biosafety potential denisa manolescu1,2, georgiana uță1,2,*, anca șuțan3, cătălin ducu1, alin din1, sorin moga1, denis negrea1, andrei biță4, ludovic bejenaru4, cornelia bejenaru5, speranța avram2 polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel (foeniculum vulgare mill.) egizia falistocco methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. sazada siddiqui*, sulaiman a. alrumman comparative study and genetic diversity in malva using srap molecular markers syamand ahmed qadir1, chnar hama noori meerza2, aryan mahmood faraj3, kawa khwarahm hamafaraj4, sherzad rasul abdalla tobakari5, sahar hussein hamarashid6,* nuclear dna 2c-values for 16 species from timor-leste increases taxonomical representation in tropical ferns and lycophytes inês da fonseca simão1, hermenegildo ribeiro da costa1,2,3, helena cristina correia de oliveira1,2, maria helena abreu silva1,2, paulo cardoso da silveira1,2,* nuclear dna content and comparative fish mapping of the 5s and 45s rdna in wild and cultivated populations of physalis peruviana l. marlon garcia paitan*, maricielo postillos-flores, luis rojas vasquez, maria siles vallejos, alberto lópez sotomayor identification of genetic regions associated with sex determination in date palm: a computational approach zahra noormohammadi1,*, masoud sheidai2, seyyed-samih marashi3, somayeh saboori1, neda moradi1, samaneh naftchi1, faezeh rostami1 comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(3): 145-157, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1831 caryologia international journal of cytology, cytosystematics and cytogenetics citation: neslihan taşar, i̇lhan kaya tekbudak, i̇brahim demir, mikail açar, murat kürşat (2022). comparative karyological analysis of some turkish cuscuta l. (convolvulaceae). caryologia 75(3): 145-157. doi: 10.36253/caryologia-1831 received: september 13, 2022 accepted: november 25, 2022 published: april 5, 2023 copyright: © 2022 neslihan taşar, i̇lhan kaya tekbudak, i̇brahim demir, mikail açar, murat kürşat. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid nt: 0000-0002-0417-4660 i̇kt: 0000-0002-2754-2489 i̇d: 0000-0003-1533-556x ma: 0000-0003-3848-5798 mk: 0000-0002-0861-4213 comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 1 munzur university, department of plant and animal production, tunceli vocational school of higher education, tunceli, 62000, turkey 2 van yüzüncü yıl university, faculty of agriculture, department of plant protection, van, turkey 3 bitlis eren university, faculty of arts and sciences, department of biology, bitlis, turkey *corresponding author. e-mail: mikailacar@munzur.edu.tr abstract. this study investigated the somatic chromosome numbers and morphometric properties of 11 different taxa belonging to the genus cuscuta l., which is one of the parasitic flowering plants and causes significant economic losses on agricultural products. for this purpose, the species were examined karyologically and compared statistically. belonging to the genus cuscuta, c. campestris yunck., c. hyalina roth, c. kotschyana boiss., c. babylonica aucher ex choisy, c. europaea l., c. kurdica engelm., c. brevistyla a.braun ex a.rich, c. planiflora ten., c. approximata bab., c. lupuliformis krock. c. palaestina boiss. the chromosome number and morphology of the species were investigated using karyological techniques. chromosome numbers of the species; c. kotschyana, c. babylonica, c. europaea, c. kurdica, c.planiflora 2n=14; c. campestris, c. hyalina, c. approximata, c. lupuliformis, and c. palaestina 2n=28 and c. brevistyla 2n=42 is determined. also, the species’ chromosome number, total chromosome length, relative length, arm ratio, centromere index and centromere states, and karyotype asymmetry values were determined. chromosome numbers of c. kotschyana and c. kurdica taxa were defined for the first time in this study. thus, new data on the systematics of these species have been revealed. keywords: convolvulaceae, cuscuta, parasitic plant, chromosome number, karyotype. introduction cuscuta (dodder) is a member of the convolvulaceae family, including about 200 different root parasite species. about 15-20 of these species cause severe problems in agricultural areas (dawson et al., 1994). with this, most cuscuta species are considered extinct in the wild, and some species even require conservation measures in nature (costea and stefanovic 2009a). cuscuta species can be found in various habitats, including temperate, tropical, desert, riparian, coastal, high mountain, woodland, saltwater, and degraded environments (costea et al., 2015). like other parasitic plants, cuscuta species play an essential role in ecosystems (press et al. 1999). 146 neslihan taşar et al. yunker (1932) divided the genus cuscuta into three subgenera according to styles and stigma shapes. these; cuscuta are grammica (lour.), yunck, and monogynella (des moul.). in the flora of turkey, 15 species of these subgenera and two unknown species (c. aratica butk. and c. subuniflora k. koch), and one suspicious (c. epilinum wiehe) species have been identified (plitman, 1978). since the vegetative parts of parasitic plants are generally reduced, flower characters are insufficient in taxonomy. this situation creates problems in diagnosis. for this reason, it is necessary to use some methods to identify species belonging to the genus cuscuta. studying chromosome numbers and structures can give valuable results in solving taxonomic problems (taşar et al., 2018a; 2018b). it has been determined that cuscuta species generally have holocentric chromosomes and undergo inverted meiosis (pazy and putmann 1987; 1991; 1994). some morphological features are overlooked in plant determinations and classifications of classical taxonomy. characteristics acquired according to environmental factors appear to be new features, confusing classification. for this reason, considering the characters in classical taxonomy, examining the chromosome numbers, structure and structures gives beneficial results in solving the problems (taşar et al., 2018a; 2018b ). in addition, statistical analyzes can be useful in morphological and anatomical studies recently (genç et al. 2021; arabacı et al. 2021; dirmenci et al. 2019; dirmenci et al. 2020; açar and satıl 2019; açar and taşar 2022). this study aims to reveal the karyological features of cuscuta species distributed in turkey, determine the relationships between them, and contribute to the genus’s taxonomic classification. material and methods material cuscuta samples, the study material, were obtained from the field. localities of taxa are given in table 1. plant taxa were identified using the genus cuscuta (yuncker 1932) and flora of turkey. (davis, 1978). the collected specimens have been turned into herbarium material and are kept in van yüzüncü yıl university, faculty of agriculture, plant protection department, and bitlis eren university, biology department. methods chromosome measurements the seeds of the plant samples were sown in petri dishes and germinated in an oven at 20-22 ºc. roots reaching 1–2 cm in length from the germinated seeds were cut, kept in colchicine for 2 hours at room temperature, and subjected to pretreatment (gedik et al., 2014). then, the root tips were placed in carnoy fixative (3:1) and fixed by keeping them in the refrigerator at +4 ºc for 24 hours. at the end of the period, root tips were hydrolyzed in 1n hcl in an oven at 60 ºc for 5-18 minutes. root tips removed from hydrolysis were stained with feulgen stain for 1 hour in a dark environment at room temperature. then it was washed 2-3 times with tap water. for preparation, the growth meristem part was cut off with a sharp razor blade in a drop of 45% acetic acid on the slide, and the coverslip was closed. the best three somatic cells for each species were photographed using an olympus bx53 microscope. the naming system of levan (1964) was used to locate the centromere. the intra-chromosomal asymmetry index (a1) was calculated according to the formula proposed by romero zarco (1986). interchromosomal asymmetry index (a2) and karyotype symmetry nomenclature were made according to stebbins (1971). statistical analisyes for analysis used, several formulas were established on chromosome characteristics. the measurements were built on haploid datasets. the calculations and abbreviations used in the analysis are as follows. tlc (total length of chromosomes), mtlc (mean of total length of chromosomes), max (maximum length of chromosome), min (minimum length of chromosomes), mla (mean of long arms), msa (mean of short arms), mrv (mean of r-value), mdv (mean of d value), mar (mean of arm ratio), mci (mean of chromosome index), mrlc (mean of the relative length of chromosomes), drl (difference of range of relative length), tf% (total form percentage), s% (relative length of the shortest chromosome), a1 (intrachromosomal asymmetry index), a2 (interchromosomal asymmetry index), and a (degree of asymmetry). both arm ratios were assumed to be equally affected (adhikary 1974). all karyotype formulas and asymmetry indexes were determined based on huziwara (1962) (tf%), levan et al. (1964) (r and d values), zarco (1986) (a1 and a2), watanabe (1999) (a), peruzzi and eroğlu (2013) (ci) as well. the abbreviations were taken from rezeai et al. (2014) (rlc%, drl, s%). the formulas are as follows. formulas 147comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) d value=length of the long arm of chromosome-length of the short arm of chromosome drl=(maximum relative length)(minimum relative length) (li = lengths of a long arm, si = lengths of a short arm, n = haploid chromosome number). (n = number of homologous chromosome pairs, bi = the average length of short arms in every homologous chromosome pair, bi = the average length of long arms in every homologous chromosome pair). (s = standard deviation of chromosome lengths, = mean of chromosome lengths). a data matrix was constructed according to 17 chromosomal traits in table 1. the principal component analysis (pca) was used based on the data matrix. next, the cluster analysis was made using the gower similarity index to determine the relationships between cuscuta taxa’s chromosome traits. also, the pearson correlation coefficient (r) analysis was performed to see strong and weak relationships between chromosome traits. at the same time, shapiro wilk normality test was performed. then, the one-way analysis of variance (anova) was performed to determine whether the difference between the data was statistically significant. all the analyses were carried out with paleontostatistics (past) (hammer et al. 2001). results in this study, the karyological characteristics of 11 different cuscuta taxa were investigated, and their details are given below. cuscuta campestris: the chromosome number of c. campestris, native to the united states of america and spread to many countries from there, and can be found almost everywhere in turkey, was found to be 2n=2x=28. the haploid karyotype formula of this species is 10 median regions (m), 2 submedian regions (cm), and 2 dotted median (m) regions. metaphase chromosome length varies between 2.48-1.48 μm. chromosome arm ratios vary between 1.43-1 μm. its centromere index ranges from 50.00 to 29.44 μm, and its relative length is between 10.93 and 18.32 μm. the intra-chromosomal asymmetric index (a1) is 0.32, and the inter-chromosomal asymmetric index (a2) is 0.04 (table 2, figure 1). cuscuta hyalina: the chromosome number of c. hyalina species, distributed in turkey’s local area (bitlis table 1. the localities of studied taxa. taxa localities voucher specimen cuscuta campestris yunck. adana, i̇mamoğlu, alaybey village 1752 cuscuta hyalina roth. bitlis, hizan, karbastı village 2101 cuscuta kotschyana boiss. bitlis, süphan mountain 2098 cuscuta babylonica aucher ex choisy. van, çatak, sırmalı village 2100 cuscuta europaea l. bitlis, hizan 1993 cuscuta kurdica engelm. hakkâri, ördekli village 14935 cuscuta brevistyla a.braun ex a.rich bitlis, hizan 1786 cuscuta planiflora ten. van, tuşba 1766 cuscuta approximata bab. denizli, honaz mountain 1801 cuscuta lupuliformis. hakkâri centre 2099 cuscuta palaestina boiss. van, gürpınar 2095 148 neslihan taşar et al. table 2. chromosomes measurements of cuscuta taxa (ch. no: chromosome no, c: total length of the chromosome, l: length of the long arm, s: length of the short arm, cp: centromeric position). ch. no c l s l/s ci rl cp cuscuta campestris 1 2,48 1,46 1,02 1,43 41,13 0,00 m 2 2,4 1,4 1 1,40 41,67 0,00 m 3 2,29 1,39 0,9 1,54 39,30 0,00 m 4 2,1 1,31 0,79 1,66 37,62 0,00 m 5 2,06 1,03 1,03 1,00 50,00 0,00 m 6 2,06 1,16 0,9 1,29 43,69 0,00 m 7 1,98 1,21 0,77 1,57 38,89 0,00 m 8 1,9 1,08 0,82 1,32 43,16 0,00 m 9 1,84 1,12 0,72 1,56 39,13 0,00 m 10 1,8 1,27 0,53 2,40 29,44 0,00 sm 11 1,67 1,03 0,64 1,61 38,32 0,00 m 12 1,54 0,84 0,7 1,20 45,45 0,00 m 13 1,51 1,03 0,48 2,15 31,79 0,00 sm 14 1,48 0,74 0,74 1,00 50,00 0,00 m cuscuta kotschyana 1 3,95 2,1 1,85 1,14 46,84 6,19 m 2 3,93 2,51 1,42 1,77 36,13 6,22 sm 3 3,51 1,89 1,62 1,17 46,15 6,96 m 4 3,47 1,93 1,54 1,25 44,38 7,04 m 5 3,36 1,68 1,68 1,00 50,00 7,27 m 6 3,18 1,61 1,57 1,03 49,37 7,69 m 7 3,04 1,52 1,52 1,00 50,00 8,04 m cuscuta europaea 1 6,48 3,60 2,88 1,25 44,44 5,25 m 2 5,58 3,42 2,16 1,58 38,71 6,10 m 3 4,72 3,00 1,72 1,74 36,44 7,21 sm 4 4,53 2,63 1,90 1,38 41,94 7,51 m 5 4,37 2,45 1,92 1,28 43,94 7,79 m 6 4,35 2,52 1,83 1,38 42,07 7,82 m 7 4,00 2,70 1,30 2,08 32,50 8,51 sm cuscuta brevistyla 1 3,95 2,10 1,85 1,14 46,84 6,19 m 2 3,93 2,51 1,42 1,77 36,13 6,22 sm 3 3,51 1,89 1,62 1,17 46,15 6,96 m 4 3,47 1,93 1,54 1,25 44,38 7,04 m 5 3,36 1,68 1,68 1,00 50,00 7,27 m 6 3,18 1,61 1,57 1,03 49,37 7,69 m 7 3,04 1,52 1,52 1,00 50,00 8,04 m 8 3,01 1,85 1,16 1,59 38,54 11,07 m 9 2,90 1,79 1,11 1,61 38,28 11,49 m 10 2,85 1,55 1,30 1,19 45,61 11,69 m 11 2,81 1,60 1,21 1,32 43,06 11,86 m 12 2,68 1,34 1,34 1,00 50,00 12,44 m 13 2,54 1,49 1,05 1,42 41,34 13,12 m 14 2,46 1,26 1,20 1,05 48,78 13,55 m 15 2,30 1,18 1,12 1,05 48,70 14,49 m 16 2,22 1,11 1,11 1,00 50,00 15,01 m ch. no c l s l/s ci rl cp 17 2,03 1,10 0,93 1,18 45,81 16,42 m 18 1,94 1,16 0,78 1,49 40,21 17,18 m 19 1,92 1,00 0,92 1,09 47,92 17,36 m 20 1,89 1,05 0,84 1,25 44,44 17,63 m 21 1,78 0,92 0,86 1,07 48,31 18,72 m cuscuta palaestina 1 4,80 2,40 2,40 1,00 50,00 10,78 m 2 4,74 2,44 2,30 1,06 48,52 10,92 m 3 4,69 2,53 2,16 1,17 46,06 11,04 m 4 4,36 2,97 1,39 2,14 31,88 11,87 sm 5 4,28 2,93 1,35 2,17 31,54 12,09 sm 6 4,13 2,91 1,22 2,39 29,54 12,53 sm 7 3,93 2,37 1,56 1,52 39,69 13,17 m 8 3,72 2,34 1,38 1,70 37,10 13,91 sm 9 3,42 1,71 1,71 1,00 50,00 15,13 m 10 3,16 1,58 1,58 1,00 50,00 16,38 m 11 2,89 1,55 1,34 1,16 46,37 17,91 m 12 2,79 1,56 1,23 1,27 44,09 18,55 m 13 2,62 1,44 1,18 1,22 45,04 19,76 m 14 2,23 1,23 1,00 1,23 44,84 23,21 m cuscuta hyalina 1 5,18 2,63 2,55 1,03 49,23 11,08 m 2 5,05 2,58 2,47 1,04 48,91 11,36 m 3 4,93 2,50 2,43 1,03 49,29 11,64 m 4 4,80 2,40 2,40 1,00 50,00 11,95 m 5 4,50 2,35 2,15 1,09 47,78 12,75 m 6 4,40 2,20 2,20 1,00 50,00 13,04 m 7 4,25 2,15 2,10 1,02 49,41 13,50 m 8 4,18 2,30 1,88 1,22 44,98 13,72 m 9 4,00 2,00 2,00 1,00 50,00 14,34 m 10 3,58 1,85 1,73 1,07 48,32 16,03 m 11 3,42 1,71 1,71 1,00 50,00 16,77 m 12 3,19 1,61 1,58 1,02 49,53 17,98 m 13 3,09 1,57 1,52 1,03 49,19 18,57 m 14 2,80 1,50 1,30 1,15 46,43 20,49 m cuscuta babylonica 1 6,23 3,48 2,75 1,27 44,14 5,45 m 2 5,32 3,12 2,20 1,42 41,35 6,38 m 3 5,22 3,50 1,72 2,03 32,95 6,51 sm 4 4,69 3,24 1,45 2,23 30,92 7,24 sm 5 4,36 2,18 2,18 1,00 50,00 7,79 m 6 4,34 2,64 1,70 1,55 39,17 7,82 m 7 3,80 2,36 1,44 1,64 37,89 8,94 m cuscuta kurdica 1 4,80 2,40 2,40 1,00 50,00 6,46 m 2 4,67 2,45 2,22 1,10 47,54 6,64 m 3 4,66 2,50 2,16 1,16 46,35 6,65 m 4 4,40 3,00 1,40 2,14 31,82 7,05 sm 149comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) province), was found as 2n=2x=28. the haploid karyotype formula of this species has 10 median regions (m) and 4 points median (m) regions. metaphase chromosome length varies between 5.18-2.80 μm. chromosome arm ratios vary between 1.03-1.15 μm. its centromere index ranges from 50.00 to 44.98 μm and relative length from 11.08 to 20.49 μm. the intra-chromosomal asymmetric index (a1) is 0.04, and the inter-chromosomal asymmetric index (a2) is 0.04 (table 2, figure 1). cuscuta kotshyana var. caudata: the chromosome number of this species was determined as 2n=2x=14. haploid karyotype formula; it has 4 median regions (m), 2 points median (m) and 1 submedian region (cm) region. metaphase chromosome length was measured in lengths ranging from 3.93-3.04 μm. chromosome arm ratios vary between 1.77-1 μm. the centromere index is 50.00-36.13 μm. its relative length was measured in the range of 6.22-8.04 μm. the intra-chromosomal asymmetric index (a1) is 0.15, and the inter-chromosomal asymmetric index (a2) is 0.07 (table 2, figure 1). cuscuta babylonica var. babylonica: the chromosome number of this species is mainly found in the eastern anatolia region of turkey, at an altitude of 850-1200 m, whose stems are between thin filamentous and medium thickness, and which is yellowish-red is 2n=2x=14. the haploid karyotype formula of this species is 4 median regions (m), 2 submedian regions (cm), and 1 dotted median (m) region. metaphase chromosome length varies between 6.23-3.80 μm. chromosome arm ratios range from 1.64 to 1 μm. its centromere index ranges from 50.00-30.92 μm, and its relative length ranges from 5.45 to 8.94 μm. the intra-chromosomal asymmetric index (a1) is 0.34, and the inter-chromosomal asymmetric index (a2) is 0.07 (table 2, figure 1). cuscuta europaea: c. europaea; has 2n=2x=14 chromosomes. the haploid karyotype formula has 5 median regions (m) and 2 submedian regions (cm). metaphase chromosome length varies between 6.48-4 μm. chromosome arm ratios vary between 2.08-1.25 μm. its centromere index ranges from 44.44 to 32.50 μm, and its relative length ranges from 5.25 to 8.51 μm. the intrachromosomal asymmetric index (a1) is 0.33, and the inter-chromosomal asymmetric index (a2) is 0.07 (table 2, figure 1). cuscuta kurdica: the chromosome number of this species was found to be 2n=2x=14. the haploid karyotype formula has 3 median regions (m), 3 submedian regions (cm), and 1 dotted median (m) region. metaphase chromosome length varies between 4.80-3.87 μm. chromosome arm ratios vary between 2.27-1 μm. its centromere index ranges from 50.00-30.59 μm and relative length is between 6.46 and 8.01 μm. the intra-chromosomal asymmetric index (a1) is 0.34, and the interchromosomal asymmetric index (a2) is 0.07 (table 2, figure 1). cuscuta brevistyla: the chromosome number of c. brevistyla species, which is annual, parasitic, and generally distributed in the mountains, was determined as 2n=6x=42. the haploid karyotype formula has 15 medich. no c l s l/s ci rl cp 5 4,36 2,97 1,39 2,14 31,88 7,11 sm 6 4,25 2,95 1,30 2,27 30,59 7,30 sm 7 3,87 2,36 1,51 1,56 39,02 8,01 m cuscuta approximata 1 3,68 1,84 1,84 1,00 50,00 8,87 m 2 3,36 1,68 1,68 1,00 50,00 9,72 m 3 3,08 1,82 1,26 1,44 40,91 10,60 m 4 2,48 1,28 1,20 1,07 48,39 13,17 m 5 2,42 1,50 0,92 1,63 38,02 13,49 m 6 2,36 1,36 1,00 1,36 42,37 13,83 m 7 2,28 1,40 0,88 1,59 38,60 14,32 m 8 2,12 1,24 0,88 1,41 41,51 15,40 m 9 2,01 1,20 0,81 1,48 40,30 16,24 m 10 1,98 0,99 0,99 1,00 50,00 16,49 m 11 1,85 1,24 0,61 2,03 32,97 17,65 sm 12 1,84 1,06 0,78 1,36 42,39 17,74 m 13 1,62 1,00 0,62 1,61 38,27 20,15 m 14 1,57 0,84 0,73 1,15 46,50 20,80 m cuscuta lupuliformis 1 6,96 3,48 3,48 1,00 50,00 6,40 m 2 3,82 2,24 1,58 1,42 41,36 11,65 m 3 3,80 2,55 1,25 2,04 32,89 11,72 sm 4 3,20 1,60 1,60 1,00 50,00 13,91 m 5 3,14 1,82 1,32 1,38 42,04 14,18 m 6 3,07 1,83 1,24 1,48 40,39 14,50 m 7 2,98 1,76 1,22 1,44 40,94 14,94 m 8 2,94 1,80 1,14 1,58 38,78 15,14 m 9 2,76 1,70 1,06 1,60 38,41 16,13 m 10 2,52 1,56 0,96 1,63 38,10 17,67 m 11 2,43 1,33 1,10 1,21 45,27 18,32 m 12 2,40 1,20 1,20 1,00 50,00 18,55 m 13 2,30 1,26 1,04 1,21 45,22 19,36 m 14 2,20 1,10 1,10 1,00 50,00 20,24 m cuscuta planiflora 1 5,28 2,64 2,64 1,00 50,00 5,54 m 2 4,35 2,55 1,80 1,42 41,38 6,72 m 3 4,24 2,82 1,42 1,99 33,49 6,89 sm 4 4,08 2,04 2,04 1,00 50,00 7,16 m 5 3,96 2,26 1,70 1,33 42,93 7,38 m 6 3,78 2,15 1,63 1,32 43,12 7,73 m 7 3,54 2,18 1,36 1,60 38,42 8,26 m 150 neslihan taşar et al. an regions (m), 3 submedian regions (cm), and 3 dotted median (m) regions. metaphase chromosome length varies between 4.73-1.78 μm. chromosome arm ratios vary between 1.97-1 μm. its centromere index ranges from 50.00-33.63 μm, and its relative length varies between 12.6333.55 μm. the intra-chromosomal asymmetric index (a1) is 0.25, and the inter-chromosomal asymmetric index (a2) is 0.02 (table 2, figure 1). cuscuta planiflora: the chromosome number of this species was determined as 2n=2x=14. the haploid karyotype formula has 4 median regions (m), 1 submedian region (cm), and 2 dotted median (m) regions. metaphase chromosome length varies between 5.28-3.54 μm. chromosome arm ratios vary between 1.60-1 μm. its centromere index ranges from 50.00 to 38.42 μm, and its relative length ranges from 5.54 to 8.26 μm. the intrachromosomal asymmetric index (a1) is 0.24, and the inter-chromosomal asymmetric index (a2) is 0.07 (table 2, figure 1). cuscuta approximata: c. approximata has 2n=4x=28 chromosomes. the haploid karyotype formula has 10 median regions (m), 1 submedian region (cm), and 3 point median (m) regions. metaphase chromosome length varies between 3.68-1.57 μm. chromosome arm ratios vary between 1.60-1 μm. its centromere index ranges from 50.00 to 32.97 µm and its relative length from 8.87 to 20.80 µm. the intra-chromosomal asymmetric index (a1) is 0.23, and the inter-chromosomal asymmetric index (a2) is 0.04 (table 2, figure 1). cuscuta lupuliformis: the chromosome number of this species was found to be 2n=2x=28. the haploid karyotype formula has 9 median regions (m), 1 submedian region (cm), and 4 point median (m) regions. metaphase chromosome length varies between 6.96-2.20 μm. chromosome arm ratios vary between 2.04-1 μm. its centromere index ranges from 50.00-32.89 μm, and its relative length is 6.40-20.24 μm. the intra-chromosomal asymmetric index (a1) is 0.24, and the inter-chromosomal asymmetric index (a2) is 0.04 (table 2, figure 1). cuscuta palaestina: the chromosome number of this species was determined as 2n=4x=28. the haploid karyotype formula is 7 median regions (m), 4 submedian regions (cm), and 3 point median (m) regions. metaphase chromosome length varies between 4.80-2.23 μm. figure 1. mitotic metaphase chromosomes of cuscuta taxa 1. cuscuta campestris, 2. cuscuta hyalina, 3. cuscuta kotschyana, 4. cuscuta babylonica, 5. cuscuta europaea 6. cuscuta kurdica, 7. cuscuta brevistyla, 8. cuscuta planiflora, 9. cuscuta approximata, 10. cuscuta lupuliformis, 11. cuscuta palaestina (scale:10 μm). 151comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) chromosome arm ratios vary between 2.39-1 μm. its centromere index ranges from 50.00 to 44.84 µm and its relative length from 10.78 to 23.21 µm. the intra-chromosomal asymmetric index (a1) is 0.28, and the interchromosomal asymmetric index (a2) is 0.04 (table 2, figure 1). karyotypes in plants; according to the types of chromosomes, there are two types: symmetrical and asymmetrical. the symmetrical karyotype is characterized by the predominance of median and submedian chromosomes of approximately the same size. the increase in asymmetry caused by the centromere shift creates an asymmetric karyotype. chromosomes change from the median and submedian type to subterminal and terminal (babaarslan and eroğlu, 2014). when the asymmetric indices of cuscuta taxa were examined, it was seen that the tf% value changed between 0.41-0.49, the a index changed between 0.02 and 0.25, the a1 index between 0.21-0.38 and the a2 index between 0.09-0.31 (table 3). statistical findings chromosome micromorphological features of 11 cuscuta taxa were specified, and statistical analyses were performed using formulas created using various chromosome features. mitotic metaphase chromosome images of cuscuta taxa are given in figure 1, and karyotype features are given in table 2-3. one-way anova test, which is one of the analyzes made according to the chromosome characteristics of the taxa, is given in table according to the values obtained with the formulas using the micromorphological chromosome features of taxa, the data show a normal distribution according to the shapiro-wilk test (p>0.05), and the residual plot graph is shown in figure 2. then, according to the oneway anova test p-value, the difference between taxa was statistically significant (p<0.05) (table 4). correlation analysis according to the correlation analysis, there are relations between the r-values of chromosomal data according to the significance level less than p <0.05. particularly a high relationship although there was a strong positive relationship between mtlc and min, max, mla, msa, and mrv, it was observed that there was a strong negative relationship between mrlc and drl. in addition, mar and a1 and a characters are strongly positively correlated, while tf% is strongly negative; with mrlc, drl is strongly positive while a2 is strongly negative; tf% was strongly negatively correlated with mar, mdv, a1, a (figure 3). principal component analysis (pca) according to pca (figure 4), the first two components explained most of the variation according to chromosome data between taxa. while the first two components explain 87.94 and 9.80% of the variance, these characters explained 97.75% of the total variation. the characters most affected by the variation were tlc, drl%, mcl, and mrlc. the tlc value was the most influential one. the impact of other characters was very table 3. karyotype characteristics of cuscuta taxa (tlc: total lenght of chromosomes, mtlc (mean of total length of chromosomes, max: maximum length of chromosome, min: minimum length of chromosome, mla: mean of long arms, msa: mean of short arms, mrv: mean of r value, mdv: mean of d value, mar: mean of arm ratio, mci: mean of chromosome index, mrlc: mean of relative length of chromosomes, drl: difference of range of relative length, tf%: total form percentage, s%: relative length of shortest chromosome, a1: intrachromosomal asymmetry index, a2: interchromosomal asymmetry index). cuscuta taxa tlc mtlc max min mla msa mrv mdv mar mci mrlc drl tf% s% a1 a2 a c. campestris 27.11 0.97 1.46 0.48 1.14 0.78 1.92 0.36 1.51 40.69 14.37 7.39 0.41 0.33 0.32 0.04 0.19 c. hyalina 57.37 2.05 2.63 1.30 2.09 2.00 4.09 0.09 1.05 48.79 14.52 9.41 0.49 0.49 0.04 0.04 0.02 c.kotschyana 24.44 1.75 2.51 1.42 1.89 1.60 3.49 0.29 1.19 40.12 7.06 1.82 0.46 0.57 0.15 0.07 0.08 c. babylonica 33.96 2.43 3.5 1.45 2.93 1.92 4.85 1.01 1.59 39.49 7.16 3.49 0.40 0.41 0.34 0.07 0.21 c. europaea 34.03 2.43 3.6 1.30 2.90 1.95 4.85 0.95 1.53 40.01 7.17 3.26 0.40 0.36 0.33 0.07 0.20 c. kurdica 31.01 2.22 2.97 1.30 2.66 1.76 4.42 0.90 1.62 39.60 7.03 1.55 0.40 0.44 0.34 0.07 0.20 c. brevistyla 59.72 1.42 2.53 0.78 1.62 1.22 2.84 0.40 1.34 43.45 22.63 20.92 0.43 0.31 0.25 0.02 0.14 c. planiflora 29.23 2.09 2.82 1.36 2.37 1.79 4.16 0.58 1.38 42.76 7.01 2.72 0.43 0.48 0.24 0.07 0.14 c. approximata 32.65 1.17 1.84 0.61 1.31 1.01 2.32 0.30 1.37 43.87 14.89 11.92 0.43 0.33 0.23 0.04 0.13 c. lupuliformis 44.52 1.59 3.48 0.96 1.80 1.37 3.17 0.43 1.36 43.10 15.19 13.84 0.43 0.28 0.24 0.04 0.14 c. palaestina 51.76 1.85 2.97 1.01 2.14 1.55 3.69 0.59 1.43 42.48 14.80 12.43 0.42 0.34 0.28 0.04 0.16 152 neslihan taşar et al. low. while this tlc value was positively correlated with mcl, mrlc, and drl characters in the correlation analysis, it was negatively correlated with mar, a, a1, a2, and s% characters. cluster analysis according to the cluster analysis results of the upgma algorithm and gower similarity index, the taxa are divided into three main groups (figure 5). c. brevistyla, c. lupuliformis, c. palaestina, c. approximata, and c. campestris were group, c. kotschyana, c. planiflora, c. babylonica, c. europea, c. kurdica had created a group. the c. hyalina species wholly separated from these groups were a group. as stated before, the fact that c. hyalina species spread in a local area directly correlates with the analysis result. discussion cuscuta species show wide variation in chromosome numbers ranging from 2n = 8 to 2n = 60. therefore, the genus is generally a polyploid complex resulting from two basic chromosome numbers x = 7 and x = 15 (pazy & plitmann, 1995; hunziker, 1949-50). the first step in combating parasitic plants is their correct diagnosis, as with other weeds. due to the lack of true root and leaf structure of dodder, diagnosis is mainly made according to flower and fruit characteristics. these features are sometimes insufficient for diagnosis. diagnosis of this genus is problematic in the world and turkey. therefore, determining the chromosome number and chromosomal morphology of the species belonging to this genus is of great importance in determining the systematic location of the species, identifying the species, and, when necessary, agriculturally struggling with these species. according to the karyotype analysis results of cuscuta taxa, the primary chromosome number was determined as x=7. among the study samples, c. brevistyla is polyploid, c. campestris, c. hyalina, c. approximata, c. lupuliformis, c. palaestina tetraploid, and other taxa are diploid. according to the total length of chromosomes, the species with the longest chromosome length is c. lupuliformis, with 6.96 mμ lengths. this species was morphological; c. campestris, with a total chromosome length of 2.48 mμ was determined to be the shortest chromofigure 2. shapiro wilk normality test(p=0.4809>0.05)-residual plot. table 4. one way anova test results. test for equal means sum of sqrs df mean square f p (same) between groups: 45343.3 16 2833.96 351.6 8.149e-179 within groups: 2329.64 289 8.06104 permutation p (n=99999) total: 47672.9 305 1e-05 omega2: 0.9483 153comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) some length. the chromosome number of c. campestris was first determined by ward(1984) as 2n=28; later, aryavand and garcía & castroviejo (1987) 2n=56; khatoon & ali(1993) determined it as 2n=14, 28. according to our research results, the chromosome number of the species is 2n=28. it has been shown that the haploid karyotype formula is 10m+2sm +2m. the morphometric characteristics of the species were first revealed in this study. singh and roy(1970). collected c. hyalina from india; the chromosome number of the species is 2n=30; vu et al. determined as 2n=28. according to our study results, the chromosome number of the species is 2n=28. the haploid karyotype formula is 10m+4m. this study first revealed the chromosome number and morphometric characteristics of c. kotschyana species. chromosome number 2n=14; haploid karyotype formula; it has 4 median regions (m), 2 points median (m), and 1 submedian region (cm) region (figure 6). figure 3. correlation analysis between karyotype characteristics. figure 4. pca analysis scatter plot (same colors are the same subgenus). 154 neslihan taşar et al. pazy and plitmann (2002) determined the chromosome number of c. babylonica as 2n=8, where they specified israel as the locality. however, according to our research results, the chromosome number of the species is 2n=14, and the haploid karyotype formula is 4m + 2 cm + 1m. the chromosome number of the c. europaea species was previously reported by albers and pröbsting(1998) and garcía and castroviejo(2003) as 2n=14. our research data also confirm this result. the haploid karyotype formula of the species, in which we found the chromosome number as 2n=14, is 5m + 2 cm. regarding chromosome number and morphology, the chromosome number of c. kurdica species, which was first discussed in this study, was determined as 2n=14. the haploid karyotype formula is 3m + 3 cm + 1 m. pazy and plitman (1994) and feinbrun and taub(1978) found the chromosome number of c. brevistyla as 2n=42, where they specified israel as a locality. according to our study results, the chromosome number of this species is 2n=42. the haploid karyotype formula is 15m + 3 cm + 3 m. the chromosome number of c. planiflora has been determined by many researchers. singh and roy determined the chromosome number of this species as 2n=14; pazy and plitmann. (1991) 2n=14; garcía and castroviejo. (2003) 2n=26, 28; aryavand(1987) reported 2n=28 and vasudevan 2n=14. as a result of our research, the chromosome number of the species was determined as 2n=14. the haploid karyotype formula was 4m + 1 cm + 2 m. c. approximata; garcía and castroviejo (2003) and guerra(2004). 2n=28 chromosomes have reported it. our studies also confirm this result, and the chromosome number of this species is 2n=28. the haploid karyotype formula is 10m + 1 cm + 3m.. the chromosome number of c. lupuliformis was determined as 2n=28 by vasudevan. according to our research results, the chromosome number of this species is 2n=28. the haploid karyotype formula is 9m + 1 cm + 4m. plazy and plitmann (1991) showed the c. palaestina species as 2n=28 chromosomes. our research confirms this result. we found the chromosome number of 2n = 28 of this species. the haploid karyotype formula is 7m + 4 cm + 3m. various karyological studies have been carried out on the chromosome number of species belonging to the cuscuta genus. as a result of these studies, the chromosome number of cuscuta japonica choisy. species is 2n= 32 (leusova et al., 2005); the chromosome number of cuscuta epithymum l. species is 2n= 14 (montgomery et al., 2003); the chromosome number of cuscuta australis r. br. species is 2n=56 (yeh et al., 1995); chromosome figure 5. cluster analysis according to karyotype characteristics show that 3 main groups (same colored taxa are located in the same section). 155comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) number of cuscuta triumvirati lange. species 2n= 14 (garcía et al., 2003); chromosome number of cuscuta pentagona engelm. is 2n= 44 (pazy et al., 1995); the chromosome number of cuscuta pedicellata ledeb. species was determined as 2n= 10 (pazy et al., 1991), and the chromosome number of cuscuta chinensis lam. species was determined as 2n= 60 (mesĭcek et al., 1995). according to cluster analysis, taxa were divided into 3 main groups. it is noteworthy that although c. campestris and c. hyalina are in the grammica subgenus, they are in different groups according to chromosome micromorphological data. here, it is estimated that some chromosomal features (according to pca, such as tlc) may have differentiated over time, as the c. hyalina species was distributed in a local region in turkey. it is seen that c. babylonica and c. europea species in the cuscuta subgenus and c. kurdica species are closely related. according to their morphological similarities, c. europea and c. kurdica species show very close similarities. according to pca, the most important character explaining the differentiation between taxa was seen as tlc (total lenght of chromosomes) character. in addition, when the distribution of taxa in the diagram is examined, it is a compatible image with cluster analysis. in this study, 11 species belonging to the genus cuscuta, an essential part of turkey’s biological richness and consists of parasitic plants, were discussed in detail in terms of chromosome number and chromosome morphology and compared statistically. these karyological studies reveal the karyological differences and similarifigure 6. haploid idiogram in cuscuta taxa 1. c. campestris, 2. c. hyalina, 3. c. kotschyana, 4. c. babylonica, 5. c. europaea 6. c. kurdica, 7. c. brevistyla, 8. c. planiflora, 9. c. approximata, 10. c. lupuliformis 11. c. palaestina.. 156 neslihan taşar et al. ties between the infrageneric and species. the results obtained increase our knowledge about these species. thus, obtaining new data that can be used in the systematics of these species aims to reveal basic information about the systematics, karyology, and morphological features of taxa. in addition, it will form a fundamental step for future breeding and hybridization studies related to this genus and contribute to other biological research. references açar m, satıl f. 2019. distantes r. bhattacharjee (stachys l. /lamiaceae) altseksiyonu taksonları üzerinde karşılaştırmalı anatomik ve mikromorfolojik çalışmalar. 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(convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(4): 87-92, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1939 caryologia international journal of cytology, cytosystematics and cytogenetics citation: simona ceraulo, francesca dumas (2022). mapping cap-a satellite dnas by fish in sapajus cay paraguay and s. macrocephalus (platyrrhini, primates). caryologia 75(4): 87-92. doi: 10.36253/caryologia-1939 received: september 15, 2022 accepted: december 24, 2022 published: april 28, 2023 copyright: © 2022 simona ceraulo, francesca dumas. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. mapping cap-a satellite dnas by fish in sapajus cay paraguay and s. macrocephalus (platyrrhini, primates) simona ceraulo, francesca dumas* department of biological, chemical and pharmaceutical sciences and technologies (stebicef), university of palermo, 90100, palermo, italy *corresponding author. e-mail: francesca.dumas@unipa.it abstract. satellite dnas such as cap-a sequences are potentially informative taxonomic and phylogenetic markers useful for characterizing primate genomes. they have also been used as cytogenetic markers facilitating species identification in many taxa. the aim of this work is to map cap-a sequences by fish (fluorescent in situ hybridization) on two platyrrhini (primates) species genomes, sapajus cay paraguay and s. macrocephalus, in order to study their distribution pattern on chromosomes. the capa probes showed bright signals with almost the same interstitial pattern of distribution in correspondence with c and cma3 rich regions on six pairs of chromosomes in both sapajus species. an additional pair was detected on s. macrocephalus. the analysis of the results, compared with previous literature data on other phylogenetically close new world species, shows that cap-a satellite sequences have a genus-specific pattern, but with slight species-specific patterns that are useful as phylogenetic and taxonomic markers. keywords: heterochromatin, karyotype, genome, new world monkeys. introduction apart from coding regions (about 2%), the human genome includes highly repetitive sequences (about 98%) which are usually underestimated in genome analyses due to their complexity; these sequences are known as the dark matter of the genome (ahmad et al. 2020) and consist of satellite dna (satdna), defined as tandemly arranged repeats that represent a considerable proportion of the heterochromatic portion of chromosomes in the eukaryotic genome. satdna, at first seen as serving no useful purpose, is now known to be associated with genome function, chromosome evolution, speciation, and diversity, comprising different kind of elements such as satellite dnas, sines (short interspersed nuclear elements), line retrotransposons (long interspersed nuclear elements), and rdna repeats (ahmad et al. 2020, ceraulo et al. 2021 a, dumas et al. 2022). thus, satdnas are potentially informative cytogenetic markers which can be used to study karyotype evolution and address taxonomic issues. they 88 simona ceraulo, francesca dumas display high evolutionary rates and consist of tandem repeats organized in the type of large arrays (up to mb size) typically associated with chromosome landmarks such as centromeres, telomeres, and heterochromatic regions (ahmed 2020). they evolve by mechanisms of gene conversion, and unequal crossing-over which are involved in what is known as concerted evolution (sander lower et al. 2018). satdnas have high intraspecific sequence homogeneity and interspecific differences, making satdnas potential taxonomic markers and, in some cases, allowing their use for phylogenetic inference. furthermore, satdnas have been used as cytogenetic markers facilitating species identification in many taxa (prakhongcheep et al. 2013 a,b, cacheux, et al. 2018). among repetitive sequences, cap-a is a satdna that has been analyzed in many mammals through molecular comparative sequence analysis (valeri et al. 2018), and their history has been reconstructed in mammals. this analysis led researchers to show that a capa like sequence is present as a single monomer in most eutherians such as chiroptera, some eulipotyphla, and some rodentia, and also in homo sapiens. indeed, in h. sapiens, it is only a sequence within the intron of the nos1ap (nitric oxide synthase 1 adaptor protein) gene. no cap-a like sequence was found among marsupialia or monotremata, the sister clades to eutheria, presumably due to the occurrence of low copy numbers or because the sequence has diverged (valeri et al. 2018, valeri et al. 2020). on the other hand, cap-a duplication and expansion have been shown in new world monkeys (nwms); this amplification may be explained by a mechanism in which the cap-a intronic segment was transferred to heterochromatic regions in the ancestral platyrrhini genome followed by a hyper-expansion through unequal crossing (valeri et al. 2020). comparative cy togenetics using different kinds of repetitive sequence probes mapped by fluorescence in situ hybridization (fish) on chromosomes led us to study sequence pattern distribution among species allowing genomic comparison (ceraulo et al. 2021 b, c). cap-a sequence probes have been mapped by fish in many taxa, including primates (valeri et al. 2018, valeri et al. 2020), in order to study their distribution pattern. this work permits researchers to show that cap-a is an abundant satdna in platyrrhini with a high accumulation in blocks in some genomes. in particular, cap-a has been found in representatives of the three platyrrhini families (cebidae, with the exception of callitrichines, atelidae and pitheciidae, with the exception of the callicebus genus), with genome abundance ranging from less than 1% up to 5%, and chromosome localization which is always associated with non-centromeric constitutive heterochromatin (valeri et al. 2018, 2020). furthermore, intragenus research analyzing cap-a distribution on four saimiri species has also been performed (valeri et al. 2020) showing slight pattern differences between species. the fact that cap-a is present across platyrrhini led researchers to show its utility as a marker for chromosome and genome evolution studies in nwms (valeri et al. 2018). this is especially important because of the extinction to an alarmingly large number of nwm species due to rapid habitat loss. the cap-a sequence was first described in the tufted capuchin monkey sapajus apella (previously classified as cebus apella); cap-a was identified after digestion of genomic dna with restriction enzymes and with dna– dna hybridization (malfoy et al. 1986, fanning et al. 1993). thus, in order to extend previous studies using capa as a marker in platyrrhines, two additional sapajus species, s. cay paraguay and s. macrocephalus (cebidae), were analyzed, mapping the cap-a probe by fish. this study will help clarify sapajus chromosome evolution and add potentially useful data for taxonomic, systematics, and conservation issues. indeed, the cytogenetic information about sapajus is poor, whereas more species from the phylogenetically close cebus have been analyzed (garcia et al. 2002). the number of specimens karyotypically analyzed is low, and most samples have not been studied, especially from the recently recognized sapajus genus. karyotypes among the two taxa are very similar, and these species have been hypothesized to be distinguishable for the non-centromeric heterochromatin block of some chromosomes, with a differential chromosomal position in each of them (mudry, 1990, garcia et al. 2002). material and methods peripheral blood from male samples of s. cay paraguay and s. macrocephalus (cebidae) was collected from primates at the istc-cnr of rome, in accordance with international and institutional ethics rules. metaphases were obtained from lymphoblast cell cultures in rpmi culture medium, following standardized protocols. cell harvesting was performed after 3 h incubation with colcemid 10 μl (10 μg/ml gibco), followed by hypotonic treatments of 0.075 m kcl for 20 min at 37 °c, following standard protocols (dumas et al. 2022). metaphases of the analyzed species were stained preand post-fish using chromomycin a3 (cma3) and 4’,6-diamidino-2-phenylindole (dapi) 89mapping cap-a satellite dnas by fish in sapajus cay paraguay and s. macrocephalus (platyrrhini, primates) staining, according to a recent protocol (lemskaya et al. 2018), with some adjustments. cma3 staining of gc-rich regions and dapi staining of at-rich regions were useful for identifying chromosomes and preferential insertion sites of cap-a sequences. dapi images were inverted with a photo editing program (adobe photoshop c 2022 v23.3.2); inverted gray bands generally correspond to dark g-bands or light r bands; the dapi inverted karyotypes for the s. cay paraguay and s. macrocephalus species were compared with previously published banded karyotypes of the phylogenetically close species s. apella and cebus capucinus (garcia et al. 2002, milioto et al. 2022). human dna extraction from lymphoblast cell lines was performed using the pure link dna kit (invitrogen), according to the basic dna extraction protocol. cap-a was amplified by polymerase chain reaction (pcr) from human dna; the following universal set of primers, developed for the pcr of cap-1 repeats in primates, were used: (cap-a f: acttcctcactgacctgtctt; cap-a r:gggctgatgcttaatgtagca). genomic dna was amplified in 50 μl pcr-reactions: five units of taq gold dna polymerase (invitrogen), the template dna, 500 nm of each primer, 200 μm each of datp, dctp, dttp, and dgtp in 10 mm tris-hcl, ph 8.3, 1.5 mm mgcl2, 50 mm kcl. pcr reactions were performed using an applied biosystems simplyamp (thermo fisher scientific) with the following cycling parameters: 30 cycles each of 94 °c, 60 s; 55 °c, 60 s; 72 °c, 60 s, following a 3 min denaturation at 94 and with a final elongation step of 72 °c, 10 min. a bright band of about 1500 pb was visualized on 1% agarose gel. the pcr products were directly labeled through nick translation using 11-dutp-fluorescein (green) (invitrogen). fish was performed following previously described protocols (dumas and sineo 2014, dumas et al. 2015) using cap-a probes obtained by pcr as previous described (valeri et al. 2018, 2020). the hybridization mix consisted of 2.5 ng/l of probe, 50% formamide, 10% dextran sulfate, and 2xssc, with an incubation time of 18 h at 37 c. detection was performed at medium stringency, with washing at low temperatures (45 °c) and at high saline buffer concentration of ssc 0.1 tween, 15 min, pbs 1min. c banding was done sequentially post-fish through a protocol which included denaturation with formamide (fernàndez et al. 2002). after fish, the metaphases were analyzed under a zeiss axio2 epifluorescence microscope. images were captured using a coupled zeiss digital camera. at least ten metaphase spreads were analyzed for each sample. results sequential staining, banding, and fish mapping were performed for the two sapajus species. the inverted dapi karyotypes of s. cay paraguay and s. macrocephalus were almost the same as those of the other congeneric species previously published (garcia et al. 2002, milioto et al. 2022), with both species having the diploid number 2n = 54; for the karyotype reconstruction, we followed a previous publication (garcia et al. 2002), with ten pairs of meta/submetacentric chromosomes in s. cay paraguay (pairs 1–10), eight pairs in s. macrocephalus (1–7, 9), and fourteen and sixteen acrocentric chromosomes, respectively, thus differing over chromosome pairs 8 and 10, which are subtelocentric in the former and acrocentric in the latter. dapi/cma3 staining was helpful for identifying chromosomes and preferential sites of capa insertion (fig. 1, 2). figure 1. metaphases of s. cay paraguay in dapi (a), in dapi inverted (b), cap-a probe mapping (c), cma3/dapi stains (d), the reconstructed karyotype of s. cay paraguay from the metaphase in a) after sequential cma3/ dapi, dapi inverted, fish with cap-a probes (e). 90 simona ceraulo, francesca dumas cap-a probe mapping revealed bright signals on the metaphases of the two species analyzed, with a similar accumulation pattern and slight differences (fig. 1, 2): twelve signals were on six chromosome pairs: 5 acrocentric and a subtelocentric chromosome pairs, respectively pairs 11, 17-20 and 8. additional signals were found on acrocentric chromosome pairs 23 in s. macrocephalus, for a total of fourteen (fig. 2). the post-fish c banding pattern obtained was compared with previously published c banding of phylogenetically close species such as s. apella (dumas et al. 2022). chromosomes pairs with evident c bands were: 8, 11, 18-20; other c bands were at the centromeres of acrocentric chromosomes (fig. 3), as in the previously analyzed sapajus species. discussion cap-a satdna have been mapped in many mammals, including in primates; previous works have shown that cap-a is highly amplified in nwms, localized at non-centromeric positions, with different patterns in the different platyrrhini species (valeri et al. 2018, 2020). to extend cap-a distribution analysis to more primate samples, we used fish to map cap-a probes in two species of the genus sapajus. in the two species, chromosome pairs having these signals were identified as 8, 11, 17-20, (fig. 1, 2); an additional signal was detected on chromosome pair 23 in s. macrocephalus (fig 2); the probe signals fall on cma3 rich regions in correspondence to the big interstitial c bands (fig. 3). our results for s. cay paraguay and s. macrocephalus were compared with previous cap-a mapping data on other platyrrhine species (sapajus xanthosternos, saimiri boliviensis, aotus infulatus, alouatta guariba, lagotrix lagotricha, brachyteles hypoxanthus, callicebus nigrifrons, chiropotes satanas, pithecia irrorata, s. boliviensis, s. sciureus, s. vanzolinii and s. ustus) (valeri et al. 2018, 2020). the analysis of the results compared with the one from the previous species of the same genus s. xanthosternos permitted us to hypothesize that the same chromosome pairs would have these signals. indeed, in s. xanthosternos, six pairs showed signals plus an addtional figure 2. metaphases of s. macrocephalus in dapi (a), in dapi inverted (b), cap-a probe mapping (c), cma3/dapi stains (d), the reconstructed karyotype of s. macrocephalus from the metaphase in a) after sequential cma3/ dapi, dapi inverted, fish with cap-a probes (e). figure 3. metaphases of s. cay paraguay with c bands. example of representative chromosome pairs with evident bands are indicated with numbers. 91mapping cap-a satellite dnas by fish in sapajus cay paraguay and s. macrocephalus (platyrrhini, primates) on a single chromosome, for a total of thirteen signals. whereas twelve signals were detected on chromosome pairs in s. cay paraguay: on acrocentric chromosome pairs 11, 17-20, and on the subtelocentric chromosome pair 8; moreover, additional signals were found on the acrocentric chromosome pair 23 in s. macrocephalus, with a total of fourteen cap-a signals. however, one pair seems to have a different cap-a pattern; indeed, in the previously analyzed s. xanthosternos species, the cap-a probe signal covers both arms, almost all the q and the p arm, while in s. cay paraguay and s. macrocephalus all the cap-a probe signals cover just part of the q arm. this difference could presumably be due to an intrachromosomal rearrangement such as an inversion that has amplified and dislocated the sequences differently (presumably on the subtelocentric/ acro chromosome pair 8). analyzing our results in relation to all the previous available data from different taxa (valeri et al. 2018, 2020), it is possible to underline that cap-a localization has high interspecific repeat homogeneity within a genus; indeed, the saimiri species have almost the same chromosomes harboring the cap-a sequences, with slight differences (valeri et al. 2020), as it also occurs in the species from the sapajus genus as shown above. cap-a probe signals are abundant, around fourteen or fifteen, among saimiri species and are, in the distal regions of the short arms, and in the interstitial heterochromatin of five to seven chromosome pairs. among saimiri species, signals are on the same chromosome pairs, while others are additional or absent in same specimens. this slightly different location of the cap-a probe found between the samiri species is particularly evident, especially on chromosomes involved in rearrangements, such as chromosomes 5 and 15. these differences in the cap-a hybridization pattern in squirrel monkeys has been reported in captivity and in nature; for this reason, it has been hypothesized that cap-a mapping patterns may be useful in revealing the origin of chromosome sets in hybrids more precisely than chromosome morphology or banding patterns (valeri et al. 2020). the link between cap-a distribution and rearrangements in saimiri is in agreement with the different cap-a position shown on subtelocentric chromosome between the sapajus species analyzed here and the previously analyzed s. xanthosternos (valeri et al. 2018). furthermore, saimiri species also show additional chromosomes with cap-a signals, just as it occurs on s. microcephalus in our study. thus, it can be confirmed the hypothesis that new acquisition of cap-a occurs; it is presumably due to unequal crossing-over and concerted evolution as previous suggested (sander lower et al. 2018). we observed a slight, variable chromosomal localization of cap-a signals among the species of the sapajus genus, thus we hypothesized that these differences can be used as taxonomic markers for species identification, in agreement with what was previously shown among saimiri species. this evidence is in agreement with the hypothesis that satdna sequences, in general, can be used as cytogenetic markers facilitating species identification in many taxa (prakhongcheep et al. 2013 a,b, cacheux, et al. 2018). fur t hermore, t hrough t he classic cy togenetic approach, detecting heterochromatin with differential chromosomal position has already been hypothesized as distinguishing species (mudry, 1990, garcia et al. 2002). the correspondence of the cap-a signal with heterochromatin block extends the hypothesis regarding the possibility of distinguishing species not only by c bands but also through the cap-a pattern. in conclusion, this work demonstrates the presence of cap-a satellite sequences on chromosomes of sapajus genomes, with a genus specific pattern interstitially in correspondence with c and cma3 rich regions, but with slight species-specific patterns that can be useful as phylogenetic and taxonomic markers. acknowledgements we are grateful to elsa addessi, serena gastaldi, and arianna manciocco for providing the sapajus blood samples from primates of the istc-cnr of rome, italy. literature cited ahmad sf, singchat w, jehangir m, suntronpong a, panthum t, malaivijitnond s, srikulnath k. 2020. dark matter of primate genomes: satellite dna repeats and their evolutionary dynamics. cells, 9, 2714. doi. org/10.3390/cells9122714. cacheux l, ponger l, gerbault-seureau m, loll f, gey d, richard fa, escudé c. 2018. the targeted sequencing of alpha satellite dna in cercopithecus pogonias provides new insight into the diversity and dynamics of centromeric repeats in old world monkeys. genome biol. evol.,10, 1837–1851. ceraulo s, milioto v, dumas f. 2021. centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates). caryo92 simona ceraulo, francesca dumas logia, 74, 111–119. https://doi.org/10.36253/caryologia-1296. ceraulo s, perelman lp, mazzoleni s, rovatsos m, dumas f. 2021. repetitive sequence distribution on saguinus, leontocebus and leontopithecus tamarins (platyrrhine, primates) by mapping telomeric (ttaggg) motifs and rdna loci. biology, 10, 844. https://doi.org/10.3390/biology10090844 ceraulo s, perelman pl, dumas f. 2021. massive line-1 retrotransposon enrichment in tamarins of the cebidae family (platyrrhini, primates) and its significance for genome evolution. j. zool. syst. evol. res., 59, 2553–2561 https://doi.org/10.1111/jzs.12536 dumas f, perelman pl, biltueva l, roelke me. 2022. retrotransposon mapping in spider monkey genomes of the family atelidae (platyrrhini, primates) shows a high level of line-1 amplification. j biol res doi: 10.4081/jbr.2022.10725 dumas f, sineo l, ishida t. 2015.taxonomic identification of aotus (platyrrhinae) through cytogenetics| identificazione tassonomica di aotus (platyrrhinae) mediante la citogenetica. j. biol. res 88: 65-66. dumas f, sineo l. 2014. the evolution of human synteny 4 by mapping sub-chromosomal specific probes in primates. caryologia. 67, 281–291. https://doi.org/10 .1080/0144235x.2014.974357. 30. fanning tg, seuanez hn, forman l. 1993. satellite dna sequences in the new world primate cebus apella (platyrrhini, primates). chromosoma 102,306– 311. 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firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1432 caryologia international journal of cytology, cytosystematics and cytogenetics citation: isara patawang, suphat prasopsin, chatmongkon suwannapoom, alongklod tanomtong, puntivar keawmad, weera thongnetr (2022) chromosomal description of three dixonius (squamata, gekkonidae) from thailand. caryologia 75(2): 101-108. doi: 10.36253/caryologia-1432 received: october 22, 2021 accepted: october 22, 2021 published: september 21, 2022 copyright: © 2022 isara patawang, suphat prasopsin, chatmongkon suwannapoom, alongklod tanomtong, puntivar keawmad, weera thongnetr. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* 1 department of biology, faculty of science, chiang mai university, muang chiang mai, chiang mai, thailand 2 research academic supports division, mahidol university, kanchanaburi campus, saiyok, kanchanaburi, thailand 3 department of fishery, school of agriculture and natural resources, university of phayao, muang phayao, phayao, thailand 4 program of biology, faculty of science, khon kaen university, muang khon kaen, khon kaen, thailand 5 major of biology, faculty of science and technology, mahasarakham rajabhat university, muang mahasarakham, maha sarakham, thailand 6 walai rukhavej botanical research institute, mahasarakham university, kantharawichai, maha sarakham, thailand *corresponding author: weeraatah@hotmail.com abstract. chromosomal characteristics and karyological analysis of three dixonius, including d. hangseesom, d. siamensis and d. melanostictus, from thailand were studied. chromosome preparations were conducted by squash technique from bone marrow and testis. conventional giemsa’s staining and ag-nor banding techniques were applied to stain the chromosome. the results showed that the diploid chromosomes are 2n=40, for d. hangseesom and d. siamensis; and 2n=42, for d. melanostictus. the fundamental number (nf) is 42 in d. hangseesom and d. siamensis and 44 in d. melanostictus. the types of chromosomes were 2 metacentrics and 38 telocentrics for d. hangseesom and d. siamensis, while the karyotype of d. melanostictus comprised 2 acrocentrics and 40 telocentrics. in the d. hangseesom and d. siamensis, nors are located to the near centromere on long arm of the telocentric chromosome pair 13. although, the nors of d. melanostictus are situated on the subtelomeric region of telocentric chromosome pair 8. there are no sex differences in karyotypes between males and females of these three geckos species. we found that during metaphase i and metaphase ii on male meiosis of the d. hangseesom and d. siamensis, the homologous chromosomes showed synapsis of 20 bivalents and 20 haploid chromosomes (n=20). moreover, metaphase i and metaphase ii of the male d. melanostictus showed synapsis of 21 bivalents and 21 haploid chromosomes (n=21). their karyotype formulas is as follows: d. hangseesom (2n=40): lm2 + lt2 + mt4 + st32, d. siamensis (2n=40): lm2 + lt6 + mt4 + st28, and d. melanostictus (2n=42): la2 + lt12 + mt4 + st24. keywords: dixonius, chromosome, karyotype, nucleolar organizer region (nor). 102 isara patawang et al. introduction dixonius is a genus of asian geckos or commonly known as leaf-toed geckos that belong to the class reptilia, order squamata, and family gekkonidae. the dixonius was first chosen to accommodate southeast asian leaf-toed geckos previously to the polyphyletic and nearly cosmopolitan phyllodactylus (bauer et al. 1997). the d. siamensis is the type species of the genus dixonius, which receive name from the first zoologist that note siamensis (dixon 1964). in thailand, total 7 species of dixonius were found including d. dulayaphitakorum, d. hangseesom, d. kaweesaki, d. mekongensis, d. melanostictus, d. pawangkhananti and d. siamensis (sumontha et al. 2017; pauwels et al. 2020, 2021). only about 10% of gekkonid species have been karyotyped and were studied with classical cytogenetic methods, including routine staining, as well as r, nor and c banding (moritz 1984; shibaike et al. 2009). the series from 2n=28 to 46 of the diploid chromosomes is characteristic of the gekkonid lizard’s karyotype (gorman 1973; king 1987; schmid et al. 1994). the typical karyotype consists of a gradual series of mono-armed chromosome (sometimes with a few bi-armed chromosome), and there is no distinction between macroand microchromosomes are present, the centromere is often subterminal (gorman 1973). karyotype evolution within the group is accompanied by robertsonian fusions, fissions and pericentric inversions (gorman 1973; king 1987). examples of the gekkonid’s chromosome study in thailand that were reported: gekko gecko, 2n=38=12biarmed+26mono-armed (patawang et al. 2014); hemidactylus frenatus, 2n=40=34bi-armed+6mono-armed; h. platyurus, 2n=46=2bi-armed+44mono-armed (patawang and tanomtong 2015); cyrtodactylus kunyai, 2n=40=12bi-armed+28mono-armed; c. interdigitalis, 2n=42=10bi-armed+32mono-armed (thongnetr et al. 2019); c. jarujini, 2n=40=28bi-armed+12mono-armed; and c. doisuthep, 2n=34=28bi-armed+6mono-armed (thongnetr et al. 2021) etc. in thailand, there is only one previous report on dixonius species chromosome. ota et al. (2001) demonstrated that d. siamensis’ karyotype by conventional staining technique is as 2n=42, male and female specimen from mae yom national park, phrae province, thailand; and 2n=40, male from phu wua wild life reserve area, nongkhai province, thailand. the present study of the karyological analysis of d. hangseesom, d. siamensis and d. melanostictus, provides the first report on the ag-nor banding technique, chromosome size, chromosome type, karyotype formula, and standardized idiogram which are compared to earlier reports. materials and methods sample collection both male and female of three dixonius species (figure 1) were collected from three sites in thailand, d. hangseesom from kanchanaburi province; d. siamensis from chiang mai province; and d. melanostictus from saraburi province. the geckos were transferred to the laboratory and kept under standard conditions for one day prior to the experimentation. chromosome preparation chromosomes were directly prepared in vivo (patawang et al. 2014) by injecting phy tohemagglutinin (pha) solution into its abdominal muscle. after ten hours, colchicine is injected to animal intramuscular and its abdominal cavity and left for 8-10 hours. bone marrow (in male and female) and testis (in male) are cut figure 1. general characteristic of the d. hangseesom (a), d. siamensis (b) and d. melanostictus (c) from thailand. scale bars indicate 1 centimeter. 103chromosomal description of three dixonius (squamata, gekkonidae) from thailand into small pieces then mix squash with 0.075m potassium chloride (kcl). after discarding all large cell pieces, 15 ml of cell sediment is transferred to a centrifuge tube and incubated for 30-40 minutes. cells were fixed in fresh cool canoy fixative (3 absolute methyl alcohol: 1 glacial acetic acid) gradually added up to 8 ml before centrifuging again at 3,000 rpm for 10 minutes, whereupon the supernatant was discarded. fixation was repeated until the supernatant was clear and the pellet was mixed with 1 ml fixative. the mixture was dropped onto a clean and cold slide by micropipette followed by the air-drying. chromosome staining conventional staining (gosden 1994) the mixture is dropped onto a clean and cold slide by micropipette followed by the air-dry technique. the slide is conventionally stained with 20% giemsa’s solution for 30 minutes. ag-nor banding (howell and black 1980) the two drops of each 50% silver nitrate and 2% gelatin were added on slides, respectively. then it was sealed with cover glasses and incubated at 60 °c for 5-10 minute. there after that it was soaked in distilled water until cover glasses are separated. chromosome checking ten clearly observable cells with well spread chromosomes of each male and female were selected and photographed. the length of short arm chromosome (ls) and long arm chromosome (ll) were measured and the length of total arm chromosome (lt, lt = ls+ll) was calculated. the relative length (rl), the centromeric index (ci), and standard deviation (sd) of rl and ci were estimated (turpin and lejeune 1965; chaiyasut 1989). the ci (q/[p+q]) between 0.500-0.599, 0.6000.699, 0.700-0.899, and 0.900-1.000 were described as metacentric, submetacentric, acrocentric and telocentric chromosomes, respectively. the fundamental number (nf, number of chromosome arms) was obtained by assigning a value of 2 to the metacentric, submetacentric and acrocentric chromosomes and 1 to the telocentric chromosome. all data were used in karyotyping and idiograming. results and discussion diploid number and chromosome characteristics karyomorphology of the d. hangseesom and d. siamensis revealed that the diploid chromosome number (2n) is 40 and the d. melanostictus showed 2n=42. the karyotypes of male and female d. hangseesom composed 2 large metacentrics, 2 large telocentrics, 4 medium telocentrics and 32 small telocentrics (table 1 and figures 2a-b, 4a). for the karyotypes of male and female d. siamensis comprised 2 large metacentrics, 6 large telocentrics, 4 medium telocentrics and 28 small telocentrics (table 2 and figures 2c-d, 4b). though, both sexs of d. melanostictus’s karyotypes consisted 2 large acrocentrics, 12 large telocentrics, 4 medium telocentric and 24 small telocentrics (table 3 and figures 2e-f, 4c). all three dixonius species exhibit no sex differences in karyotypes between males and females (figures 2a-f ). the chromosomal characteristic of d. hangseesom and d. siamensis showed more the closed relationship than d. melanostictus. karyotypes of the d. hangseesom and d. siamensis revealed the same of diploid number and chromosome type, which has only different chromosome size. their karyotype formulas is as follows: 2n=40=lm2 + lt2 + mt4 + st32 (d. hangseesom) 2n=40=lm2 + lt6 + mt4 + st28 (d. siamensis) 2n=42=la2 + lt12 + mt4 + st24 (d. melanostictus). the chromosome character of d. hangseesom (2n=40) and d. melanostictus (2n=42) from this study is the first report of both species. however, the diploid result of d. siamensis (2n=40) in this report both showed difference and accordance with d. siamensis in the reported of ota et al. (2001). it is possible that reported by ota et al. (2001) may have studied more than one with the complex species. however, overall of these karyotypes of d. hangseesom, d. siamensis and d. melanostictus resemble to other gekkonid, which comprised many gradient mono-armed (telocentric) and few bi-armed chromosomes (meta-, submetaor acrocentric). proximity of chromosome number and karyotype feature within genus dixonius represents a close evolutionary line in the group. nucleolar organizer regions and haploid number this study is the first report of nucleolar organizer regions in d. hangseesom, d. siamensis and d. melanostictus. in both sexs of the d. hangseesom and d. siamensis, we found the clearly observable nors on the region 104 isara patawang et al. adjacent to subcentromeric of the telocentric chromosome pair 13th (figures 3a-d, 4a-b). whereas, the nors of male and female d. melanostictus are situated on the subtelomeric region of telocentric chromosome pair 8th (figures 3e-f, 4c). the nors characteristic of d. hangseesom and d. siamensis showed more the closed relationship than d. melanostictus. position of nucleolar organizer regions of the d. hangseesom and d. siamensis revealed the same located, subcentromeric of telocentric chromosome pair 13th. compared with other geckos, most showed two nors appearing near terminal region (centromere or telomere) of small bi-armed or small mono-armed chromosome. an example of the previous reports of the geckos’ nor localization included in the genus cyrtodactylus (thongnetr et al. 2019, 2021), gehyra (king 1983), gekko (chen et al. 1986; shibaike et al. 2009; patawang et al. 2014), hemidactylus (patawang and tanomtong 2015), and lepidodactylus (trifonov et al. 2015). these previous studies showed the nor appearing near terminal region of one homologous small chromosome. the metaphase i (meiosis i, reductional division) was found which can be defined as the 20 bivalents for d. hangseesom (figure 5a) and d. siamensis (figure 5c), and 21 bivalents for d. melanostictus (figure 5e). no metaphase i cells with partially paired bivalents, which are speculated to be male heteromorphic sex chromosomes in these three dixonius species. moreover, haploid chromosome of n=20 in d. hangseesom (figure 5b), n=20 in d. siamensis (figure 5d) and n=21 in d. melanostictus (figure 5f ) were found at metaphase ii (meiosis ii, equational division) of spermatid cells. for these results, behavior and number of chromosomes in metaphase i and metaphase ii confirmed of each other’s accuracy and also verified the accuracy of diploid chromosome in somatic cells. an overview of dixonius chromosomal feature and their chromosome evolution gekkonid chromosome that has been reported in the past, most species show the gradient karyotype, which comprising of many mono-armed chromosomes and few bi-armed chromosomes. present results of d. hangseesom, d. siamensis and d. melanostictus agree with chromosomal evolution line hypothesis within the gekkonid group. the karyotype of these three dixonius showed the gradient of major telocentric, while just comprised 2 bi-armed chromosomes. these features conform to the table 1. mean length of short arm chromosome (ls), long arm chromosome (ll), length of total chromosomes (lt), centromeric index (ci), relative length (rl) and standard deviation (sd) of ci and rl from 20 metaphases of male and female yellow-tailed leaf-toed gecko (dixonius hangseesom) 2n=40. chr pair ls ll lt ci±sd rl±sd chr size chr type 1 6.156 6.718 12.874 0.522±0.024 0.143±0.002 large metacentric 2 0.000 10.305 10.305 1.000±0.000 0.114±0.002 large telocentric 3 0.000 7.204 7.204 1.000±0.000 0.080±0.001 medium telocentric 4 0.000 6.470 6.470 1.000±0.000 0.072±0.003 medium telocentric 5 0.000 5.445 5.445 1.000±0.000 0.060±0.002 small telocentric 6 0.000 4.924 4.924 1.000±0.000 0.055±0.003 small telocentric 7 0.000 4.434 4.434 1.000±0.000 0.049±0.003 small telocentric 8 0.000 4.037 4.037 1.000±0.000 0.045±0.003 small telocentric 9 0.000 3.661 3.661 1.000±0.000 0.040±0.001 small telocentric 10 0.000 3.484 3.484 1.000±0.000 0.039±0.002 small telocentric 11 0.000 3.297 3.297 1.000±0.000 0.037±0.002 small telocentric 12 0.000 3.036 3.050 1.000±0.000 0.034±0.001 small telocentric 13* 0.000 3.050 3.036 1.000±0.000 0.034±0.002 small telocentric 14 0.000 3.007 3.007 1.000±0.000 0.033±0.001 small telocentric 15 0.000 2.724 2.724 1.000±0.000 0.030±0.003 small telocentric 16 0.000 2.661 2.671 1.000±0.000 0.030±0.003 small telocentric 17 0.000 2.671 2.661 1.000±0.000 0.029±0.003 small telocentric 18 0.000 2.434 2.434 1.000±0.000 0.027±0.001 small telocentric 19 0.000 2.273 2.273 1.000±0.000 0.025±0.001 small telocentric 20 0.000 2.239 2.239 1.000±0.000 0.025±0.002 small telocentric abbreviations: chr, chromosome; *, nors bearing chromosomes. 105chromosomal description of three dixonius (squamata, gekkonidae) from thailand hypothesis of rearrangement from ancestral karyotype by robertsonian fusions, fissions or pericentric inversions (gorman 1973; king 1987). in this study, from all chromosome characters by conventional giemsa’s staining and ag-nor banding techniques, we suggest that the chromosome of dixonius showed a high genetic conservation and there were only a few changes at the chromosome structure level. however, the chromosome of d. hangseesom and d. siamensis showed more the closed evolutionary relationship than d. melanostictus. acknowledgements this research was financially supported by sharing from the thailand science research and innovation (tsri) 2021, mahasarakham university and the unit of excellence 2022 on biodiversity and natural resources management, university of phayao (ff65-uoe003). the project was approved by the institute of animals for scientific purpose development of national research council of thailand (resolution u1-02740-2559). figure 2. metaphase chromosome plates and karyotypes using conventional giemsa’s staining of male (a) and female (b) d. hangseesom (2n=40); male (c) and female (d) d. siamensis (2n=40); and male (e) and female (f ) d. melanostictus (2n=42). scale bars indicate 10 micrometers. figure 3. metaphase chromosome plates and karyotypes using agnor staining of male (a) and female (b) d. hangseesom (2n=40); male (c) and female (d) d. siamensis (2n=40); and male (e) and female (f ) d. melanostictus (2n=42). arrows indicate nucleolar organizer regions (nors) and scale bars indicate 10 micrometers. 106 isara patawang et al. table 2. mean length of short arm chromosome (ls), long arm chromosome (ll), length of total chromosomes (lt), centromeric index (ci), relative length (rl) and standard deviation (sd) of ci and rl from 20 metaphases of male and female siamese leaf-toed gecko (dixonius siamensis), 2n=40. chr pair ls ll lt ci±sd rl±sd chr size chr type 1 5.925 6.102 12.027 0.507±0.032 0.125±0.002 large metacentric 2 0.000 10.928 10.928 1.000±0.000 0.113±0.001 large telocentric 3 0.000 8.012 8.012 1.000±0.000 0.083±0.002 large telocentric 4 0.000 7.072 7.072 1.000±0.000 0.073±0.003 large telocentric 5 0.000 6.821 6.821 1.000±0.000 0.071±0.003 medium telocentric 6 0.000 6.121 6.121 1.000±0.000 0.064±0.002 medium telocentric 7 0.000 5.814 5.814 1.000±0.000 0.060±0.002 small telocentric 8 0.000 5.214 5.214 1.000±0.000 0.054±0.001 small telocentric 9 0.000 4.012 4.012 1.000±0.000 0.042±0.002 small telocentric 10 0.000 3.628 3.628 1.000±0.000 0.038±0.003 small telocentric 11 0.000 3.421 3.421 1.000±0.000 0.036±0.002 small telocentric 12 0.000 3.214 3.214 1.000±0.000 0.033±0.001 small telocentric 13* 0.000 3.042 3.042 1.000±0.000 0.032±0.001 small telocentric 14 0.000 2.988 2.988 1.000±0.000 0.031±0.002 small telocentric 15 0.000 2.756 2.756 1.000±0.000 0.029±0.003 small telocentric 16 0.000 2.524 2.524 1.000±0.000 0.026±0.003 small telocentric 17 0.000 2.326 2.326 1.000±0.000 0.024±0.001 small telocentric 18 0.000 2.214 2.214 1.000±0.000 0.023±0.001 small telocentric 19 0.000 2.112 2.112 1.000±0.000 0.022±0.002 small telocentric 20 0.000 2.042 2.042 1.000±0.000 0.021±0.002 small telocentric abbreviations: chr, chromosome; *, nors bearing chromosomes. table 3. mean length of short arm chromosome (ls), long arm chromosome (ll), length of total chromosomes (lt), centromeric index (ci), relative length (rl) and standard deviation (sd) of ci and rl from 20 metaphases of male and female dark-sides ground gecko (dixonius melanostictus), 2n=42. chr pair ls ll lt ci±sd rl±sd chr size chr type 1 2.019 5.945 7.964 0.746±0.029 0.101±0.002 large acrocentric 2 0.000 7.363 7.363 1.000±0.000 0.094±0.002 large telocentric 3 0.000 7.241 7.241 1.000±0.000 0.092±0.001 large telocentric 4 0.000 7.026 7.026 1.000±0.000 0.089±0.003 large telocentric 5 0.000 6.736 6.736 1.000±0.000 0.086±0.003 large telocentric 6 0.000 6.159 6.159 1.000±0.000 0.078±0.002 large telocentric 7 0.000 4.739 4.739 1.000±0.000 0.060±0.002 large telocentric 8* 0.000 4.506 4.506 1.000±0.000 0.057±0.001 medium telocentric 9 0.000 4.102 4.102 1.000±0.000 0.052±0.003 medium telocentric 10 0.000 3.824 3.824 1.000±0.000 0.049±0.002 small telocentric 11 0.000 3.256 3.256 1.000±0.000 0.041±0.003 small telocentric 12 0.000 2.109 2.109 1.000±0.000 0.027±0.003 small telocentric 13 0.000 2.047 2.047 1.000±0.000 0.026±0.001 small telocentric 14 0.000 2.024 2.024 1.000±0.000 0.026±0.001 small telocentric 15 0.000 1.812 1.812 1.000±0.000 0.023±0.001 small telocentric 16 0.000 1.556 1.556 1.000±0.000 0.020±0.002 small telocentric 17 0.000 1.508 1.508 1.000±0.000 0.019±0.003 small telocentric 18 0.000 1.375 1.375 1.000±0.000 0.017±0.002 small telocentric 19 0.000 1.130 1.130 1.000±0.000 0.014±0.002 small telocentric 20 0.000 1.062 1.062 1.000±0.000 0.014±0.002 small telocentric 21 0.000 1.057 1.057 1.000±0.000 0.013±0.001 small telocentric abbreviations: chr, chromosome; 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21 haploid chromosomes, f ). scale bars indicate 5 micrometers. caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 2 2022 firenze university press cytogenetic studies of six species in family araceae from thailand piyaporn saensouk1, surapon saensouk2,*, rattanavalee senavongse2 effect of ag nanoparticles on morphological and physio-biochemical traits of the medicinal plant stevia rebaudiana sherzad r. abdull, sahar h. rashid*, bakhtiar s. ghafoor, barzan s. khdhir morphometric analysis and genetic diversity in hypericum l. using sequence related amplified polymorphism wei cao1, xiao chen2,*, zhiwei cao3 population differentiation and gene flow of salicornia persica akhani (chenopodiaceae) xiaoju zhang1, li bai2,*, somayeh esfandani-bozchaloyi3 scot molecular markers are efficient in genetic fingerprinting of pomegranate (punica granatum l.) cultivars shiva shahsavari1, zahra noormohammadi1,*, masoud sheidai2,*, farah farahani3, mohammad reza vazifeshenas4 first record of nucleus migration in premeiotic antherial cells of saccharum spontaneum l. (poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(1): 29-39, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1419 caryologia international journal of cytology, cytosystematics and cytogenetics citation: tao shu, chao li, chen she, huan-ping zhao (2022) morphometric analysis and genetic diversity in glaucium (papaveraceae) using sequence related amplified polymorphism. caryologia 75(1): 29-39. doi: 10.36253/caryologia-1419 received: september 29, 2021 accepted: january 25, 2022 published: july 6, 2022 copyright: © 2022 tao shu, chao li, chen she, huan-ping zhao. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. morphometric analysis and genetic diversity in glaucium (papaveraceae) using sequence related amplified polymorphism tao shu1,*, chao li2,+, chen she3,*, huan-ping zhao4 1 modern information technology center, sichuan vocational and technical college, suining 629000, china 2 school of information engineering and automation, kunming university of science and technology, kunming, 650500,china 3 school of economics and management, tiangong university, tianjin, 300387, china 4 school of computer and software, nanyang institute of technology, henan 473004, china +corresponding author. e-mail: super123sb@163.com *co-first author abstract. glaucium belongs to the papaveraceae family. glaucium is a genus of annual, biennial, and perennial herbaceous plants that thrive on salty soils and near the sea. glaucium is represented by a total of 10 taxa in iran. sequence-related amplified polymorphism was used to estimate genetic diversity. a combination of morphological and genomic data was used to identify genetic diversity and species features in glaucium species. in eight provinces, 65 people connected to five glaucium were gathered. through polymerase chain reaction (pcr) amplification of five glaucium species, a total of 144 (number of total loci) (ntl) dna bands were obtained. these bands were created by combining 10 different selective primers. the total number of amplified fragments varied from seven to twenty-six. the expected unbiased heterozygozity (h) ranged from 0.19 (g. grandiflorum subsp. grandiflorum var. grandiflorum) to 0.33 (g. grandiflorum subsp. grandiflorum var. grandiflorum) (g. oxylobum var. oxylobum). the genetic similarities between five species range from 0.63 to 0.88. the findings of clustering revealed two large groupings. the srap (sequence-related amplified polymorphism) markers study revealed that g. grandiflorum and g. oxylobum var. oxylobum had the least similarity. this investigation also discovered a substantial indication of distance isolation (mantel test results). the current findings indicate that sequencerelated amplified polymorphism can discover and understand genetic affinity in glaucium species. the current findings have consequences for biodiversity and conservation efforts. aside from that, the current findings may pave the way for identifying acceptable ecotypes for grazing and pasture uses in iran. keywords: population structure, gene flow, network, genetic admixture. 30 tao shu, chao li, chen she, huan-ping zhao introduction: srap (sequence-related amplified polymorphism) is a pcr-based marker system (gondal et al 2021; dadzie et al 2021; chimwamurombe et al 2020; abeshu & zewdu 2020). it is one of the most efficient and straightforward marker systems for studying gene mapping and gene tagging in plant species (si et al, 2020; sun et al, 2021; sun and khayatnezhad 2021; tao et al., 2021; wang et al., 2021), and srap are potential markers for plant systematics and genetic diversity studies (robarts and wolfe 2014 khayatnezhad and gholamin 2021; gholamin and khayatnezhad 2020; 2021; guo et al., 2021). poppy family (papaveraceae) comprises of approximately 26 to 42 genera and 690 to 800 species in the world (judd et al., 1999). the members of papaveraceae are shrub, herbaceous perennials and annuals distributed in the temperate and the subtropical regions of the world. among five genera of family papaveraceae in iran, glaucium, hypecoum, chelidonium and roemeria consist of 10, 1, 1 and 2 species, respectively (rechinger and cullen, 1966). glaucium is found mostly in atlantic europe and central asia (kaderiet 1993). the genus is divided into two sections, each containing four species, four subspecies, and two varieties: sect. acropetala mory has four species, four subspecies, two varieties and sects. glaucium, which has 19 species, eight subspecies, and 16 variants (mory 1979). it was represented by 11 (cullen 1966) to 13 in iran (mobayen 1985; gran and sharifnia 2008). morover, mobayen (1985) introduced two subspecies g. fimbrilligerum boiss. subsp. annuum and g. fimbrilligerum subsp. ophyocarpum. azizian and alishahi norani (1997) studied anatomical characteristics of fruit and blade with emphasis on latex tubes in species of glaucium. furthermore, carlquist and hoekman (1985) studied anatomical structure of wood in romneya and dendromecon. carlquist and zona (1988) continued his studies in cooperation with zona on structure of wood in papaveraceae. some anatomical features of midrib and fruit of glaucium are of diagnostic value (solereder, 1908; metcalfe and chalk, 1950). several taxonomic investigations have demonstrated that seed and trichome micromorphology may be used for taxonomic categorization and delimitation at all taxonomic levels and across plant families (ma et al., 2021a; 2021b; peng et al., 2021; ren et al., 2021). arabi et al., 2017; tavakkoli and assadi, 2016). gran and sharifnia also researched the seed ornamentations of 14 glaucium species in iran (2008). light microscopy (lm) and scanning electron microscopy (sem) was used to examine the seeds and trichomes of 15 species of the genus glaucium found in iran (tavakkoli and assadi 2019). the seeds are semicircular to reniform in shape. however reniform and elongated reniform seeds have been identified in g. oxylobum and g. elegans, respectively. the most common types of testa surface sculpturing include verrucate–rugulate, verrucate–granulate, verrucate–perforate, verrucate–lineolate, rugulate–granulate, rugulate, and ocellate. their findings reveal that the micro-morphological properties of seed and ovary trichomes give important and substantial information for species and taxa within species separation, as well as a diagnostic key to the taxa. glaucium taxa were studied in terms of morphological, palynological, and phylogenetic characteristics, according to fatma mungan kiliç et al. (2019). their findings reveal that several of these features change across species, particularly in micromorphology and the development of clades in phylogenetic trees based on matk and its3-6 dna sequence data. the genus glaucium of turkey was separated into subsections glabrousae and pubescentae based on dna investigations backed by morphological evidence (stem trichomes). the present study investigated the molecular variation of five species in iran. objectives of the study were; a) to estimate genetic diversity; b) to evaluate population relationships using ward approaches. there are consequences for breeding and conservation initiatives based on current findings. materials and methods: plants collection sixty-five (65) individuals were sampled. five glaucium species in west azerbaijan, mazandaran, hamadan, kurdistan, esfahan, semnan, khorasan and razavi khorasan provinces of iran were selected and sampled during may-august 2014-2020 (table 1). morphometric and srap analyses on sixty five plant accessions were carried out. based on additional eco-geographic criteria, five to twelve samples from each population belonging to five distinct species were chosen. five samples were stored at 20 °c till further use. detailed information about locations of samples and geographical distribution of species are mentioned (table 1 and figure 1). morphological studies each species was subjected to morphometric analysis and twelve samples per species were processed. qualitative (12) and quantitative (14) morphological characters 31morphometric analysis and genetic diversity in glaucium using sequence related amplified polymorphism table 1. list of the investigated taxa including origin of voucher specimens. taxa locality latitude longitude altitude(m) g. fimbrilligerum boiss. kurdestan, sanandaj 35°19’18.75’’ 46°59’10.194’’ 1538 g. corniculatum var. corniculatum (l.) curtis west-azarbaijan, urumieh, silvana 37.552673 45°4’33.7656’’ 1344 g. oxylobum var. oxylobum boiss. & buhse kurdestan, sanandaj 38°22’18” 46°37’10” 1523 g. grandiflorum subsp. grandiflorum var. grandiflorum boiss. & a.huet a.huet semnan, 20km nw of shahrud 36°25’14” 54°15’32” 1345 g. contortuplicatum var. cantortuplicatum boiss. mazandaran, 40 km tonekabon to janat abad 35°46’56” 51°23’29” 2383 figure 1. provinces and collection sites of glaucium species. 32 tao shu, chao li, chen she, huan-ping zhao were studied. data were transformed before calculation. different morphological characters of flowers, leaves, and seeds were studied. ordination analyses were conducted while using euclidean distance (podani 2000). sequence-related amplified polymorphism method one to twelve plants’ worth of fresh leaves were utilized at random. silica gel powder was used to dry them. following the prior technique, the dna was extracted (esfandani-bozchaloyi et al. 2019). according to the protocol, we ran the srap assays (li and quiros 2001). ten srap were employed with various primer combinations (table 2). single primers, 20 ng of genomic dna, and 3 u of taq dna polymerase (bioron, germany) were used in 25l of tris-hcl buffer at ph 8; 50 mm of kcl; 1.5 mm of mgcl2; 10 mm of tris-hcl buffer at ph 8 and 3 u taq dna polymerase (bioron, germany) were used in pcr reactions. the total volume of the reaction was 25 l. a techne thermocycler was used for this pcr experiment (germany). data analyses to evaluate morphological characteristics, the upgma (unweighted paired group using average) ordination approach was used. to analyze morphological differences across species, an anova (analysis of variance) was used. to find variable morphological features in glaucium species, principal component analysis (pca) was used. past software version 2.17 was used to conduct multivariate statistical studies, often known as pc analysis (hammer et al. 2001). molecular analyses sequence-related amplified polymorphism (srap) bands were recorded. presence and absence of bands were scored present (1) and absent (0), respectively. total loci (ntl) and the number of polymorphism loci (npl) for each primer were calculated. mantet test was performed with 5000 permutations in past, version 2.17 (hammer et al. 2001). comparing genetic divergence or genetic distances, as assessed by pairwise fst and related statistics, with geographical distances, as evaluated by the mantel test, is one of the most used tools for examining spatial dynamics driving population structure. the mantel test, as originally formulated in 1967, where gij and dij are, are the genetic and geographical distances between populations i and j, respectively. respectively, the genetic and geo-graphic distances between populations i and j, considering populations. because zm is is defined as the sum of product distances, its value is affected by the number of populations analyzed as well as the size of their distances. the zm-value may be compared to a null distribution, and mantel initially advocated using the standard normal deviation (snd), which is defined as snd =zm/var(zm)1/2 (mantel 1967). past ver. 2.17 (hammer et al. 2012) and darwin ver. 5 (2012) software were used for these investigations. the amova (analysis of molecular variance) test (with 1000 permutations) created in genalex 6.4 4 (peakall and smouse 2006) was used to reveal genetic differences across the populations. results morphometery the anova findings showed substantial differences (p<0.01) between the species in terms of quantitative morphological characteristics. principal component analysis results explained 68% cumulative variation. the first pca axis accounted for 59% of the overall variance. the highest correlation (> 0.7) was shown by morphological characters such as calyx length, calyx width, corolla length, corolla color. the morphological characters of glaucium species are shown in pcoa plot (figure 2). each species formed separate groups based on morphological characters. the morphometric analysis showed clear difference among glaucium species and separated each groups. species identification and genetic diversity ten (10) suitable primer combinations (pcs), out of 25 pcs were screened in this research. figure 3 illustrates the banding pattern of em2-me4, em3-me1 and em5-me1 primer by the srap marker profile. one hundered and thirty six (136) amplified polymorphic bands (number of polymorphic loci) were produced. these bands (fragments) had different range i.e. 150bp to 3000 bp. maximum and minimum numbers of polymorphic bands were 22 for em2-me4 and 7 em5-me2, respectively. each primer produced 13 polymorphic bands on average. the pic ranged from 0.14 (em4-me1) to 0.63 (em1-me4) for the 10 srap primers, with an average of 0.42 for each primer the primers’ rp varied from 12.24 (em3-me4) to 56.55 (em3-me1), with an average of 32.25. (figure 3, table 2). 33morphometric analysis and genetic diversity in glaucium using sequence related amplified polymorphism the calculated genetic parameters of glaucium species are shown (table 3). the unbiased heterozygosity (h) varied between 0.19 (g. grandiflorum subsp. grandiflorum var. grandiflorum) and 0.33 (g. oxylobum var. oxylobum) with a mean of 0.28. shannon’s information index (i) was maximum in g. grandiflorum subsp. grandiflorum var. grandiflorum (0.444), where as we recorded minimum shannon’s information index in g. oxylobum var. oxylobum (0.231). the observed number of alleles (na) ranged from 0.22 in g. oxylobum var. oxylobum to 1.445 in g. corniculatum var. corniculatum. the significant number of alleles (ne) ranged from 1.029 (g. grandiflorum subsp. grandiflorum var. grandiflorum) to 1.88 (g. oxylobum var. oxylobum). molecular variance analysis reveals a substantial genetic difference (p = 0.01) between glaucium species. the bulk of genetic diversity was found between species. figure 2. morphological characters analysis of glaucium species by pca plot. figure 3. electrophoresis gel of studied ecotypes from dna fragments produced by srap profile with primer em2-me4. 34 tao shu, chao li, chen she, huan-ping zhao analysis of molecular variance results amova findings revealed that 77% of the total variation was between species and comparatively less genetic variation was recorded at the species level (table 4). genetic difference between glaucium species was highlighted by genetic statistics (nei’s gst), as evident by significant p values i.e. nei’s gst (0.699, p = 0.01) and d_est values (0.196, p = 0.01) because several clustering and ordination approaches yielded comparable findings, nj clustering is provided here (figure 4). plant samples from each species, which belong to a different part, were grouped together and created a single cluster. this finding indicates that the molecular characteristics analyzed may separate glaucium species into two primary clusters or groupings. we found no transitional forms among the specimens analyzed. in general, two large clusters emerged in the nj tree (figure 4), populations g. fimbrilligerum; g. contortuplicatum and g. oxylobum were put in the first main cluster and were separated from the other species by a large distance. the second major cluster included two sub-clusters. plants of g. corniculatum var. corniculatum comprised the first sub-cluster, while plants of g. grandiflorum subsp. grandiflorum var. grandiflorum formed the second sub-cluster. we detected strong correlation between geographical and genetic distances (r = 0.29, p=0.0002) and gene flow (nm) score of 0.388 was reported among species. detailed information about genetic distances and genetic identity (nei’s) are described (supplementary table). the results indicated that g. oxylobum var. oxylobum and g. fimbrilligerum had the greatest degree of genetic similarity (0.88). on the contrary to this, g. grandiflorum and g. oxylobum var. oxylobum (0.63) had lowest genetic resemblance. to determine the ideal number of genetic groups, we used structure analysis followed by the evanno test. in the species analyzed, we employed the admixture model to show interspecific gene flow or / and ancestrally shared alleles. according to pseudo-f, k-means clustering yielded k = 5 and bic yielded k = 3. k = 5 is contable 2. srap primer information and results. primer name ntla nplb pc picd rpe em1-me1 10 8 94.31% 0.33 23.77 em2-me2 17 17 100.00% 0.26 39.77 em1-me4 11 10 96.4% 0.63 20.46 em2-me4 22 22 100.00% 0.29 13.76 em2-me5 9 9 100.00% 0.34 40.99 em3-me4 13 13 100.00% 0.51 12.24 em3-me1 26 18 73.00% 0.20 56.55 em4-me1 11 11 100.00% 0.14 34.23 em5-me1 15 15 100.00% 0.57 48.55 em5-me2 7 7 100.00% 0.45 19.65 mean 15 13 92.00% 0.42 32.25 total 144 136 322.99 a: number of total loci (ntl); b: number of polymorphic loci (npl); c: polymorphic ratio(p %); d: polymorphic information content (pic); e: resolving power (rp). table 3. genetic diversity parameters in the studied glaucium species. sp n na ne i he uhe %p g. fimbrilligerum 16.000 0.113 1.099 0.292 0.27 0.32 48.23% g. corniculatum var. corniculatum 12.000 1.445 1.190 0.271 0.284 0.292 55.91% g. oxylobum var. oxylobum 12.000 0.228 1.880 0.444 0.40 0.33 66.50% g. grandiflorum subsp. grandiflorum var. grandiflorum 10.000 0.288 1.029 0.231 0.17 0.19 44.38% g. contortuplicatum var. cantortuplicatum 15.000 0.772 1.095 0.288 0.35 0.27 62.05% abbreviations: (n = number of samples, na= number of different alleles; i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations). 35morphometric analysis and genetic diversity in glaucium using sequence related amplified polymorphism sistent with the nj grouping and amova. k = 5 indicates the existence of five genetic groups. the evanno test on structure analysis yielded a similar result, with a large peak at k = 5. the organization plot (fig. 5, 6) revealed further information about the genetic structure of the species analyzed, as well as common ancestral alleles and/or gene flow among glaucium species. this plot demonstrated the genetic difference between species 1 and 2 (which were colored differently), as well as 3 and 4, 5. this is consistent with the neighbor joining dendrogram that was previously provided. the other species’ allele compositions are diverse, and they vary genetically from one another. the low nm value (0.388) indicates limited gene flow or ancestrally shared alleles between the species studied and supports genetic stratification as indicated by k-means and structure analyses. population assignment test also agreed with nm result and could not identify significant gene flow among members of the studied species. discussion we employed morphological and molecular (srap) data to determine species relationships in glaucium spefigure 4. dendrograms of glaucium species. figure 5. evanno’s test of srap data in glaucium populations studied. figure 6. structure plot of srap data in glaucium populations studied. 36 tao shu, chao li, chen she, huan-ping zhao cies in this work. morphological analyses of glaucium species showed that quantitative indicators (anova test results) and qualitative characteristics are well differentiated from each other. pca analysis suggests that morphological characters such as corolla color, pedicel hair, stem hair, leaf hair, petiole hair, width of petal have the potentials to identify and delimitate glaucium species. principal component analysis results suggests the utilization of morphological characters to identify and delimitate glaucium species. morphological characters including corolla color, the pedicel hair, the stem hair, the leaf hair, the petiole hair,width of petal play key role in plant systematics and taxonomy. our work also highlighted the significance of morphological characters and molecular data to identify and study species genetic diversity. in general, genetic relationships obtained from srap data coincides with morphometric results. this is in accordance with the parameters of amova and genetic diversity results. srap molecular markers detected clear genetic difference among species. these results indicate that srap have potentials to study plant systematics and taxonomy in glaucium members. given the negative impact of biodiversity threats and overexploitation of glaucium plant species in iran, it is necessary to conduct genetic diversity studies on glaucium species. genetic diversity based studies pave our understanding to develop conservation strategies (esfandani-bozchaloyi et al. 2017). genetic diversity studies are conducted through appropriate selection of primers and indexes including polymorphic information content (pic) and marker index (mi) are important indexes to fathom genetic variation in species (hou et al., 2021; huang et al., 2021). common logic suggests that different makers have different abilities to assess genetic diversity, and usually, genetic diversity is linked with polymorphism (jia et al., 2020; karasakal et al., 2020a; 2020b; khayatnezhad and gholamin 2020a; 2020b). in this research, we reported pic values of srap primers from 0.14 to 0.63, with a mean value of 0.42. pic values indeed show low and high genetic diversity among genotypes. values between zero and 0.25 indicate minimal genetic diversity; values between 0.25 and 0.50 indicate moderate genetic diversity. additionally, values greater than 0.5 are linked with a high level of genetic diversity (tams et al. 2005; wasana et al 2021; hopla et al 2021; fikirie et al 2020). present results highlighted the efficiency of srap markers to estimate genetic diversity in glaucium species. in our study, srap markers detected average percentage of polymorphism (92%). additionally, the current study findings indicated the average pic values of srap makers (0.42) and the average rp (resolving power) values of srap markers (32.25). current research results also described average pic values of srap glaucium species have a lot more markers that show how well they’re doing now than other species have had (maria et al. 2007; dana et al. 2007). these current reported values are higherin the recent study, low gene flow (nm) was detected among glaucium species. the present study also depicted a significant correlation between genetic and geographical distances. our findings revealed that isolation by distance (ibd) existed between glaucium species (mantet test results). several mechanisms, such as isolation, local adaptation, and genetic drift, shape the species or population differentiation (frichot et al. 2013; de kort et al. 2014). the amount of variation in na, ne, h, and i indices showed that there was a lot of genetic variation in glaucium species. the magnitude of variability among dendrogram and principal component analysis results showed clear difference among glaucium species. this shows the high utilization of the srap technique to identify glaucium species. our results have implications for conservation and breeding programs. furthermore, it may identify suitable ecotypes for forage and pasture. there are two possible explanations for why isolated populations don’t have any differences from each other. the first hypothesis said that genetic diversity within and between populations shows how gene flow happens, which led to smaller populations (dostálek et al., 2010). the second hypothesis is that people who live close to each other are better connected through gene flow than people who live far away. the morphological, palynological, and phylogenetic features of ten glaucium taxa were studied (fatma mungan kiliç et al., 2019). a total of 10 although some of the morphological characters of the taxa examined were following the information contained in flora of turkey (cullen 1965), it was noticed that some of their properties were different. in addition, the data yielded from mory’s (1979) study and those yielded as a result of our measurements were compared. in this comparison, the major similarity was observed in terms of the morphotable 4. molecular variance analysis source df ss ms est. var. % φpt among pops 11 1221.364 88.789 12.164 77% 77% within pops 170 114.443 6.88 5.238 23% total 181 1385.807 17.060 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance; φpt: proportion of the total genetic variance among individuals within an accession, (p < 0.001). 37morphometric analysis and genetic diversity in glaucium using sequence related amplified polymorphism logical and palynological characters. in a micromacromorphological study performed by gran and sharifnia (2008) of 18 glaucium taxa, the species g. haussknechtii has been recognized as synonymous with g. grandiflorum based on the analyses of 28 qualitative and 37 quantitative characters. according to fatma mungan kiliç et al (2019) the glaucium taxa were divided into two groups with respect to stem hairs. taxa with pubescence stems were g. corniculatum subsp. corniculatum and g. corniculatum subsp. refractum, g. grandiflorum var. grandiflorum, g. grandiflorum var. torquatum, g. grandiflorum var. haussknechtii and g. secmenii, while the taxa with hairless stems were g. flavum, g. leiocarpum, g. acutidentatum and g. cappadocicum. the findings of phylogenetic analysis revealed that the glaucium taxa were classified into two major clades using matk and its3-6 dna sequences, which is consistent with the hairiness of their stems, petal color, and seed testa outline. the taxa included in these two sub-clades were also compatible with ovary tubercle. acknowledgement funding: the science and technology research project of henan province(no: 142102210554). references abeshu, y., zewdu, a. 2020. developing calibration model for prediction of malt barley genotypes quality traits using fourier transform near infrared spectroscopy. agriculture and food sciences research, 7(1), 38-45. arabi, z. et al. 2017. seed micromorphology and its systematic significance in tribe alsineae (caryophyllaceae). flora 234: 41-59. azizian, d. and alishahi norani, f. 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management. wasana, w.l.n., ariyawansha, r., basnayake, b. 2021. development of an effective biocatalyzed organic fertilizer derived from gliricidia sepium stem biochar. current research in agricultural sciences, 8(1), 11–30. yeh fc, yang r, boyle t (1999). popgene. microsoft windows-based freeware for population genetic analysis. release 1.31. university of alberta, 1-31. caryologia. international journal of cytology, cytosystematics and cytogenetics 72(4): 61-67, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-160 citation: m. alemdag, r.c. ozturk, s.a. sahin, i. altinok (2019) karyotypes of danubian lineage brown trout and their hybrids. caryologia 72(4): 61-67. doi: 10.13128/caryologia-160 published: december 23, 2019 copyright: © 2019 m. alemdag, r.c. ozturk, s.a. sahin, i. altinok. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyotypes of danubian lineage brown trout and their hybrids melike alemdag, rafet cagri ozturk, sebnem atasaral sahin, ilhan altinok* department of fisheries technology engineering, surmene faculty of marine sciences, karadeniz technical university, 61530 surmene, trabzon, turkey *corresponding author: ialtinok@ktu.edu.tr abstract. cytogenetic analysis of brown trout, salmo trutta, have been described for different populations and morphs; however, cytogenetic analysis of interspecific brown trout hybrids is unknown. cultured kidney cells from four brown trout subspecies (salmo trutta abanticus, s.t. caspius, s.t. fario and s.t. labrax) and their reciprocal hybrids were karyotyped using conventional staining, c-banding and ag-nor staining techniques. chromosome number (2n) and chromosome arm number (nf) ranged from76 to 80 and 98 to 102, respectively. silver staining revealed the presence of nor sites on the short arm of the submetacentric chromosome. the size and number of nor sites showed uniformity. the presence of heterochromatin on different chromosome arms was confirmed by c-banding. the presence and position of constitutive heterochromatin showed variability among individuals. chromosome structures of purebred brown trout subspecies belonging to the danubian linage and their hybrids were similar, and no distinctive characteristics were observed in any of the species. the results of this study are applicable to the development of improved conservation and management strategies for brown trout. keywords. cytogenetic, karyotype, salmo trutta, ag-nor, c-banding. introduction brown trout, salmo trutta (linnaeus, 1758), is a polymorphic and widespread species. its historic geographic range covers europe, western asia and northern africa. during the past century, salmo trutta have been introduced to different parts of the world, and the range of brown trout has been extended to all continents except antarctica (elliott, 1989). the systematic classification of salmo trutta is plagued by many nomenclatural issues. salmo trutta was once recognized as a polymorphic species with three morphs based on life-history variation: resident trout, lake trout and river trout (ferguson, 2004). mitochondrial dna (mtdna) sequence variation analysis revealed the existence of five major phylogenetic groups, which are believed to have been separated for some 500,000 to 2 million years (bernatchez, 1995). over the years, distinct species or nominal subspecies have 62 melike alemdag et al. been described based on morphological and molecular analysis (kottelat & freyhof, 2007; turan, kottelat, & engin, 2014). however, s. trutta subspecies such as s.t. abanticus, s.t. caspius, s.t. fario and s.t. labrax belonging to danubian lineage have been proved to be a single biological species called salmo trutta. thus, it was recommended that strains should be named according to location, such as abant, caspian, anatolian and black sea (kalayci et al., 2018). interand intraspecific hybridization experiments in fish are often less concerned with identification of the genomic composition than with the evolution of performance and survival (johnson & wright, 1986). morphology and variation in chromosome number have been proven useful in identifying fish populations (phillips, 2005). cytogenetically, the salmo trutta complex is one of the best analyzed salmonid. the karyotype of salmo trutta consists of 80 chromosomes with a fundamental arm number (nf) ranging from 98 to 102 (amaro, abuin, & sanchez, 1996; woznicki, jankun, & luczynski, 1998; woznicki, sanchez, martinez, pardo, & jankun, 2000). although salmo trutta have been subjected to numerous cytogenetic analyses, and karyotypes have been described for different populations and morphs, (caputo, giovannotti, cerioni, splendiani, & olmo, 2009; jankun, 2000; kalbassi, dorafshan, tavakolian, khazab, & abdolhay, 2006; northland-leppe, lam, jara-seguel, & capetillo-arcos, 2009; woznicki, jankun, & luczynski, 1997; woznicki et al., 1998), the chromosome complement of interspecific brown trout hybrids seems to be comparatively less studied (polonis, fujimoto, dobosz, zalewski, & ocalewicz, 2018; ziomek, debowska, hliwa, & ocalewicz, 2016). a cytogenetic characterization of hybrids and parental species would aid in a better understanding of their species status. therefore, the aim of the present study was 1) to determine the chromosomal characteristics of abant trout (s.t. abanticus), black sea trout (s.t. labrax), caspian trout (s.t. caspius), anatolian trout (s.t. fario) and their reciprocal hybrids and 2) to determine if the nf of chromosomes varies among purebred and hybrid trout. materials and methods fish abant, anatolian, black sea and caspian trout were crossed to each other to produce the f1 generation of all possible reciprocal crossing combinations (16 crosstypes) (table 1). after fertilization, each family was separately incubated in a vertical incubator and transferred to a separate flow-through indoor tank after hatching. this study was approved by the institutional animal care and use committee at karadeniz technical university (approval #14/2013). chromosome preparation five fish from each cross-type were used in chromosome analysis (table 1). fish were anaesthetized with ice, and their anterior kidney tissue was sampled on ice. tissue was cut into small pieces and incubated in 1.5 ml of rpmi media supplemented with penicillin g (75 u/ml), fungizone (1.5 μg/ml), gentamycin sulphate (30 μg/ml) and streptomycin sulphate (75 μg/ml) for 24 h at room temperature. supplementing the culture media with antibiotics eliminated any growth of fungi, yeasts, mycoplasma and gram-positive and gram-negative bacteria. after incubation of the tissue with colchicine (0.1%) for 1 h, samples were centrifuged at 1000 x g for 10 min, and the supernatant was removed. pellets were resuspended in 3 ml ice-cold 0.075 mol/l kcl solution, incubated at 4ºc for 30 min and then four drops of ice-cold carnoy fixative (methanol: acetic acid, 3:1) were added. samples were centrifuged at 1000 x g for 10 min, and the supernatant was removed. after that, 5 ml of fixative was added to the sample, which was then centrifuged at 1000 x g for 10 min. this step was repeated three times to wash the cells. tissues were transferred to a petri dish with one milliliter of fixative and then cut into small pieces with a surgery blade. slides were placed over boiled table 1. cross-types of fish and their abbreviation, mean length and weight. crosses (female x male) family abbreviation mean length (cm) mean weight (gr) s.t labrax x s.t. labrax ll 18.63±1.41 69.18±5.25 s.t. labrax x s.t. abanticus la 19.70±1.50 71.51±5.31 s.t. labrax x s.t. caspius lc 24.36±1.81 156.0±10.12 s.t. abanticus x s.t. abanticus aa 17.37±1.28 38.84±3.00 s.t. abanticus x s.t. labrax ll 16.45±1.11 48.58±3.41 s.t. abanticus x s.t. caspius la 15.20±1.08 34.78±2.04 s.t. caspius x s.t. labrax lc 15.88±1.12 41.70±3.06 s.t. caspius x s.t. abanticus aa 11.62±0.84 13.67±0.07 s.t. caspius x s.t. caspius ll 12.54±0.92 18.30±1.025 s.t. fario x s.t. fario ff 7.15±0.41 5.11±1.01 s.t. fario x s.t. abanticus fa 6.01±0.28 5.09±0.09 s.t. fario x s.t. caspius fc 5.12±0.17 4.81±0.41 s.t. fario x s.t. labrax fl 6.57±0.65 4.51±0.46 s.t. abanticus x s.t. fario af 7.24±0.47 5.11±1.06 s.t. caspius x s.t. fario cf 5.03±0.21 4.24±0.38 s.t. labrax x s.t. fario lf 7.31±0.58 5.19±0.91 63karyotypes of danubian lineage brown trout and their hybrids water steam, and three drops of cell suspension were dropped onto slides from a height of 30–40 cm. for each fish species, a total of 15 slides were prepared and air dried, and 5 of them were stained with 10% giemsa. the remaining 10 were used for c-banding (5 slides) and agnors analysis as explained below. c-banding was performed according to the method described by sumner (1972), with slight modifications. slides containing the chromosome preparation were treated with 0.2 mol/l hcl solution at 37ºc for 1 h and rinsed with distilled water. washed slides were incubated in 2x ssc (ph 7.0) at 60ºc for 1 h, rinsed with distilled water and finally stained with 10% giemsa for 20 min. silver staining of nucleus organizer regions (agnors) were performed according to the method described by howell and black (1980). two drops of colloidal developer and a single drop of aqueous silver nitrate were dropped onto a slide on which the chromosome preparation was mounted and covered with a cover glass. the slide was incubated at 70ºc until the silverstaining mixture turned a golden-brownish color. the slides were then rinsed with distilled water, air dried and stained with 10% giemsa. metaphase cells were screened with a fully automated karyotyping software system (cytovision ver. 3.92) connected to an olympus light microscope. metaphase cell photos were captured at 100x magnification for further analysis. ten high-quality metaphase spreads from each slide were used in chromosome analysis. image-pro premier (media cybernetics), smarttype 3.1.0.43 (digital scientific, cambridge, uk) and tpsdig2 v2.26 (new york state university, stony brook, usa) were used in karyotyping. the nf value was estimated by counting biarmed (metacentric and submetacentric) and unarmed (acrocentric and subtelocentric) chromosomes and calculated according to the formula given by naran (1997). results the chromosome numbers and structures of four subspecies of brown trout and their cross-types (n = 16) were successfully determined. furthermore, karyogram and chromosome measurement tables were generated. about 500 metaphase plates from 80 individuals were examined. cross-types were karyotyped based on the representative chromosome image (fig. 1) and chromosome arm scale (table 2). diploid chromosome numbers (2n) of all examined cross-types ranged from 76 to 80, but the majority of cross-types had 2n = 80 chromosomes (table 3). the pure breed ll (see table 1 for abbreviation) and the hybrid ca had 76 chromosomes, while cl had 78 chromosomes the nf varied from 96 to 102, the lowest being obtained from cl (96) followed by cc, ll and ca (98) (table 3). metacentric (m), submetacentric (sm) and acrocentric/telocentric (a/t) chromosome numbers varied from 14 to 18, 4 to 8, 2 to 14 and 46 to 56, respectively, among cross-types (table 3). ag-nor staining revealed the presence of one pair of nor sites on the short arm of the sm chromosome in all the analyzed specimens (fig 2). c-banding showed constitutive heterochromatin at the centromeres and arms of most of the chromosomes (fig. 3) and the presence and position of constitutive heterochromatin within cross-types were variable even in pure breeds (fig. 3). c-banding was not discriminative for brown trout subspecies. discussion several cytogenetic methods of chromosome isolation have been developed. the main objective of all such methods is to obtain cells at the metaphase stage by disrupting the cell spindle (pack, 2002). solid tissues and cultured cells, together with colchicine treatment, are the most common sources of samples for the preparation of slides of fish chromosomes. spleen, kidney, liver, gills and scales are the preferred sources of chromosomes. to prepare chromosomes, we first used the solid-tissue technique by harvesting various fish tissues and then empirically tested the colchicine concentration, exposure method (injection and bath) and fixation duration to obtain the most efficient means of chromosome preparation. despite our efforts, we were unable to prepare metaphase plates for all but a couple of samples. with figure 1. karyotype of abant trout salmo t. abanticus (2n=80) stained conventionally with giemsa. metacentric (m), submetacentric (sm), subtelocentric (st), acrocentric and telocentric chromosome (a/t) of cross-types. 64 melike alemdag et al. the cell culture technique as described in the materials and methods section, we were able to obtain numerous well-spread metaphase chromosomes. the solid-tissue technique is applicable to various eukaryotic organisms (kligerman & bloom, 1977), but we favor the culture technique when working with salmonid fish, especially salmo trutta. the ty pical kar yoty pes of all three ecological forms of salmo trutta (2n = 80 and nf = 100 – 102) were found, in agreement with numerous other studies table 2. relative arm lenght (µ), total lenght (µ), arm ratio (p/q) and chromosome type of abant trout. chromosome number (2n) short arm length (p) long arm length(q) total lenght arm ratio (q/p) chromosome type 1 0.12 0.12 0.24 1.00 m 2 0.12 0.12 0.24 1.00 m 3 0.12 0.12 0.24 1.00 m 4 0.12 0.12 0.24 1.00 m 5 0.90 0.90 1.80 1.00 m 6 0.10 0.10 0.20 1.00 m 7 0.80 0.80 1.70 0.89 m 8 0.05 0.12 0.17 2.40 sm 9 0.07 0.13 0.20 1.86 sm 10 0.05 0.10 0.15 2.00 sm 11 0.03 0.12 0.15 4.00 st 12 0.06 0.19 0.25 3.17 st 13 0.02 0.13 0.15 6.50 st 14 0.00 0.22 0.22 ∞ a 15 0.00 0.09 0.09 ∞ a 16 0.00 0.14 0.14 ∞ a 17 0.00 0.12 0.12 ∞ a 18 0.00 0.14 0.14 ∞ a 19 0.00 0.15 0.15 ∞ a 20 0.00 0.11 0.11 ∞ a 21 0.00 0.11 0.11 ∞ a 22 0.00 0.11 0.11 ∞ a 23 0.00 0.11 0.11 ∞ a 24 0.00 0.11 0.11 ∞ a 25 0.00 0.12 0.12 ∞ a 26 0.00 0.10 0.10 ∞ a 27 0.00 0.11 0.11 ∞ a 28 0.00 0.10 0.10 ∞ a 29 0.00 0.08 0.08 ∞ a 30 0.00 0.10 0.10 ∞ a 31 0.00 0.12 0.12 ∞ a 32 0.00 0.12 0.12 ∞ a 33 0.00 0.11 0.11 ∞ a 34 0.00 0.11 0.11 ∞ a 35 0.00 0.07 0.07 ∞ a 36 0.00 0.08 0.08 ∞ a 37 0.00 0.08 0.08 ∞ a 38 0.00 0.10 0.10 ∞ a 39 0.00 0.08 0.08 ∞ a 40 0.00 0.13 0.13 ∞ a table 3. chromosome number (n) fundamental number (nf) and structure [metacentric (m), submetacentric (sm), subtelocentric (st), acrocentric and telocentric chromosome (a/t)] of cross-types. crosstype m sm st a/t n nf aa 14 8 2 56 80 102 cc 14 4 4 58 80 98 ll 16 6 4 50 76 98 ff 14 6 4 56 80 100 ac 16 4 8 52 80 100 al 16 6 2 56 80 102 ca 16 6 6 48 76 98 cl 14 4 8 52 78 96 la 16 4 14 46 80 100 lc 18 4 2 56 80 102 af 18 4 2 56 80 102 fa 16 4 4 56 80 100 fc 18 4 4 54 80 102 cf 16 4 6 54 80 100 lf 16 6 4 54 80 102 fl 16 4 6 54 80 100 figure 2. karyotype of abant trout salmo t. abanticus with silver staining. presence of nor sites on the short arm of the submetacentric chromosome indicated with red ring. 65karyotypes of danubian lineage brown trout and their hybrids (woznicki et al., 1998). this study documented slight karyotype variation among cross-types, with a diploid chromosome number and nf ranging from 76 to 80 and 98 to 102, respectively, while the majority of the crosstypes exhibited 2n = 80, in agreement with previous reports (woznicki et al., 1998). intra-specific variation in both chromosome number and nf was previously documented among different fish species, including brown trout (gjedrem, eggum, & refstie, 1977). intra-specific variation in chromosome numbers in these trout forms and their hybrids suggest centric fusion between acrocentric chromosome pairs during the karyotype evolution of robertsonian translocation. loss of chromosome number due to counting errors and chromosome loss during preparation of slides is within the bounds of possibility (gold & gall, 1975; zenzes & voiculescu, 1975). allopolyploids have genomes from different species; therefore, it is associated with hybridization. allopolyploidy can be occurred in the nature as a results of interspecific or intergeneric hybridizations and offspring holds two different diploid chromosome sets (zhou & gui, 2017). consequence of interhomolog recombination in genomic rearrangements can cause gene losses, and gametic aneuploidy (hollister, 2015). polymorphic nor size is common in fish and particularly in salmonids (gold, 1984; woznicki & jankun, 1994). the nors are commonly located on chromosome pair number 11 in salmo trutta, but multichromosomal nor-site polymorphism and variation in nor size has also been reported (sanchez, martinez, vinas, & bouza, 1990; schmid et al., 1995; zhuo, reed, & phillips, 1995). in our study, the positions of nors showed remarkable uniformity among individuals and cross-types. we could not detect any variation in the size and number of nors. chromosomal characteristics of brown trout hybrids were studied for the first time in the present study. chromosome structures of purebred brown trout subspecies (s.t. abanticus, s.t. caspius, s.t. fario and s.t. labrax) belonging to the danubian linage and their hybrids were similar, and no distinctive characteristic was observed in any of the species. therefore, they should be the same species but different strains. this statement was confirmed by kalayci et al. (2018). they found that s.t. abanticus, s.t. caspius, s.t. fario and s.t. labrax are single biological species which should be called salmo trutta. the results of this study are applicable to the development of improved conservation and management strategies for brown trout. brown trout population in the nature is very low and governmental fisheries agencies are releasing hatchery reared brown trout to the stream or rivers to restore the population. therefore, extra precaution should be should be taken in order to protect local brown trout population genetics acknowledgements this study was funded by the scientific and technological research council of turkey (tubitak: 214o595). disclosure statement the authors declare that they have no conflict of interest references amaro, r., abuin, m., & sanchez, l. 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(1995). hypervariability of ribosomal dna at multiple chromosomal sites in lake trout (salvelinus-namaycush). genome, 38(3), 487-496. doi: 10.1139/g95-064 zhou, l., & gui, j. 2017. natural and artificial polyploids in aquaculture. aquaculture and fisheries 2, 103-111. doi: 10.1016/j.aaf.2017.04.003 ziomek, e., debowska, m., hliwa, p., & ocalewicz, k. (2016). impaired gonadal development in the sea trout (salmo trutta) x atlantic salmon (salmo salar) f1 hybrid females. oceanological and hydrobiological studies, 45(3), 337-343. doi: 10.1515/ohs-2016-0028 substantia an international journal of the history of chemistry vol. 2, n. 1 march 2018 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 75(2): 23-31, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1515 caryologia international journal of cytology, cytosystematics and cytogenetics citation: wei cao, xiao chen, zhiwei cao (2022) morphometric analysis and genetic diversity in hypericum l. using sequence related amplified polymorphism. caryologia 75(2): 23-31. doi: 10.36253/caryologia-1515 received: december 01, 2021 accepted: july 06, 2022 published: september 21, 2022 copyright: © 2022 wei cao, xiao chen, zhiwei cao. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. morphometric analysis and genetic diversity in hypericum l. using sequence related amplified polymorphism wei cao1, xiao chen2,*, zhiwei cao3 1 college of information science and engineering, tongji university, shanghai city, china 2 koal software co., ltd, shanghai city, china 3 the third research institute of the ministry of public security, shanghai city,china *corresponding author. e-mail: ruirui5349@163.com abstract. there are about 484 species of hypericum in the guttiferae family, which includes hypericoideae.  in iran, species of this genus are mainly found in the north, northwest, and center of the country, and they are key contributors to the floral elements of the hyrcanian mountains, irano-turanian, and mediterranean regions (such as the zagros).  medicinal, commercial, and horticultural values are associated with these plants.the genetic diversity was assessed through sequence-related amplified polymorphism. to uncover genetic diversity and species characteristics in hypericum species, were studied through a combination of morphological and molecular data. eighty-five individuals related to 7 hypericum were collected in 6 provinces. a total of 76 (number of total loci) (ntl) dna bands were produced through polymerase chain reaction amplifications (pcr) amplification of seven hypericum species. these bands were produced with the combinations of 5 selective primers. the total number of amplified fragments ranged from 10 to 20. according to the srap (sequence-related amplified polymorphism) markers analysis, h. perforatum and h. asperulum had the lowest similarity. this study also detected a significant signature of isolation by distance (mantel test results). present results showed that sequence-related amplified polymorphism have the potential to identify and decipher genetic affinity in hypericum species. current results have implications in biodiversity and conservation programs. besides this, present results could pave the way for selecting suitable ecotypes for forage and pasture purposes in iran. keywords: sequence-related amplified polymorphism, gene flow, genetic diversity, morphometric analysis, hypericum. introduction: genetic diversity is a basic component of biodiversity and its conservation is essential for survival of any species in the changing environments (si et al. 2020; liu et al. 2021). most authors agree that genetic diversity is necessary to preserve the long-term evolutionary potential of a species (peng et al. 2021; ma et al. 2021). this is very important in fragmented populations, 24 wei cao, xiao chen, zhiwei cao because they are more vulnerable due to the loss of allelic richness and increased population differentiation by genetic drift (decreased heterozygosity and eventual fixation of alleles) and inbreeding depression (increased homozygosity within populations; chen et al. 2021; bi et al. 2021). therefore, understanding the genetic variability and diversity within and among different populations is crucial for their conservation and management (e.g., esfandani-bozchaloyi et al., 2018a, 2018b, 2018c). sequence-related amplified polymorphism (srap) is pcr –based marker system. it is one of the efficient and simple marker systems to study gene mapping and gene tagging in plant species (li and quiros 2001), and srap are potential markers to assess plant systematics and genetic diversity studies (wang et al. 2021; yin et al. 2021; zhao et al. 2021). previously, wu et al. (2010) assessed genetic diversity and population structure in pogostemon cablin  with the aid of srap markers. srap markers were successfully implemented in lamiaceae family to study natural populations and variations within the family. these past studies showed that molecular markers, including srap markers, are efficient to investigate genetic diversity analyses and phylogenetic relationship among hypericum species in guttiferae, hypericoideae family. there are about 484 species of hypericum in the guttiferae family, which includes hypericoideae.  in iran, species of this genus are mainly found in the north, northwest, and center of the country, and they are key contributors to the floral elements of the hyrcanian mountains, irano-turanian, and mediterranean regions (such as the zagros).  medicinal, commercial, and horticultural values are associated with these plants. they prefer steep-sloped rocky and calcareous cliffs, as well as the edges of highland woods (robson 1968; azadi 1999). robson (1968) expanded the flora iranica region by 21 species. h. fursei n. robson and h. dogonbadanicum were described by robson (1977) and assadi (1980), respectively (1984). assadi can only be found in iran’s north and southwestern regions. azadi (1999) identified 19 species in the flora of egypt, four subspecies divided into five sections (campylosporus (spach) r. keller, hypericum, hirtella stef., taeniocarpum jaub. & spach., and drosanthe (spach) endl.) and two doubtful species (h. heterophyllum vent. and h. olivieri) and two doubtful species (h (spach) the term “hofariqun” was used by bo ebn sina (or bo ali sina) to denote hypericum species in iran (rechinger, 1986). st. john’s wort (hypericum perforatum l.) is the most important medicinal species of the genus and its main uses in medicine includes treatment of mild and moderate depression, skin wounds and burns (barnes et al. 2001). the plant contains a vast array of secondary metabolites, among which naphthodianthrones (hypericin and pseudohypericin), acylphloroglucinols (hyperforin and adhyperforin) and essential oil can be mentioned (jia et al. 2020; shi et al. 2021; zheng et al. 2021; zhu et al. 2021). the present study investigated the molecular variation of seven species in iran. objectives of the study were; a) to estimate genetic diversity; b) to evaluate population relationships using nj approaches. current results have implications in breeding and conservation programs. materials and methods plants collection eighty-five (85) individuals were sampled. seven hypericum species in east azerbaijan, esfahan, hamedan, tehran, mazandaran, kermanshah and kohgilouyeboirahmad provinces of iran were selected and sampled during may-august 2014-2020 (table 1). we employed 85 plant accessions (five to twelve samples from each community) from 7 distinct populations with various eco-geographic features for srap analysis, which were sampled and kept in -20 till further use. table 1 provide further information on the geographical distribution of accessions. morphological studies each species was subjected to morphometric analysis and twelve samples per species were processed. qualitative (10) and quantitative (11) morphological characters were studied. data were transformed before calculation. different morphological characters of flowers, leaves, and seeds were studied. ordination analyses were conducted while using euclidean distance (podani 2000). sequence-related amplified polymorphism method in each of the tested populations, fresh leaves were taken at random from one to twelve plants. silica gel powder was used to dry them. to extract genomic dna, the ctab activated charcoal procedure was applied (esfandani-bozchaloyi et al., 2019). srap assay was performed as described previously (li and quiros 2001). five srap in different primer combinations were used (table 2). a 25μl volume containing 10 mm of trishcl buffer at ph 8; 50 mm of kcl; 1.5 mm of mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of 25morphometric analysis and genetic diversity in hypericum l. using sequence related amplified polymorphism single primer; 20 ng of genomic dna and 3 u of taq dna polymerase (bioron, germany) were subjected to pcr reactions. the overall reaction volume consisted of 25 μl. this pcr reaction was carried out in techne thermocycler (germany). the following cycles and programs were observed. the initial denaturation step was performed for 5 minutes at 94°c. the initial denaturation step was followed by 40 cycles for 1 minute at 94°c; 1 minute at 52-57°c, and 2 minutes at 72°c. the reaction was completed by a final extension step of 7-10 min at 72°c. staining was performed with the aid of ethidium bromide. dna bands/fragments were compared against a 100 bp molecular size ladder (fermentas, germany). data analyses morphological characteristics were first normalized (mean = 0, variance = 1) before being utilized to calculate euclidean distance between taxonomic pairs (podani 2000). the upgma (unweighted paired group using average) ordination techniques were utilized to group the plant specimens (podani 2000). molecular analyses sequence-related amplified polymorphism (srap) bands were coded as binary characters (presence = 1, absence = 0). total loci (ntl) and the number of polymorphism loci (npl) for each primer were calculated. furthermore, the polymorphic ratio was assessed based on npl/ntl values. polymorphism information content was calculated as previously suggested by roldan-ruiz et al. (2000). resolving power for individual marker system was calculated as: rp = σib. ib (band informativeness) was estimated while following equation: proposed as: ib= 1 [2 x (0.5-p)]. in the equation, p indicates the presence of bands (prevost and wilkinson, 1999). to quantify the capability of each primer to identify polymorphic loci among the genotypes, two measures, polymorphism information content (pic) and marker index (mi) were utilized to assess its discriminatory ability (powell et al. 1996). the mantel test was used to see whether there was a link between the analyzed populations’ geographical and genetic distances (podani 2000). past ver. 2.17 (hammer et al. 2012) and darwin ver. 5 (2012) software were used to conduct these studies. table 1. voucher details of hypericum species in this study from iran. no section sp. locality sp1 hypericum h. perforatum l. esfahan:, ghameshlou, sanjab sp2 hirtella stef. h. lysimachioides boiss. & noe in boiss. kermanshah, islamabad sp3 h. asperulum jaub. & spach. hamedan, nahavand sp4 h. helianthemoides (spach) boiss. tehran, damavand sp5 h. vermiculare boiss. & hausskn hamedan, alvand sp6 taeniocarpium h. hirsutum l. mazandaran, nowshahr sp7 h. linarioides bosse. azarbaiejan, west of tabriz table 2. srap primer information and results. primer name ntla nplb pc picd rpe em1-me1 25 20 88.22% 0.29 34.71 em2-me2 14 14 100.00% 0.46 32.16 em1-me4 16 13 83.4% 0.35 40.16 em2-me4 13 13 100.00% 0.22 31.30 em2-me5 10 10 100.00% 0.40 49.94 mean 17 16 93.50% 0.33 39.14 total 76 70 198.33 a: number of total loci (ntl). b: number of polymorphic loci (npl). c: polymorphic ratio(p %). d: polymorphic information content (pic). e: resolving power (rp). 26 wei cao, xiao chen, zhiwei cao to reveal genetic differences across the populations, the amova (analysis of molecular variance) test (with 1000 permutations) was utilized, which was implemented in genalex 6.4 (peakall & smouse 2006). gene flow was calculated by i using popgene ver. 1.32 (1997) to calculate nm, an estimate of gene flow from gst, as follows: nm = 0.5(1 gst)/gst. this method takes into account the same amount of gene flow in all populations. gene flow was conducted in popgene software, version 1.32 (yeh et al. 1999). results mophometery the anova findings showed substantial differences (p<0.01) between the species in terms of quantitative morphological characteristics. principal component analysis results explained 77% cumulative variation. the first pca axis explained 43% of the total variation. the highest correlation (> 0.7) was shown by morphological characters such as calyx length, calyx width, corolla length, corolla color. the morphological characters of hypericum species are shown in upgma tree (figure 1). each species formed separate groups based on morphological characters. the morphometric analysis showed clear difference among hypericum species and separated each groups. species identification and genetic diversity five (5) suitable primer combinations (pcs), out of 10 pcs were screened in this research. figure 2 illustrates the banding pattern of em2-me4, em1-me1 and em2-me2 primer by the srap marker profile. seventy (70) amplified polymorphic bands (number of polymorphic loci) were produced. these bands (fragments) had different range i.e. 150bp to 3000 bp. maximum and minimum numbers of polymorphic bands were 20 for em1-me1 and 10 em2-me5, respectively. each primer produced 16 polymorphic bands on average. the pic ranged from 0.22 (em2-me4) to 0.46 (em2-me2) for the 5 srap primers, with an average of 0.33 per primer. rp of the primers ranged from 31.30 (em2-me4) to 49.94 (em2-me5) with an average of 39.14 per primer (figure 2, table 2). the calculated genetic parameters of hypericum species are shown (table 3). the unbiased heterozygosity (h) varied between 0.17 (h. helianthemoides) and 0.32 (h. hirsutum) with a mean of 0.32. shannon’s information index (i) was maximum in h. hirsutum (0.49), where as we recorded minimum shannon’s information index in h. helianthemoides (0.18). the observed number of alleles (na) ranged from 0.113 in h. perforatum to 1.222 in h. lysimachioides. the significant number of alleles (ne) ranged from 1.011 (h. helianthemoides) to 1.190 (h. lysimachioides). analysis of molecular variance results in significant genetic difference (p = 0.01) among hypericum species. the majority of genetic variation occurred among species. amova findings revealed that 75% of the total variation was between species and comparatively less genetic variation was recorded at the species level (table 4). genetic difference between hypericum species was highlighted by genetic statistics (nei’s gst), as evident by significant p values i.e. nei’s gst (0.476, p = 0.01) and d_est values (0.843, p = 0.01) . different clustering and ordination methods produced similar results therefore, ward clustering are presented here (figure 3). in general, plant samples of each species belong to a distinct section, were grouped figure 1. morphological characters analysis of hypericum species by upgma tree. figure 2. electrophoresis gel of studied ecotypes from dna fragments produced by srap profile; 1,8: h. perforatum; 2, 9: h. lysimachioides; 3,10: h. asperulum ; 4, 11: h. helianthemoides; 5,12: h. vermiculare; 6,13: h. hirsutum; 7,14: h. linarioides. 27morphometric analysis and genetic diversity in hypericum l. using sequence related amplified polymorphism together and formed separate cluster. this result show that molecular characters studied can delimit hypericum species in two different major clusters or groups. in the studied specimens we did not encounter intermediate forms. in general, two major clusters were formed in ward tree (figure 3), populations of h. perforatum were placed in the first major cluster and were placed with great distance from the other species. the second major cluster included two sub-clusters. plants of h. hirsutum and h. linarioides comprised the first sub-cluster, while plants of h. lysimachioides, h. asperulum, h. helianthemoides and h. vermiculare formed the second subcluster. we detected strong correlation between geographical and genetic distances (r = 0.88, p=0.0002) and gene flow (nm) score of 0.265 was reported among species. detailed information about genetic distances and genetic identity (nei’s) are described (supplementary table). the findings suggested that there was the highest degree of genetic similarity (0.89) between h. hirsutum and h. linarioides. on the contrary to this, h. perforatum and h. asperulum (0.68) had lowest genetic resemblance. we performed structure analysis followed by the evanno test to identify the optimal number of genetic groups. we used the admixture model to illustrate interspecific gene flow or / and ancestrally shared alleles in the species studied. k-means clustering showed k = 7 according to pseudo-f and k = 5 according to bic. k = 7 is in agreement with ward grouping and amova. k = 7 reveal the presence of 7 genetic group. similar result was obtained by evanno test performed on structure analysis which produced a major peak at k = 7. the structure plot (figure not included) produced more detailed information about the genetic structure of the species studied as well as shared ancestral alleles and / or gene flow among hypericum species. this plot revealed that genetic difference of species 1 and 2 (differently colored), as well as 3 and 4. this is in agreement with neighbor joining dendrogram presented before. the other species are distinct in their allele composition and differed genetically from each other. table 3. genetic diversity parameters in the studied hypericum species. sp n na ne i he uhe %p h. perforatum l. 20.000 0.113 1.099 0.262 0.27 0.22 38.23% h. lysimachioides boiss. & noe in boiss. 17.000 1.222 1.190 0.211 0.284 0.292 25.91% h. asperulum jaub. & spach. 12.000 0.228 1.180 0.414 0.22 0.25 46.50% h. helianthemoides (spach) boiss. 15.000 0.288 1.011 0.181 0.19 0.17 16.11% h. vermiculare boiss. & hausskn 9.000 0.352 1.083 0.27 0.29 0.24 45.05% h. hirsutum l. 8.000 0.333 1.016 0.492 0.33 0.32 48.23% abbreviations: n = number of samples, i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations. table 4. molecular variance analysis. source df ss ms est. var. % φpt among pops 22 1116.114 77.111 24.100 75% 75% within pops 112 55.455 18.27 10.133 25% total 134 1656.127 34.022 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance; φpt: proportion of the total genetic variance among individuals within an accession, (p < 0.001). figure 3. ward tree of srap data revealing species delimitation in the hypericum species. 28 wei cao, xiao chen, zhiwei cao the low nm value (0.265) indicates limited gene flow or ancestrally shared alleles between the species studied and supports genetic stratification as indicated by k-means and structure analyses. population assignment test also agreed with nm result and could not identify significant gene flow among members of the studied species. discussion in the present study, we used morphological and molecular (srap) data to evaluate species relationships in hypericum species. morphological analyses of hypericum species showed that quantitative indicators (anova test results) and qualitative characteristics are well differentiated from each other. pca analysis suggests that morphological characters such as corolla color, pedicel hair, stem hair, leaf hair, petiole hair, width of petal have the potentials to identify and delimitate hypericum species. principal component analysis results suggests the utilization of morphological characters to identify and delimitate hypericum species. morphological characters including corolla color, pedicel hair, stem hair, leaf hair, petiole hair, width of petal play key role in plant systematics and taxonomy. our work also highlighted the significance of morphological characters and molecular data to identify and study species genetic diversity. in general, genetic relationships obtained from srap data coincides with morphometric results. this is in accordance with the parameters of amova and genetic diversity results. srap molecular markers detected clear genetic difference among species. these results indicate that srap have potentials to study plant systematics and taxonomy in hypericum members. given the negative impact of biodiversity threats and overexploitation of hypericum plant species in iran, it is necessary to conduct genetic diversity studies on hypericum species. genetic diversity based studies pave our understanding to develop conservation strategies (esfandani-bozchaloyi et al. 2017a,b,c,d). genetic diversity studies are conducted through appropriate selection of primers and indexes including polymorphic information content (pic) and marker index (mi) are important indexes to fathom genetic variation in species (sivaprakash et al. 2004). common logic suggests that different makers have different abilities to assess genetic diversity, and usually, genetic diversity is linked with polymorphism (sivaprakash et al. 2004). in this research, we reported pic values of srap primers from 0.22 to 0.46, with a mean value of 0.33. pic values indeed show low and high genetic diversity among genotypes. values are ranging from zero to 0.25 show low genetic diversity; in contrast to this, 0.25 to 0.50 highlight mid-level of genetic diversity. in addition to this, values higher than 0.5 are associated with high genetic diversity (tams et al. 2005). present results highlighted the efficiency of srap markers to estimate genetic diversity in hypericum species. in our study, srap markers detected average percentage of polymorphism (93.50%). current research results also described average pic values of srap makers (0.33) and average rp (resolving power) values i.e. 39.14 of srap markers. these current reported values are higher than other reported markers on hypericum species (maria et al. 2007; dana et al. 2007). in the recent study, low gene flow (nm) was detected among hypericum species. the present study also depicted a significant correlation between genetic and geographical distances. our findings revealed that isolation by distance (ibd) existed between hypericum species (mantet test results). several mechanisms, such as isolation, local adaptation, and genetic drift, shape the species or population differentiation (frichot et al. 2013; de kort et al. 2014). the magnitude of variability among na, ne, h, and i indices demonstrated a high level of genetic diversity among hypericum species. dendrogram and principal component analysis results showed clear difference among hypericum species. this shows the high utilization of the srap technique to identify hypericum species . our results have implications for conservation and breeding programs. furthermore, it may identify suitable ecotypes for forage and pasture. a high level of variation among h. perforatum populations was also reported by percifield et al. (2007) which confirms results of the present study. similar results have been reported on this species using the rapd markers by hazler pilepic et al. (2008). the high genetic diversity of h. perforatum populations is as a result of its mating systems. in fact, propagation method(s) of plant species is considered as one of the most important factors determining their levels of genetic diversity (hamrick 1982). self-incompatibility is a wide spread phenomenon in the genus hypericum (robson 1981), resulting in the high levels of genetic variability (borba et al. 2001). furthermore, this perennial plant produces a great number of seeds every year in favor of the high amounts of diversity in this species (zhao et al. 2007). bi et al (2021) were conducted to study hypericum genetic diversity by random amplified polymorphic dna (rapd) from seventy plant specimens. they showed significant differences in quantitative morphological characters in plant species. h. dogonbadanicum depicted unbiased expected heterozygosity (uhe) in the range of 0.10. shannon information was high (0.32) in h. 29morphometric analysis and genetic diversity in hypericum l. using sequence related amplified polymorphism perforaturm. h. dogonbadanicum showed the lowest value, 0.17. the observed number of alleles (na) ranged from 0.22 to 0.53 in h. dogonbadanicum and h. elongaturn. gene flow (nm) was relatively low (0.87) in hypericum. ma et al. (2021) conducted a study in iran on identification of hypericum population through morphological and issr markers. they observed 10 primers produced 141 bands, of which 127 were polymorphic (95.78%). the obtained high average pic and mi values revealed high capacity of issr primers to detect polymorphic loci among hypericum species. the genetic similarities of 17 collections were estimated from 0.617 to 0.911. according to inter-simple sequence repeats (issr) markers analysis, h. androsaemum and h. hirtellum had the lowest similarity and the species of h. perforatum and h. triquetrifolium had the highest similarity. since widespread species may possess the higher levels of genetic diversity than narrowly distributed plants (singh et al. 1998), the wide range of h. perforatum distribution is an important factor in this respect. considering the low level of gene flow rate among studied wild populations of h. perforatum, therefore, genetic drift might be inevitable. in h. perforatum, the low rate of gene flow may be due to factors such as prevailing apomixes and short distance of seed dispersal as stated by hazler pilepic et al. (2008). molecular markers have been used to investigate the genetic diversity, population structure, and reproductive biology of h. perforatum. high among-population variation was previously reported in hypericum species by percifield et al. (2007), pilepić et al. (2008), and farooq et al. (2014). high differentiation among populations is mostly coupled with limited gene flow among them. the low gene flow and the high differentiation among populations has been explained mainly by founder events such as time since colonization (jacquemyn et al., 2004). conclusions the present study investigated the molecular variation of seven species. molecular and morphometric analysis confirmed morphological and genetical difference between hypericum species. this was first attempt to assess genetic diversity through sequence-related amplified polymorphism and morphometrics analysis in iran. current study reported two major clusters. these two major groups were separated on the basis of genetic and morphological characters. the genetic similarities between four species was estimated from 0.68 to 0.89. srap (sequence-related amplified polymorphism) markers analysis, showed that h. perforatum and h. asperulum had the lowest similarity. current study also reported correlation between genetic and geographical distances. this clearly indicated isolation mechanism envloved in the ecology of hypericum species. present results indicated the potential of sequence-related amplified polymorphism to assess genetic diversity and genetic affinitiy among hypericum species. current results have implications in biodiversity and conservation programs. besides this, present results could pave the way for selecting suitable ecotypes for forage and pasture purposes in iran. references azadi, r. 1999: guttiferae. in: assadi, m. 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(poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(3): 159-167, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1560 caryologia international journal of cytology, cytosystematics and cytogenetics citation: masoud sheidai, mohammad mohebi anabat, fahimeh koohdar, zahra noormohammadi (2022). identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach. caryologia 75(3): 159-167. doi: 10.36253/caryologia-1560 received: january 31, 2022 accepted: october 02, 2022 published: april 5, 2023 copyright: © 2022 masoud sheidai, mohammad mohebi anabat, fahimeh koohdar, zahra noormohammadi. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 1 department of plant sciences and biotechnology, faculty of life sciences and biotechnology, shahid beheshti university, tehran, iran 2 department of biology, school of basic science, science and research branch, islamic azad university, tehran, iran *corresponding author. e-mail: msheidai@sbu.ac.ir abstract. the genus crocus l. of iridaceae family contains about 160 species and is considered as a complex group of plant taxa with regard to evolutionary and phylogenetic events. inter-specific hybridization and gene flow contribute to species genetic homogeneity in one hand and high within species genetic variability and species genetic content overlaps caused species resolution a problem. in spite of extensive molecular phylogenetic studies in this genus, nothing is known about dna sequences or single nucleotide polymorphisms (snps) which are of adaptive nature. moreover, nothing is known about which geographical or environmental factors plays role in species local adaptation and speciation events within crocus l. genus. therefore, the present study was conducted to answer the above said questions. we used a combined molecular data set of internal transcribed spacer (its) nuclear gene and trnl-f intergenic spacer (trnl-f) sequences of chloroplast genome. a multiple analytical method of canonical correlation (cca), redundency analysis (rda), and latent factor mixed model (lfmm)identified a few potential adaptive snps. moreover, population criterions like tajimas’ d, molecular clock test, as well as skyline-plot revealed a smooth and continuous genetic changes for most of the crocus species, but the occurrence of a sudden deep nucleotide substitution for crocus taxa of iran. the impact of latitude was significantly higher on nucleotide changes compared to that of longitudinal distribution of crocus species. keywords: crocus, adaptive divergence, snps, speciation. 1. introduction the genus crocus l. (family iridaceae), has about 100 species and contains an economically important species crocus sativus l., the edible saffron. the species of this genus are distributed from western europe and north160 masoud sheidai et al. western africa to western china. though the asia minor is the center of genus diversity (sheidai et al., 2017), many species grow in the mediterranean region (saxena, 2010). several studies were concerned with molecular phylogeny and dna barcoding of this genus which produced valuable information on different molecular aspects of genus. aghighiravan et al. (2019), reported that its barcode is the best molecular marker for phylogenetic investigation on crocus l. genus. similarly, sheidai et al. (2017), reported a high degree of genetic variability both within and among the studied species in the genus and that issr molecular markers are useful in crocus species delineation. along with the species relationships, these authors also reported population fragmentation and inter-specific gene flow in these taxa. in a recent investigation, mohebi et al. (2021), presented both dna barcode and chromosome number variation in the genus. these authors suggested that molecular events like horizontal gene transfer (hgt) and deep coalescence may be associated with geographical distribution and crocus taxa diversification. due to importance of this genus and also lack of knowledge on geographical association of the genetic differences in crocus species, we carried out a detailed bioinformatic analyses of a combined molecular data set of its nuclear dna sequences and trnl-f chloroplast sequences, to : 1identify discriminating nucleotide sequences among crocus species, 2illustrate if these sequences are significantly associated with geographical coordinates, 3 identify nucleotide sequences with phylogenetic importance. for bioinformatic studies, we used different analytical approaches like discriminate analysis of principal components analysis (dapc), which is suitable for snp sequences, as well as both cca (canonical correspondence analysis), and rda (redundancy analysis). moreover, some data on crocus species expansion were also produced by using population genetics analysis methods of tajimas’ d value, molecular clock test, and mismatch nucleotide pair test. the findings of this research are new to crocus science. 2. material and methods in this study, its nuclear dna and trnl-f sequences of 68 crocus species were obtained from national center for bioinformatic information (ncbi). in addition, we used two species of the genus romulea as outgroup taxa because of the high similarity to crocus (goldblatt et al., 2006; petersen et al., 2008) (table 1). 2.1. data analyses sequence alignment and curation was done by muscle program implemented out in molecular evolutionary genetics analysis (mega) 7 program. mismatch analysis and skyline plot was constructed in r package 4.2. these sequences were then used to construct maximum likelihood phylogenetic tree (ml tree), by mega 7 program based on kimura-two parameters distance. the following analyses were performed to identify the snps which show association with geographical coordinates of crocus species distribution. we should mention that these analytical approaches have different assumptions and may differ to some extent in their results. therefore, comparing obtained results are important for drawing a solid conclusion. 2.2. canonical correspondence analysis in the first approach we used cca (canonical correspondence analysis). this method is based on regression of the snps and ecological features and uses an approach similar to principal components analysis (pca), but it is utilized for discrete characteristics like snps (podani, 2000; sheidai et al., 2020). this method differs from pca in the way that, pca tries to maximize the variance of data in a reduced space, while cca tries to maximize the association of data (snps), to ecological features studied (podani, 2000; sheidai et al., 2020). cca was performed in past ver. 4., program. 2.3. latent factor mixed model (lfmm) latent factor mixed model is a method for testing associations between loci and environmental gradients using latent factor mixed models. lfmm implements an mcmc algorithm for regression analysis in which the confounding variables are modeled with unobserved (latent) factors. the program estimates correlations between environmental variables and allelic frequencies, and simultaneously infers the background levels of population structure (frichot et al., 2013, frichot and francois, 2015). lfmm was performed by lfmm package in r. 4.2. 2.4. redundency analysis (rda) redundancy analysis (rda), a form of constrained ordination which is fit for genomic data sets, where we are interested in understanding how the multivariate environment shapes patterns of genomic composition across geographical areas. rda is based on multivariate 161identifying potential adaptive snps within combined dna sequences in genus crocus table 1. the accession numbers and chromosome number of taxa in for the genus crocus and outgroup representatives. number taxa accession number(its) accession number(trnl-f) chromosome number country 1 c. veneris he801061.1 he864222.1 2n= 16 cyprus 2 c. etruscus hg518187.1 hg518216.1 2n= 8 italy 3 c. kosaninii hg518189.1 hg518206.1 2n= 14 serbia 4 c. baytopiorum ls398370.1 lt991646.1 2n= 28 turkey 5 c. scardicus he663961.1 he864166.1 2n= 36 macedonia 6 c. versicolor he801142.1 he864249.1 2n= 26 italy 7 c. malayi he801170.1 he864246.1 2n= 30 croatia 8 c. imperati he801131.1 he864231.1 2n= 26 italy 9 c. minimus he801140.1 he864247.1 2n= 24 italy 10 c. corsicus he801096.1 he864241.1 2n= 18 italy 11 c. cambessedesii he801105.1 he864228.1 2n= 16 spain 12 c. nudiflorus he801146.1 he864253.1 2n= 48 spain 13 c. serotinus he801125.1 he864225.1 2n= 22 portugal 14 c. niveus he801081.1 he864219.1 2n= 28 greece 15 c. goulimyi he801130.1 he864230.1 2n= 12 greece 16 c. ligusticus he801167.1 he864234.1 2n= 24 italy 17 c. kotschyanus he664000.1 he864256.1 2n= 8 turkey 18 c. scharojanii he801135.1 hg518229.1 2n= 8 russia 19 c. vallicola he801168.1 he864238.1 2n= 8 russia 20 c. gilanicus he801172.1 he864255.1 2n= 24 iran 21 c. sativus he801172.1 lt991682.1 2n= 24 iran 22 c. pallasii sub sp. hausknechtii ls398387.1 lt991663.1 2n= 14 iran 23 c. thomasii ls398411.1 lt991688.1 2n= 16 italy 24 c. cartwrightianus ls398376 lt991648.1 2n= 16 greece 25 c. moabiticus ls398392.1 lt991669.1 2n= 14 jordan 26 c. oreocreticus ls398397.1 lt991674.1 2n= 16 greece 27 c. asumaniae ls398366.1 lt991641.1 2n= 26 turkey 28 c. mathewii he801089.1 he864217.1 2n= 70 turkey 29 c. reticulatus lm993447.1 lm993633.1 2n= 10 moldova 30 c. cvijicii lt222444.1 he864276.1 2n= 18,20,22 albania 31 c. dalmaticus he801137.1 he864242.1 2n= 24 croatia 32 c. sieberi subsp. sieberi he663966.1 he864171.1 2n= 22 greece 33 c. robertianus he801134.1 he864236.1 2n= 20 greece 34 c. cancellatus subsp. pamphylicus he801128.1 he864229.1 2n= 12 turkey 35 c. hermoneus he801163.1 he864268.1 2n= 12 jordan 36 c. abantensis he664019.1 he864239.1 2n= 8,16 turkey 37 c. angustifolius he801136.1 lm993589.1 2n= 20 russia 38 c. ancyrensis he663987.1 lm993597.1 2n= 10 turkey 39 c. gargaricus sub sp. gargaricus he801138.1 he864243.1 2n= 30 turkey 40 c. sieheanus he801157.1 he864263.1 2n= 16 turkey 41 c. rujanensis lt222441.1 he864280.1 2n= 22 serbia 42 c. biflorus sub sp. biflorus he801121.1 he864220.1 2n= 8 italy 43 c. almehensis he801162.1 he864271.1 2n= 20 iran 44 c. danfordiae he664007.1 he864201.1 2n= 8 turkey 45 c. pestalozzae he801141.1 he864248.1 2n= 28 turkey 46 c. cyprius he663962.1 he864168.1 2n= 10 greece 47 c. hartmannianus he801173.1 he864264.1 2n= 20 cyprus 48 c. leichtlinii ln864711.1 he864277.1 2n= 20 turkey 49 c. kerndorffiorum he801159.1 he864213.1 turkey 162 masoud sheidai et al. regression, and models linear combinations of the environmental predictors that explain linear combinations of the snps. this method effectively identifies covering loci associated with the multivariate environmental features (legendre and legendre, 2012). redundency analysis is a highly flexible framework, and produce answers on: 1what environmental conditions cause genetic divergence among the studied taxa? and 2. what is the genetic basis of local adaptation to the environment? rda identifies linear relationships among the response and predictor matrices; if non-linear relationships are expected, other statistical frameworks may be more suitable. rda was performed in paleontological statistics (past) ver. 4, program. mantel test was performed with 1000 times permutations as implemented in past ver. 4., program to study correlation between genetic distance and geographical distance of the studied species. phylogenetically important snps was determined by character mapping of 110 snps obtained based on parsimony criterion as performed in mesquite 3.6 program. we performed tajima’s d test (tajima, 1989) to reveal if crocus species dna sequences evolved randomly (“neutrally”), or under a non-random process, including directional or balancing selection, demographic expansion or contraction. moreover, we also carried out the molecular clock test, to show if snp changes occurred in accordance with a uniform clock rate model of evolution during crocus genus speciation events. these tests were performed by mega 7 program. 3. results 3.1. the species genetic difference the preliminary analysis of combined sequences obtained after sequence alignments and curation, produced a dna segment of 110 base pair length. the average p dis of the studied species was 0.126. based on kimura 2-parameters, the studied taxa differed in genetic distance from 0. 01 to 0.30. the paired mismatch plot of nucleotide difference is presented in fig. 1. this plot shows a normal distribution in genetic difference of these species, which indicates that genetic divergence occurred in a continuous and steady mode in evolution of the genus crocus l. skyline-plot (fig. 2), of the same species also revealed a smooth and continuous species expansion in the genus crocus, with two sudden changes in population demography and sequence change which are related to the speciation events in iran crocus taxa. number taxa accession number(its) accession number(trnl-f) chromosome number country 50 c. nerimaniae he663977.1 he864181.1 2n= 10 turkey 51 c. korolkowii he801139.1 he864244.1 2n= 20 uzbekistan 52 c. michelsonii ky797650.1 he864278.1 2n= 20 iran 53 c. caspius he801171.1 he864266.1 2n= 24 iran 54 c. alatavicus he801116.1 he864273.1 2n= 20 uzbekistan 55 c. naqabensis ls398395.1 lt997016.1 2n= 14 jordan 56 c. antalyensis he664015.1 he864209.1 2n= 8 turkey 57 c. olivieri he8011 he864216.1 2n= 6 turkey 58 c. candidus he663981.1 he864186.1 2n= 6 turkey 59 60 c. hyemalis he801060.1 he864215.1 2n= 6 plestin 61 c. aleppicus he801175.1 he864267.1 2n= 16 jordan 62 c. veneris he801062.1 he864222.1 2n= 16 cyprus 63 c. carpetanus he801071.1 he864265.1 2n= 64 turkey 64 c. nevadensis he663960.1 he864170.1 2n= 28, 30 spain 65 c. fleischeri he663983.1 he864188.1 2n= 20 turkey 66 c. pulchellus he801145.1 he864252.1 2n= 12 greece 67 c. laevigatus he801166.1 he864233.1 2n= 30 greece 68 c. banaticus he801147.1 he864254.1 2n= 26 romania 69 romulea ramiflora he664012.1 he864206.1 2n= 36 turkey 70 r. bulbocodium he664012.1 he864202.1 2n= 34,36,42 turkey 163identifying potential adaptive snps within combined dna sequences in genus crocus 3.2. genetic grouping of taxa ml (maximum likelihood) phylogenetic tree of the studied crocus species based on combined molecular data set and the species geographical distribution, is presented in fig. 3. we can place the studied species in three to four major clades. at the first glance, it is evident that species with mediterranean distribution and those of south-west asia (iran, iraq, and afghanistan), and the neighboring regions, comprise adjacent clades, while the species growing in europe are showing closer genetic affinity. genetic grouping of these species by linear discriminating analysis (lda), as performed in dapc analysis is provided in fig. 4. this plot also supports the presence of four genetic groups in the studied taa. the assignment test for the studied crocus species based on dapc analysis identified the species with genetic affinity (fig. 5). the species n1-n68, are scattered in four major genetic groups as revealed by different cluster colors. linear discriminating analysis revealed that the first three discriminating analysis (da) axes, comprise about 80% of total variation, and the first two axes have significant contribution with high fst value (fig. 6). da loading obtained revealed that snps 74, 75 have the highest figure 1. mismatch plot of nucleotide difference among crocus species. figure 2. skyline-plot of crocus species based on combined sequence data set. figure 4. genetic groups identified based on lda analysis. figure 3. ml phylogenetic tree of the studied crocus species and their geographical distribution. (n1-n70, as in table 1). figure 5. assignment plot of crocus species based on dapc analysis (individuals from left to wright are n1 to n 68 of table 1). 164 masoud sheidai et al. discriminating power in the first lda axis, followed by snps 31, and 109, in the second axis. similarly, snp 53 has high discriminating power in the third da axis. the following analyses were performed to identify the snps which show association with geographical coordinates of crocus species distribution. we should mention that these analy tical approaches have different assumptions and may differ to some extent in their results. therefore, comparing obtained results are important for drawing a solid conclusion. 3.3. canonical correspondence analysis cca plot of crocus species and 110 snps used is provided in fig. 7. the analysis produced two cca axes with eigenvalue% of 99.97and 0.028, respectively. distribution of 110 snps used shows association between snps 31, 70, 71, 74, and 75, with latitude distribution of crocus species of these three snps. viz. 31, 74, and 75, were identified as discriminating loci among crocus taxa, by dapc analysis. these snps have high association value as are placed in the first cca axis. the snps 2, 93 and 94 of the second cca axis, show a lower degree of association with longitude distribution of the studied crocus taxa. from these results, we may conclude that, genetic changes of crocus species towards latitude distribution was accompanied to these snps, which probably were associated with some important adaptive genes during crocus speciation. it becomes interesting when we plot the selected countries (geographical regions), by cca (fig. 8). we observe that countries like iran, russia, and georgia, become separated from the other studied countries towards latitude. that means snps’ changes occurred in these regions. the skyline plot presented before also revealed a sudden change in nucleotide substitution and population size in iran. 3.4. latent factor mixed model (lfmm) manhattan plot of lfmm analysis is presented in fig. 9. it identified snps 2, 9, 63, and 79, showed a significant association with environmental features. figure 8. cca plot of geographical regions showing separation of countries towards altitude and longitude crocus species. figure 7. cca plot of crocus species showing association of few snps with geographical factors. figure 6. f-statistics of lda analysis showing significant contribution of the first two axes in discriminating crocus species. figure 9. manhattan plot of lfmm analysis identifying four snps associated with environmental features. 165identifying potential adaptive snps within combined dna sequences in genus crocus 3.5. redundency analysis (rda) redundancy analysis (r da) was performed to detect the roles of geographical variables in crocus species genetic subdivision, as well as the relative contribution of each variable to the population genetic structure. rda plot is presented in fig. 10. the snps 22, 71, 74, 75, 98, and 103, show association with latitude which occurs in rda axis one with about 85% of total variance, followed by snps 9, 93 and 94, associated with longitude and second rda axis with only 14% of total variance. therefore, if we consider different association approaches utilized in this study, we can consider a few snps which are significantly associated with geographical factors studied. these snps occurred during species divergence within the genus crocus. a negative tajima’s d = -1.2, was obtained for the studied snps in crocus species. this signifies an excess of low frequency polymorphisms relative to expectation, indicating population size expansion after a bottleneck or a selective sweep, which result in reduction in genetic diversity and formation of adaptive genotypes (species), in different geographical areas. the molecular test showed that snp changes within the genes crocus did not occurred under uniform rate of evolution and different phylogenetic clades differed in their genetic changes. this results also agree with the earlier result of skyline plot showing a deep change in snp substitution and population size change in crocus species of iran and neighboring regions. mantel test performed after 1000 times permutations, produced non-significant correlation between genetic distance and geographical distance of the studied species (r = 0.03, p = 0.7). this result indicates that nucleotide difference and change in crocus taxa is not due to mere geographical distance and as indicated by different analyses reported here, genetic changes are. mainly associated with latitude distribution of these taxa. 3.6. phylogenetically important snps character mapping of the snps (fig. 11), based on parsimony criterion, revealed that some of these snps are of phylogenetic importance as they differentiate almost a particular clade of the studied species. interesting enough, snps 74 and 75. are also among these phylogenetic important snps. these two snps were identified as discriminating snps among crocus species and also they are associated with latitude distribution of taxa, particularly crocus species of iran and neighboring areas. 4. discussion speciation within the genus crocus is complex. a combination of diploid and polyploidisation events as well as inter-specific hybridization have been postulated for crocus genus evolution (mosolygo-l et al., 2016). complexity at the species level has been reported by seberg and petersen (2009), as these authors could not delineate crocus species even by utilizing different barcoding markers. however, some authors, could resolve crocus species of balkan (mosolygo-l et al., 2016) and iran (sheidai et al., 2018), by using different molecular markers. figure 10. rda plot of crocus species showing association of few snps with geographical factors. figure 11. character mapping of snps by parsimony criterion showing that these snps can differentiate different phylogenetic clades. 166 masoud sheidai et al. recently, mohebi et al. (2020), provided dna barcode of chloroplast dna (trnh-psba) region, which differentiated saffron genotypes of iran from the other imported genotypes. moreover, the same authors (unpublished data), provided some dna barcode which illustrate genetic differentiation between crocus taxa growing in different geographical regions and not for a particular crocus species. nine crocus l. species have been reported from persia and some adjacent areas (wendelbo, 1977; matine, 1978). taxonomy of the genus is controversial as evidenced by difficulties in crocus species delineation. in spite of extensive efforts on the phylogenetic aspects of crocus genus, there has been now report on ecological or geographical association of the genetic or dna sequence changes with speciation events in this genus. the present study revealed that dna nucleotides of both nuclear and chloroplast origin can efficiently differentiate some of the phylogenetic clades of crocus taxa. moreover, some of these sequences may be associated with geographical distribution of crocus species. some nucleotide seems to be tightly associated with latitudinal distribution of these taxa. tajima’s test of these sequences produced a negative tajima’s d, which indicates an excess of low frequency polymorphisms relative to a selective sweep and speciation events (tamura and nei, 1993). we also observed almost a continuous and gradual nucleotide substitution for most of the species growing in other parts of the world, but a sudden deep change in dna sequences of iran crocus species, which may be related to geographical adaptation as also evidenced by cca and rda analyses. different approaches used to identify the nucleotides associated with geographical variables, revealed some degree of difference. it is due the fact that cca and rda methods are based on linear association (regression), with different approaches, while lfmm method is a bayesianmodel approach (podani, 2000; frichot and francois, 2015). it seems therefore, using different approaches may improve understanding of associated snps with geographical and ecological variables. such combined data evaluations, give insights into contemporary processes, and may explain how environmental factors influence selective and neutral genomic diversity within and among related species or different geographical populations within a single species (segovia et al., 2020). presence of heterogenous environmental conditions are known to cause changes in genetic diversity of plant species and result in local adaptations even in the populations of a single species (segovia et al., 2020). understanding the genetic basis of local adaptation is one of the main concern of evolutionary biologists, as identifying adaptive genetic loci or snps improves our knowledge of the genetic mechanism of local adaptation and probably species diversification within a genus (zhang et al., 2019). recent studies which are concerned with genetics of local adaptations try to answer two major questions: 1 which environmental variables play key role in the adaptive genetic divergence of a species or different species within a particular genus and shape its landscape genetic structure, and, 2-which genes or loci on the genome undergo adaptive genetic differentiation (li et al., 2017, zhang et al., 2019). in general, populations’ local adaptation which leads to speciation thing a genus is the act of natural selection in oppose to continuous gene flow. in plant groups such as crocus genus in which species differentiation is vague due to inter-specific hybridization and a high degree of genetic affinity, local adaptation, may be expected to happen for a few genes or nucleotides, as also we demonstrated in this study. the latitude occurrence of nucleotide changes and species diversification in crocus genus, may be related to a warmer and drier environmental conditions of iran, and afghanistan and neighboring regions in compare to those prevailing in mediterranean countries and europe. in a similar study, ingvarsson et al. (2006), characterized patterns of dnasequence variation at the putative candidate gene phyb2 in 4 populations of european aspen (populous tremula) and scored single-nucleotide polymorphisms in an additional 12 populations collected along a latitudinal gradient in sweden. they utilized a sliding-window scan of phyb2 and identified six putative regions with enhanced population differentiation and four snps showed significant clinal variation. therefore, they suggested that the clinal variation at individual snps is an adaptive response in phyb2 to local photoperiodic conditions. it has been suggesting that divergent selection enhances the levels of genetic differentiation not only for the sites that are the direct target of selection but also for neutral sites in the vicinity of the site(s) under selection (charlesworth et al., 1997; nordborg and innan, 2003). 5. conclusion in conclusion, the present study provide data on dna sequences changes in association with geographical variables in the genus crocus and suggest that latitudinal distribution has a more profound effect on these genetic changes. moreover, we also suggest utilizing a multiple analytical approaches for identification of both discriminating dna nucleotides/ snps within a genus 167identifying potential adaptive snps within combined dna sequences in genus crocus and for illustrating snps association with geographical or ecological variables. references aghighiravan, f., shokrpour, m., nazeri, v., naghavi, m.r., 2019. phylogenetic assessment of some species of crocus genus using dna barcoding. j. genet. resour. 5(2), 118-129. charlesworth, b., nordborg, m., charlesworth,d., 1997. the effects of local selection, balanced polymorphism and background selection on equilibrium patterns of genetic diversity in subdivided populations. genet. res. 70, 155–174. frichot, e., schoville, s.d., bouchard, g., francois, o., 2013. testing for associations between loci and environmental gradients using latent factor mixed models. mol. biol. evol. 30 (7), 1687-1699. frichot, e., francois, o., 2015. lea: an r package for landscape and ecological association studies. methods in ecology and evolution: 6 (8), 925-929 ingvarsson, p. k., garcı´a, m.v., david hall, d., luquez, v., jansson, s., 2006. clinal variation in phyb2, a candidate gene for day-length-induced growth cessation and bud set, across a latitudinal gradient in. european aspen (populus tremula). genetics. 172, 1845–1853. doi: 10.1534/genetics.105.047522 legendre, p., legendre, l., 2012. numerical ecology. 3 editions. oxford, uk. li, y., zhang, x.x., mao, r.l., yang, j., miao, c.y., li, z., qiu, x.y., 2017 ten years of landscape genomics: challenges and opportunities. front. plant. sci. 8, 2136. matine, f., 1978. liste des plantes de l’herbierd’. vine (herbarium ministerii iranici agriculturae). iridaceae, tehran. mohebi anabat, m., riahi, h., sheidai, m., koohdar, f., 2020. population genetic study and barcoding in iran saffron (crocus sativus l.). ind. crop. prod. 143, 111915. mohebi anabat, m., riahi, h., sheidai, m., koohdar, f., 2021. a new look at the genus crocus l. phylogeny and speciation: insight from molecular data and chromosome geography. genet. resour. crop. accepted paper. mosolygó-l, á., sramkó, g., barabás, s., czeglédi, l., jávor, a., molnár, a., surányi, g., 2016. molecular genetic evidence for allotetraploid hybrid speciation in the genus crocus l. 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(convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(3): 101-108, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1533 caryologia international journal of cytology, cytosystematics and cytogenetics citation: syamand ahmed qadir, chnar hama noori meerza, aryan mahmood faraj, kawa khwarahm hamafaraj, sherzad rasul abdalla tobakari, sahar hussein hamarashid (2022). comparative study and genetic diversity in malva using srap molecular markers. caryologia 75(3): 101-108. doi: 10.36253/caryologia-1533 received: january 01, 2022 accepted: november 23, 2022 published: april 5, 2023 copyright: © 2022 syamand ahmed qadir, chnar hama noori meerza, aryan mahmood faraj, kawa khwarahm hamafaraj, sherzad rasul abdalla tobakari, sahar hussein hamarashid. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. comparative study and genetic diversity in malva using srap molecular markers syamand ahmed qadir1, chnar hama noori meerza2, aryan mahmood faraj3, kawa khwarahm hamafaraj4, sherzad rasul abdalla tobakari5, sahar hussein hamarashid6,* 1 medical laboratory techniques department/halabja technical institute, research center/ sulaimani polytechnic university, sulaymaniyah, iraq 2 food science and quality control department, bakrajo technical institute, sulaimani polytechnic university, sulaymaniyah, iraq 3 medical laboratory science department/ technical college of applied science/sulaimani polytechnic university, sulaymaniyah, iraq 4 nursing department/halabja technical institute/ sulaimani polytechnic university, sulaymaniyah, iraq 5 medical laboratory techniques department/halabja technical institute, research center/ sulaimani polytechnic university, sulaymaniyah, iraq 6 agricultural project management department/ technical college of applied science/ sulaimani polytechnic university, sulaymaniyah, iraq *corresponding author. e-mail: sahar.rashid@spu.edu.iq abstract. the malva genus has 25-40 species and it can be considered as an annual and/or biannual herb. malva species are indicated with potential therapeutic as cicatrizing and analgesic by the ministry of health. the aim of this study was to analyze srap (sequence-related amplified polymorphism) markers in a total of 70 accessions of malva species, which included five species malva neglecta wallr., malva pusilla sm., malva sylvestris l., malva verticillata l., malva nicaeensis all.. a total of 89 (number of total loci) (ntl) dna bands were produced through polymerase chain reaction amplifications (pcr) amplification of five malva species. these bands were produced with the combinations of 5 selective primers. the total number of amplified fragments ranged from 10 to 27. the predicted unbiased gene diversity (uhe) varied between 0.077 (malva sylvestris) and 0.382 (malva pusilla). the genetic similarities between three species are estimated from 0.70 to 0.91. neighbor-joining tree results showed two major clusters. according to the srap (sequence-related amplified polymorphism) markers analysis, malva pusilla and malva aegyptia had the lowest similarity. our results provided great molecular identification of all assayed genotypes, which have shown that there is large quantity of genetic diversity among the malva accessions. objectives of the study were; a) to estimate genetic diversity; b) to evaluate population relationships using nj approaches. current results have implications in breeding and conservation programs. keywords: sequence-related amplified polymorphism, genetic diversity, medicinal plants malva,taxonomy. 102 syamand ahmed qadir et al. introduction the use of medicinal plants can be influenced by the economic condition, the high cost of medicines and the difficult access to public consultations. in addition to that, there is difficulty of access by residents in rural areas to health care units located in urban areas. moreover, the increase the trend for considering traditional knowledge that supports using natural resources as an alternative to synthetic drugs (battisti et al., 2013). given the significance of genetic diversity in conservation strategies, it is of utmost importance to disentangle genetic diversity in plant species, particularly threatened and rare species (esfandani-bozchaloyi et al. 2018a, 2018b, 2018c, 2018d). the indiscriminate use of plants due to the lack of phytochemical, pharmacological and mainly toxicological knowledge is of great concern for public health. the correct identification of medicinal plant species is necessary, especially when they are processed in order to avoid misuse of medicinal plants (romitelli & martins, 2013). the malva genus presents different species with therapeutic potential and inadequate consumption can occur due to the incorrect identification of the plant in the market. malvaceae or the mallow family is the family of flowering plants containing over 200 genera with close to 2300 species (la duke and dobley 1995). many researches have been published on the ecology, taxonomy, genetic, cytology, chemotaxonomy, physiology, seed germination and economic uses of family malvaceae such as (el-rjoob and omari 2009) in ecology; in taxonomy in chemotaxonomy (blunden et al., 2001) and in genetic researches (baum et al., 2004) studied the pollen. malva l. (mallow) is the genus within the malvaceae juss. family, which includes 25–40 species and several hybrids (ray 1995). this genus contains herbaceous annual, biennial, and perennial species that are native to regions of africa, asia, and europe (shaheen et al., 2009). in medicine, mallow species are used in the treatment of respiratory, urinary, and digestive problems as they have high bactericidal, antiulcerogenic, anti-inflammatory, hepatoprotective, and antidiabetic activities (pandey et al, 2012). the malva genus is morphologically very diverse, but some species are hardly distinguishable based on morphological features (escobar et al., 2009). several studies have been conducted to clarify the taxonomic affiliation of malva species using different features, such as molecular data (nuclear ribosomal dna (rdna), internal transcribed spacer (its) region, intron–exon splice junction (isj), and inter simple sequence repeat polymerase chain reaction (issr) markers), differentiation of seed and seed coat structure (el naggar, 2001), morphology of pollen grains (el naggar, 2004), epidermal structures and stem hairs (akçin and özbucak, 2006), and plant morphological traits (michael et al., 2009). the variability in mallow species is due, at least in part, to hybridization. natural crossings between m. pusilla sm. and m. neglecta, m. alcea l., and m. moschata l. as well as m. sylvestris and m. neglecta were found in europe. ray (1995) stated that hybridization or polyploidy is probably a factor in the evolution of these species, but this aspect has not been investigated so far. the taxonomy and systematics of the malva genus are still unclear and very complicated. taxonomic doubts have appeared because of the high level of homoplasty in morphological traits that are usually used as diagnostic features (chen et al. 2021 ; bi et al. 2021). sequence-related amplified polymorphism (srap) is pcr –based marker system. it is one of the efficient and simple marker systems to study gene mapping and gene tagging in plant species (li and quiros 2001), and srap are potential markers to assess plant systematics and genetic diversity studies (robarts and wolfe 2014). previously, wu et al. (2010) assessed genetic diversity and population structure in pogostemon cablin  with the aid of srap markers. srap markers were successfully implemented in lamiaceae, geraniacea, caryophyllacea and rosaceae family to study natural populations and variations within the family (peng et al., 2021; ma et al.,2021). objectives of the study were; a) to estimate genetic diversity; b) to evaluate population relationships using nj approaches. current results have implications in breeding and conservation programs. the present study is the first report on genetic diversity and phylogenetic relationships between and within malva species in iraq using srap markers. materials and methods plants collection five wild malva species (malva neglecta wallr., malva pusilla sm., malva sylvestris l., malva vericillata l., malva nicaeensis all..) in halabja, sulaimanieh, kalar, chamchamal and basreh provinces of iraq were selected and sampled during july-august 2015-2020. morphometric and srap analyses on 70 plant accessions were carried out. five to twelve samples from each population belonging to three different species were selected based on other eco-geographic characteristics. detailed information about locations of samples and geographical distribution of species are mentioned. 103comparative study and genetic diversity in malva using srap molecular markers morphological studies five to twelve samples from each species were used for morphometry. in total 36 morphological (13 qualitative, 23 quantitative) characters were studied. data obtained were standardized (mean= 0, variance = 1) and used to estimate euclidean distance for clustering and ordination analyses (podani 2000). morphological characters studied are: corolla shape, bract shape, calyx shape, calyx length, calyx width, calyx apex, calyx margins, bract length, corolla length, corolla width, corolla apex, leaf length and leaf width, leaf apex, leaf margins, leaf shape, leaf gland and bract margins. sequence-related amplified polymorphism method fresh leaves were used randomly from one to twelve plants. these were dried with silica gel powder. genomic dna was extracted while following previous protocol. srap assay was performed as described previously (li and quiros 2001). five srap in different primer combinations were used (table 1). the overall reaction volume consisted of 25 μl. this pcr reaction was carried out in techne thermocycler (germany). the following cycles and programs were observed. the initial denaturation step was performed for 5 minutes at 94°c. the initial denaturation step was followed by 40 cycles for 1 minute at 94°c; 1 minute at 52-57°c, and 2 minutes at 72°c. the reaction was completed by a final extension step of 7-10 min at 72°c. staining was performed with the aid of ethidium bromide. dna bands/fragments were compared against a 100 bp molecular size ladder (fermentas, germany). data analyses anova (analysis of variance) was conducted to assess morphological differences among species. principal component analysis (pca) was implemented to identif y variable morphological characters in malva species. multivariate statistical analyses i.e., pc analysis, were performed in past software version 2.17 (hammer et al. 2001). molecular analyses sequence-related amplified polymorphism (srap) bands were recorded. presence and absence of bands were scored present (1) and absent (0), respectively. total loci (ntl) and the number of polymorphism loci (npl) for each primer were calculated. furthermore, the polymorphic ratio was assessed based on npl/ntl values. polymorphism information content was calculated as previously suggested by roldan-ruiz et al. (2000). parameter like nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism (p% = number of polymorphic loci/number of total loci) were determined. nei’s genetic distance among populations was used for neighbor joining (nj) clustering and neighbor-net networking. mantel test checked the correlation between geographical and genetic distances of the studied populations (podani 2000). these analyses were done by past ver. 2.17 (hammer et al. 2012), darwin ver. 5 (2012) and splitstree4 v4.13.1 (2013) software. to assess the population structure of the pistachio genotypes, a heuristic method based on bayesian clustering algorithms were utilized. the clustering method based on the bayesian-model implemented in the software program structure () was used on the same data set to better detect population substructures. this clustering method is based on an algorithm that assigns genotypes to homogeneous groups, given a number of clusters (k) and assuming hardy-weinberg and linkage equilibrium within clusters, the software estimates allele frequencies in each cluster and population memberships for every individual (pritchard et al. 2000). the number of potential subpopulations varied from two to ten, and their contribution to the genotypes of the accessions was calculated based on 50,000 iteration burn-ins and 100,000 iteration sampling periods. the most probable number (k) of subpopulations was identified following evanno et al. (2005). in k-means clustering, two summary statistics, pseudo-f, and bayesian information criterion (bic), provide the best fit for k. pairwise genetic similarity between species was evaluated to reveal genetic affinity between species (jaccard, 1908). unbiased expected heterozygosity and shannon information index were calculated in genalex 6.4 software (peakall and smouse, 2006). table 1. srap primer information and results. primer name ntla nplb pc picd rpe em3-me4 27 27 100.00% 0.55 33.24 em3-me1 16 10 75.00% 0.11 55.55 em4-me1 17 17 100.00% 0.39 11.23 em5-me1 10 10 100.00% 0.50 38.55 em5-me2 19 13 66.00% 0.32 44.65 mean 19 15 83.10% 0.44 39.23 total 89 80 104 syamand ahmed qadir et al. results morphometry the anova findings showed substantial differences (p<0.01) between the species in terms of quantitative morphological characteristics. principal component analysis results explained 60% cumulative variation. the first pca axis explained 40% of the total variation. the highest correlation (> 0.7) was shown by morphological characters such as corolla apex, seed length; number of segment stem leaves; calyx length, calyx width; bract length and leaf shape. the morphological characters of five malva species are shown in pca plot (figure 1). each species formed separate groups based on morphological characters. the morphometric analysis showed clear difference among malva species and separated each groups. species identification and genetic diversity five (5) suitable primer combinations (pcs), out of 25 pcs were screened in this research. figure 2 illustrates the banding pattern of em2-me4 and em4-me1 primer by the srap marker profile. eighty (80) amplified polymorphic bands (number of polymorphic loci) were produced. these bands (fragments) had different range i.e. 100bp to 3000 bp. maximum and minimum numbers of polymorphic bands were 27 and 10 for em3-me4 and em5-me1, respectively. each primer produced 15 polymorphic bands on average. the pic ranged from 0.11 (em3-me1) to 0.55 (em1-me4) for the 5 srap primers, with an average of 0.44 per primer. rp of the primers ranged from 11.23 (em4-me1) to 55.55 (em3-me1) with an average of 39.23 per primer (table 2). the calculated genetic parameters of malva species are shown (table 2). the unbiased heterozygosity (h) varied between 0.077 (malva sylvestris) and 0.382 (malva pusilla) with a mean of 0.23. shannon’s information index (i) was maximum in malva nicaeensis (0.395), where as we recorded minimum shannon’s information index in malva sylvestris (0.13). the observed number of alleles (na) ranged from 1.11 in malva aegyptia to 1.580 in malva nicaeensis. the significant number of alleles (ne) ranged from 1.077 (malva nicaeensis) to 1.432 (malva pusilla). figure 1. morphological characters analysis of the malva species by pcoa plot. table 2. genetic diversity parameters in the different malva populations, species, and cultivars; abbreviations. population %p n na ne i he uhe malva pusilla sm. 53.00% 15.000 1.500 1.432 0.388 0.310 0.382 malva sylvestris l. 22.11% 10.000 1.333 1.177 0.130 0.033 0.077 malva vericillata l. 33.33% 12.000 1.300 1.388 0.271 0.167 0.288 malva nicaeensis all. 49.00% 17.000 1.580 1.077 0.395 0.156 0.277 malva aegyptia l. 30.33% 13.000 1.110 1.366 0.299 0.238 0.144 105comparative study and genetic diversity in malva using srap molecular markers analysis of molecular variance results in significant genetic difference (p = 0.01) among malva species. the majority of genetic variation occurred among species. amova findings revealed that 66% of the total variation was between species and comparatively less genetic variation was recorded at the species level (table 3). genetic difference between malva species was highlighted by genetic statistics (nei’s gst), as evident by significant p values i.e. nei’s gst (0.578, p = 0.01) and d_est values (0.829, p = 0.01) . nj tree and upgma clustering produced similar results therefore only nj tree is presented and discussed (figure 3). this result show that molecular characters studied can delimit malva species in two different major clusters or groups. in general, two major clusters were formed in nj tree (fig. 3), 20 individual of malva nicaeensis and malva aegyptia formed a single cluster. cluster ii contained two sub-clusters, and most of individual malva pusilla; malva sylvestris and malva vericillata formed cluster ii. there were 50 individuals in this cluster. we detected strong correlation between geographical and genetic distances (r = 0.45, p=0.0002) and gene flow (nm) score of 0.48 was reported among species. detailed information about genetic distances and genetic identity (nei’s) are described (table not included). the findings suggested that there was the highest degree of genetic similarity (0.91) between malva vericillata and malva nicaeensis. on the contrary to this, malva pusilla and malva aegyptia (0.70) had lowest genetic resemblance. the evanno test δk =5 (figure not included), showed the genetic details of the malva species. according to structure analysis, the malva species are genetically differentiated due to different allelic structures (figure not included). limited gene flow results were supported by k-means and structure analyses too. we could not identify substantial gene flow among the malva species. this result is in agreement with grouping we obtained with nj tree (figure 3), as these populations were placed close to each other. as evidenced by structure plot based on admixture model, these shared alleles comprise very limited part of the genomes in these populations and all these results are in agreement in showing high degree of genetic stratification within malva populations. figure 2. electrophoresis gel of studied ecotypes from dna fragments produced by srap profile; 1, 7: malva neglecta 2,8: malva parviflora 3,9: malva pusilla. 4,10: malva sylvestris 5,11: malva vericillata 6,12: malva nicaeensis = ladder 100 bp. table 3. analysis of molecular variance (amova) of the studied species. source df ss ms est. var. % φpt among pops 70 2701.394 77.782 10.166 66% 66% within pops 10 111.449 390.19 27.833 34% total 80 2875.807 37.060 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance; φpt: proportion of the total genetic variance among individuals within an accession, (p < 0.001). figure 3. neighbor-joining tree of populations in malva species based on srap molecular markers. 106 syamand ahmed qadir et al. discussion in the present study, we used morphological and molecular (sr ap) data to evaluate species relationships in malva. morphological analyses of malva species showed that quantitative indicators (anova test results) and qualitative characteristics are well differentiated from each other. pca analysis suggests that morphological characters such as corolla shape, bract shape, calyx shape, calyx length, calyx width, calyx apex, calyx margins, bract length have the potentials to identify and delimitate malva species. principal component analysis results suggests the utilization of morphological characters to identify and delimitate malva species. morphological characters including corolla length, corolla width, corolla apex, leaf length and leaf width, leaf apex, leaf margins, leaf shape, leaf gland and bract margins play key role in plant systematics and taxonomy. our work also highlighted the significance of morphological characters and molecular data to identify and study species genetic diversity. in general, genetic relationships obtained from srap data coincides with morphometric results. this is in accordance with the parameters of amova and genetic diversity results. srap molecular markers detected clear genetic difference among species. these results indicate that srap have potentials to study plant systematics and taxonomy in malva members. given the negative impact of biodiversity threats and overexploitation of malva plant species in iran, it is necessary to conduct genetic diversity studies on malva species. genetic diversity based studies pave our understanding to develop conservation strategies (jia et al. 2020; shi et al., 2021; zheng et al., 2021; zhu et al., 2021). genetic diversity studies are conducted through appropriate selection of primers and indexes including polymorphic information content (pic) and marker index (mi) are important indexes to fathom genetic variation in species (wang et al., 2021; yin et al., 2021; zhao et al., 2021). common logic suggests that different makers have different abilities to assess genetic diversity, and usually, genetic diversity is linked with polymorphism (sivaprakash et al. 2004). in the present work, 5 malva species were characterized with 5 srap markers. the results confirm the efficiency of microsatellite markers for fingerprinting purposes. our results demonstrated that the pic ranged from 0.11 (em3-me1) to 0.55 (em3-me4) for the 5 srap primers, with an average of 0.44 per primer. rp of the primers ranged from 11.23 (em4-me1) to 55.55 (em3me1) with an average of 39.23 per primer. diversity study in malva species malvaceous germplasm has been variously investigated by different molecular marker techniques but the earlier studies either focused on the comparison of the malvaceae with other families in the order malvales or to explore the genetic relationships and diversity within and among population and limited number of species in the same genus. very little attention has been given to the analysis at interspecific and intergeneric levels. la duke and dobley (1995) has the only worth mentioning work in this regard. their results showed that, the genetic relationships and diversity within and between 12 malvaceous species belonging to five genera are investigated by using the amplified fragment length polymorphism (aflp). shaheen et al., (2009) with used aflp (amplified fragment length polymorphism) marker to explore phenetic relationships and diversity within and between 13 malvaceae species belonging to 5 different genera. their primary objective of the study was to evaluate the taxonomic potential, usefulness and applicability of aflp marker system to reconstruct genetic relationships at interspecific and intergeneric level in malvaceae. two primer pairs produced a total of 73 bands, of which 70 were polymorphic. according to celka et al (2010) two categories of dna markers were used to determine genetic relationships among eight malva taxa. a maximum parsimony analysis validated the division of the genus malva into the sections bismalva and malva. the species classified into those sections formed separate clusters. m. moschata was a distinctive species in the section bismalva, as confirmed by previous genetic research based on its and cpdna sequence analyses. the applied markers revealed a very high level of genetic identity between m. alcea and m. excisa and enabled molecular identification of m. alcea var. fastigiata. jedrzejczyk and rewers (2020) applied flow cytometry and inter simple sequence repeat polymerase chain reaction (issr-pcr) for fast and accurate species identification. genome size estimation by flow cytometry was proposed as the first-choice method for quick accession screening. out of the 12 tested accessions, it was possible to identify six genotypes based on genome size estimation, whereas all species and varieties were identified using issr markers. flow cytometric analyses revealed that malva species possessed very small (1.45–2.77 pg/2c), small (2.81–3.80 pg/2c), and intermediate (11.06 pg/2c) genomes, but the majority of accessions possessed very small genomes. the relationships between the investigated accessions showed the presence of two clus107comparative study and genetic diversity in malva using srap molecular markers ters representing malvoid and lavateroid group of species. their results showed that flow cytometry and issr molecular markers can be effectively used in the identification and genetic characterization of malva species. conclusions the present study investigated the molecular variation of five species. molecular and morphometric analysis confirmed morphological and genetical difference between malva species. this was first attempt to assess genetic diversity through sequence-related amplified polymorphism and morphometrics analysis in iraq. current study reported two major clusters. these two major groups were separated on the basis of genetic and morphological characters. the genetic similarities between three species was estimated from 0.70 to 0.91. current study also reported correlation between genetic and geographical distances. this clearly indicated isolation mechanism envloved in the ecology of malva species . present results indicated the potential of sequencerelated amplified polymorphism to assess genetic diversity and genetic affinitiy among malva species. current results have implications in biodiversity and conservation programs. besides this, present results could pave the way for selecting suitable ecotypes for forage and pasture purposes in iraq. references akçin, ö.e.; 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genetika-belgrade, 53(2): 559-574 zhu, k., l. liu, s. li, b. li, m. khayatnezhad and a. shakoor. 2021. “morphological method and molecular marker determine genetic diversity and population structure in allochrusa.” caryologia 74(2): 121-130. caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 3 2022 firenze university press chromosome mapping of repetitive dnas in the picasso triggerfish (rhinecanthus aculeatus (linnaeus, 1758)) in family balistidae by classical and molecular cytogenetic techniques kamika sribenja1, alongklod tanomtong1, nuntaporn getlekha2,* chromosome number of some satureja species from turkey esra kavcı1, esra martin1, halil erhan eroğlu2,*, fatih serdar yıldırım3 l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations selma tabur1,*, nai̇me büyükkaya bayraktar2, serkan özmen1 characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques kristel ramírez-matadamas1, elva irene cortés-gutiérrez2, sergio moreno-limón2, catalina garcía-vielma1,* contributions of species rineloricaria pentamaculata (loricariidae:loricariinae) in a karyoevolutionary context a cius¹, ca lorscheider2, lm barbosa¹, ac prizon¹, ch zawadzki3, la borin-carvalho¹, fe porto4, alb portela-castro1,4 cadmium induced genotoxicity and antioxidative defense system in lentil (lens culinaris medik.) genotype durre shahwar1,2,*, zeba khan3, mohammad yunus khalil ansari1 biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts and evaluation of their biosafety potential denisa manolescu1,2, georgiana uță1,2,*, anca șuțan3, cătălin ducu1, alin din1, sorin moga1, denis negrea1, andrei biță4, ludovic bejenaru4, cornelia bejenaru5, speranța avram2 polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel (foeniculum vulgare mill.) egizia falistocco methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. sazada siddiqui*, sulaiman a. alrumman comparative study and genetic diversity in malva using srap molecular markers syamand ahmed qadir1, chnar hama noori meerza2, aryan mahmood faraj3, kawa khwarahm hamafaraj4, sherzad rasul abdalla tobakari5, sahar hussein hamarashid6,* nuclear dna 2c-values for 16 species from timor-leste increases taxonomical representation in tropical ferns and lycophytes inês da fonseca simão1, hermenegildo ribeiro da costa1,2,3, helena cristina correia de oliveira1,2, maria helena abreu silva1,2, paulo cardoso da silveira1,2,* nuclear dna content and comparative fish mapping of the 5s and 45s rdna in wild and cultivated populations of physalis peruviana l. marlon garcia paitan*, maricielo postillos-flores, luis rojas vasquez, maria siles vallejos, alberto lópez sotomayor identification of genetic regions associated with sex determination in date palm: a computational approach zahra noormohammadi1,*, masoud sheidai2, seyyed-samih marashi3, somayeh saboori1, neda moradi1, samaneh naftchi1, faezeh rostami1 comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(2): 59-69, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1493 caryologia international journal of cytology, cytosystematics and cytogenetics citation: zhu lin, hamed khodayari (2022) genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna. caryologia 75(2): 59-69. doi: 10.36253/caryologia-1493 received: november 19, 2021 accepted: july 06, 2022 published: september 21, 2022 copyright: © 2022 zhu lin, hamed khodayari. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 1 sias university, xin zheng city, henan province, 451150, china 2 department of biology, faculty of science, lorestan university, 5km khorramabad toward tehran, khorramabad, iran *corresponding author. e-mail: masxiongg@163.com abstract. salicornia is a wild and annual (biennial, in some species) halophytic plant from chenopodiaceae family that grows near salt marshes and salted wetlands or the vicinity of coastal areas in asia, north america, and the middle east. it is also grown in semi-arid areas of iran such as isfahan, fars, and yazd provinces. genetic variability and populations, structure were studied in 10 geographical populations of salicornia persica. genetic diversity parameters were determined in these populations. 10 of 20 random amplified polymorphic dna (rapd) primers produced 146 reproducible bands with average of 14.6 bands per primer and 88.67% of polymorphism. opa10 primer showed the highest number of effective allele (ne), shannon index (i) and genetic diversity (h). the highest values of genetic diversity in rapd markers were obtained in esfahan, nain, it is 70km to varzaneh populations. ward trees of rapd data grouped the populations in two different clusters/groups, indicating their genetic difference which is discussed in details. the results of this study showed that the level of genetic variation in salicornia persica is relatively low. ward-based dendrogram showed a close relationship between members of esfahan, varzaneh and esfahan, the river of zayanderud at varzaneh while the yazd, meybud and yazd, nádüshan protected population differ the most from the other populations. principal component analysis, however, showed some minor differences with ward-based dendrograms. keywords: gene flow, salicornia persica, random amplified polymorphic dna (rapd). introduction genetic diversity is one aspect of biological diversity that is extremely important for conservation strategies, especially in rare and narrowly endemic species (wang et al., 2021; yin et al., 2021; zhao et al., 2021). most of the authors agree that genetic diversity is necessary to preserve the longterm evolutionary potential of a species (jia et al. 2020; shi et al., 2021; zheng et al., 2021; zhu et al., 2021). salicornia is a wild and annual (biennial, in some species) halophytic plant from chenopodiaceae family that grows near salt marshes and salted 60 zhu lin, hamed khodayari wetlands or the vicinity of coastal areas in asia, north america, and the middle east. it is also grown in semiarid areas of iran such as isfahan, fars, and yazd provinces (akhani, 2003). salicornia varieties are often multipurpose (al-oudat and qadir, 2011; si et al, 2020) and it is very rich in vitamins, minerals and highly unsaturated oils. salicornioideae species are also rich in dietary fiber and many bioactive substances, such as phytosterols, polysaccharides, and phenolic compounds mainly flavonoids and phenolic acids (choi et al., 2014). salicornia, commonly called saloni is a member of chenopodiaceae (amaranthaceae). a family that constitute among it vegetables like spinach and beets etc. salicornia is a halophyte which tolerates extreme saline conditions and grows along the coastlines and feeds on salt water. it is a leaf less annual succulent salt marsh halophyte considered to be a potential alternative crop of seawater agriculture, due to its economic potential (glenn et al. 1991). the salicornia species are small, usually less than 30 cm tall, succulent herbs with a jointed horizontal main stem and erect lateral branches. the leaves are small and scale-like and as such the plant may appear leafless. many species are green, but their foliage turns red in autumn. the hermaphrodite flowers are wind pollinated, and the fruit is small and succulent and contains a single seed. salicornia species can generally tolerate immersion in salt water. salicornia has leafless stems with branches that resembles asparagus, hence the plants other common name sea asparagus. common names for the genus include glasswort, pickleweed, and marsh samphire. according to a molecular phylogenetic study by kadereit et al. (2006), salicornia is monophyletic and nested within the morphologically and ecologically closely related sarcocornia. salicornia and sarcocornia differ from all other salicornioideae by seeds that lack perisperm (shepherd et al., 2005). salicornia split from sarcocornia during the middle miocene (14.2– 9.4 mya), but its extant lineages started to diversify only in the early pleistocene (1.4–1.8 mya; kadereit et al., 2006; liu et al., 2021). according to zhang and bai (2022) a total of 72 randomly collected plants from 8 natural populations salicornia persica akhani in 2 provinces were evaluated using issr markers and morphological traits. salicornia persica is frequent along several salt marshes, river salt margins, and salty river estuaries in the provinces esfahan, fars and yazd. however, populations in these areas are threatened because many important wetlands (e.g., gavkhooni salt swamp) and rivers (e.g., zayandeh rud) are drying out. observations around tashk lake show that the species is grazed by goats. the species is characterized by having ascending habit, verticillate inflorescence branches, and reversed pentagonal central flowers that are truncated at the apex and reach to the upper segments. the molecular markers are extensively used in germplasm characterization, fingerprinting, genetic analysis, linkage mapping, and molecular breeding. rapd (random amplified polymorphic dna) analysis using pcr in association with short primers of arbitrary sequence has been demonstrated to be sensitive in detecting variation among individuals. the advantages of this technique are: a) a large number of samples can be quickly and economically analyzed using only micro-quantities of material; b) the dna amplicons are independent from the ontogenetic expression; and c) many genomic regions can be sampled with a potentially unlimited number of markers (peng et al., 2021; ma et al., 2021 esfandanibozchaloyi et al., 2017a, b, c, d). we have no information on genetic variability, gene flow and genetic structure of salicornia persica populations. moreover, due to extensive morphological variability of this species in the country, there is possibility of having infra-specific taxonomic forms in this species. therefore, we carried out population genetic analysis and morphometric study of 10 geographical populations for the first time in the country. materials and methods plant materials a total of 60 individuals were sampled representing 10 natural populations of salicornia persica in esfahan, fars and yazd provinces of iran during may-agust 2016-2020 (table 1, fig. 1). according to previous references, all the population were identified (kadereit et al., 2006; akhani 2003). morphological studies a total of 19 metric and 6 multistate characterers were used for measurements in different combinations (table 2), modified from the character list detailed by ingrouille and pearson (1987). of these 25 characters, 15 covered the overall vegetative morphology, and 10 were characteristics of the fertile spike, fertile spike segments and f lowers. though vegetative morphology may be partly uninformative due to the wide phenotypic plasticity, both vegetative and fertile spike characteristics were used, because some vegetative traits have been shown useful in separating populations and taxa at least in single cases (ingrouille and pearson 1987, ). 61genetic characterization of salicornia persica assessed using random amplified polymorphic dna dna extraction and rapd assay fresh leaves were used randomly from 5-10 plants in each of the studied populations. these were dried by silica gel powder. ctab activated charcoal protocol was used to extract genomic dna (esfandanibozchaloyi et al., 2019). the quality of extracted dna was examined by running on 0.8% agarose gel. 22 decamer rapd primers of operon technology (alameda, canada) belonging to opa, opb, opc, oph, opi, opm sets were used in this study. pcr reactions were carried in a 25μl volume containing 10 mm tris-hcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). the amplifications, reactions were performed in techne thermocycler (germany) with the following program: 5min initial denaturation step 94°c, followed by 40 cycles of 1min at 94°c; 1 min at 52-57°c and 2 min at 72°c. the reaction was completed by final extension step of 7-10 min at 72°c. the amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated by using a 100 bp molecular size ladder (fermentas, germany). data analyses morphological studies morphological characters were first standardized (mean = 0, variance = 1) and used to establish euclidean distance among pairs of taxa (podani, 2000). for grouping of the plant specimens, the upgma (unweighted paired group using average) and ward (minimum spherical characters) as well as ordination methods of mds (multidimensional scaling) and pcoa table 1. location and herbarium accession numbers of the studied populations of salicornia persica in iran. pop. no locality 1 esfahan, varzaneh 2 esfahan, the river of zayanderud at varzaneh 3 esfahan, nain, it is 70km to varzaneh 4 esfahan; varzaneh to kashan 5 fars, tashk lake 6 fars, lake bakhtegan 7 fars, abadeh tashk 8 yazd, kavire marvast 9 yazd, meybud 10 yazd, nádüshan table 2. evaluated morphological characters for populations of salicornia persica 1. height of plant from rooting point to apex, mm 2. number of sterile internodes below the terminal spike 3. number of 1st order branches 4. number of 2nd order branches 5. number of 3rd order branches 6. number of 4th order branches 7. length of longest basal 1st order branch, mm 8. number of sterile internodes on longest basal 1st order branch 9. number of fertile segments on main axis of longest basal 1st order branch 10. branching angle of longest basal 1st order branch, 11. branching habit of longest basal 1st order branch, 12. length of longest (regular) ultimate 1st order branch, mm 13. number of fertile segments on longest (regular) ultimate 1st order branch 14. branching angle of longest (regular) ultimate 1st order 15.branching habit of longest (regular) ultimate 1st order 16. length of spike, mm 17. number of fertile segments 18. length of 2nd fertile segment measured over the central flower of cyme, mm 19. height of triangular apex of 2nd fertile segment, mm 20. width of 2nd fertile segment at base, mm 21. maximum width of 2nd fertile segment, mm 22. place of the widest point of 2nd fertile segment, 23. length of visible part of central flower of cyme, 2nd fertile segment, mm 24. width of central flower of cyme, 2nd fertile segment, mm 25. place of the widest point of central flower of 2nd figure 1. map of distribtion of populations salicornia persica. 62 zhu lin, hamed khodayari (principal coordinate analysis) were used (podani, 2000). anova (analysis of variance) were performed to show morphological difference among the populations while, pca (principal components analysis) biplot was used to identify the most variable morphological characters among the studied populations (podani 2000). past version 2.17 (hammer et al., 2012) was used for multivariate statistical analyses of morphological data. random amplified polymorphic dna (rapd) analysis rapd bands obtained were treated as binary characters and coded accordingly (presence =1, absence = 0). parameter like nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism (p% = number of polymorphic loci/number of total loci) were determined (weising et al, 2005, freeland et al. 2011). shannon’s index was calculated by the formula: h’ = -σpiln pi. rp is defined per primer as: rp = ∑ ib, were “ib” is the band informativeness, that takes the values of 1-(2x [0.5-p]), being “p” the proportion of each genotype containing the band. the percentage of polymorphic loci, the mean loci by accession and by population, uhe, h’ and pca were calculated by genalex 6.4 software (peakall and smouse 2006). nei’s genetic distance among populations was used for neighbor joining (nj) clustering and neighbor-net networking (freeland et al. 2011, ). mantel test checked the correlation between geographical and genetic distances of the studied populations (podani 2000). these analyses were done by past ver. 2.17 (hammer et al. 2012), darwin ver. 5 (2012) software. amova (analysis of molecular variance) test (with 1000 permutations) as implemented in genalex 6.4 (peakall and smouse 2006) were used to show genetic difference of the populations. the genetic structure of populations was studied by bayesian based model structure analysis (pritchard et al. 2000; chen et al. 2021 ; bi et al. 2021 ), and maximum likelihood-based method of k-means clustering of genodive ver. 2. (2013). for structure analysis, data were scored as dominant markers . the evanno test was performed on structure result to determine proper number of k by using delta k value (evanno et al., 2005). in k-means clustering, two summary statistics, pseudo-f, and bayesian information criterion (bic), provide the best fit for k . gene flow was determined by (i) calculating nm an estimate of gene flow from gst by popgene ver. 1.32 (1997) as: nm = 0.5(1 gst)/gst. this approach considers equal amount of gene flow among all populations. (ii) population assignment test based on maximum likelihood as performed in genodive ver. in genodive ver. 2. (2013). the presence of shared alleles was determined by drawing the reticulogram network based on the least square method by darwin ver 5. (2012). results morphometric analyses in present study 60 plant samples were collected from 10 geographical populations. anova test revealed significant difference in quantitative morphological characters among the studied populations (p < 0.05). clustering and pca plot of salicornia persica populations based on morphological characters produced similar results therefore only pca plot is presented and discussed (fig. 2). the result showed morphological difference/ divergence among most of the studied populations. this morphological difference was due to quantitative characters only. for example, character (length of longest basal 1st order branch), separated population no. 1-4, character (length of longest (regular) ultimate 1st order branch) separated population no. 8, while character length of 2nd fertile segment measured over the central flower of cyme, separated populations 5 and 6,7 from the other populations. rapd analysis out of 20 rapd primers used, 10 primers produced reproducible bands. in total, 146 rapd bands (loci) were obtained with average of 14.6 bands per primer and only 10 bands ranging in size from 100– 2000 bp were common in all 10 populations studied and 129 bands were polymorph. t he mea n percentage of poly mor phism was 88.67%. r apd primers including opb-05, opa-13, produced the highest polymorphism (85-100%), while opa-10, opc-01 produced the lowest percentage of polymorphism (69-77%). the mean value of effective alleles, shannon index and genetic diversity of rapd loci studied were 1.06, 0.20 and 0.25, respectively, while opa-10 primers showed the highest number of effective alleles (1.098), shannon index (0.377) and genetic diversity (0.34) (table 3). among the primers used opa-10 produced the highest number of bands (24), while primers opa-04 produced the lowest number of bands (7). the primer opa-04 also produced the highest number of polymorphic bands (22). the primer opc-01 produced 8 specific bands, primer oph-07 produced 6 specific bands, while some 63genetic characterization of salicornia persica assessed using random amplified polymorphic dna other primers produced 4 and 3 specific bands and the primers opa-04, opm-10 and opa-10 produced 1 specific band. some of the populations showed the presence of specific bands, for example only esfahan, the river of zayanderud at varzaneh population had band 1100 bp of rapd primer opa-10, band 900 bp of the primer opm-10 and band 730 bp of the primer opa-04. the fars, lake bakhtegan population showed specific bands of 420 bp of the primer opb-05, while esfahan, nain, it is 70km to varzaneh population was the only population having band 150 bp of the primer opa-09, band 635 bp of the primer opc-01 and 220 bp of oph-07. yazd, kavire marvast population had the band of 370 bp of the primer opa-13. some bands were present in all except one population, for example bands 930 bp of the figure 2. pca plot of morphological data in salicornia persica populations studied; 1. esfahan, varzaneh ; 2. esfahan, the river of zayanderud at varzaneh; 3. esfahan, nain, it is 70km to varzaneh; 4. esfahan; varzaneh to kashan, 5. fars, tashk lake 6. fars, lake bakhtegan; 7. fars, abadeh tashk.8. yazd, kavire marvast .9. yazd, meybud. 10. yazd, nádüshan. table 3. genetic diversity parameters in the studied populations of salicornia persica (n = number of samples, ne = number of effective alleles, i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations). rapd loci total band na ne i he uhe %p opb05 19 0.201 1.00 0.29 0.21 0.22 42.23% opa04 7 0.341 1.058 0.24 0.20 0.20 53.75% opa10 24 0.455 1.098 0.377 0.34 0.32 55.05% opm10 10 0.499 1.067 0.24 0.23 0.24 49.26% oph07 11 0.555 1.020 0.22 0.24 0.28 43.53% opa13 15 0.431 1.088 0.29 0.25 0.25 41.53% opa05 20 0.255 1.021 0.23 0.28 0.22 47.15% opc01 11 0.724 1.067 0.143 0.095 0.180 38.74% opi12 16 1.115 1.003 0.257 0.251 0.280 52.57% opa09 12 1.215 1.075 0.157 0.271 0.270 40.57% mean 14.6 0.77 1.05 0.28 0.25 0.25 49.13% 64 zhu lin, hamed khodayari primer opa-05 and 870 bp of opm-10 were absent only in yazd, kavire marvast population. population genetic differentiation amova (phipt = 0.66, p = 0.010), and gst analysis (0.279, p = 0.001) revealed significant difference among the studied populations (table 4). it also revealed that, 41% of total genetic variability was due to within population diversity and 59% was due to among population genetic differentiation. pairwise amova produced significant difference among the studied populations. moreover, we got high values for hedrick standardized fixation index after 999 permutation (g’st = 0.279, p = 0.001) and jost, differentiation index (d-est = 0.777, p = 0.001). these results indicate that the geographical populations of salicornia persica are genetically differentiated from each other. populations, genetic affinity upgma tree and neighbor-net network produced similar results therefore only neighbor-net network is presented and discussed (fig. 3). we have almost complete separation of the studied population in the network, supporting amova result. the populations 3 and 4 are distinct and stand separate from the other populations with great distance. the populations 1 and 2, as well as populations 6 and 7 show closer genetic affinity and are placed close to each other. nei’s genetic identity and genetic distance for rapd data determined among the salicornia persica are given in table 5. the value of genetic identity varied from 0.552 between esfahan, varzaneh and yazd, meybud to 0.918 between esfahan, nain, it is 70km to varzaneh and esfahan; varzaneh to kashan. the mean value of genetic identity for rapd markers was 0.75. ward and nj dendrograms of rapd data produced similar results supported by pca ordination plot (fig. 2). the cophenetic correlation of ward tree was higher (r = 0.95) and therefore, it is discussed below. in general, two major clusters were obtained. the populations of esfahan, varzaneh ; 2. esfahan, the river of zayanderud at varzaneh; 3. esfahan, nain, it is 70km table 4. analysis of molecular variance (amova) of the studied species. source df ss ms est. var. % φpt among pops 18 226.576 22.327 19.082 59% 59% within pops 12 104.767 18.530 21.530 41% total 30 330.342 40.613 100% figure 3. neighbor-net of rapd data in salicornia persica populations studied. 1. esfahan, varzaneh ; 2. esfahan, the river of zayanderud at varzaneh; 3. esfahan, nain, it is 70km to varzaneh; 4. esfahan; varzaneh to kashan, 5. fars, tashk lake 6. fars, lake bakhtegan; 7. fars, abadeh tashk.8. yazd, kavire marvast .9. yazd, meybud. 10. yazd, nádüshan. table 5. nei’s genetic identity (above diagonal) for rapd data among salicornia persica. pop1 pop2 pop3 pop4 pop5 pop6 pop7 pop8 pop9 pop10 1.000 pop1 0.882 1.000 pop2 0.838 0.755 1.000 pop3 0.883 0.683 0.918 1.000 pop4 0.594 0.591 0.714 0.741 1.000 pop5 0.719 0.591 0.808 0.871 0.750 1.000 pop6 0.652 0.737 0.764 0.735 0.609 0.794 1.000 pop7 0.706 0.573 0.837 0.744 0.621 0.784 0.724 1.000 pop8 0.552 0.737 0.764 0.735 0.609 0.729 0.875 0.868 1.000 pop9 0.534 0.581 0.714 0.641 0.621 0.639 0.661 0.635 0.829 1.000 pop10 65genetic characterization of salicornia persica assessed using random amplified polymorphic dna to varzaneh; 4. esfahan; varzaneh to kashan, 5. fars, tashk lake 6. fars, lake bakhtegan; 7. fars, abadeh tashk.8. yazd, kavire marvast protected formed the first major cluster, out of which, esfahan, varzaneh and esfahan, the river of zayanderud at varzaneh show more rapd similarity and the esfahan; varzaneh to kashan, 5. fars, tashk lake 6. fars, lake bakhtegan; 7. fars, abadeh tashk.8. yazd, kavire marvast protected population differ the most from the other populations. two other populations form the second major cluster, which, yazd, meybud and yazd, nádüshan showed close genetic affinity. mantel test after 5000 permutations produced significant correlation between genetic distance and geographical distance in these populations (r = 0.55, p = 0.001). therefore, the populations that are geographically more distant have less amount of gene flow, and we have isolation by distance (ibd) in the salicornia persica. populations genetic structure k = 2 reveal the presence of 2 genetic group. similar result was obtained by evanno test performed on structure analysis which produced a major peak at k = 2 (fig. 5). both these analyses revealed that salicornia persica populations show genetic stratification. structure plot based on k = 2, revealed genetic affinity between populations 1: esfahan, varzaneh; 2. esfahan, the river of zayanderud at varzaneh; 3. esfahan, nain, it is 70km to varzaneh; (red colored), also population 9: yazd, meybud. 10. yazd, nádüshan (green colored), as well as populations 5 and 7 (green colored). the mean nm = 0.33 was obtained for all rapd loci, which indicates low amount of gene flow among the populations and supports genetic stratification as indicated by k-means and structure analyses. population assignment test also agreed with nm result and could not identify significant gene flow among these populations. however, reticulogram obtained based on the least square method (figure not included), revealed some amount of shared alleles among populations 7 and 8, and between 10 and 6 and 7, also between 8, and 9. this result is in agreement with grouping we obtained with pca plot, as these populations were placed close to each other. as evidenced by structure plot based on admixture model, these shared alleles comprise very limited part of the genomes in these populations and all these results are in agreement in showing high degree of genetic stratification within salicornia persica populations. discussion nature is having a hard time where human activities, global environmental changes, habitat loss and species extinction often lead to a loss of biodiversity. for example, habitat fragmentation and population decline could reduce the effective population size and threaten the viability of the target species (falk and holsinger, 1991; paul et al. 2021; salami et al. 2021; wasana et al. 2021). many biologists argue that establish correct conservation strategies minimizing biodiversity lossand a good example is conserve geographically-rare species (esfandani-bozchaloyi et al., 2018a, 2018b, 2918c, 2018d). programs to conserve rare and endemic plants should take into account the use of molecular markers because can contribute to the setting of conservation priorities (frankham et al., 2004). unfortunately, limited information is available regarding the population genetics of rare, endemic, threatened or endangered species. endemic (and rare) plants with narrow distrifigure 5. structure plot of rapd data in salicornia persica populations studied. 1. esfahan, varzaneh ; 2. esfahan, the river of zayanderud at varzaneh; 3. esfahan, nain, it is 70km to varzaneh; 4. esfahan; varzaneh to kashan, 5. fars, tashk lake 6. fars, lake bakhtegan; 7. fars, abadeh tashk.8. yazd, kavire marvast .9. yazd, meybud. 10. yazd, nádüshan. figure 4. nj tree of rapd data in salicornia persica populations studied. 1. esfahan, varzaneh ; 2. esfahan, the river of zayanderud at varzaneh; 3. esfahan, nain, it is 70km to varzaneh; 4. esfahan; varzaneh to kashan, 5. fars, tashk lake 6. fars, lake bakhtegan; 7. fars, abadeh tashk.8. yazd, kavire marvast .9. yazd, meybud. 10. yazd, nádüshan. 66 zhu lin, hamed khodayari bution range have been analyzed traditionally within the framework of the theoretical predictions of small populations. in these taxa, the lowest population genetic diversity levels are expected, and many study cases confirm such predictions (cole, 2003). our study of the genetic structure of salicornia persica populations has important implications for the conservation and management of this narrowly distributed. molecular analyses salicornia species can generally tolerate immersion in salt water. they use the c 4 to take in carbon dioxide from the surrounding atmosphere. salicornia has leafless stems with branches that resembles asparagus. taxonomic studies of the genus by moss (1954), duval-jouve (1868), scott (1977), ball (1964) and others ball and tutin (1959), ball and brown (1970) and the recent revision of eurasian salicornias by kadereit and her school (kadereit et al.,2012), are notable. there are three regional studies of salicornia that were based on molecular data (papini et al., 2004; murakeözy et al., 2007;). papini et al. (2004) found that diploid and tetraploid accessions of salicornia resolved as sister clades. the study was based on its sequences of twelve samples of salicornia (all but one from italy) representing four species (three tetraploid, one diploid). the presence of rapd polymorphic bands in the populations studied indicates the presence of genetic polymorphism in these populations. moreover, the occurrence of specific bands/loci only in some of the populations illustrates the occurrence of unique insertion/deletion in dna material of these genotypes. as stated before, there were bands which occurred in some of the populations but were absent in the others. since, even single base change at the primer annealing site is manifested as appearance or disappearance of rapd and issr bands, these bands may indicate the occurrence of genetic changes in the genome of the populations through the loss or rearrangement of some of their nucleotides (smith et al. 1996; dadzie et al. 2021; fikirie et al. 2020; hindersah & kalay 2021; hindersah et al. 2021; mieso & befa 2020). both rapd and morphological trees obtained are in general agreement separating the populations studied in two groups, indicating their genetic difference, which is also partly supported by issr analysis. diversity study in salicornia species in field growing plants morphological variations can be observed in growth pattern, plant canopy and number of other features. genetic diversity of the parental lines is good indicator of progeny performance. success through hybridization and subsequent selection depends primarily on the selection of parents having high genetic variability for various agronomic traits. sanane et al. (2003) conducted a study in japan on identification of salicornia population through morphological and rapd fingerprinting. they observed variations in plant length, segment number, length and number of branches, and incidence of the secondary branches etc. on the basis of genotype based on the rapd marker they identified five groups in three selected populations. gohil and pandya (2006) conducted study to find out the degree and the nature of genetic divergence among salicornia brachiata (roxb.) genotypes. gohil and pandya (2006) found a significant difference amongst the salicornia genotypes for all the phenological characters, (like height, canopy, main branch, segment, spike length, spike/branch and seed yield) indicating high genetic variability present in the population. the genotypes under study were grouped into five clusters, indicating wide diversity in the material for majority of the characters. zhang and bai (2022) studied in iran on population differentiation and gene flow of salicornia persica using issr markers and morphological traits. analysis of molecular variance (amova) test revealed significant genetic difference among the studied populations, and also showed that 45% of total genetic variability was due to the diversity within the population, while 55% was due to the genetic differentiation among populations. previous results from molecular studies imply near 100% inbreeding in salicornia, which certainly contributes greatly to the taxonomic difficulties in the group because of inbreeding lines with minute but fixed phenotypic differences (noble et al., 1992). dna polymorphism was detected among the three spanish populations of salicornia using random amplification of polymorphic dna (rapd) approach (luque et al., 1995). the other study using rapd technique showed correlations between dna polymorphism and geographical distribution in s. ramosissima (kruger et al., 2002). according to kadereit et al. (2007), the main reason for the taxonomic confusion are the young age of the extant lineages, the rampant dispersal of salicornia which has led to widespread genotypes with high phenotypic plasticity. this is the reason why salicornia plants have different names in different regions, and morphological parallelism resulted in the fact that different genotypes have the same name in one region. anita k. badlani, (2011) was undertaken to assess the genetic diversity among germplasm of salicornia collected from 11 different locations using rapd and issr marker system. 67genetic characterization of salicornia persica assessed using random amplified polymorphic dna this study will provide the genetic back ground of s. brachiata populations and extent of molecular diversity existing among them. the characterized diversity and identified polymorphic markers can be a good source of plant genetic resources and can be further exploited for genetic improvement of the species through marker assisted breeding. conclusions this study proved the potential of rapd marker analyses in the identification and evaluation of genetic relationships between populations of salicornia persica. this is the first report about the application of these techniques to estimate the genetic variation and genetic characterization of salicornia persica accessions. the results of this study can be useful in breeding programs, planning of conservation strategies, germplasm collection, and taxonomy of the genus. acknowledgment moe (ministry of education in china) project of humanities and social sciences “research on the organization mode and governance mechanism of shared recycling recreation” (20yjc630241); soft science research program of department of science and technology of henan province “research on organizational optimization and regulation of remanufacturing industry based on sharing economy”(202400410134) references akhani h. 2003. salicornia persica akhani (chenopodiaceae), a remarkable new species from central iran. linz. biol. beit., 35: 607-612. al-oudat, m. qadir, m. 2011. the halophytic flora of syria. international center for agricultural research in the dry areas (icarda), aleppo, syria. ball, p. w. 1964. a taxonomic review of salicornia in europe. feddes repert. 69: 1–8. ball, p. w. and tutin, t. g. 1959. notes on annual species of salicornia in britain. watsonia 4: 193–205. ball, p. w. and brown, k. g. 1970. a biosystematic and ecological study of salicornia in the dee estuary. watsonia 8: 27–40. bi, d., c. dan, m. khayatnezhad, z. sayyah hashjin, z. y. ma. 2021. molecular identification and genetic diversity in hypericum l.: a high value medicinal plant using rapd markers markers. genetika 53(1): 393-405. chen, w., khayatnezhad, m., sarhadi, n. 2021. protok gena i struktura populacije kod allochrusa (caryophylloideae, caryophyllaceae) pomocu molekularnih markera genetika 53(2): 799-812. cole, c.t. 2003. genetic variation in rare and common plants. annu. rev. ecol. evol. syst. 34: 213-237. choi, d., lim, g. s., piao, y. l., choi, o. y., cho, k. a., park, c. b., chang, y. c., song, y. i., lee, m. k. & cho, h. 2014. characterization, stability, and antioxidant activity of salicornia herbaciea seed oil. korean journal of chemical engineering, 31, 2221-2228. dadzie, r. g., amoah, r. s. ., ampofo-asiama, j., quaye, b., kizzie-hayford, n., & abano, e. e. 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(poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(3): 31-38, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1856 caryologia international journal of cytology, cytosystematics and cytogenetics citation: kristel ramírez-matadamas, elva irene cortés-gutiérrez, sergio moreno-limón, catalina garcía-vielma (2022). characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques. caryologia 75(3): 31-38. doi: 10.36253/caryologia-1856 received: september 29, 2022 accepted: november 25, 2022 published: april 5, 2023 copyright: © 2022 kristel ramírezmatadamas, elva irene cor tésgutiérrez, sergio moreno -limón, catalina garcía-vielma. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid kr-m: 0000-0001-6767-1683 eic-g: 0000-0002-5025-1284 sm-l: 0000-0002-8983-5914 cg-v: 0000-0003-3078-5761 characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques kristel ramírez-matadamas1, elva irene cortés-gutiérrez2, sergio moreno-limón2, catalina garcía-vielma1,* 1 departamento de citogenética, centro de investigación biomédica del noreste, instituto mexicano del seguro social. monterrey, n.l., méxico 2 facultad de ciencias biológicas, universidad autónoma de nuevo león, san nicolás de los garza, n.l., méxico *corresponding autor. e-mail: katygarcia2@hotmail.com; catalina.garciav@imss.gob.mx abstract. sotol (dasylirion cedrosanum trel.) is a perennial species with numerous grayish-to-green leaves that grow symmetrically from the base of the stem outward. its inflorescences can measure up to 3 m in height and contain membranous bracts that enclose seeds. it is a species that has been scarcely studied at the cytogenetic level, with only one report available in the literature. in mexico, it has economic importance because it is used to prepare the alcoholic beverage sotol. in the present work, the chromosomes of dasylirion cedrosanum trel. were obtained and analyzed using different cytogenetic banding techniques and morphometric analysis to construct the first karyotype for this species. chromosomes were obtained by germinating plant seeds collected in the locality of las adjuntas, santiago, nuevo león, mexico. treatment with colchicine as an antimitotic was performed, followed by enzymatic treatment with pectinase and cellulase, to eliminate the cell walls. chromosome slides were stained with giemsa, gtg banding technique, cbw banding, and the 4’,6-diamidino2-phenylindole fluorescence dye, and observed under a microscope. a chromosomal number 2n of 38 chromosomes, as previously reported, was confirmed. using the different banding techniques, we observed that all chromosomes exhibited a submetacentric morphology with a fundamental number of 76, and it was possible to visualize the pattern of gtg and cbw bands; these findings are reported for the first time for this species. morphometric analysis established that the average length of the chromosomes was between 5.09 and 9.84 mm. keywords: dasylirion cedrosanum trel., sotol, karyotype, morphology, cytogenetics. introduction sotol (dasylirion cedrosanum trel.) belongs to the asparagaceae family and was first described in 1838. sotol is perennial and polycarpic in nature, with a semicylindrical and apical morphology (zuccarini, 1838). the name of the genus means “thick lily” and it has numerous pointed and thorny 32 kristel ramírez-matadamas, elva irene cortés-gutiérrez, sergio moreno-limón, catalina garcía-vielma grayish-to-green leaves measuring from 30 to 170 cm with a spoon shape in their lower part that grows from the base of the stem outward in a symmetrical way. its stem is thick and fibrous, and up to 1 m in height (sierra tristán, 2008). it presents dioecious inflorescences with a single type of gametes in stamens (male scape) or pistils (female scape) that reach 3 m in height (flores-gallegos, 2019) (figure 1). the geographic distribution of sotol ranges from the southwestern united states to oaxaca, mexico. sixteen species were originally described, most of which are endemic to mexico (bogler, 1995), and additional species were later identified in northeastern mexico (bogler, 1998). dasylirion cedrosanum trel. is the species with the greatest economic importance in mexico, from which a drink called sotol is prepared via the fermentation of the stem of the plant (hernández-quintero, 2015). this drink is used for recreational purposes and as a medicinal remedy for diabetes and stomach ailments (goverment of mexico, 2022). the genus dasylirion has rarely been studied at the cytogenetic level. previous studies reported a diploid chromosome number of 38 in the species dasylirion texanum and dasylirion wheeler in specimens grown under greenhouse conditions, and also described multiple submetacentric chromosomes and two acrocentric chromosomes exclusively in dasylirion texanum (sato, 1935). for dasylirion cedrosanum trel., there is only one report of the gametic (n = 19) and somatic (2n = 38) chromosome number in plants collected in saltillo, coahuila, mexico (hernández-quintero, 2015). moreover, it has not been reported in the plant chromosomal number index (ipcn: index to plant chromosome numbers) of the missouri botanical garden, usa, where most of the records of the chromosome numbers of various plant species worldwide can be found (goldblatt, 2021). using different cytogenetic staining techniques, the chromosomes of a species can be observed and characterized. the usual giemsa and 4’,6-diamidino-2-phenylindole (dapi) staining allows the observation of the number and structure of each chromosome. the gtg banding technique (g bands with trypsin and giemsa) helps to visualize the shape and pattern of the light bands (euchromatin) and dark bands (heterochromatin) present in each chromosome; and the cbw banding technique (c bands with barium and wright’s stain) specifically stains the centromeres and heterochromatic regions of the chromosomes (barch, 1997). the morphometric measurements of chromosomes are another important tool in this context. images of the chromosomes are acquired under a bright-field or fluorescence microscope, and are analyzed using software that reports the results of the measurements in micrometers. the morphometric measurements that can be used are as follows: the total length of the chromosome (tcl), the length of the short (sal) and long (lal) arms, and the average length of the short (asal) and long (alal) arms. the centromere index (ci), which is the relationship between the length of the short arm of the chromosome and the tcl (peruzzi, 2013), is expressed as a percentage (0%–50%). the relative longitude (rl), which is defined as the tcl divided by the sum of the total length of the karyotype, is also expressed as a percentage (jabeen, 2012), whereas the fundamental number (fn) is the number of short and long arms present in a karyotype (matthey, 1945; matthey, 1965) and helps to determine the type of chromosomes present (nirchio, 2014). the objective of this study was to obtain and characterize the chromosomes of dasylirion cedrosanum trel. to develop a karyotype of the species based on the morphological characteristics of its chromosomes. figure 1. dasylirion cedrosanum trel. (photograph by arturo cruz anaya, https://www.naturalista.mx/observations/21031288) 33characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques materials and methods collection site we used dasylirion cedrosanum trel. seeds collected in the locality of las adjuntas, santiago, nuevo león, mexico (25°18’03.6”n, 100°08’27.3”w) (figure 2). preparation seeds the seeds were washed and disinfected with 1% sodium hypochlorite (sigma-aldrich, st. louis, usa), and 10 seeds were placed in each petri dish (pyrex, tehama, ca, usa) in triplicate, and transferred to a bioclimatic chamber (stemcells technologies, vancouver, canada) at 25-30°c for approximately 10 days for germination. cytogenetics to obtain chromosomes, the technique of hernández-quintero et al. (2015), with some modifications, was used. the apical meristems were cut with a scalpel, preferably between 08:00 and 10:00 am, and incubated in 2% colchicine (sigma-aldrich) at 37°c for 48 h, followed by washing for 15 min with distilled water. farmer’s fixative solution (methanol:glacial acetic acid, 3:1) (ctr scientific, mexico) was then added, and the solution was incubated for 24 h at 4°c, washed in distilled water for 15 min, vortexed for 5 min, and centrifuged at 7440’ g for 1 min. subsequently, the supernatant was decanted and a mixture of pectinase (plantmedia, ohio, usa) and cellulase (plantmedia) at a ratio of 1:1 at 0.2% was added, followed by resuspension of the pellet in distilled water and incubation at 37°c for 2 h. the cell button was dripped onto slides with a layer of glacial acetic acid, and heat was then applied. different staining and cytogenetic banding techniques were performed on the chromosomes obtained. 1) giemsa staining: the chromosomes were incubated in giemsa solution (sigma-aldrich) (1:20) for 5 min, rinsed in distilled water, and allowed to dry. 2) gtg banding: the chromosomes were incubated in 0.025 m trypsin at 37°c for 1 min, stained with giemsa stain 1:20 for 5 min, rinsed, and allowed to dry. 3) cbw banding: the figure 2. dasylirion cedrosanum trel. seed collection site, las adjuntas, santiago, n.l., mexico (25°18’03.6’’n, 100°08’27.3’’w), as indicated by the red globe (inegi map, 2021 https://www.inegi.org.mx/app/mapas/). 34 kristel ramírez-matadamas, elva irene cortés-gutiérrez, sergio moreno-limón, catalina garcía-vielma chromosomes were incubated in 0.2 n hcl (sigmaaldrich) for 60 min at room temperature, rinsed with dnase-free water, and allowed to dry; then they were immersed in 5% baoh (sigma-aldrich) for 40 min and rinsed, passed through 70% and 100% ethanol, immersed in 2× ssc (3 m sodium chloride and 300 mm sodium citrate dihydrate, ph 7.0, sigma-aldrich) for 60 min at 60°c, rinsed, dried, and stained with 1:3 wright’s stain for 2 min. 4) fluorescent staining with dapi (sigma-aldrich): 7 µl of dapi at 2.5 mg/ml were added to chromosomes, which were then incubated for 15 min at 4°c. the slides were observed using an axioscope a1 microscope (zeiss, göttingen, germany) with a 100× objective and coupled to an axiocam 502 mono camera (zeiss) coupled to zen blue (version 3.3.89.0000) software (zeiss). images were acquired with an axiophot hxp 120 v fluorescence lamp with a dapi filter (zeiss). the karyotype was organized based on the size of the chromosomes, from longest to shortest, in descending order, and pairing the homologues based on the observed gtg bands (levan, 1964). morphometry drawid (version 0.26) software (kirov et al., 2017) was used to perform the morphological measurements of tcl, sal, lal, asal, alal and ci, and for the elaboration of the ideogram of the chromosomes. the images of the metaphases were opened in the software and each chromosome was measured, starting from the long arm toward the middle region, where the position of the centromere was marked (centromere button), and continuing with the short arm at the lower end, thus ending the measurement. the process was repeated individually for each chromosome. rl was obtained using the formula: rl = (tcl / stcl) (100); tcl was obtained by adding the measurements of sal and lal; asal and alal were obtained based on the lengths determined for the 38 chromosomes; whereas ci was obtained from the formula: ci = sal / (sal + lal). all measurements were performed individually for each of the 38 chromosomes in each of the cells analyzed. the fn of the karyotype was calculated using as a criterion the presence of two arms in the chromosomes. descriptive statistics were applied, such as mean and standard deviation, using spss statistics, version 22 (ibm, armonk, ny, usa). results cytogenetics thirty metaphases were analyzed, which led to the identification of 38 chromosomes (2n) in 100% of the analyzed cells. all chromosomes were of the submetacentric type, that is, with the centromere displaced toward one of the ends, in which the arms differed in length. using the gtg banding technique, it was possible to establish a pattern of bands in each chromosome, with the presence of dark bands in the telomeric regions of most of them. based on the gtg banding patterns, the karyotype depicted in figure 3 was established. using cbw banding, the centromere was observed in a displaced position toward one of the ends of the arms of the chromosomes. concomitantly, heterochromatin regions were observed at the ends of some chromosomes (figure 4). figure 3. karyotype of dasylirion cedrosanum trel. stained with gtg banding. the pattern of bands in each chromosome and the dark bands in the telomeric region (arrow) are shown. figure 4. chromosomes of dasylirion cedrosanum trel. stained with cbw banding, in which the presence of telomeric heterochromatin is confirmed (arrow). 35characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques visualization using dapi allowed us to observe the morphology of the chromosomes in greater detail, while also confirming their number and submetacentric shape (figure 5). morphometry tcl average of the chromosomes was in the range of 5.09 mm for the shortest chromosome and 9.84 mm for the longest. the remaining chromosomes ranged from 5.47 to 8.62 mm in length. the average of the sal indicated a length of 2.96 ± 0.58 mm, whereas the average of the lal was 3.93 ±0.73 mm. the rl average of the 38 chromosomes ranged from 1.94% to 3.76%, whereas the ci average was between 39.98% and 44.66 %. fn of the karyotype of dasylirion cedrosanum trel. was 76, considering as a criterion that each chromosome had two arms (table 1). based on the results of staining, banding, and previous measurements, and using the image analysis software, an ideogram of the dasylirion cedrosanum trel. chromosomes was constructed with gtg bands (figure 6). figure 5. chromosomes of dasylirion cedrosanum trel. stained with dapi. more intensely marked areas of constitutive heterochromatin can be observed (chromosomes marked with an asterisk). in the upper-right corner, the cbw bands can be compared with the zones detected using dapi, thus confirming the distribution in the chromosomes of the constitutive heterochromatin. table 1. mean length of short arm chromosome (sal), long arm chromosome (lal), total arm chromosome (tcl), relative length (rl), and centromeric index (ci) from 30 metaphases of dasylirion cedrosanum trel (2n=38). chromosome sal x̄±sd (µm) lal x̄±sd (µm) tcl x̄ (µm) rl % ci % type of chromosome 1 4,44±1,42 5,79±1,63 9,84 3,76 44,66 submetacentric 2 3,66±0,44 5,06±1,44 8,62 3,29 43,76 submetacentric 3 3,81±1,25 4,54±0,46 8,30 3,17 43,24 submetacentric 4 3,48±0,83 4,64±0,84 8,01 3,06 42,21 submetacentric 5 3,48±0,77 4,31±0,68 7,72 2,95 43,31 submetacentric 6 3,26±0,72 4,32±0,64 7,52 2,87 43,64 submetacentric 7 3,04±0,84 4,23±0,45 7,19 2,74 43,11 submetacentric 8 2,77±0,66 4,22±0,51 6,96 2,66 39,98 submetacentric 9 2,73±0,30 4,07±0,90 6,77 2,59 41,96 submetacentric 10 2,89±0,49 3,78±0,66 6,64 2,54 44,47 submetacentric 11 2,81±0,47 3,74±0,66 6,53 2,49 43,34 submetacentric 12 2,80±0,63 3,63±0,52 6,41 2,45 42,57 submetacentric 13 2,58±0,27 3,72±0,76 6,26 2,39 42,73 submetacentric 14 2,70±0,49 3,48±0,57 6,16 2,35 43,12 submetacentric 15 2,66±0,33 3,38±0,70 5,99 2,29 43,27 submetacentric 16 2,39±0,10 3,48±1,03 5,84 2,23 41,65 submetacentric 17 2,31±0,70 3,44±0,34 5,69 2,17 41,64 submetacentric 18 2,49±0,56 3,02±0,44 5,47 2,09 43,79 submetacentric 19 2,23±0,60 2,94±0,52 5,09 1,94 43,85 submetacentric asal= 2.96±0.58 alal=3.93±0.73 σtcl=261.88 sd = standard deviation, asal= average short arm, alal= average long arms. 36 kristel ramírez-matadamas, elva irene cortés-gutiérrez, sergio moreno-limón, catalina garcía-vielma discussion in the present work, we found a somatic number of 38 chromosomes in the studied specimens of dasylirion cedrosanum trel. these results coincide with those reported by hernández-quintero et al. (2015), thus reaffirming that the chromosome number is highly conserved among the species of the genus dasylirion (sato, 1935). in some plant species, there is a natural selection process, karyotypic orthoselection, which consists of the presence of the same type of chromosome rearrangement in specimens of the same species, thus maintaining the chromosome number, which is involved in the process of evolution (palomino, 2010). this process occurs via the amplification of noncoding regions of dna at chromosome crossover sites, resulting in a uniform basic number and karyotypes that maintain chromosome number and structure (flores-maya et al., 2015). additional cytogenetic and molecular biology studies are required to determine if the same process is present in dasylirion cedrosanum trel. in plants, it is difficult to obtain chromosomal bands because of the presence of a cell wall and the characteristics of the tissue (chattopadhyay, 1988). in plant chromosomes, the pattern of gtg bands observed in mammals has not been reported. however, in this work, modifications of the original technique allowed the resolution of light and dark chromosomal bands, leading to the identification of seven bands on large chromosomes and five bands on smaller chromosomes. in addition, dark telomeric bands were detected in most of the chromosomes, which represents the first report of a banding pattern for this species. previous work on c. pubescens indicated the presence of dark bands at the telomere level (guevara, 2000) and suggested that these bands represent the constitutive heterochromatin—as corroborated here in dasylirion cedrosanum trel.—with the implementation of the cbw bands. plant chromosomes contain much more dna than do vertebrate chromosomes, with a comparable length and with a higher degree of compaction, which explains the presence of these dark bands at the telomere level (argüelles saenz, 2018). using different cytogenetic banding techniques and morphometric analysis, we established that all chromosomes were of the submetacentric type, which differs from the report of subtelocentric chromosomes previously (hernández-quintero et al., 2015). therefore, it was possible to establish an fn of 76 for dasylirion cedrosanum trel., and represents the first report defining the fn in this species. the tcl of the chromosomes was significantly higher than those obtained in previous work (hernández-quintero et al., 2015). although hydroxyquinoline is widely used in plants and is especially suitable for species with large chromosomes (sharma, 2014), in the present work, colchicine was used as an antimitotic agent, with modification of the exposure time and concentration, and afforded more elongated chromosomes, which assisted observation of the bands in each of them. the ci average was between 39.98 % and 44.66 %, and the rl was between 1.94 % and 3.76 % for each chromosome, which can be considered as an approximation of the contribution of each of them to the total content of the dasylirion cedrosanum trel. genome. figure 6. ideogram of the chromosomes of dasylirion cedrosanum trel., with gtg bands. 37characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques although all chromosomes present in the karyotype of dasylirion cedrosanum trel. were of the submetacentric type, in some plant species, transcriptional activity has been observed in nonacrocentric chromosomes. as a future perspective of this study, agnors banding or fluorescent in situ hybridization using specific probes for the 5s and 45s regions should be carried out. it is important to determine the number of chromosomes and the ploidy level of a species, especially in those of economic importance. in the case of dasylirion cedrosanum trel., the lack of cytogenetic studies is well known. to our knowledge, this is the first time that chromosomal characteristics have been reported and a karyotype constructed for this species in specimens from the state of nuevo león. in addition, these data are very useful in the phylogenetic and taxonomic study of a species to analyze the evolutionary mechanisms involved in speciation and diversity. author’s contributions c.g.v, e.i.c.g and s.m.l designed the study, k.r.m. and c.g.v. performed analyses, k.r.m., c.g.v, e.i.c.g and s.m.l collected data, k.r.m., c.g.v and e.i.c.g led the writing of the manuscript. all authors contributed critically to the drafts and gave final approval for publication. geolocation information h t t p s : // w w w . j o u r n a l m a p . o r g / s e a r c h # l i s t ? b o u n ds=57.98481,164.35547|-24.36711,44.47266&precision=1& query=sotol acknowledgments in memory of dr. sergio moreno-limón, thank you for your hard work and dedication for imparting the knowledge of botany. to arturo cruz anaya for giving us permission to use one of his photographs of dasylirion cedrosanum trel. references argüelles saenz, f. v. 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(1838). ueber eine neue gattung aus der familie der bromeliaceae. allgemeine gartenzeitung, 33, 257-259. caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 3 2022 firenze university press chromosome mapping of repetitive dnas in the picasso triggerfish (rhinecanthus aculeatus (linnaeus, 1758)) in family balistidae by classical and molecular cytogenetic techniques kamika sribenja1, alongklod tanomtong1, nuntaporn getlekha2,* chromosome number of some satureja species from turkey esra kavcı1, esra martin1, halil erhan eroğlu2,*, fatih serdar yıldırım3 l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations selma tabur1,*, nai̇me büyükkaya bayraktar2, serkan özmen1 characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques kristel ramírez-matadamas1, elva irene cortés-gutiérrez2, sergio moreno-limón2, catalina garcía-vielma1,* contributions of species rineloricaria pentamaculata (loricariidae:loricariinae) in a karyoevolutionary context a cius¹, ca lorscheider2, lm barbosa¹, ac prizon¹, ch zawadzki3, la borin-carvalho¹, fe porto4, alb portela-castro1,4 cadmium induced genotoxicity and antioxidative defense system in lentil (lens culinaris medik.) genotype durre shahwar1,2,*, zeba khan3, mohammad yunus khalil ansari1 biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts and evaluation of their biosafety potential denisa manolescu1,2, georgiana uță1,2,*, anca șuțan3, cătălin ducu1, alin din1, sorin moga1, denis negrea1, andrei biță4, ludovic bejenaru4, cornelia bejenaru5, speranța avram2 polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel (foeniculum vulgare mill.) egizia falistocco methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. sazada siddiqui*, sulaiman a. alrumman comparative study and genetic diversity in malva using srap molecular markers syamand ahmed qadir1, chnar hama noori meerza2, aryan mahmood faraj3, kawa khwarahm hamafaraj4, sherzad rasul abdalla tobakari5, sahar hussein hamarashid6,* nuclear dna 2c-values for 16 species from timor-leste increases taxonomical representation in tropical ferns and lycophytes inês da fonseca simão1, hermenegildo ribeiro da costa1,2,3, helena cristina correia de oliveira1,2, maria helena abreu silva1,2, paulo cardoso da silveira1,2,* nuclear dna content and comparative fish mapping of the 5s and 45s rdna in wild and cultivated populations of physalis peruviana l. marlon garcia paitan*, maricielo postillos-flores, luis rojas vasquez, maria siles vallejos, alberto lópez sotomayor identification of genetic regions associated with sex determination in date palm: a computational approach zahra noormohammadi1,*, masoud sheidai2, seyyed-samih marashi3, somayeh saboori1, neda moradi1, samaneh naftchi1, faezeh rostami1 comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(4): 123-131, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1678 caryologia international journal of cytology, cytosystematics and cytogenetics citation: seyed mahmood ghaffari¹, seyed mohsen hesamzadeh hejazi² (2022). cytogenetic studies in the centaurea aucheri group (sect. phaeopappus). caryologia 75(4): 123-131. doi: 10.36253/caryologia-1678 received: june 06,2022 accepted: december 06, 2022 published: april 28, 2023 copyright: © 2022 seyed mahmood ghaffari¹, seyed mohsen hesamzadeh hejazi². this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid smg: 0000-0002-6777-6992 cytogenetic studies in the centaurea aucheri group (sect. phaeopappus) seyed mahmood ghaffari¹,*, seyed mohsen hesamzadeh hejazi² ¹ institute of biochemistry and biophysics, university of tehran, tehran, iran ² research education and extension organization (areeo), tehran, iran *corresponding author. e-mail: mghaffary@ut.ac.ir abstract. in this study, it was aimed to evaluation of the revision in centaurea aucheri group by the cytogenetical studies and determines the chromosome number for c. albonitens species, to determine chromosome morphology and karyotype analysis. results of meiotic behavior and karyomorphologycal parameters of centaurea aucheri group indicated that raised this group in five distinct species are correct. somatic and gametic chromosome numbers indicated that c. albonitens, c. assadii, c. farsestanica, c. indistincta and c. phaeopappa are diploid with n=9 and 2n=18 and c. aucheri is tetraploid with n=18 and 2n= 4x= 36+0-2b. gametic chromosome number and karyomorphology of c. aucheri and c. indistincta species is reported here for the first time. also, meiotic behavior and karyomorphology parameters of c. albonites, c.assadi , c. farsestanica and c. phaeopappa are newly reported here. analysis of karyotype and behavior of meiosis indicated that c. aucheri is a natural allotetraploid species. karyotype symmetry parameters showed that all the studied species were classified in the class 2a except of c. aucheri which was located in class 2b. keyword: allotetraploid, centaurea aucheri, cytogenetic, karyomorphology, meiotic behavior. introduction centaurea, as the fourth largest genus among the genera in asteraceae and also the second largest genus in the tribe cardueae consist of 200–250 species placed in 40 sections (wagenitz and hellwig 1997; lópez et al. 2011 hilpold et al. 2014 ). the present classification of centaurea in 40 sections is highly problematic (wagenitz 1975) and does not take into account all of the pollen, karyological and carpological evidence (susanna et al. 1995). in flora iranica 88 species belong to 28 sections are reported (wagenitz 1980). centaurea section phaeopappus (dc.) o. hoffm.is one of the section comprising of five species in flora iranica. of these, 3 taxa viz: c. albonitens, c. aucheri and c. spectabilis are distributed in iran (wagenitz 1980). ranjbar and heydari (2016) elevated this section to 14 species in the world, which 12 are belongs to iran (10 species are endemic to the country). in flora of iran 5 subspecies has been cited for c. aucheri viz: subsp. aucheri, subsp. elbur124 seyed mahmood ghaffari, seyed mohsen hesamzadeh hejazi sensis, subsp. farsistanica, subsp. indistincta and subsp. szowitsii (wagenitz 1980; mozafarian 2018). ranjbar and negaresh (2013) raised these five subspecies to the c. aucheri (dc.) wagenitz, c. assadii ranjbar and negaresh, c. farsistanica (wagenitz) ranjbar & negaresh, c. indisticta (wagenitz) and c. phaeopappa (dc.) schultz bipontinus respectively based on the morphological characteristics. the aims of this present paper is evaluation of the revision in centaurea aucheri group by the cytogenetical studies. materials and methods the origin of the plant material studied here is shown in table 1. for meiotic studies floral buds of plants found in nature were collected and immediately fixed in piennr’s fluid containing ethanol 96%, chloroform, propionic acid, 6:3:2 (v/v/v)for 24 hours at room temperature. anthers dissected out from the buds were squashed and stained with 2% acetocarmine. chromosome counts obtained from a minimum of 50 pollen table 1. the species and origin of the material examined. taxon origin altitude(m) longitude and latitude h. code centaurea albonitens turrill azerbaijan: tabriz, 20 km towards marand 1360 e:46°03’57”; n:38°15’48” 109080 azerbaijan: between khoy and salmas 1530 e: 44°54’15”; n: 38°24’39” 109081 zanjan, 10 km towards miyaneh 1500 e: 47°27’35”; n: 37°12’36” 109101 zanjan, 30 km towards miyaneh 1600 e: 47°30’53”; n: 37°14’41” 109102 centaurea aucheri kordestan:saqqez to divandareh 2200 e: 46°54’15”; n: 36°24’39” 109091 kordestan: w saqqez 2160 e: 46°16’13”; n: 36°15’39” 109092 qazvin: avaj, 10 km. to hamadan 2400 e: 49°09’38”; n: 35°32’17” 109201 qazvin: neck of avaj 2320 e: 49°12’05”; n: 35°35’35” 109202 qazvin: abgarm to avaj 2300 e: 49°14’07”; n: 35°42’58” 109203 hamadan: 90 km. to qazvin 2070 e: 49°02’19”; n: 35°27’54” 109301 lorestan: doroud, gahar, saravand 2110 e: 49°10’03”; n: 33°22’26” 109421 markazi: tafresh, noghre-kamar 2080 e: 50°08’13”; n: 34°41’53” 109501 centaurea assadii azerbaijan:miyaneh to bostanabad 1720 e: 47°18’27”; n: 37°32’05” 109087 tehran: kouhdashteh 1800 e: 51°20’13”; n: 35°44’23” 109701 tehran: kouhdashteh 1950 e: 51°23’50”; n: 35°44’29” 109702 tehran: sorkheh hesar 1500 e: 51°36’03”; n: 35°43’12” 109714 tahran: sorkheh hesar 1700 e: 51°35’46”; n: 35°43’09” 109712 tehran: jajroud 2200 e: 51°29’45”; n: 35°42’39” 109754 tehran: abali 2400 e: 51°57’55”; n: 35°45’54” 109718 centaurea farsistanica shiraz: bamoo park 1750 e: 52°36’52”; n: 29°42’48” 109614 shiraz: bamoo park 1820 e: 52°37’15”; n: 29°43’11” 109615 shiraz: bamoo park 2000 e: 52°36’33”; n: 29°12’41” 109617 fars: dasht arjan, khers dareh 2400 e: 51°58’56”; n: 29°39’47” 109687 yasouj, kakan 2300 e: 51°35’57”; n: 30°38’18” 109813 yasouj: kamehr 2400 e: 51°35’38”; n: 30°39’55” 109815 centaurea indistincta tehran: kouhdashteh 1800 e: 51°20’13”; n: 35°44’23” 109706 tehran: kouhdashteh 2300 e: 51°23’50”; n: 35°44’30” 109709 lorestan: doroud, gahar 2100 e: 49°10’03”; n: 33°22’25” 109432 zanjan 1630 e: 47°33’53”; n: 37°14’41” 109132 centaurea phaeopappa karaj, eshtehard, dakin 1550 e: 50°27’36”; n: 35°37’18” 109745 qazvin: abgarm to avaj 2300 e: 49°14’07”; n: 35°42’58” 109208 tahran: kohdashteh 1800 e: 51°20’13”; n: 35°44’24” 109784 karaj, ziyaran 2000 e: 50°31’43”; n: 36°07’06” 109764 azerbaijan: oroumiyeh to mahabad 1330 e: 45°19’22”; n: 37°13’52” 109075 azarbaijan: salmas to oroumiyeh 1375 e: 45°36’08”; n: 37°34’46” 109094 azerbaijan: mishodagh mt. 2040 e: 45°38’23”; n: 38°17’50” 109049 qazvin: takestan to hamadan 2350 e: 49°01’44”; n: 35°20’46” 109257 125cytogenetic studies in the centaurea aucheri group (sect. phaeopappus) mother cells within each collection. actively growing root tips were used for mitotic analysis. roots were pretreated with 0.002m, 8-hydroxyquinoline at 20˚c for 3 hr, and then fi xed in 3:1 (ethanol: acetic acid). staining was carried out with the feulgen reaction enhanced by squashing in 2% acetocarmine. nomenclature adopted by levan et al. (1964) was followed for recognizing chromosome types. both mitotic and meiotic slides were made permanent by the venetain turpentine (wilsom 1945). voucher specimens are preserved in the herbarium of research institute of forests and rangelands (rifr). results centaurea albonitens turrill centaurea albonitens is widely distributed in western iran, especially zanjan, west and east azerbaijan provinces. five samples of this taxon showed chromosome numbers of n=9 and 2n=18 in both meiosis and mitotic respectively. meiosis in this taxon showed 9 bivalents at diakinesis which ring tetravalent in some cells were observed (figure 1a). also, two bivalents were associated with the nucleolus at diakinesis, which is confi rmed to presence of two satellite chromosomes in this species. first metaphase indicated 9 bivalents, which most of them were in rod shape (figure 1b). anaphase i showed (9-9) chromosomes segregation (figure 1c). somatic chromosome counts in 50 root tips disclosed a chromosome number of 2n=18 (figure 1d, e). th e representatives of chromosome set at mitotic metaphase are shown in figure 1f and table 2. th e karyotype of c. albonitens consists of one pair of metacentric chromosome and 8 pairs of sub-metacentric chromosomes. th e total length of the chromosomes varied from 5.18 µm to 2.96 µm (table 2). th is count (2n=18) agrees with previous reports by garcia-jacas et al. (1998) and ranjbar and negaresh (2013). karyotype analysis and meiotic count for this taxon is reported here for the fi rst time. c. aucheri (dc.) wagenitz eight samples of this taxon showed chromosome numbers of n=18 and 2n=36 in both meiosis and mitotic respectively. meiosis showed some clamping of chromosomes at fi rst metaphase. th e bivalents at metaphase i were usually in the shape of rod with one terminal chiasmata (figure 2a). many cells were observed in order to ascertain the correct chromosome count. th e number c f figure 1. meiosis and mitotic micrographs of centaurea albonitens. a; diakinesis, showing ring tetravalent (arrow). b: metaphase i (n=9). c: anaphase i (9-9). d: late prophase (2n=18). e: metaphase (2n=18). f: karyotype showing nine pairs of chromosomes. scale= 5μm. 126 seyed mahmood ghaffari, seyed mohsen hesamzadeh hejazi n=18 observed at diakinesis (figure 2b) and (18-18) segregation at first anaphase (figure 2c). in the first meiosis stage, we did not observe any tetravalents at metaphase i and diakinesis. also we did not found any abnormality at first and second anaphase of meiosis, which is prevalent in autopolyploids species. these results indicated that this taxon is natural allotetraploid species. somatic chromosome counts of 50 root tips disclosed a chromosome number of 2n= 36+0-2b (figure 2d,e). these b-chromosomes were not found at meiosis stage. the karyotype consisted of 15 metacentric pairs, and 3 submetacentric pairs (figure 3a). the total length of the chromosomes varied from 6.11 µm to 2.85 µm (table 3). different chromosome number of this taxon (2n=36) with others subspecies (2n=18) and allotetraploid behavtable 2. measurement of somatic chromosomes in a diploid c. albonitens (obtained from 50 cells). no. of chromosome total length (µm) long arm (µm) short arm (µm) arm ratio l/s = r 1 5.18 2.59 2.59 1 2 4.44 2.59 1.85 1.4 3 3.89 2.22 1.67 1.33 4 3.33 2.22 1.11 2 5 3.33 2.04 1.29 1.85 6 3.33 2.04 1.29 1.85 7 3.14 1.85 1.29 1.43 8 2.96 1.85 1.11 1.67 9 2.96 1.85 1.11 1.67 figure 2. meiosis and mitotic micrographs of centaurea aucheri group ae. c. aucheri, a: metaphase i. b: diakinesis. c: anaphase i. d: mitotic metaphase, showing b-chromosome (arrow). e: mitotic metaphase, showing 2b-chromosomes (arrows). scale = 5μm. 127cytogenetic studies in the centaurea aucheri group (sect. phaeopappus) ior indicated that upgrade to species rank (c. aucheri (dc.) wagenitz ) by ranjbar and negaresh (2013) is correct. in addition, we found some specimens of this taxon together with c. aucheri subsp. szowitsii (c. phaeopappa) in one place (abgarm to avaj, table 1), that chromosome examination showed independence in chromosome number for each species ( subsp. aucheri , n=18, 2n=36: subsp. szowitsii n=9, 2n=18) and did not show any hybrid adjective between this two taxon. c. assadii ranjbar & negaresh wagenitz (1980) is introduced the c. aucheri subsp. elbursensis (c. assadii) as a new endemic subspecies to iran for the first time. previous chromosome number report for this taxon is n=9 by ghaffari (1986). the results obtained in this study showed nine bivalents in pollen mother cells at diakinesis (figure 2f). another stages of meiosis showed chromosome segregation (9-9) at anaphase i and 9 chromatid segregation at anaphase ii (figure 2g,h). chromosome complement at metaphase of mitotic was 2n=18 (figure 2i). karyotype consisted of seven pairs of metacentric and two pairs of submetacentric chromosomes (figure 3b). the total length of the chromosomes varied from 4.74 µm to 2.79 µm (table 4). this taxon distributed in azerbaijan, mazandaran, qazvin, and tehran provinces. we found overlapping of this species with c. indistincta and c. phaeopappa in one place (tehran, kouhdashteh, see table 1) and did not see any hybrid between them. lack of geographical distribution independence, joint with other subspecies, morpho figure 3. a: karyotype of c. aucheri. b: karyotype of c. assadii. c: karyotype of c. farsestanica. d: karyotype of c. indistincta. e: karyotype of c. phaeopappa scale bar = 10 μm. table 3. measurement of somatic chromosomes in a tetraploid c. aucheri (obtained from 12 cells). no. of chromosome total length (µm) long arm (µm) short arm (µm) arm ratio l/s = r 1 6.11 3.67 2.44 1.50 2 5.61 3.35 2.26 1.48 3 5.11 3.02 2.08 1.45 4 4.76 2.49 2.26 1.10 5 4.41 2.49 1.91 1.30 6 4.38 2.47 1.91 1.29 7 4.23 2.44 1.79 1.36 8 4.20 2.61 1.58 1.64 9 4.16 2.84 1.32 2.14 10 4.06 2.44 1.61 1.51 11 3.91 1.97 1.94 1.01 12 3.55 2.14 1.41 1.52 13 3.52 2.23 1.29 1.72 14 3.50 2.17 1.32 1.64 15 3.32 2.11 1.20 1.75 16 3.23 1.82 1.41 1.29 17 2.91 1.76 1.14 1.53 18 2.85 1.61 1.23 1.30 128 seyed mahmood ghaffari, seyed mohsen hesamzadeh hejazi logical characteristics and difference in chromosomal characteristics with c. aucheri, indicated that this taxon is a distinct species. c. farsistanica (wagenitz) ranjbar & negaresh meiosis in this taxon was regular and showed nine bivalents at metaphase i which more of them were in rod shape (figure 2j). anaphase i indicated (9-9) chromosomes segregation (figure 2k). mitotic study showed 2n=18 chromosomes at metaphase (figure 2l). this diploid species has a symmetrical karyotype with six pairs of metacentric and three pairs of submetacentric chromosomes (figure 3c). the total length of the chromosomes varied from 3.98 µm to 2.25 µm (table 5). this taxon is completely different with others subspecies in morphological characteristic and pattern of distribution (ranjbar and negaresh 2013), which are introduced by wagenitz (1980) and mozaffarian (2018). centaurea indistincta (wagenitz) ranjbar & negaresh this taxon is reported by wagenitz (1980) as a new endemic subspecies species (c. aucheri subsp. indistincta) for flora of iran, which was introduced by ranjbar and negaresh (2013) as a distinct species. meiosis in this species showed nine bivalents at metaphase i and (9-9) univalents segregation at first anaphase (figure 2m,n). chromosome complement in this species was 2n=18 (figure 2o). karyotype consisted of five pairs of metacentric and four pairs of submetacentric chromosomes (figure 3e). the total length of the chromosomes varied from 4.08 µm to 2.52 µm (table 6). centaurea phaeopappa (dc.) schulpz & bipontinus meiotic and mitotic divisions were examined on eight samples of this species (table 1). meiosis showed nine bivalents at diakinesis and metaphase i (figure 2p,q). nondisjunction of (8-10) segregation at anaphase i was observed (figure 2r). also, in some cells laggard chromosomes at anaphase ii were observed (figure 2s). mitotic stages in this species indicated chromosome complement of 2n= 18 (figure 2t) which is agrees with the previous report by ghaffari (1988). the karyotype consisted of six pairs of metacentric and three pairs of submetacentric chromosomes (figure 3f). the total length of the chromosomes varied from 4.21 µm to 2.70 µm (table 7). discussion the study has detected the somatic and gametic chromosome number and karyomorphology of c. table 4. measurement of somatic chromosomes in a diploid c. assadii (obtained from 9 cells). chromosome no total length (µm) long arm (µm) short arm (µm) arm ratio l/s = r 1 4.74 2.43 2.30 1.05 2 4.67 2.51 2.15 1.16 3 4.10 2.45 1.65 1.48 4 3.87 2.43 1.43 1.69 5 3.43 1.94 1.48 1.31 6 3.23 2.28 0.95 2.40 7 3.19 1.84 1.35 1.36 8 3.02 1.80 1.22 1.47 9 2.79 1.54 1.24 1.23 table 5. measurement of somatic chromosomes in a diploid c. farsistanica (obtained from 14 cells). chromosome no total length (µm) long arm (µm) short arm (µm) arm ratio l/s = r 1 3.08 2.24 1.73 1.29 2 3.34 1.95 1.39 1.39 3 3.09 1.82 1.27 1.43 4 2.71 1.90 0.80 2.36 5 2.54 1.31 1.23 1.06 6 2.46 1.39 1.06 1.31 7 2.38 1.27 1.11 1.14 8 2.29 1.44 0.85 1.69 9 2.25 1.49 0.76 1.94 table 6. measurement of somatic chromosomes in a diploid c. indistincta (obtained from 8 cells). chromosome no total length (µm) long arm (µm) short arm (µm) arm ratio l/s = r 1 4.08 2.21 1.86 1.19 2 3.72 2.30 1.42 1.62 3 3.15 1.95 1.37 1.41 4 2.92 2.08 1.06 1.96 5 2.92 1.59 1.33 1.20 6 2.75 1.55 1.37 1.12 7 2.57 1.73 1.02 1.69 8 2.13 1.64 0.93 1.76 9 2.52 1.59 0.93 1.71 129cytogenetic studies in the centaurea aucheri group (sect. phaeopappus) aucheri and c. indistincta species for the first time. also, meiotic behavior and karyomorpholog y parameters of c. albonites, c.assadi and c. phaeopappa are newly reported here. c. albonitens, c. assadii, c. indistincta , c. farsestanica and c. phaeopappa are diploid with n= 9, 2n=2x=18 and c. aucheri is tetraploid with 2n=4x=36+02b. all taxa had the basic chromosome number of x= 9. by definition, a subspecies designation is applied to a plant that is geographically isolated from other members of its species in habitat and therefore does not interbreed for this reason. five subspecies (c. aucheri subsp. aucheri, c. aucheri subsp. elbursensis, c. aucheri subsp. farsestanica, c.aucheri subsp. indistincta, c. aucheri subsp. szowitsii) which is introduced by wagenitz (1980) and mozafarian (2018) for flora of iran, were often adjacent to each other [see table 1 and pattern of distribution of these subspecies in the research article by ranjbar and negaresh (2013)]. therefore, they cannot be considered as subspecies. eight samples of the c. aucheri showed chromosome numbers of n=18 and 2n=36 in both meiosis and mitotic respectively. meiosis in pollen mother cells in this taxon showed that the most common chromosome configurations were bivalents at diakinesis and first metaphase. analysis of karyotype and behavior of meiosis indicated that this taxon is a natural allotetraploid species. therefore, the results of meiotic behavior and karyomorphologycal parameters of five subspecies which are introduced by wagenitz (1980) and mozaffarian (2018) are not correct and revision of them by ranjbar and negaresh (2013) in five independence species are correct. in this study, most of the chromosomes of the eva luated species were metacent ric (m) or sub metacentric(sm). kar yot y pe sy mmetr y parameters showed that all the studied species were classified in the class 2a of the category of stebbins (1971), except of c. aucheri which was located in class 2b (table 8). total form percentage (tf%) for c. indistincta and c. phaeopappa were 40.41 and 40.02 respectively, that indicated similarity between them. also, this tf% similarity can be seen between c. assadii and c. farsistanica with 41.75 and 40.82 respectively. according to the data obtained from the five species (c. aucheri, c. assadii , c. farsistanica, c. indisticta and c. phaeopappa), the karyotype formulas were different between them (table 8). also, the inter and intrachromosomal asymmetry index (a1 and a2) for five taxa were different (table 8). in this study, c. farsistanica had a higher di value, which is associated with an enhanced order of karyotypic specialization. c. aucheri had the highest a2 values therefore its karyotype was more asymmetric than the other species. to analyze the variability of the karyotypes among species, all karyotype characteristics of centaurea species (table 8) were compared by one-way design. using principal components analysis (pca), the first two independent components accounted for about 81.68% of total variation (table 9). the first component indicated that tl, la, sa, ci, tf% and a2 were important characters for classification of species with about 61% of total variation. ar, la%, sa%, drl, a1 and di were important traits in the second component (21%) (table 9). table 7. measurement of somatic chromosomes in a diploid c. phaeopappa (obtained from 23 cells). chromosome no total length (µm) long arm (µm) short arm (µm) arm ratio l/s = r 1 4.21 2.13 2.08 1.02 2 3.72 2.21 1.50 1.47 3 3.59 2.04 1.55 1.31 4 3.55 2.21 1.33 1.66 5 3.51 2.21 1.29 1.71 6 3.46 2.17 1.28 1.68 7 2.88 1.68 1.19 1.40 8 2.75 1.86 0.88 2.10 9 2.70 1.68 1.02 1.65 table 8. karyotype parameters of centaurea taxa; total length of chromosome(tl), long arm (la), short arm (sa), arm ratio(ar), centromeric index(ci ), long arm percent(la % ), short arm percent(sa %); total form percent (tf % ), difference of relative length (drl), intrachromosome asymmetry index(a1 ), interchromosome asymmetry index(a2), dispersion index(di), symmetry classes of stebbins(sc), haploid karyotype formula(h.k.f.) (m: metacentric , sm: submetacentric). species tl la sa ar ci %la %sa %tf drl a1 a2 di sc h.k.f c. assadi 3.68 2.14 1.54 1.47 0.41 6.47 4.64 41.75 5.89 0.28 0.19 8.11 2a 7m+2sm c. aucheri 4.11 2.43 1.68 1.48 0.41 3.29 2.27 40.83 4.41 0.31 0.22 8.41 2b 15m+3sm c. farsistanica 2.79 1.65 1.14 1.52 0.41 6.58 4.54 40.82 6.87 0.30 0.21 9.12 2a 6m+3sm c. indistincta 3.11 1.85 1.26 1.52 0.40 6.62 4.49 40.41 5.55 0.32 0.17 7.65 2a 5m+4sm c. phaeopappa 3.38 2.03 1.35 1.56 0.40 6.66 4.45 40.02 4.96 0.33 0.15 5.47 2a 6m+3sm 130 seyed mahmood ghaff ari, seyed mohsen hesamzadeh hejazi th e tree phylogeny (figure 4) of the fi ve species indicated two major clades. th e fi rst major clade consists of two species (c. indistincta and c. phaeopappa) showed a degree of affi nity and were placed close to each other, while, c. aucheri joined the other species at a great distance. th e second clade contained c. assadii and c. farsestanica. th us, these studies could greatly help us in the classifi cation and taxonomic studies. th e diagram of species’ dispersion, based on two fi rst components, showed that the species separated in three groups, which completely fi ts with results obtained through the grouping analysis by ward’s method (figure 5). references garcia-jacas n, susanna a, mozaff arian, v. 1998. new chromosome counts in the subtribe centaureinae (asteraceae, cardueae) from west asia, iii. botanical journal of the linnean society 128: 413–422. ghaff ari sm. 1986. chromosome number reports xciii. taxon 35(4):900-901. ghaff ari sm. 1988. chromosome number reportsxcix .taxon 37(2):397. hilpold a, garcia-jacas n, vilatersana r, susanna a. 2014. taxonomical and nomenclatural notes on centaurea: a proposal of classifi cation, a description of new sections and subsections, and a species list of the redefi ned section centaurea. collectanea botanica 33: e001. pp. 1-29.doi: 10.3989/ collectbot.2013. v33.001. levan a, fredga k, sandberg aa. 1964. nomenclatureforcentromeric position on chromosomes. hereditas 52:201–220. lópez, e, devesa ja, arnelas i. 2011. taxonomic study in the centaurea longei complex (asteraceae). annales botanici fennici 48: 1–12.http://dx.doi. org/10.5735/085.048.0101 mozafarian v. (2018). tribus cardueae cass. in: mozafarian, v. & al. (eds.), flora of iran, research institute of forests and ranglands. ranjbar m, negaresh k. 2013. a revision of centaurea sect. phaeopappus (asteraceae, cardueae). phytotaxa 123 (1): 1–40. ranjbar m, heydari r. 2016. a taxonomic contribution to yellow-fl owered members of centaurea sect. phaeopappus (asteraceae) in iran. phytotaxa 277: 182–190. table 9. eigenvectors from the fi rst two principal components for 12 karyotype parameters to classify centaurea species. parameters first component second component tl 0.328 -0.054 la 0.336 -0.050 sa 0.316 -0.061 ar 0.294 0.308 ci -0.320 -0.206 la% -0.275 0.381 sa% -0.304 0.316 tf% -0.326 -0.169 drl -0.204 0.528 a1 0.133 -0.176 a2 0.332 0.218 di 0.215 0.471 eigen value 7.3257 2.4761 percentage of variance 61.0479 20.6346 cum percentage of variance 61.0479 81.6824 c. phaeopappa figure 4. dendrogram of cluster analysis (ward) of centaurea species based on karyotype characteristics. cophenetic correlation r=0.94. -3 -1 1 c. assadiis c. aucheri c. farsistanica c. indistincta c.phaeopappa figure 5. scatter plot of centaurea species for the fi rst two principal components. 131cytogenetic studies in the centaurea aucheri group (sect. phaeopappus) stebbins g.l. 1971. chromosomal evolution in higher plsnts. edward arnold ltd. london. susanna a, garcia-jacas n, soltis de, soltis p.s. 1995. phylogenetic relationship in tribe carduae (asteraceae) based on its sequence. american journal of botany 82: 1056-1068. wagenitz g. 1980. centaurea. in: rechinger kh (ed.), flora iranica, lieferung 138b. pp. 313-420. graz: akademische druck-und verlagsanstalt. wagenitz g, hellwig fh. 1997. eine neue und eine verschollene centaurea-art aus der türkei und eine neue volutaria-art (compositae-cardueae). annalen des naturhistorischen museums in wien 98b: 175–181. wilson gb. 1945. the venetian turpentine mounting medium. stain technol. 20: 133-135. caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 4 2022 firenze university press avicennia genus molecular phylogeny and barcoding: a multiple approach laleh malekmohammadi1, masoud sheidai1,*, farrokh ghahremaninejad2, afshin danehkar3, fahimeh koohdar1 studying some morphological responses of stevia (stevia rebaudiana bertoni) to some elicitors under water deficiency basoz sadiq muhealdin1, sahar hussein hamarashid1,*, fairuz ibrahim ali1, nakhshin omer abdulla1, syamand ahmad qadir2 morphological and cytogenetic characterization in experimental hybrid aloe jucunda reyn. x aloe vera (l.) burm. f. (asphodelaceae) wendy ozols-narbona*, josé imery-buiza assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions seyedeh mahsa hosseini1, sepideh kalatejari1, mohsen kafi2,*, babak motesharezadeh3 assessment of protein and dna polymorphisms in corn (zea mays) under the effect of non-ionizing electromagnetic radiation ekram m. abdelhaliem1,*, hanan m.abdalla1, ahmed a. bolbol1, rania s. shehata1,2 chromosome counts of some species of wetland plants from northwest iran saeedeh sadat mirzadeh vaghefi*, adel jalili delimiting species using dna and morphological variation in some alcea (malvaceae) species based on srap markers chnar hama noori meerza1, basoz sadiq muhealdin2, sahar hussein hamarashid2,*, syamand ahmad qadir3, yusef juan4 mapping cap-a satellite dnas by fish in sapajus cay paraguay and s. macrocephalus (platyrrhini, primates) simona ceraulo, francesca dumas* determination of genome size variation among varieties of ilex cornuta (aquifoliaceae) by fow cytometry peng zhou1, jiao li2, jing huang1, fei li1, qiang zhang2,*, min zhang1,* first report of chromosome and karyological analysis of gekko nutaphandi (gekkonidae, squamata) from thailand: neo-diploid chromosome number in genus gekko weera thongnetr1, suphat prasopsin2, surachest aiumsumang3,*, sukhonthip ditcharoen4, alongklod tanomtong5, prayoon wongchantra6, wutthisak bunnaen7, sumalee phimphan3 intraspecific karyomorphological and genome size variations of in vitro embryo derived iranian endemic asafoetida (ferula assa-foetida l., apiaceae) narges firoozi, ghasem karimzadeh*, mohammad sadegh sabet, vahid sayadi cytogenetic studies in the centaurea aucheri group (sect. phaeopappus) seyed mahmood ghaffari¹,*, seyed mohsen hesamzadeh hejazi² karyomorphology, genome size, and variation of antioxidant in twelve berry species from iran saeed mohammadpour1, ghasem karimzadeh1,*, seyed mahmood ghaffari2 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1848 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(2): 3, 2022 editorial dear readers, as you know the publishing of scientific articles is under constant change over the years, partly due to the introduction of many new journals, most of them published online and open access. while the introduction of new scientific journals should be seen in general as positive news, this way of publishing, as a matter of fact, caused a relevant increase in the costs of publication for researchers, and such costs are subtracted from other possible uses. the increase in the number of scientific journals corresponded also to a huge increase in submitted articles, partly due to the increase in researchers (a very positive fact), but also to the use of publishing parameters for teaching/research habilitations and funding applications. more published articles means more funding and a faster career, and the increase in quantity very rarely leads to an increase in quality. for this reason, some (few?) researchers submit articles that result too carelessly written, and in some cases even contain some parts of the text (partially) copied from somewhere else, figures already used in other articles, and even wrong (?) figures. also multiple submissions may be another issue, that is the same article is sent to more journals. in many cases of these misconducts, the reviewers (and the editors) have large difficulties intervening. in some cases, one can remember very similar sentences read somewhere else (it occurred to me a couple of times) or notice a strange change in style from one paragraph to another, but in most cases, the misconduct is noticed only after the article has already been published (for multiple submission it would be impossible doing otherwise), often leading to retraction (steen et al. 2013). for this reason, the editorial committee decided, from now on, to indicate directly in the journal which articles published in caryologia (fortunately very very few) resulted to have some flaws like those listed above. alessio papini, editor-in-chief of caryologia literature cited steen rg, casadevall a, fang fc. (2013) why has the number of scientific retractions increased?los one 8, e68397. caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 2 2022 firenze university press cytogenetic studies of six species in family araceae from thailand piyaporn saensouk1, surapon saensouk2,*, rattanavalee senavongse2 effect of ag nanoparticles on morphological and physio-biochemical traits of the medicinal plant stevia rebaudiana sherzad r. abdull, sahar h. rashid*, bakhtiar s. ghafoor, barzan s. khdhir morphometric analysis and genetic diversity in hypericum l. using sequence related amplified polymorphism wei cao1, xiao chen2,*, zhiwei cao3 population differentiation and gene flow of salicornia persica akhani (chenopodiaceae) xiaoju zhang1, li bai2,*, somayeh esfandani-bozchaloyi3 scot molecular markers are efficient in genetic fingerprinting of pomegranate (punica granatum l.) cultivars shiva shahsavari1, zahra noormohammadi1,*, masoud sheidai2,*, farah farahani3, mohammad reza vazifeshenas4 first record of nucleus migration in premeiotic antherial cells of saccharum spontaneum l. (poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(2): 15-22, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1447 caryologia international journal of cytology, cytosystematics and cytogenetics citation: sherzad r. abdull, sahar h. rashid, bakhtiar s. ghafoor, barzan s. khdhir (2022) effect of ag nanoparticles on morphological and physio-biochemical traits of the medicinal plant stevia rebaudiana. caryologia 75(2): 15-22. doi: 10.36253/caryologia-1447 received: november 04, 2021 accepted: may 24, 2022 published: september 21, 2022 copyright: © 2022 sherzad r. abdull, sahar h. rashid, bakhtiar s. ghafoor, barzan s. khdhir. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. effect of ag nanoparticles on morphological and physio-biochemical traits of the medicinal plant stevia rebaudiana sherzad r. abdull, sahar h. rashid*, bakhtiar s. ghafoor, barzan s. khdhir technical college of applied science, sulaimani polytechnic university, iraq *corresponding author. e-mail: sahar.rashid@spu.edu.iq abstract. nowadays, overproduction of secondary metabolites in remedial herbs through giving biotic/abiotic stresses is an interesting area of research. in the current study, the influences of various concentrations of silver nanoparticles (ag nps) were evaluated on several morphological and physio-biochemical traits, such as the steviol glycosides level in stevia. the findings showed that the herbs incubated with 400-ppm ag nps own the highest dry and fresh weight of shoot, while those incubated with 80up to 200ppm ag nps own the highest steviol glycosides content. as a result, we successfully improve the content of stevioside glycoside up to 1.75-fold by applying the 80up to 200-ppm ag nps in stevia medicinal plant. moreover, our findings revealed that low concentrations the ag nps lead to an increase of glutathione content and total antioxidant capacity, and a decrease of mda, whereas treatments at higher concentrations induced adverse effects for the plant. as a result, the treatment with ag nps low concentrations had a favorable efficacy on physio-biochemical and morphological characteristics of stevia. these achievements are very promising, because they revealed a considerable capability for the ag nps application in enhancing the secondary metabolites in stevia remedial herb. the present study is the first case assessing the desirable influences of ag nps on the stevia, in regard with shifting of biosynthetic pathway of steviol glycosides in a concentration-dependent manner. keywords: ag nanoparticles, medicinal plant, stevia, steviol glycosides. introduction stevia (stevia rebaudiana bertoni) is a medicinal perennial herb sweet in taste. this herb is a member of asteraceae family and native to paraguay as well as brazil (shivanna et al. 2012). stevia gives rise to steviosides and rebaudiosides as zerocalorie diterpene glycosides, and naturally keeps safe from obesity, hypertension, and diabetes mellitus (thiyagarajan and venkatachalam 2012; goyal et al. 2010; geuns 2003). stevia can be propagated through tissue culture techniques for producing elite varieties (yucesan et al. 2016). the less efficiency of stem cutting and poor seeds germination actually led to difficulties for in vitro large-scale propagation of this herb (hendaw16 sherzad r. abdull, sahar h. rashid, bakhtiar s. ghafoor, barzan s. khdhir ey et al. 2015). up to now a number of approaches have been established to achieve an increased content of secondary metabolites from the leaf tissues of stevia (javed et al. 2017a; si et al. 2020; liu et al. 2021). biotic and abiotic elicitors own the potential of bringing about the larger level of secondary metabolites as well as sweetening compounds through modification of metabolic cycles (sabzehzari and naghavi 2018, 2019; sabzehzari et al. 2020, 2019; gupta et al. 2015; peng et al. 2021; ma et al. 2021). the elicitors, however, are beneficial up to particular threshold levels because being cytotoxic at much higher concentration (javed et al. 2017a; hendawey et al. 2015; chen et al. 2021; bi et al. 2021). the cytotoxic influences of various nanoparticles as abiotic elicitors have been recorded in a variety of crops/plants (javed et al. 2017b, c; shaw and hossain 2013; lin and xing 2008; lee et al. 2008, 2010; spanò et al. 2020). in terms of stevia, only a few types of nanoparticles have been evaluated. for instance, rezaizad et al. (2019) observed that the plants incubated with 200 ppm tio2 nps had the lowest mda extent and the highest steviol glycosides content, while those incubated with 400 ppm tio2 nps had the highest fresh and dry weights of shoot. accordingly, the authors suggested that the treatment with tio2 nps leads to a positive influence on phytochemical and morphological attributes in stevia herb. javed et al. (2018) observed that total reducing power (trp), total antioxidant capacity (tac), scavenging activity of free radical (dpph), and total phenolic content (tpc) were highest at 10 ppm of cuo nps, while the highest level of total flavonoid content (tfc), dpph, and tpc were registered at 100 ppm concentration of zno nps. their results clearly showed that cuo nps are more cytotoxic to stevia plant when compared to zno nps. thus, the authors proposed a promising way for future research using cuo or zno nps for increasing commercially significant secondary metabolites in various medicinal herbs. in terms of ag nps, kaveh et al. (2013) observed that exposure to higher concentration of these nanoparticles (up to 20 ppm) led to a decrement of the biomass in arabidopsis. similarly, dimkpa et al. (2013) recorded that ag nps treatment decreased the roots and shoots length in a dose-dependent way in wheat. nair and chung (2014a) also recorded that ag nps treatment decreased root and shoot weight and root elongation in rice. al-huqail et al. (2018) registered a decrease in the total protein content, total chlorophyll content, fresh weight, and root and shoot elongation after exposure to ag nps in lupinus termis. patlolla et al. (2012) reported that ag nps treatment enhanced the micronuclei and chromosomal aberrations and declined the mitotic index in root tips of broad bean, proposing that mitosis and cell cycle in root tips was disrupted by silver nanoparticles. however, there are several studies that documented the positive effect of silver nanoparticles on plant growth and development in a plant-dependent manner (reviewed in yan and chen, 2019). however, there is no report on the effect of silver nanoparticles on stevia plant. based on what has been mentioned about the value of stevia secondary metabolites and the effect of silver nanoparticles, the present study was focused on the evaluation of the photocatalytic efficacy of ag nps on the phytochemical as well as morphological characteristics of stevia plant under controlled conditions. material and methods plant material and growth conditions the seeds of stevia were provided by the college of agriculture and natural resources, university of tehran, iran. the current research was carried out through a completely randomized design with three replications. after germination, the seedlings were moved to pot comprising pittmoss and perlite (1:1) (each pot including three samples), and then put in growth chambers for 10 days under 18-h light at 25°c to grow and take root. the seedlings were moderately irrigated on the first day, and then watered every two days through 50% hoagland solutions. the treatments in this study were performed at increasing concentrations (0, 20, 40, 60, 80, 100, 200, 400 and 800) of ag nps. after being developed, the leaves of all plants were sprayed by ag nps on the 11th day. the subsequent spraying operations were performed one week later, followed by the last spraying operations two weeks later. at the end of the treatments some morphological and physio-biochemical traits like the dry and fresh weights of shoot, mda level as a measure of membrane lipid peroxidation, glutathione content, total antioxidant capacity and the steviol glycosides content were assayed in the stevia leaves. morphological evaluation to estimate the fresh weights, the shoots (leaves as well as stems) were washed, cut into pieces, and eventually the shoot fresh weights were registered in g. to measure the dry weights, the samples were dried at 60°c by uing an oven, and ultimately the weights were registered in g. 17effect of ag nanoparticles on morphological and physio-biochemical traits of the medicinal plant stevia rebaudiana physio-biochemical analysis the estimation of the level of malondialdehyde (mda), as output derived from lipid peroxidation, has been performed by the method described by cakmak and horst (1991). the absorbance at 532 and 600 nm of cell extracts was recorded and the average of the readings in triplicate was utilized for estimating the level of mda by 155 mm-1cm-1 extinction coefficient as follows: malondialdehyde (nm)= δa(532-600)/1.56*105. the glutathione content (gsh) was calculated through the procedure as elucidated by moron et al. (1979). for evaluation of total antioxidant capacity, 100 μl stock solution of each specimen (5 mg/ml in dimethyl sulfoxide) was combined with 1000 μl reagent solution, including 0.7 m of sulfuric acid, 5 mm of ammonium molybdate, and 30 mm of sodium phosphate. the reaction admixture was maintained for an hour and a half at 95°c, followed by cooling at 25°c. the absorbance of specimens was recorded at 695 nm through micro plate reader in triplicates. vitamin c was utilized as standard. the data was represented as μg aa/mg (i.e., mg ascorbic acid equivalent) (ali et al. 2015). measurement of steviol glycosides to calculate the steviol glycosides content in leaves, 100mg of dry leaves was incubated in 10 ml methanol for 15 min. the residual solvent was removed and the solid residue was solubilized in 5ml water/acetonitrile mix (20:80). the extract (20μl/ml) was injected into the hplc column (cosmosoil 5 nh2-ms with particle size of 5 μm, 4.5 mm in diameter, and 15 cm in length) linked to the hplc (rezaizad et al. 2019).the moving phase of 80% of acetonitrile as well as 20% distilled water was set at a rate of 1 ml/min. statistical analysis the q data was analyzed through spss ver. 20. in variance analysis, p=0.05 was taken into consideration as significant level. results and discussion influence of ag nps on the dry and fresh weights of shoot, membrane lipid peroxidation, glutathione content, total antioxidant capacity and the steviol glycosides content was assayed in the stevia plants. the findings revealed that ag nps own a considerable positive efficacy on the analyzed traits. influence of ag nps on the dry and fresh weights of shoot a comparison of mean dry and fresh weight of shoot in ag nps-treated stevia plants indicated that the 0 ppm ag nps (control) sample has the lowest shoot weight, whereas the 400-ppm concentration of ag nps has the highest (figures 1, 2). the fresh and dry weights were increased up to 5and 3-fold in the 400-ppm ag nps-treated stevia plants when compared to control, respectively. in order to explain our observations, we can suppose that ag nps can stimulate root strength and enhance the capability of the root to uptake nutrients and water, finally leading to an increment in the fresh and dry weights of the shoot, as reported by vannini et al. (2013) in eruca sativa. moreover, it was found that the using of ag nps in the early developmental stages has ability to increases the carbon fixation efficiency and photosynthesis rate in the plants, resulting in enhancing of the yield of dry matter (rezaizad et al., 2019). however, the 800-ppm ag nps treatment led to a considerable decrease in dry and fresh weight of shoot in treated stevia plants. similarly, in sorghum bicolor, 0 0,5 1 1,5 2 2,5 3 0 20 40 60 80 100 200 400 800 fr es h w ei gh ts o f s ho ot s (g ) concentrations of ag nanoparticles (ppm) 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 0 20 40 60 80 100 200 400 800 d ry w ei gh ts o f s ho ot s (g ) concentrations of ag nanoparticles (ppm) figure 1. efficacy of various concentrations of ag nanoparticles on fresh weight of shoots in stevia plant. figure 2. efficacy of various concentrations of ag nanoparticles on dry weight of shoots in stevia plant. 18 sherzad r. abdull, sahar h. rashid, bakhtiar s. ghafoor, barzan s. khdhir krishnaraj et al. (2012) observed at low concentrations of ag nps, an increase in the shoot and root length and weight, while at high concentrations a decline in the length and weight. influence of ag nps on the mda the production rate of free oxygen radicals has a close relationship with the strength of the membrane (shivanna et al. 2012). malondialdehyde, as a reactive oxygen species leads to peroxidation of membrane lipid, enhancing membranes permeability, whereas declining membranes strength. thus, the content of mda serves as a measure of the lipid peroxidation, offering an image of cell damages (yan and chen, 2019). however, the 200up to 800-ppm ag nps treatments resulted in an increase in mda level in treated stevia plants (figures 3). similarly, thiruvengadam et al. (2015) evaluated the effect of ag nps exposure in turnip seedlings, observing that a higher concentration of ag nps led to overproduction of superoxide radicals and increased the lipid peroxidation; hydrogen peroxide production was also enhanced after exposure to silver nanoparticles. nair and chung (2014b) recorded that exposure to ag nps leads to an increment in lipid peroxidation and hydrogen peroxide production in rice root and shoot in a dose-dependent way. nair and chung (2014a) documented that lipid peroxidation increases after exposure to silver nanoparticles in arabidopsis. de la torre-roche et al. (2013) also documented that ag nps exposure with concentration at 500 up to 2000 ppm caused a considerable increase in mda level in soybean. overall, our results revealed that the 100-ppm ag nps treatment can decline the level of mda, whereas ag nps treatment with a concentration above 100-ppm represents an adverse effect likely because of damaging to thylakoid membrane structure (nair and chung 2014a; shivanna et al. 2012). influence of ag nps on the glutathione content in light of our findings, glutathione content was enhanced in stevia plants after 100-ppm ag nps treatments when compared to the 0 ppm (control) concentration. however, the 200up to 800-ppm ag nps treatments led into a decrease in glutathione content in treated stevia plants. the glutathione extent was increased up to 2-fold in the 100-ppm ag nps-treated herbs when compared to control conditions (figures 4). similarly, nair and chung, (2014b) reported an increased glutathione content after exposure arabidopsis thaliana seedlings to the high concentration of ag nps. the enhanced level of glutathione may be resulted from the increased glutathione biosynthesis, expression of glutathione s-transferase and glutathione reductase genes, and sulfur assimilation after exposure to ag nps (nair and chung, 2014b). after silver nps exposure, a considerable increase in shoots glutathione content was observed, proposing that plant employs glutathione to decrease the effect of ros derived from the high concentration of silver nanoparticles (mirzajani et al. 2013). these outputs are in coincidence with jiang et al. (2014), who showed that silver own an important function in the increase of antioxidant potentials like glutathione content in spirodela polyrhiza plant. in fact, antioxidants such as glutathione present a key role in detoxification of toxic metal ions (pompella et al. 2003). indeed glutathione is a key antioxidant in crops/plants, and preserves significant cellular components from reactive oxygen species (singh and sinha, 2005). influence of ag nps on the total antioxidant capacity we found that total antioxidant capacity enhanced in stevia plants after incubation with the 20up to 200-ppm ag nps concentrations in contrast to the con0 5 10 15 20 25 0 20 40 60 80 100 200 400 800 m em br an e lip id p er ox id at io n nm .g -1 f. w concentrations of ag nanoparticles (ppm) figure 3. efficacy of various concentrations of ag nanoparticles on the mda (membrane lipid peroxidation) in stevia plant. 0 0,5 1 1,5 2 2,5 0 20 40 60 80 100 200 400 800 g lu ta th io ne c on te nt (μ m ol / g fw ) concentrations of ag nanoparticles (ppm) figure 4. efficacy of various concentrations of ag nanoparticles on glutathione content in stevia plant. 19effect of ag nanoparticles on morphological and physio-biochemical traits of the medicinal plant stevia rebaudiana trol conditions. however, the 400and 800-ppm ag nps treatment resulted in a decrease in total antioxidant capacity in the stevia herbs. the total antioxidant capacity was increased up to 1.65-fold in the 100and 200-ppm ag nps-treated stevia plants when compared to control (figures 5). in line with our findings, jiang et al. (2014) reported ag nps can induce accumulation of ros, and alter antioxidant system in the spirodela polyrhiza aquatic plant. qian et al. also observed that ag nps accumulated in the leaves of arabidopsis and changed the transcription of aquaporin and antioxidant genes, proposing that ag nanoparticles can alter the balance between antioxidant as well as oxidant systems (qian et al. 2013). influence of ag nps on the stevioside glycoside content analysis of the stevioside glycoside content revealed that 80up to 200-ppm ag nps concentrations led into the highest stevioside glycoside content, whereas spraying the ag nps at 400and 800-ppm concentrations lead to a decrease in percentage weight of this compound (figures 6). as a result, we successfully improve the content of stevioside glycoside up to 1.75fold by applying the 80up to 200-ppm ag nps in stevia medicinal plant. to the best of our knowledge, no research has ever been documented the ag nps application on stevia medicinal herb so far. however, previous findings applying copper, silicon, iron, as well as tio2 nps on this herb showed that the higher concentrations of copper and iron nanoparticles lead to the higher levels of stevioside when compared to control (rezaizad et al. 2019). for silicon nps, the findings indicated that the highest stevioside content is generated at low concentrations (0.20 up to 2 mg/l) and the least extent of stevioside glycoside is produced at the 4 up to 8 mg/l concentrations of silicon nps (hendawey et al. 2015). conclusion many studies showed the adverse effect of silver nanoparticles on various crops/plants at molecular, cellular, physiological, and morphological level (reviewed in yan and chen 2019; 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(poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 72(4): 3-13, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-196 citation: a. çetinbaş-genç, f. yanık, f. vardar (2019) histochemical and biochemical alterations in the stigma of hibiscus syriacus (malvaceae) during flower development. caryologia 72(4): 3-13. doi: 10.13128/cayologia-196 published: december 23, 2019 copyright: © 2019 a. çetinbaş-genç, f. yanık, f. vardar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. histochemical and biochemical alterations in the stigma of hibiscus syriacus (malvaceae) during flower development aslıhan çetinbaş-genç*, fatma yanık, filiz vardar department of biology, marmara university, istanbul, turkey *corresponding author: aslihan.cetinbas@marmara.edu.tr abstract. the aim of this study is to determine the histochemical and biochemical changes that occurred during flower development in the stigmas of hibiscus syriacus. the flower development of h. syriacus was divided into three successive stages; preanthesis, anthesis, post-anthesis, and stigma development was examined in parallel with these stages. at pre-anthesis, the stigmatic papillae cells covering the surface of the stigma were ovoid and their dense cytoplasm were rich in insoluble polysaccharide, protein and lipid. at anthesis, papillae cells grew and the pellicle layer becomes clear indicating dry-typed stigma. meanwhile some sub-papillae cells, which accumulate dense organic matter from the beginning of development, began the process of autolysis and release their cellular content into the intercellular space. whereas the organic matter content of papillae decreased at post-anthesis, it was still more than pre-anthesis stage. similarly, peroxidase and non-specific esterase activity were very intensive at anthesis stage and activities were still remarkable at post-anthesis stage. the maximum cat, sod activity, h2o2 and mda content were also determined at anthesis. our results revealed that stigma of h. syriacus is receptive at anthesis and still conserve its receptivity at post anthesis assisting pollen germination and pollen tube growth. keywords. anthesis, antioxidant enzyme, lipid peroxidation, papillae, stigma. introduction malvaceae family comprises 244 genera and more than 4225 species (paoletti et al. 2009). genus hibiscus of malvaceae has attracted considerable attention due to its large and attractive flowers. hibiscus syriacus is the most popular species of this genus and has hermaphrodite flowers with white, pink, red, lavender, or purple color, depending on their cultivar (punasiya and pillai 2015). it consists of a tubular group of stamens surrounding the style which ends with the five branched stigma (klips and snow 1997; çetinbaş-genç and ünal 2017). hibiscus syriacus has also a great social and economic importance whose flowers and seeds are frequently used in industry. it is mainly used in pharmaceutical products for the treatment of cardiovascular, urinary tract, skin, 4 aslıhan çetinbaş-genç, fatma yanık, filiz vardar and reproductive system diseases. besides, it is often used in the production of hair-skin care products, perfumes, and used as a natural colorant in the beverage industry, and a source of fiber in the paper industry (hsu et al. 2015). to gain knowledge about the plant reproductive biology concerning pollination and fertilization helps to improve reproductive success which has direct effect on yield quality and economic benefits. pollination involves a complex set of cell-cell communications that enable pollen-pistil interaction. this molecular interaction between the pollen wall and components of the stigmatic surface determines whether fertilization will take place (mcinnis et al. 2006). besides, stigma receptivity is one of the essential events for the start of pollen-pistil interaction. in many angiosperms, the stigma is receptive during anthesis but in some cases, the stigma may be still receptive after or even before anthesis (brito et al. 2015). therefore, the developmental characteristics of stigma are the focal point for pollination biology studies. despite their morphological diversity, the stigma of angiosperms is divided into two main groups: wet and dry typed. although wet stigmas produce large surface secretions, dry stigmas are lack of secretions and its cuticle is coated with a proteinaceous surface layer. despite these fundamental differences between wet and dry stigmas, high level of enzymatic activities indicates that stigmatic enzymes are essential for stigma function in both stigmatic types (souza et al. 2016). stigma includes heterogeneous components such as proteins, lipids, carbohydrates, amino acids and phenolic acids playing an important role in the pollen germination and tube growth on the stigma surface. these compounds present as a content of the exudate in wet stigmas, but in dry type stigmas they take place as a dry extracellular layer on the cuticle (edlund et al. 2004). the receptive surface of stigma is also characterized by the expression of biomolecules such as the peroxidases, esterases and reactive oxygen species (mcinnis et al. 2006). it has been revealed that stigma shows high peroxidase and esterase activity in many plants when it gains receptivity for pollination (hiscock et al. 2002). besides, peroxidases and non-specific esterases are functional on the pollen-stigma interaction to loosen cell wall components of stigma cells with the aim of allowing pollen tubes to penetrate, and grow into the stigma (hiscock et al. 2002; mcinnis et al. 2006). peroxidases generally catalyze the reduction of a wide range of organic substrates using hydrogen peroxide (h2o2) (mcinnis et al. 2006). stigmatic peroxidases and indirectly h2o2 metabolism form the components of signaling systems that mediate the identification of the proper pollens during pollen-stigma interaction (cheong et al. 2002; delannoy et al. 2003; do et al. 2003). in many cellular processes including development and tolerance to environmental stress, reactive oxygen species (ros) also play a role as secondary messengers at low concentrations (yanık et al. 2018). however, high concentrations of ros cause harmful chain effects in the cell. detoxification of ros is carried out with antioxidant enzymes and non-enzymatic antioxidant systems. superoxide dismutase, catalase, and peroxidase are some of the important antioxidant enzymes involved in the detoxification of ros (yanık et al. 2018). ros have a role in signaling networks promoting pollen germination and pollen tube growth on stigma (mcinnis et al. 2006; hiscock and allen 2008; zafra et al. 2010). the concentration of ros on the stigma affects the stigma receptivity, pollen germination and as a result the success of pollination. therefore, in order to understand the pollination biology in detail, it is very important to examine the balance between production and detoxification of ros during the development of the stigma. the aim of the current study is to evaluate the histochemical and biochemical alterations of stigma in h. syriacus during the defined flowering stages; pre-anthesis (the period before anthesis), anthesis (the period in which flower is fully open and functional), and post-anthesis (the period after anthesis). knowledge on stigmatic development will improve our information about the pollination success in plants as well as in hibiscus varieties which have agronomic and ornamental potential. material and methods flowers of hibiscus syriacus l. were collected in june-august (2016-2018) in the vicinity of göztepei̇stanbul (turkey). pistils at the different developmental stages were determined by stereomicroscope ez4hd (leica, germany) and photographed by las ez software. pistils were removed from flowers and fixed overnight in 3% (w/v) paraformaldehyde in 0.05 m sodium cacodylate buffer (ph 7.4) at 4 °c. after dehydration process with ethanol series and embedded in epoxy resin using propylene oxide. semi-thin sections (1-2 μm) were stained with periodic acid-schiff (pas) (feder and o’brien 1968) for insoluble polysaccharides, with coomassie brilliant blue (cbb) (fisher et al. 1968) for proteins and, with sudan black b (sbb) for lipids (pearse 1961). the optical density of organic content in papillae and sub-papillary cells was measured at different developmental stages according to rodrigo et al. (1997). 5histochemical and biochemical alterations in the stigma of hibiscus syriacus (malvaceae) during flower development images were converted to 8-bit gray-scale and the optical density was quantized from black-and-white images using the image j software. the mean and standard deviation of 5 images captured over an area of 300 µm² for papillae and 100 µm² for sub-papillary cells were computed. for determination of qualitative peroxidase activity, fresh stigmatic tissue was incubated in sodiumphosphate buffer (pbs0.1 m, ph 5.8) containing 15 mm guaiacol and 5 mm h2o2 for 60 min (birecka et al. 1973). to establish qualitative non-specific esterase activity, fresh stigmatic tissue was incubated in incubation buffer containing 1 mm α−naphthol  acetate, 0.06 m na2po4, 0.01 m nano2 and 2 m pararosaniline chloride for 10 min at 37 °c (gomori 1950). after washing with dh2o for 5 min, the stigmatic tissue squashed gently. all of the preparations were photographed with the kameram software, assisted by a kameram digital camera and an olympus bx-51 microscope. to evaluate the superoxide dismutase (sod) and catalase (cat) activity, 100 mg fresh stigmatic tissue were homogenized with 1 ml of cold pbs (50 mm, ph 7.0). after centrifugation at 14 000 rpm for 20 min at +4 °c, the supernatant was used as enzyme source. for cat activity, 25 µl of the supernatant and 1 ml reaction mixture (20 mm pbs, ph 7.0 and 6 mm h2o2) were mixed and measured by the decrease in absorbance for 2 min at 240 nm, spectrophotometrically (cho et al. 2000). for sod activity, 2 µl of the supernatant and 2 ml reaction mixture (100 mm ph 7.0 pbs, 2 m na2co3, 0.5 m edta, 300 mm l-methionine, 7.5 mm nitro blue tetrazolium, 0.2 mm riboflavin) were mixed. after incubation under 15 w fluorescent lamps for 10 min, the mixture was measured at 560 nm, spectrophotometrically (cakmak and marschner 1992). to measure the amount of hydrogen peroxide (h2o2) 300 mg fresh stigmatic tissue were homogenized with 2 ml extraction buffer (0.1% tca, 1 m ki, 10 mm pbs) and centrifuged at 12 000 g for 15 min at 4 °c. after incubation in dark for 20 min, the supernatants were measured at 390 nm, spectrophotometrically (junglee et al. 2014). lipid peroxidation (lpo) was determined by the production of malondialdehyde (mda) level. 200 mg fresh stigmatic tissue were homogenized with 1 ml 0.1% tca and centrifuged at 12 000 g for 20 min at +4 °c. 250 µl supernatant and 1 ml reaction mixture (0.6% tba in 20% tca) were mixed and incubated for 30 min at 95 °c. after cooling on ice, the mixture was centrifuged at 12 000 g for 10 min and the supernatant was measured at 532 and 600 nm, spectrophotometrically (cakmak and horst 1991). all measurements and quantifications were repeated at least 3 times. statistical analysis was performed using one-way analysis of variance (anova), (spss 16.0 software). the significance of the applications was designated at the p < 0.05 level using the tukey’s test. all data presented are means ± sd. results in the current study, the stigma of hibiscus syriacus analyzed in three successive stages (pre-anthesis, anthesis, and post-anthesis) correlated with some morphological markers such as color, the position of calyx and corolla, anther dehiscence, and the absence or presence of pollen on it. in the stage of pre-anthesis, the flower buds of h. syriacus were ovoid with calyx covering half of the bud. five stigmatic branches were very close to each other. there were no pollen grains on the stigma, because the anthers were still indehiscent (figure 1 a,d,g). at anthesis stage, the flower was fully opened and their petals elongated. stigma presented five distinctly separated branches with yellowish color. a lot of pollen grains were visible on the stigma due to anther dehiscence (figure 1 b,e,h). at post-anthesis stage, the color of the petals began to fade and turned to brown, however a lot of pollen grains were still deposited on the stigma surface (figure 1 c,f,i). hibiscus syriacus has capitate type stigma with five branches. the receptive surface of the stigma was covered with the tissue of unicellular papillae that are short, ovoid shaped, thin walled and tightly packed cells (figure 2 a,f,k). papillae cells lost their tight alignment with the increase of their width and length and their tips began to tape at anthesis. in the course of post-anthesis, papillae cells were much extended and thorn-shaped cells. at all stages of development, the dense cytoplasm of papillae cells was rich in insoluble polysaccharide, protein and lipids (figure 2, figure 3). the pellicle layer which was not very distinct at pre-anthesis became evident at anthesis (figure 2 f ). the papillae surfaces were covered with continuous pellicle and showed intense cbb staining indicating the dry-type stigma (figure 2 g). the content of organic material in the papillae cells reached at maximum during anthesis (figure 2 b,g,l). according to the optical density results, the insoluble polysaccharide content of papillae increased by 43 % (figure 3 a), the protein content increased by 40 % (figure 3 b), and lipid content increased by 77 % (figure 3 c) at anthesis when compared to pre-anthesis. besides, some of the sub-papillary cells accumulated a large 6 aslıhan çetinbaş-genç, fatma yanık, filiz vardar figure 1. determination of development stages of flower and stigma in hibiscus syriacus. flower morphology at pre-anthesis (a), anthesis (b), post-anthesis (c). stigma morphology at pre-anthesis (d), anthesis (e), post-anthesis (f ). stigmatic surface at pre-anthesis (g), anthesis (h), post-anthesis (i). scale: 1 cm in a-c, 1 mm in d-f and 100 µm in g-i. 7histochemical and biochemical alterations in the stigma of hibiscus syriacus (malvaceae) during flower development amount of organic matter during the transition from the pre-anthesis to anthesis. when sub-papillary cells started to degenerate at anthesis, their rich organic contents spread into the intercellular space indicating positive reaction with the pas, cbb, and sbb staining (figure 2 c,h,m)). at-post anthesis, the content of organic material papillae reduced in compare to anthesis (figure 2 d,i,n)). the insoluble polysaccharide content of papillae was decreased by 10 % (figure 3 a), the protein content was decreased by 18 % (figure 3 b) and lipid content was decreased by 27 % (figure 3 c) at post-anthesis with regard to anthesis. at this stage the pellicle got thinner (figure 2 i). after the degeneration of sub-papillary cells, the accumulation of organic material at the intercellular space became more intense (figure 2 e,j,o). to measure the amount of starch granules in subpapillary cells, the optical density of starch granules was measured after pas staining. although papillae cells had very few starch granules at all developmental stages, subpapillary cells contained a large amount of dense starch granules representing a peak at anthesis (figure 4 a-c). the starch content was increased by 109 % at anthesis (figure 4 d) with regard to pre-anthesis. despite the starch granule content decreased by 25 % at post-anthesis, it was still 55 % more than the pre-anthesis stage (figure 4 c,d). based on our squash preparation results, non-specific esterase and peroxidase activity were not observed in stigmatic papillae cells at pre-anthesis (figure 5 a,d). however, both enzyme activities gave progressive positive reaction at anthesis (figure 5 b,e)). these positive activities indicated the stigma gains receptivity at this. although the reduction of stigma receptivity at postanthesis detected by poor reaction, it was still receptive in contrast to pre-anthesis (figure 5 c,f)). according to antioxidant enzyme activity results, the maximum cat activity was determined at anthesis by 53 % while the minimum was observed at postanthesis by 20 % in compare to pre-anthesis (figure 5 g). besides, the highest h2o2 production was observed at anthesis by 118 %, and lowest was observed at post anthesis by 6 % (figure 5 h). moreover, the sod activity increased by 118 % at anthesis and 68 % at post-anthesis in compare to pre-anthesis (figure 5 i). mda one of the last products of lipid peroxidation was very high at anthesis and post-anthesis showing increase by 150 % and 116 %, respectively (figure 5 j). figure 2. semi-thin longitudinal sections of stigma of hibiscus syriacus stained by pas (a-e), cbb (f-j) and sbb (k-l). short and ovoid papillae cells at anthesis (a, f, k). papillae cells with dense organic material content and prominent pellicle layer (arrows) at anthesis (b, g, l). sub-papillary cells that accumulate a large amount of organic material (c, h, m). thorn shaped papillae with dense cytoplasm at postanthesis (d, i, n). degenerated subpapillary cells and their diffused contents to intercellular space (arrows) (e, j, o). p: papillae cell, sp: subpapillary cell, dsp: degenerated sub-papillary cell. scale: 20 µm. 8 aslıhan çetinbaş-genç, fatma yanık, filiz vardar figure 3. organic material dynamics of papillae cells during stigma development of hibiscus syriacus. a. insoluble polysaccharide content, b. protein content, c. lipid content. data are expressed as optical density per area unit (300 µm²). the data with different letters are significantly different according to tukey’s test at p < 0.05 for independent samples. results are expressed as mean ± sd. figure 4. starch dynamics at sub-papillary cells during stigma development of hibiscus syriacus. a. very few starch granules in sub-papillary cells at pre-anthesis, b. large and dense starch granules in sub-papillary cells at anthesis, c. starch granule content at post-anthesis (note the reduction in compare to anthesis), d. starch synthesis/degradation pattern of sub-papillary cells, data are expressed as optical density per area unit (100 µm²). the data with different letters are significantly different according to tukey’s test at p < 0.05 for independent samples. results are expressed as mean ± sd. scale: 5 µm. 9histochemical and biochemical alterations in the stigma of hibiscus syriacus (malvaceae) during flower development figure 5. enzymatic activities in stigma of hibiscus syriacus. non spesific esterase activity at pre-anthesis (a), anthesis (b), post-anthesis (c). peroxidase activity at pre-anthesis (d), anthesis (e), post-anthesis (f ), cat activity (g), sod activity (h), h2o2 content (i) and lipid peroxidation (j) of stigmas at different developmental stages. the data with different letters are significantly different according to tukey’s test at p < 0.05 for independent samples. results are expressed as mean ± sd. scale: 50 µm. 10 aslıhan çetinbaş-genç, fatma yanık, filiz vardar discussion flower development which is closely related to stigma differentiation may be categorized into different stages considering stigma (suarez et al. 2012). annahwi et al. (2017) previously defined the flower development stages of hibiscus rosa-sinensis especially focusing on flower phenology. according to the researchers, we specified three stigma development stages in hibiscus syriacus as a result of the examined morphological markers. h. syriacus and h. rosa-sinensis had similarities in flowering features. calyx was longer than the corolla bud at preanthesis. flowers were fully opened and anthers were dehiscent at anthesis. annahwi et al. (2017) stated the last stage of flower development as fertilization, which was marked with the fall of the corolla, column, and pistil. but we preferred to name the last stage as postanthesis, which was marked with shrinkage and discoloration of the corolla. the mature pistils of malvaceae have been characterized as having five-branched and capitated stigma which was separated to each other during maturation in h. syriacus as it was in h. rosa-sinensis (annahwi et al. 2017). as a result of increased metabolic activity, it has been known that stigmatic papillae cells begin to accumulate polysaccharide, lipid, and protein throughout their development (neil et al. 2002; zafra et al. 2010). lipids are reported to be effective in pollen hydration and growing pollen tube orientation. in addition, polysaccharides are known to form a suitable medium for pollen germination (herrero and dickinson 1979; wolters-arts et al. 1998). so, lipid and polysaccharides are found to be abundant on stigma, especially when it is ready for the pollination. consistent with this, it was determined that stigmatic papillae cells of h. syriacus had an abundant polysaccharide, protein, and lipid content at all stages of development. however, their amounts were found to be at the maximum level at anthesis stage in which the stigma was the most receptive. edlund et al. (2004) also stated that at the receptive stigma the formation of pellicle layer usually occurs. in h. syriacus, pellicle of papillae was formed at anthesis and it remained intact during the development. however, when stigma receptivity reduced at post-anthesis, the pellicle became thinner. degenerated sub-papillary cells were detected previously by losada and herrero (2012) in malus domestica as it was in h. syriacus. the researcher stated that these cells contribute to the formation of stigmatic secretion. the release of their contents into the intercellular space provides the increment in the amount of secretion on the stigma surface and the intercellular space. similar to m. domestica, organic material content in the intercellular space increased by degeneration of the sub-papillary cells in h. syriacus. however, there was no degeneration-based secretion on the stigmatic surface. although degeneration-based organic material release started at post-anthesis, pellicle prevented the release of substances onto the stigma. according to the researchers the intercellular secretion has important roles in pollen recognition and germination (heslop-harrison 2000; mcinnis et al. 2006). besides, the accumulation of organic substances usually associated with stigma maturation and receptivity. it can be suggested that the accumulation of the substance in the intercellular space in h. syriacus was related to both maturity and receptivity at anthesis, however the continuation of the accumulation at postanthesis may be associated with organ senescence rather than receptivity (hiscock and allen 2008). starch is principal storage carbohydrates having important roles on pollen tube growth, ovule and fruit formation and determination of flower quality in angiosperms (chapin et al. 1990; rodrigo et al. 2000; reale et al. 2009; alcaraz et al. 2010). suarez et al. (2012) stated that the amount of starch increased in the sub-papillary cells and style channel during pollination in olea europaea. in h. syriacus, whereas sub-papillary cells contained plenty of starch granules at pre-anthesis stage, along with pollination starch accumulation increased. at post-anthesis stage starch granules were still existed. similarly, researchers noted that starches are reduced in stigmatic cells after pollination consuming as a source of energy for pollen tube growth (rodriguez-garcia et al. 2003). esterase and peroxidase are the major constituent of the stigma surface proteins. they are functional on the pollen-stigma interaction to loosen cell wall components of stigma cells with the aim of allowing pollen tubes to penetrate and grow into the stigma (hiscock et al. 2002; mcinnis et al. 2006). their activity were determined on the surface of the receptive stigma in many species (seymour and blaylock 2000; shakya and bhatla 2010). in h. syriacus, the stigma exhibited poor non-specific esterase and peroxidase activity at pre-anthesis. but, intensive non-specific esterase and peroxidase activity detected at anthesis, indicating the stigma gained receptivity at this stage (serrano and olmedilla 2012). although the expression of peroxidase decreased at post-anthesis with compare to the anthesis, it was more than pre-anthesis demonstrating that the stigma still continued to receptivity at post-anthesis. however, even in the case of a reduction in stigma receptivity, dense pollen grains, and continuing enzymatic activities represented that pollination still proceeds. 11histochemical and biochemical alterations in the stigma of hibiscus syriacus (malvaceae) during flower development in recent years, many studies have reported that ros function as signal molecules during the stigma pollen interaction (mcinnis et al. 2006; zafra et al. 2010; allen et al. 2011; serrano and olmedilla 2012). the most occurring ros are superoxide anion (o2.-), hydroxyl radical (.oh) and hydrogen peroxide (h2o2). among them h2o2 which is produced by sod from reduction of superoxide anions is the most stable ros and it can cross the membranes. in angiosperm stigma, accumulation of h2o2 during the stigma receptivity is known to be a common feature. in parallel, h2o2 accumulation and sod activity which is h2o2 catalyzer was at maximum level during the anthesis in h. syriacus. high concentrations of h2o2 may be involved in signaling networks that promote pollen germination and/or pollen tube growth on stigma as it was in arabidopsis thaliana (mcinnis et al. 2006). furthermore, high levels of h2o2 can be produced as a result of increased metabolic activity in stigma papillae and surrounding tissues starting to collect pectin, arabinogalactan, proteins and other organic components, as well as starch and lipids (neil et al. 2002; zafra et al. 2010). it can be suggested that h2o2 concentration increased due to the accumulation of organic matter serving as a signal that promotes pollen germination and pollen tube entry at anthesis stage. it has been known that cat breakdown h2o2 to h2o and o2 (yanık et al. 2018). although cat activity increased at anthesis, h2o2 is still at high concentrations. this situation suggests that cat activity is not sufficient to scavenge with over accumulated h2o2. ros accumulation in papillae of h. syriacus indicates that stigma gains receptivity at anthesis. supporting results were also stated in amygdalolia and o. europaea cultivars (aslmoshtaghi and shahsavar 2016). however, high concentrations of h2o2 cause oxidative stress resulting in biomolecular damage (quan et al. 2008; schieber and chandel 2014). when the amount of ros exceeds the threshold value, lpo occurs in both cell and organelle membranes and oxidative stress increases (yanik et al. 2018). lpo can be monitored by the level of mda that end product of lpo (halliwell and gutteridge 1989). considering the highest h2o2 accumulation at anthesis, lpo was at highest level at anthesis in h. syriacus stigma. furthermore, high mda content may also relate to loosening of cell membrane components of papillae due to germinated pollen tubes at anthesis. in conclusion, stigma of h. syriacus is receptive at anthesis stage and still receptive at post anthesis even its performance has fallen. at anthesis stage organic material synthesis, enzymatic activity, lipid peroxidation and h2o2 accumulation are very progressive assisting stigma receptivity, pollen germination and pollen tube growth. our data will help improve our knowledge on pollination success in plants as well as in hibiscus. disclosure statement no potential conflict of interest was reported by the authors. references alcaraz ml, hormaza ji, rodrigo j. 2010. ovary starch reserves and pistil development in avocado (persea americana). physiol plant. 140(4):395–404. allen am, thorogood cj, hegarty mj, lexer c, hiscock sj. 2011. pollen–pistil interactions and self-incompatibility in the asteraceae: new insights from studies of senecio squalidus (oxford ragwort). ann bot. 108(4):687–698. annahwi d, ratnawati r, budiwati b. 2017. flower and fruit development phenology and generative reproduction success of hibiscus rosa-sinensis spp.  yru journal of science and technology. 2(2):19–30. aslmoshtaghi e, shahsavar ar. 2016. biochemical changes involved in self-incompatibility in two cultivars of olive (olea europaea l.) during flower development. j hortic sci biotechnol. 91(2):189–195. birecka h, briber ka, catalfamo jl. 1973. comparative studies on tobacco pith and sweet potato root isoperoxidases in relation to injury, indoleacetic acid, and ethylene effects. plant physiol. 52(1):43–49. brito ms, bertolino lt, cossalter v, quiapim ac, paoli hc, goldman gh, teixeira sp, goldman mhs. 2015. pollination triggers female gametophyte development in immature nicotiana tabacum flowers. front plant sci. 6:1–10. cakmak i, horst jh. 1991. effects of aluminum on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (glycine max). physiol plant. 83(3):463–468. cakmak i, marschner h. 1992. magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. plant physiol. 98(4):1222– 1227. çetinbaş-genç a, ünal m. 2017. timing of reproductive organs maturity in proterandrous malva sylvestris l.. not sci biol. 9(2):287–295. chapin iii fs, schulze ed, mooney ha. 1990. the ecology and economics of storage in plants.  annu rev ecol evol syst. 21(1):423–447. 12 aslıhan çetinbaş-genç, fatma yanık, filiz vardar cheong yh, chang hs, gupta r, wang x, zhu t, luan s. 2002. transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in arabidopsis. plant physiol. 129(2):661–677. cho yw, park eh, lim cj. 2000 glutathione s-transferase activities of s-type and l-type thiol transferase from arabidopsis thaliana. biochem mol biol j. 33(2):179–183. delannoy e, jalloul a, assigbetse k, marmey p, geiger jp, lherminier j, daniel jf, martinez c, nicole m. 2003. activity of class iii peroxidases in the defense of cotton to bacterial blight. mol plant microbe interact. 16(11):1030–1038. do hm, hong jk, jung hw, kim sh, ham jh, hwang bk. 2003. expression of peroxidase-like genes, h2o2 production, and peroxidase activity during the hypersensitive response to xanthomonas campestris pv. vesicatoria in capsicum annuum. mol plant microbe interact. 16(3):196–205. edlund af, swanson r, preuss d. 2004. pollen and stigma structure and function: the role of diversity in pollination. plant cell. 16(1):84–97. feder n, o’brien tp. 1968. plant microtechnique: some principles and new methods. am j bot. 55(1):123–142. fisher db, jensen wa, ashton me. 1968. histochemical studies of pollen: storage pockets in the endoplasmic reticulum. histochemie. 13(2):169-182. gomori g. 1950. an improved histochemical technique for acid phosphatase. stain technol. 25(2):81–85. halliwell b, gutteridge jm. 1989. 1 iron toxicity and oxygen radicals. baillieres clin haematol. 2(2), 195–256. herrero m, dickinson hg. 1979. pollen-pistil incompatibility in petunia hybrida: changes in the pistil following compatible and incompatible intraspecific crosses. j cell sci. 36(1):1–18. heslop-harrison y. 2000. control gates and micro-ecology: the pollen–stigma interaction in perspective. ann bot. 85(1):5–13. hiscock sj, hoedemaekers k, friedman we, dickinson hg. 2002. the stigma surface and pollen-stigma interactions in senecio squalidus l. 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l.). acta bot croat. 77(1):45–50. zafra a, rodriguez-garcia mi, alche jd. 2010. cellular localization of ros and no in olive reproductive tissues during flower development. bmc plant biol. 10(1):36–50. substantia an international journal of the history of chemistry vol. 2, n. 1 march 2018 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 75(2): 33-43, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1541 caryologia international journal of cytology, cytosystematics and cytogenetics citation: xiaoju zhang, li bai, somayeh esfandani-bozchaloyi (2022) population differentiation and gene flow of salicornia persica akhani (chenopodiaceae). caryologia 75(2): 33-43. doi: 10.36253/caryologia-1541 received: january 18, 2022 accepted: may 24, 2022 published: september 21, 2022 copyright: © 2022 xiaoju zhang, li bai, somayeh esfandani-bozchaloyi. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. population differentiation and gene flow of salicornia persica akhani (chenopodiaceae) xiaoju zhang1, li bai2,*, somayeh esfandani-bozchaloyi3 1 college of humanities and management, xi’an traffic engineering institute, xi’an 710300, shaanxi, china 2 department of mechanical and electrical technology, xijing university, xi’an 710123, shaanxi, china 3 department of plant sciences, university shahid beheshti, iran *corresponding author. e-mail: baili891066611@163.com abstract. the genus salicornia (amaranthaceae) was established by linnaeus. commonly known as ‘glassworts’, the species of the genus are articulated succulent herbs with cortical palisade, opposite decussate scale-leaves, thyrsoid cymes, flowers packed in cauline depressions and the diaspore composed of l-seeded utricle. therefore, due to the importance of the plant species, we performed a combination of morphological and molecular data analyses on this species. a total of 72 randomly collected plants from 8 natural populations in 2 provinces were evaluated using issr markers and morphological traits. analysis of molecular variance (amova) test revealed significant genetic difference among the studied populations, and also showed that 45% of total genetic variability was due to the diversity within the population, while 55% was due to the genetic differentiation among populations. a total number of 158 bands were detected by issr primers, of which 144 (89%) bands with an average of 14.4 bands per primer were polymorphic. the percentage of polymorphic bands (ppb) ranged from 70% (issr-7) to 100% (issr-1, issr-4 and issr-5). the average polymorphic information content (pic), shannon’s information index (i), and number of effective alleles (ne) were 0.49, 0.28, and 1.09, respectively. keywords: genetic diversity, gene flow, genetic differentiation, salicornia persica, inter simple sequence repeat (issr). introduction genetic diversity is a basic component of biodiversity and its conservation is essential for survival of any species in the changing environments (si et al. 2020; liu et al. 2021). most authors agree that genetic diversity is necessary to preserve the long-term evolutionary potential of a species (peng et al. 2021; ma et al. 2021). this is very important in fragmented populations, because they are more vulnerable due to the loss of allelic richness and increased population differentiation by genetic drift (decreased heterozygosity and eventual fixation of alleles) and inbreeding depression (increased homozygosity within populations; chen et al. 2021; bi et al. 2021). therefore, 34 xiaoju zhang, li bai understanding the genetic variability and diversity within and among different populations is crucial for their conservation and management (e.g., esfandani-bozchaloyi et al., 2018a, 2018b, 2018c). the genus salicornia (amaranthaceae) was established by linnaeus (1753). commonly known as ‘glassworts’, the species of the genus are articulated succulent herbs with cortical palisade, opposite decussate scaleleaves, thyrsoid cymes, flowers packed in cauline depressions and the diaspore composed of l-seeded utricle. many species are green, but their foliage turns red in autumn. the hermaphrodite flowers are wind pollinated. the species inhabit saline habitats such asinland saltmarshes, saline seasonal river banks and tidal coastlines, but all tidal coasts and salines are not home to glassworts. salicornia species can generally tolerate immersion in salt water. they use the c 4 to take in carbon dioxide from the surrounding atmosphere. salicornia has leafless stems with branches that resembles asparagus. the halophyte salicornia supports soil microbial growth and boosts the tphs degradation in saline oilcontaminated soils. combining salicornia and p.  aeruginosa accelerates tph degradation and reduces saline oilcontaminated soils phytotoxicity (ebadi et al. 2018). the first comprehensive account of family chenopodiaceae in flora iranica (hedge et al. 1997) provides very useful and fundamental data on the arid flora of the old world. the genus salicornia is among the most diverse genera of the salicornieae tribe. the genus currently comprises 25 to 30 species (kadereit et al. 2007). the taxonomy of the genus salicornia is still far from satisfactory, although numerous species aggregates, species and microspecies have been described over the last 250 years. frequently the name salicornia europaea is used in a very broad sense to include most of the species of the genus. additionally, the plants show a high level of phenotypic plasticity (ingrouille and pearson 1987). the salinity of their habitats fluctuates greatly due to different factors tidal cycles, evapotranspiration, precipitation and availability of fresh groundwater. this is the reason why salicornia develops high physiological plasticity which causes phenotypic variation (kadereit et al. 2007). morphological distinction between the taxa is only possible when the plants are fresh, between flowering and fruiting (gehu et al. 1979). morphometric studies using all phenotypic differences available, irrespective of whether they have a genetic basis or not, could not reveal distinct taxa even on a small regional scale (ingrouille and pearson 1987). salicornia plants tend to have phenotypic variations depending on environmental conditions such as temperature, quality of soil, concentration of salt and population density. ball and akeroyd (1993) suggested that the specific limits of classification of the salicornia plants based on morphological features, especially those of dried salicornia plants, are obscure. to prove the relevance between the genotype and phenotype in salicornia plants, genetic variability was analyzed by rapd fingerprinting. the use of molecular markers is considered to be the best for genetic diversity analysis since it has proved to be non-invasive in the sense that there are no negative effects on the stage of development, environment or management practices. furthermore, these kinds of studies can be applied even on dead plants when the genomic dna is extractable (choudhury et al. 2001). molecular markers play a significant role in the protection of biodiversity, identification of promising cultivars, quantitative trait loci (qtl) mapping, etc. various pcr-based markers such as issr, scot, srap, etc. have been effectively used for the quantification of genetic diversity. recent issr studies of natural populations have demonstrated the hypervariable nature of the markers and their potential use for the population-level studies (hultén and fries 1986). in the present study, the issr markers and morphologic traits were used for the first time in iran to analyze the genetic diversity in 72 salicornia persica accessions belonging to 8 different populations. materials and methods plant materials for the morphometric and issr analyses, we used 72 plant accessions (four to twelve samples from each population) belonging to 8 different populations of salicornia persica in esfahan and tehran provinces of iran during july-agust 2018-2020 (table 1). more information about the geographical distribution of the accessions are given in table 1 and fig. 1. the plant individuals were identified morphologically using different literature (kadereit et al. 2007; akhani 2003). dna extraction and issr analysis fresh leaves were randomly used from four to twelve samples for each of the studied populations. they dried by silica gel. the ctab-activated charcoal protocol was used to extract genomic dna (esfandani-bozchaloyi et al. 2019). for the issr analysis, 22 primers from the ubc (university of british columbia) series were tested 35population differentiation and gene flow of salicornia persica akhani (chenopodiaceae) for the dna amplification. ten primers were chosen for the issr analysis of genetic variability based on band reproducibility (table 2). the pcr reactions were carried out in a 25μl volume containing 10 mm tris-hcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 ng of genomic dna, and 3 u of taq dna polymerase (bioron, germany). the amplifications’ reactions were performed in techne thermocycler (germany) with the following program: 5min initial denaturation step at 95°c, followed by 37 one-minute cycles at 95°c, 1 min at 50-56°c, and 1 min at 72°c. the reaction was completed by the final 5-10 min extension step at 72°c. the amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated using a 100bp molecular size ladder (fermentas, germany). data analysis morphological studies a total of 19 metric and 6 multistate characterers were used for measurements in different combinations (table not included), modified from the character list detailed by ingrouille and pearson (1987). of these 25 characters, 15 covered the overall vegetative morphology, and 10 were characteristics of the fertile spike, fertile spike segments and flowers. though vegetative morphology may be partly uninformative due to the wide phenotypic plasticity, both vegetative and fertile spike characteristics were used, because some vegetative traits have been shown useful in separating populations and taxa at least in single cases (ingrouille and pearson 1987). the data obtained were standardized (mean= 0, variance = 1) and used to estimate the euclidean distance for clustering and ordination analyses (podani 2000). to group the plant specimens, the upgma (unweighted pair group with arithmetic mean), ward’s (minimum spherical traits), and mds (multidimensional scaling) ordination methods were used (podani 2000). past software version 2.17 (hammer et al. 2012) was used for the multivariate statistical analyses of the morphological data. molecular analysis the issr profiles obtained for each sample were scored as binary traits. the discriminating capability of the used primers was evaluated by means of two paramtable 1. voucher details and diversity within iranian populations and subspecies of salicornia persica in this study. no subspecies locality pop1 subsp. persica akhani esfahan,the river of zayanderud at varzaneh pop2 subsp. persica akhani esfahan,nain, it is 70km to varzaneh pop3 subsp. persica akhani fars, tashk lake pop4 subsp. persica akhani esfahan, northern coasts of batlaq-e gavkhooni pop5 subsp. rudshurensis akhani tehran ;60 km west of tehran, 25 km se of karaj pop6 subsp. rudshurensis akhani tehran: ca. 60 km w tehran, mardabad salt flats, along rude shur pop7 subsp. rudshurensis akhani tehran, karaj located in 25 km nw rude shur pop8 subsp. rudshurensis akhani tehran ; rude-shur river, which is located 40 km west of tehran figure 1. distribution map of studied populations of salicornia persica in iran. 36 xiaoju zhang, li bai eters, polymorphism information content (pic) and marker index (mi), to characterize the capacity of each primer to detect polymorphic loci among the genotypes. the number of polymorphic bands (npb) was calculated for each primer. the parameters like nei’s genetic diversity (h), shannon’s information index (i), number of effective alleles, and percentage of polymorphism (p% = number of polymorphic loci/number of total loci) were determined (weising et al. 2005; freeland et al. 2011). shannon’s index was calculated by the following formula: h’ = -σpiln pi. rp is defined per primer as: rp = ∑ ib, where “ib” is the band informativeness, which takes the values of 1-(2x [0.5-p]), and “p” is the proportion of each genotype containing the band. the percentage of polymorphic loci, the mean loci by accession and by population, uhe, h’ and pca were calculated by genalex 6.4 software (peakall and smouse 2006). nei’s genetic distance among the populations was used for neighbor joining (nj) clustering and neighbor-net networking (freeland et al. 2011). mantel test checked the correlation between geographical and genetic distances of the studied populations (podani 2000). the analyses were done by past ver. 2.17 (hammer et al. 2012), darwin ver. 5 (2012), and splitstree4 v4.13.1 (2013) software. amova (analysis of molecular variance) test (with 1000 permutations) implemented in genalex 6.4 (peakall and smouse 2006) and nei’s gst analysis implemented in genodive ver.2 (2013) were used to show the genetic difference of the populations. moreover, the populations’ genetic differentiation was studied by g(st)est = standardized measure of genetic differentiation (hedrick 2005), and dest = jost measure of differentiation. to assess the population structure of the salicornia persica accessions, a heuristic method based on the bayesian clustering algorithms was utilized. the clustering method based on the bayesian model implemented in the structure software (falush et al., 2007) was used on the same data set to better detect the population substructures. this clustering method is based on an algorithm that assigns genotypes to homogeneous groups based on the number of clusters (k). assuming hardy-weinberg and linkage equilibrium within the clusters, the software estimates allele frequencies in each cluster and population membership for each individual (pritchard et al. 2000). the number of potential subpopulations varied from two to ten, and their contribution to the genotypes of the accessions was calculated based on 50,000 iteration burnins and 100,000 iteration sampling periods. the most probable number (k) of subpopulations was identified following evanno et al. (2005). in k-means clustering, two summary statistics, pseudo-f and bayesian information criterion (bic), provide the best fit for k. gene flow (nm) was calculated using popgene (version 1.31) software. gene flow was estimated indirectly using the following formula: nm = 0.25(1 fst)/fst. in order to test for a correlation between pair-wise genetic distances (fst) and geographical distances (in km) among the populations, mantel test was performed using tools for population genetic analysis (tfpga; miller, 1997) (computing 999 permutations). this approach considers an equal amount of gene flow among all populations. population assignment test based on maximum likelihood was performed in genodive ver. 2 (2013). the presence of shared alleles was determined by drawing the reticulogram network based on the least square method by darwin ver 5 (2012). table 2. details about the banding pattern revealed by issr primers. primer name primer sequence (5’-3’) tnb npb ppb pic pi issr-1 dbdacacacacacacaca 10 10 100.00% 0.28 5.11 issr-2 ggatggatggatggat 9 7 93.00% 0.38 6.41 issr-3 gacagacagacagaca 24 20 87.00% 0.56 4.34 issr-4 agagagagagagagagyt 10 10 100.00% 0.49 3.88 issr-5 acacacacacacacacc 15 15 100.00% 0.41 5.66 issr-6 gagagagagagagagarc 11 9 94.00% 0.25 4.99 issr-7 ctctctctctctctctg 13 7 70.00% 0.64 4.21 issr-8 cacacacacacacacag 13 9 82.00% 0.32 4.32 issr-9 gtgtgtgtgtgtgtgtyg 12 10 93.00% 0.25 6.56 issr-10 cacacacacacacacarg 25 21 91.00% 0.57 4.11 average 15.8 14.4 89.00% 0.49 5.12 total 158 144 note: tnb the number of total bands, npb: the number of polymorphic bands, ppb (%): the percentage of polymorphic bands, pi: polymorphism index, pic, polymorphism information content for each of issr primers. 37population differentiation and gene flow of salicornia persica akhani (chenopodiaceae) results morphometry the morphological evaluation revealed considerable variations among the accessions for spike characteristics. based on the botanical traits, 41 out of 72 evaluated accessions were identified as subsp. persica and 31 accessions as subsp. rudshurensis (fig. 1). anova showed significant differences (p <0.01) in quantitative morphological traits among the populations under study. in order to determine the most variable traits among the taxa studied, the pca analysis was performed. it revealed that the first three factors comprised over 76% of the total variations. in the first pca axis with 52% of total variations, such traits as length of visible part of central flower of cyme, 2nd fertile segment; width of 2nd fertile segment at base showed the highest correlation (>0.7). length of 2nd fertile segment measured over the central; length of spike and number of fertile segments on longest were the traits influencing the pca axes 2 and 3, respectively. different clustering and ordination methods produced similar results and, therefore, the pca plot of morphological traits are presented here (fig. 2). in general, the pca plot of the studied populations did entirely delimit the studied populations and revealed that the plants in these populations are not intermixed. populations’ genetic diversity in the present study, 10 out of 22 selected issr primers amplified 158 clear discernible bands, of which 144 (80 %) were polymorphic, showing the high discriminative and resolving power of the used issrs in the studied germplasm. the total number of bands per primer ranged from 9 (issr-2) to 25 (issr-10), with an average of 15.8. the number of polymorphic bands per primer varied from 7 (issr-2, issr-7) to 21 (issr-10), with an average of 14.4. the band sizes of the amplified products were found between 100 and 3,000 bp. to characterize the capacity of each primer to detect polymorphism and to evaluate the discriminating capability of each primer in the studied germplasm, various diversity indices such as the highest percentage of polymorphic bands, ne, i and, pic were calculated. the highest percentage of polymorphic bands was produced by primers issr-1, issr-4 and issr-5 (100%), while primer issr-7 produced the lowest percentage of polymorphic bands (70%). the pic values across all primers averaged 0.49. issr-7 showed the highest (0.64) and issr-6, issr-9 the lowest (0.25) pic value, respectively (table 2). the genetic diversity parameters determined in 8 geographical populations of salicornia persica are presented in table 3. the highest value of polymorphism percentage (52.15%) was observed in esfahan, northern coasts of batlaq-e gavkhooni (population no. 4, subsp. persica), which shows a high value for the genetic diversity (0.38) and shannon’s information index (0.45). the population of tehran; rude-shur river, which is located 40 km west of tehran (no. 8, subsp. rudshurensis) has the lowest value for the percentage of polymorphism (15.91%) and the lowest value for shannon’s information index (0.17) and he (0.18). populations’ genetic differentiation amova (phipt = 0.88, p = 0.0010) revealed significant difference among the studied populations (table 4). it also revealed that 45% of total genetic variations was due to the diversity within the population and 55% was due to the genetic differentiation among the populations. the pairwise comparison of nei’s genetic identity among the studied populations salicornia persica (table table 3. genetic diversity parameters in the studied salicornia persica populations. sp n na ne i he uhe %p pop1 16.000 0.892 1.168 0.321 0.251 0.265 34.63% pop2 6.000 0.344 1.035 0.31 0.23 0.25 40.53% pop3 11.000 0.441 1.036 0.33 0.25 0.27 42.53% pop4 8.000 0.247 1.021 0.45 0.38 0.33 52.15% pop5 5.000 0.290 1.024 0.23 0.25 0.18 24.30% pop6 10.000 0.452 1.089 0.29 0.22 0.25 45.05% pop7 10.000 0.333 1.006 0.222 0.22 0.22 33.23% pop8 9.000 1.247 1.275 0.171 0.184 0.142 15.91% abbreviations: n = number of samples, na= number of different alleles; ne = number of effective alleles, i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations. 38 xiaoju zhang, li bai 5) showed a higher genetic similarity (0.93) between the populations of esfahan, nain, it is 70km to varzaneh (pop. no. 2) and fars, tashk lake (pop. no. 3), while the lowest genetic similarity value (0.712) occurred between esfahan, the river of zayanderud at varzaneh (pop. no. 1) and tehran, karaj located in 25 km nw rude shur (pop. no. 7). table 4. analysis of molecular variance (amova) of the studied salicornia persica. source df ss ms est. var. % φpt among pops 56 1221.364 52.789 21.154 55% 55% within pops 120 114.443 12.905 12.905 45% total 176 1365.807 33.060 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance; φpt: proportion of the total genetic variance among individuals within an accession, (p < 0.001). table 5. pairwise population matrix of nei unbiased genetic identity. pop1 pop2 pop3 pop4 pop5 pop6 pop7 pop8 1.000 pop1 0.833 1.000 pop2 0.810 0.933 1.000 pop3 0.875 0.873 0.830 1.000 pop4 0.818 0.896 0.874 0.812 1.000 pop5 0.852 0.858 0.844 0.838 0.884 1.000 pop6 0.712 0.846 0.800 0.796 0.881 0.794 1.000 pop7 0.779 0.855 0.809 0.794 0.874 0.752 0.862 1.000 pop8 figure 2. pca plot of salicornia persica populations based on morphological traits. 39population differentiation and gene flow of salicornia persica akhani (chenopodiaceae) populations’ genetic affinity two major clusters were formed in the upgma tree (fig. 3). the first major cluster contained two subclusters: the population of esfahan, nain, it is 70km to varzaneh; fars, tashk lake and esfahan, northern coasts of batlaq-e gavkhooni (pop. no. 2,3,4, subsp. persica) is distinct and remains separate from the other populations with a great distance and comprises the first subcluster. the second sub-cluster was formed by the other populations from subsp. rudshurensis, (pop. no. 5-8) which showed close genetic affinity. the second major cluster contained only subsp. persica, which separated from the other studied populations and joined the others with a great distance. these results show that the plant specimens of each studied subspecies were grouped together, indicating that the subspecies are delimited based on the issr molecular markers. therefore, this result confirms our morphology results. the nm analysis by popgene software also produced mean nm= 0.17, which is considered a very low value of gene flow among the studied species. mantel test after 5000 permutations produced significant correlation between genetic distance and geographical distance in these populations (r = 0.33, p = 0.001). populations’ genetic structure k = 2 reveal the presence of 2 genetic groups. similar results were obtained by evanno test performed on structure analysis, which produced a major peak at k = 2. both analyses revealed that salicornia persica populations show genetic stratification. the structure plot based on k = 2 revealed the genetic difference in population of esfahan and fars province (pop. no. 1-4; subsp. persica) (red colored) with other populations. however, it showed genetic affinity between populations 5-8 of tehran province (subsp. rudshurensis) (green colored) figure 3. upgma plot of populations in salicornia persica populations based on issr data (population numbers according to table 1). 40 xiaoju zhang, li bai the mean nm = 0.17 was obtained for all issr loci, which indicates the low amount of gene flow among the populations, and supports the genetic stratification as indicated by k-means and structure analyses. however, the reticulogram obtained based on the least square method (figure 4) revealed some amount of shared alleles among populations 1 and 2, and populations 3 and 8. this result agrees with the grouping obtained with upgma tree, as the populations were placed close to each other. as evidenced by structure plot based on the admixture model, the shared alleles comprise a very limited part of the genomes in the populations, and all the results consistently show a high degree of genetic stratification within salicornia persica populations. discussion the present study revealed interesting data about the genetic variability, genetic stratification, and morphological divergence in the central parts of iran. the genetic diversity is of fundamental importance in the continuity of a species, as it is used to bring about the necessary adaptation to cope with the changes in the environment (wang et al. 2021; yin et al. 2021; zhao et al. 2021). the degree of genetic variability within a species highly correlates with its reproduction mode. the higher degree of open pollination/cross breeding brings about a higher level of genetic variability in the studied taxon (jia et al. 2020; shi et al. 2021; zheng et al. 2021; zhu et al. 2021). the pic and mi characteristics of a primer help to figure 4. reticulogram of salicornia persica populations based on least squares method analysis of issr data (population numbers according to table 1). 41population differentiation and gene flow of salicornia persica akhani (chenopodiaceae) determine its effectiveness in the genetic diversity analysis. sivaprakash et al. (2004) suggested that the ability of a marker technique to resolve genetic diversity may be more directly related to the degree of polymorphism. generally, the pic value between 0 and 0.25 imply a very low genetic diversity among genotypes, the value between 0.25 and 0.50 shows a mid-level genetic diversity, and the value ≥0.50 suggests a high level of genetic diversity. in this research, the issr primers’ pic values ranged from 0.25 to 0.64, with a mean value of 0.49, which indicated the high capability of issr primers for determining the genetic diversity among the salicornia persica accessions. papini et al. (2004) found that diploid and tetraploid accessions of salicornia resolved as sister clades. the study was based on its sequences of twelve samples of salicornia (all but one from italy) representing four species (three tetraploid, one diploid). the presence of rapd polymorphic bands in the populations studied indicates the presence of genetic polymorphism in these populations. moreover, the occurrence of specific bands/loci only in some of the populations illustrates the occurrence of unique insertion/deletion in dna material of these genotypes. as the range of species in salicornia is imperfectly known, it is rather premature to evaluate their plant geographical importance. however, based on present data two species groups can be distinguished: the first are central and south-central iranian species including s. persica subsp. persica, s. persica subsp. rudshurensis, s. perspolitana, s. iranica and s. x tashkensis and the second group consisted only of s. sinus-persica which is endemic around the persian gulf. the central and south-central iran possess several endemic species of desert and arid flora with remarkable phytogeographic importance. furthermore the area is part of the zagros mountains which is known as a very important plant diversity center in sw asia (akhani, 2008). sagane et al. (2003) conducted a study in japan on identification of salicornia population through morphological and rapd fingerprinting. they observed variations in plant length, segment number, length and number of branches, and incidence of the secondary branches etc. on the basis of genotype based on the rapd marker they identified five groups in three selected populations. the genetic diversity of 102 individuals of s. persica (15 populations) were studied using 10 start codon targeted (scot) markers (liu & esfandani-bozchaloyi 2022). their result showed high polymorphic bands (94.18%), polymorphic information content (0.27), and allele number (1.38) showed scot as a reliable marker system for genetic analysis of this species. according to chatrenoor & akhani (2021) an integrated morpho‐ molecular study of salicornia (amaranthaceae‐chenopodiaceae) in iran proves irano‐turanian region the major center of diversity of annual glasswort species. their results (1) confirm the efficiency of plastid sequences comparing to ets sequences for clarif ying species-level phylogeny of salicornia; (2) identify the s.  persica clade as a monophyletic irano-turanian endemic lineage; (3) recognize nine origins of the iranoturanian salicornia based on nuclear ets sequences; (4) approve the monophyly of tetraploid species using plastid sequences. gohil and pandya (2006) conducted study to find out the degree and the nature of genetic divergence among salicornia brachiata (roxb.) genotypes. gohil and pandya (2006) found a significant difference amongst the salicornia genotypes for all the phenological characters, (like height, canopy, main branch, segment, spike length, spike/branch and seed yield) indicating high genetic variability present in the population. the genotypes under study were grouped into five clusters, indicating wide diversity in the material for majority of the characters. previous results from molecular studies imply near 100% inbreeding in salicornia, which certainly contributes greatly to the taxonomic difficulties in the group because of inbreeding lines with minute but fixed phenotypic differences (noble et al., 1992). the other study using rapd technique showed correlations between dna polymorphism and geographical distribution in s. ramosissima. according to kadereit et al. 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(poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(2): 45-52, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1567 caryologia international journal of cytology, cytosystematics and cytogenetics citation: shiva shahsavari, zahra noormohammadi, masoud sheidai, farah farahani, mohammad reza vazifeshenas (2022) scot molecular markers are efficient in genetic fingerprinting of pomegranate (punica granatum l.) cultivars. caryologia 75(2): 45-52. doi: 10.36253/caryologia-1567 received: february 04, 2022 accepted: july 06, 2022 published: september 21, 2022 copyright: © 2022 shiva shahsavari, zahra noormohammadi, masoud sheidai, farah farahani, mohammad reza vazifeshenas. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. scot molecular markers are efficient in genetic fingerprinting of pomegranate (punica granatum l.) cultivars shiva shahsavari1, zahra noormohammadi1,*, masoud sheidai2,*, farah farahani3, mohammad reza vazifeshenas4 1 department of biology, science and research branch, islamic azad university, tehran, iran 2 department of plant sciences and biotechnology, faculty of life sciences and biotechnology, shahid beheshti university, tehran, iran. 3 department of microbiology, qom branch, islamic azad university, qom, iran. 4 improvement plant and seed department, yazd agricultural and natural resource research center, areeo, yazd, iran. *corresponding authors. zahra noormohammadi: e-mail; marjannm@yahoo.com, z-nouri@srbiau.ac.ir. masoud sheidai: e-mail: msheidai@yahoo.com abstract. the pomegranate is an economically important fruit plant species which has been utilized since ancient time as a source of food and medicine by mankind. this plant although is cultivated in certain geographical regions, but its fruits are imported and sold throughout the world. iran is the center of origin for pomegranate and contains huge number of known cultivars. however, genetic studied on these cultivars are very limited and much detailed information has to be produced for better hybridization and breeding tasks in the country. a fingerprinting study was performed on 178 punica trees in 47 known cultivars by using scot molecular markers. we obtained 61 scot bands/ loci which were used for genetic diversity analyses and grouping of the cultivars. a low genetic variability was obtained within and among punica cultivars, but as revealed by amova, this was quiet enough to produce significant genetic difference among them. dapc analysis revealed a trace of genetic admixture among the cultivars either due to gene flow or as a result of common ancestral shared alleles. discriminating scot loci may be used in germplasm evaluation of punica. the genetic difference of these cultivars can be utilized for hybridization and breeding programs. keywords: amova, dapc, genetic diversity, pomegranate, scot. introduction pomegranate (punica granatum l., family = lythraceae) is an ancient fruit species originated from iran (graham et al., 1998). this plant species is native of iran and mediterranean region, and is also cultivated in tropical and subtropical regions (gundogdu & yilmaz, 2012, fischer et al., 2010, patil et al., 2020). one of the amazing features of pomegranate is its adaptation to wide climatic conditions and it can grow in light and heavy soils, but 46 shiva shahsavari et al. it is the only limiting factor of cold winters (vazifeshenas et al., 2012). recent investigations have shown medicinal value of pomegranate plant and particularly, its antioxidant activity and polyphenol content (li et al., 2006). interestingly enough, different parts of pomegranate tree, such as fruit peel, seeds, fruits, leaves, and flower have different and special healing (li et al., 2006). the pomegranate juices, seeds and extracts are used for treating cardiovascular disease, diabetes and prostate cancer (patel et al., 2008). based on morphological features as well as agronomical properties, several pomegranate cultivars are known in iran (beghè et al., 2019, khadivi et al., 2020, shahsavari et al., 2021a), moreover, there are two main pomegranate germplasm collection centers located in yazd, and saveh cities, which contain about 700-1000 labelled pomegranate cultivars. in spite of this huge number of pomegranate cultivars and accessions, we have very little information on their genetic structure and fingerprinting. germplasm evaluation requires a suitable system to identify parental lines, genotypes, wild relatives and released varieties. although the variability in morphological and biochemical characters are useful for the task due to easy scoring and their economical nature, they have some weakness like low variability, environmental influence, epistasis, and complex inheritance pattern. with the advent of molecular markers, dna fingerprinting became a more objective sensible, and less errorprone method of identifying plant varieties than traditional methods (poets et al., 2020). several factors together may determine the type and number of molecular markers for plant accessions’ genotyping. the number of loci required to discriminate verity depends on the diversity of the crop and its genome size. moreover, the polyploidy level as well as breeding system or pollination mechanism of the plant can affect the genetic variability of the target plant species. the estimated genome size of punica granatum is about 320. mb (luo et al., 2020), which is almost a small genome size and shows open pollination as we can obtain hybrids among different pomegranate plant trees. recent studies which are concerned with molecular assessment of different plant cultivars and accessions produce data not only on genetic finger printing of target plants but also some information may be obtained on the cultivars’ relationship, degree of gene flow among the studied samples, identify discriminating molecular loci or bands, and even identify the loci with potential adaptive value (see for example (saboori et al., 2020, 2021, sepahian et al. 2021). till present time, different dna markers, have been utilized to investigate genetic diversity within pomegranate cultivars and illustrate their relationship as well as genetic fingerprinting. these markers are random amplified polymorphic dna (rapd, (sheidai et al., 2007, hasnaoui et al., 2010, noormohammadi et al., 2012)), amplified fragments length polymorphism (aflp, (jbir et al., 2008, moslemi et al., 2010)), intersimple sequence repeats (issr, (narzary et al., 2010, ahmed, 2018)), simple sequence repeats (ssr, (noormohammadi et al., 2012, zarei et al., 2018, patil et al., 2020, 2021, shahsavari et al., 2021a, 2021b)) and start codon targeted polymorphism marker (scot, (ahmed, 2018)). scot technique has been successful in identifying cultivars and analyzing genetic diversity within and between plant species, in many different plant species including crop plants such as wheat (abdel-lateif & hewedy, 2008, collard & mackill, 2009), barley (dora et al., 2017) and potato (gorji et al., 2011) and also fruit trees such as mango (luo et al., 2010), grapes (guo et al., 2012) and date palm (saboori et al., 2020). recently breeding program on pomegranate cultivars in iran has been focused on producing hybrids with the aim to obtain elite genotypes in iran. in this regard, attempts are made to evaluate the standing genetic variability of the parental genotypes and their hybrids. the present study is a part of such investigations with the following tasks: 1produce data on genetic structure of the parental genotypes and their hybrids. 2estimating the standing genetic diversity within our germplasm collection. 3illustrate genetic affinity of the studied samples. 4compare discriminating power of scot and ssr molecular markers. materials and methods plant materials this study was performed on 187 punica tress collected randomly from 47 genotypes, which were grown in agricultural and natural resources research and training center, yazd, iran. among the studied samples, we had also 7 hybrid genotypes, each having four replicates. these were collected from the trees cultivated in mazandaran province, iran (sahebi pomegranate cooperative company -sari). details of some of these cultivars are provided in table 1. dna extraction and scot pcr amplification total genomic dna was extracted from fresh leaves by ctab with some modification based on krizman et 47scot molecular markers are efficient in genetic fingerprinting of pomegranate (punica granatum l.) cultivars table 1. genetic diversity parameters determined in punica genotypes studied. no cultivar name geographical location accession code na ne i he %p 1 rabab poostghermez fars 68-119-1 0.525 1.063 0.061 0.039 13.11% 2 vahshi poost ghermez roodbar 67-210-2 0.393 1.038 0.034 0.023 6.56% 3 goojagh shahpar vramin vramin 69-181-1 0.508 1.082 0.067 0.046 11.48% 4 makhmal shar reza esfahan 69-143-1 0.492 1.049 0.034 0.025 4.92% 5 marmar ramhormoz ramhormoz 69-161-2 0.295 1 0 0 0.00% 6 ardestani torsh semnan semnan 69-179-4 0.475 1.075 0.058 0.04 9.84% 7 golnar farsi shahdad kerman 68-541-3 0.525 1.069 0.06 0.04 11.48% 8 poostsiyah abrand abad yazd 67-233-1 0.262 1 0 0 0.00% 9 zaghe yazdi yazd 68-602-1 0.475 1.092 0.07 0.049 11.48% 10 shirin shahvar yazdi yazd 70-680-1 0.475 1.043 0.036 0.024 6.56% 11 goroch shahvar yazdi yazd 68-546-1 0.656 1.106 0.097 0.064 18.03% 12 vashik malas sistan 67-614-1 0.426 1 0 0 0.00% 13 bihaste khafri jahrom 67-215-1 0.623 1.143 0.111 0.077 18.03% 14 savehie torsh esfahan 67-209-1 0.295 1 0 0 0.00% 15 togh gardan yazdi yazd 67-203-1 0.508 1.071 0.057 0.039 9.84% 16 malas dane ghermez yazdi yazd 67-191-1 0.525 1.061 0.054 0.036 9.84% 17 faroogh ij estahban fars 67-204-1 0.541 1.066 0.05 0.035 8.20% 18 malas pishva vramin vramin 69-173-1 0.41 1 0 0 0.00% 19 sefid pooste dezfooli dezfool 69-131-1 0.426 1.016 0.011 0.008 1.64% 20 shirin poost ghermez ramsar gilan 69-168-1 0.443 1 0 0 0.00% 21 tabolarze aban mahi yazd 69-144-1 0.541 1.103 0.075 0.053 11.48% 22 vahshi jangali sisangan sisangan 69-138-1 0.344 1 0 0 0.00% 23 siyah dane shahvar kan tehran 69-132-1 0.361 1 0 0 0.00% 24 dane siyah ramhormoz khoozestan 69-120-1 0.541 1.058 0.05 0.034 8.20% 25 barge moordi charmahl bakhtiyari 69-108-1 0.426 1.058 0.05 0.034 8.20% 26 zaghe droshte hrabarjan yazd 69-151-1 0.475 1.021 0.018 0.012 3.28% 27 bagh malek ize khorasan 69-113-1 0.344 1 0 0 0.00% 28 shirin poost nazok darjezin semnan 69-174-1 0.656 1.114 0.098 0.065 18.03% 29 vahshi shirin behbahan behbahan 69-171-1 0.295 1 0 0 0.00% 30 golabi haste nazok sangan sangan 67-226-1 0.361 1 0 0 0.00% 31 fereshte ghermez sari 95-3-1 0.393 1 0 0 0.00% 32 ghandehar afghanistan 95-1-1 0.344 1 0 0 0.00% 33 totsh miankale sari 95-6-47 0.508 1.092 0.07 0.049 11.48% 34 narm haste andarab afghanistan 95-7-1 0.344 1 0 0 0.00% 35 molar spain 95-4-1 0.59 1.105 0.077 0.055 11.48% 36 wonderful zoodras usa 95-5-1 0.557 1.087 0.063 0.045 9.84% 37 wonderful dirras usa 95-9-1 0.377 1.005 0.006 0.004 1.64% 38 malas saveh saveh 92-29-1 0.361 1 0 0 0.00% 39 malas yazdi yazd 67-299-1 0.377 1 0 0 0.00% 40 sefid pooste rabi ardel boroojen 69-137-1 0.393 1 0 0 0.00% 41 code 6 sari 95-11-1 0.328 1 0 0 0.00% 42 code 16 sari 95-23-1 0.311 1 0 0 0.00% 43 code 17 sari 95-22-1 0.328 1 0 0 0.00% 44 code 18 sari 95-24-1 0.279 1 0 0 0.00% 45 code 33 sari 95-16-1 0.295 1 0 0 0.00% 46 code 40 sari 95-18-1 0.328 1 0 0 0.00% 47 code 48 sari 95-19-1 0.311 1 0 0 0.00% total 0.427 1.034 0.028 0.019 4.78% na = no. of different alleles, ne = no. of effective alleles = 1 / (p^2 + q^2, i = shannon’s information index = -1* (p * ln (p) + q * ln(q), he = expected heterozygosity = 2 * p * q, %p = percentage of polymorphic loci. 48 shiva shahsavari et al. al. (2006) (32). we used activating charcoal and polyvinyl pyrrolidone (pvp) for binding of polyphenolics during extraction. the genomic dna was examined for quality and quantity by using 0.8% agarose electrophoresis and nanodrop spetrophotometer respectively five primers (scot5, scot6, scot7, scot8, scot8) were selected based on high polymorphic genetic indices (collard & mackill, 2009). for scot amplif ication, 20 ng genomic dna and 3 u of taq dna polymerase (parstous, iran); 2 x pcr buffer, 1.5 mm mgcl2; 0.2 mm of each dntp (parstous, iran) with 0.2 µm of each primer, was implemented for 20µl polymerase chain reaction (pcr). the reactions were amplified in technethermocycler (bio-rad, usa) using the following procedure 5 min at 95°c, 40 cycles of 1 min and 15 sec at 94°c, 1 min and 30 sec at 46.9-52.9°c (scot-5 46.9°c, scot-6 49.7 °c, scot-7 50 °c, scot-8 52.6 °c, scot-9 52.9°c) and 1 min at 72°c and a final cycle of 5 min at 72°c. all pcr products were visualized on 2 % agarose gel followed by the sybr green staining. for fragment size, we used 100-base pair (bp) molecular size ladder (fermentas, germany). data analysis in total 61 scot bands/ loci were obtained in this study. these bands were coded as binary data (presence = 1, absence = 0), for further analyses. the genetic diversity parameters like, number of alleles (na), effective number of alleles (ne), shannon index (i), nei’s genetic diversity (he), unbiased he (uhe), and percentage of polymorphism (p%)) were determined for the studied cultivars by using genealex ver. 6.4 (peakall & smouse, 2006). genet ic d if ferent iat ion of t he stud ied genotypes was examined by analysis of molecular variance (amova) as implemented in genealex ver. 6.5 (peakall & smouse, 2006). the genetic distinctness of the genotypes and their replicates was determined by tcsnetworking as implemented in popart ver. 3 (hammer et al., 2001). we used principal coordinate analysis (pcoa), of past ver.3 to differentiate the genetic groups, and discriminant analysis of principal components (hammer et al., 2001). (dapc), to identify discriminating scot loci among punica genotypes (jombart et al., 2010). the assignment test of the same program was used to reveal genetic admixture in punica genotypes. these analyses were performed by adegenet package of r (jombart, 2008). results in total we obtained 61 scot bands/ loci in this study. the genetic diversity parameters determined in punica. genotypes based on scot markers are provided in table 1. the mean number of effective alleles (ne) was almost alike in all punica genotypes studied. however, the mean shanon index and gene diversity differed in these genotypes. the same holds true for genetic polymorphism as it. varied from 0.0. (complete genetic uniformity within a cultivar), to about. 18%, which is still a low value for within cultivar genetic variability. amova produced significant genetic difference among the studied punica cultivars. the analysis showed that about 9% of total genetic variability is due to within population diversity, while 91% of genetic difference. occurs due to among cultivar genetic difference. tcs network constructed based on scot loci obtained revealed a high degree of genetic uniformity within replicates of each genotype studied (fig. 1). this particularly holds true for the genotypes 12, 18, 30, 8, 27, 14, 20, 41, and 42. the replicates of these genotypes were 100% alike in scot loci and were positioned on each other in tcs network nodes. the other genotypes which showed some level of within population genetic variability were also separated from the other genotypes, and their replicates were placed closer to each other than the other genotypes. this result indicates that scot markers can differentiate the studied tunica genotypes from each other. pcoa plot of tunica genotypes (fig. 2), placed them in four different genetic groups. for example, the genotypes 2, 3, 6, 7, 9, 13, 14, 20, and 29, comprised the first genetic group due to their genetic similarity. the other genotypes formed the rest of genetic groups. figure 1. tcs network of punica genotypes based on scot data showing that these markers can differentiate the replicates of tunica genotypes from each other. 49scot molecular markers are efficient in genetic fingerprinting of pomegranate (punica granatum l.) cultivars to illustrate the genetic distance between these four genetic groups, we determined dice’ genetic similarity (s), between representative genotypes of each group and from that, we estimated the genetic distance by reducing 1-s. for example, genetic distance between genotypes number 14 and. 8, from the genetic groups. 1 and 2, produced genetic distance d = 0.55. similarly, d value between genotypes 14 and 42, was 0.62, and between genotypes 8 and 15 was 0.70. finally, d value between 10 and 42, was 0.40. therefore, genetic distance obtained between the four genetic groups ranged from 40-70%, which i a high magnitude of genetic dissimilarity, and we can use these genetic differences for further breeding tasks in punica. dapc analysis of scot data, revealed that the first five linear discriminant axes (lda), comprise the highest percentage of discrimination factors (fig. 3). lda plot constructed based on the first two la axes, grouped the studied punica genotypes in 3-4 genetic groups (fig. 4), which is in agreement with pcoa analysis presented before. lda analysis identified the scot loci with the highest discriminating power (see for exapmle, fig. 5). scot loci 7-500, and 600, as well as scot loci9-500, are important. loci of the first lda axis. similarly, scot loci5-950, and 1000, scot7-400, and. scot8-800, are important loci of the second lda axis. in the third lda axis, scot loci 5-200, 300, and 950, as well as scot7-400. therefore, of 61 scot loci obtained, a combination of scot. 5 and 7, may be used for genetic fingerprinting of punica genotypes. figure 2. pcoa plot separating punica genotypes in four main genetic groups. figure 4. dla plot of punica genotypes based on scot data. figure 3. lda. analysis of. scot data in punica genotypes, showing. the first five lda axes as discriminating factors among them. figure 5. lda loading of scot loci showing important loci of the first lda axis. figure 6. assignment plot of punica genotypes based on scot markers. similarly colored individuals have similar genetic content, while admixed colors. indicated gene flow or ancestral shared alleles. 50 shiva shahsavari et al. assignment test of dapc. analysis (fig. 6), revealed genetic affinity of the studied punica genotypes (similarly colored individuals). almost four genetic groups can be identified based on genetic content (similar colors). this plot also revealed some degree of genetic admixture (mixed colors) among punica genotypes studied. the genetic admixture may be due to cross pollination of the genotypes or due to ancestral common shared alleles discussion genetic fingerprinting as a mean for genetic equation of plants germplasm are very important and of immediate use for planning selection and hybridization programs. data obtained from these investigations illustrate molecular basis of cultivar differences and if such differences are also accompanied to important agronomic characteristics, then plant breeders have a very good source of genetic material for improvement of that target plant (nandakumar et al., 2004, gorji et al., 2011, nybom et al., 2014, saboori at al., 2021). finding the proper molecular markers for genetic fingerprinting is an essential step in genetic evaluation. for this reason, different molecular markers are used and compared in genetic fingerprinting of economically important plant species. this also holds true for punica plant. the molecular markers can be assayed for their utility in the cultivar differentiation, and also revealing genetic affinity. the present study revealed that scot markers are very efficient markers for both showing within cultivar/ population genetic variability, and also for differentiation punica cultivars. a previous study on twelve pomegranate cultivars grown in egypt (ahmed , 2018) showed a high level of genetic variability among the cultivars by using scot markers. they reported that none of scot primers were able to identify all cultivars independently while our findings on iranian pomegranate cultivars identified different alleles of scot loci that successfully isolated some cultivars. for instance, alleles in scot 5, scot 6, scot 7 and scot 9 loci distinguished poostsiyah abrand abad cultivar from other cultivars. the magnitude of genetic diversity obtained may differ in different molecular markers. however, an interesting similar results are obtained when we compare scot and ssr markers results obtained in the same genotypes. shahsavari et al. (2021), studied the same punica cultivars by ssr molecular markers and reported that these cultivars have a high genetic similarity with genetic distance ranging from 0.005 to 0.52, which is in close agreement with our study based on scot markers (10). they also reported significant genetic difference among punica cultivars and that 8% of total genetic variability was due to among genotype difference, while 92% was due to. between cultivar genetic differences, which is almost similar to the present study results of scot markers. these authors (shahsavari et al. 2021), based on ssr data, reported some degree of genetic admixture among punica. cultivars and also identified discriminating ssr loci to differentiate punica. cultivars. we could also identify a few scot loci which can discriminate punica cultivars (10). also barcoding and mini-barcode analyses based on trnh-psba and matk sequences on the same cultivars were provided better resolution of pomegranate cultivars’ assignment by shahsavari et al. 2021b (25). therefore, in conclusion we suggest that a combination of scot and ssr molecular markers may be used in punica germplasm evaluation and the results obtained on the genetic grouping and genetic difference of these cultivars can be utilized for hybridization and breeding tasks of this important fruit crop. acknowledgment the authors gratefully acknowledge science and research branch, islamic azad university (iau), and shahid beheshti university and research center and agricultural education for laboratory. also we thank natural resources yazd province and sahebi pomegranate cooperative company -sari for providing samples. author contribution statement z.n.: conceptualization of the project, data analyses; m.sh: analyses of data; sh.sh and f.f.: data collection and lab work; mr.v.: providing samples; z.n, m.sh project design. references abdel-lateif k.s and hewedy o.a (2018) genetic diversity among egyptian wheat cultivars using scot and issr markers. sabrao journal of breeding and genetics, 50(1): 36-45. ahmed a.h (2018) molecular identification and fingerprinting of some pomegranate cultivars grown in egypt using issr and scot analyses. journal of horticultural science & ornamental plants 10 (3): 179-188. 51scot molecular markers are efficient in genetic fingerprinting of pomegranate (punica granatum l.) cultivars beghè d, fabbri a, petruccelli r, marieschi m, torelli a, ganino t (2019) morphological and molecular characterization of ancient pomegranate (punica granatum l.) accessions in northern italy. adv. hort. sci., 33(4): 581592. doi: 10.13128/ahsc7908.chem. 9 collard bcy, mackill dj (2009) start codon targeted (scot) polymorphism: a simple novel dna marker technique for generating gene-targeted markers in plants. plant mol biol rep 27:86–93. dora s.a, mansour m, aboulila a.a, abdel-wahab (2017) genetic diversity and relationships among some barley genotypes for net blotch disease resistance using rapd, scot and ssr markers. egyptian journal of genetics and cytology, 46: 139-165. fischer u. a., carle r., kammerer d. r (2010) identification and quantification of phenolic compounds from pomegranate (punica granatum l.) peel, mesocarp, aril and differently produced juices by hplc-dad− esi/msn. food chem. 2011, 127, 807−821. https:// doi.org/10.1016/j.foodchem. 12.156 gorji a.m., poczai p, polgar z and taller j (2011) efficiency of arbitrarily amplified dominant markers (scot, issr and rapd) for diagnostic fingerprinting in tetraploid potato. am. j. potato res., 88: 226-237. graham s. a, thorne & reveal (1998) “validation of subfamily names in  lythraceae”.  taxon. taxon, vol. 47, no. 2. 47 (2): 435 436. doi:10.2307/1223775. jstor 1223775 gundogdu m, yilmaz h (2012) organic acid, phenolic profile and antioxidant capacities of pomegranate (punica granatum l.) cultivars and selected genotypes sci. hortic. 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pomegranate (punica granatum  l.) accessio. ns from fars province of iran using microsatellite markers. horticulture, environment, and biotechnology  volume  59,  pages239–249. https://doi.org/10.1007/ s13580-018-0019-x caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 2 2022 firenze university press cytogenetic studies of six species in family araceae from thailand piyaporn saensouk1, surapon saensouk2,*, rattanavalee senavongse2 effect of ag nanoparticles on morphological and physio-biochemical traits of the medicinal plant stevia rebaudiana sherzad r. abdull, sahar h. rashid*, bakhtiar s. ghafoor, barzan s. khdhir morphometric analysis and genetic diversity in hypericum l. using sequence related amplified polymorphism wei cao1, xiao chen2,*, zhiwei cao3 population differentiation and gene flow of salicornia persica akhani (chenopodiaceae) xiaoju zhang1, li bai2,*, somayeh esfandani-bozchaloyi3 scot molecular markers are efficient in genetic fingerprinting of pomegranate (punica granatum l.) cultivars shiva shahsavari1, zahra noormohammadi1,*, masoud sheidai2,*, farah farahani3, mohammad reza vazifeshenas4 first record of nucleus migration in premeiotic antherial cells of saccharum spontaneum l. (poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(2): 143-149, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1579 caryologia international journal of cytology, cytosystematics and cytogenetics citation: elena bonciu, mirela paraschivu, nicoleta anca șuțan, aurel liviu olaru (2022) cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system. caryologia 75(2): 143-149. doi: 10.36253/caryologia-1579 received: february 17, 2022 accepted: may 20, 2022 published: september 21, 2022 copyright: © 2022 elena bonciu, mirela paraschivu, nicoleta anca șuțan, aurel liviu olaru. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 1 university of craiova, faculty of agronomy, craiova, romania 2 university of pitesti, faculty of science, physical education and informatics, pitesti, romania *corresponding author. e-mail: paraschivumirela@yahoo.com abstract. dyes used in the food industry are an important class of food additives and are often used to make processed food more visually appealing, especially to children. the purpose of this paper was to evaluate the cytotoxic effect of sunset yellow (sy) and brilliant blue (bb) food dyes on the root meristematic cells of allium cepa l. the root tip cells of onion were exposed to aqueous solutions of dye in concentration of 50 ppm, 100 ppm and 200 ppm, respectively 150 ppm, 300 ppm and 600 ppm bb for 24 hours, at room temperature. cytogenetic tests reveal a decrease of the mitotic index and an increase of various chromosomal aberrations following food dyes treatments, in a concentration-dependent manner. types of chromosomal aberrations were varied; thus, were observed cells with irregular kinetics of chromosomes, ring chromosomes, laggards, sticky chromosomes and micronuclei. exposure of onion roots tips to both food dyes showed a large number of cells in prophase and low number of cells in anaphase, regardless of dyes concentration. the obtained results suggest caution in the consumption of foods that contain these two types of dyes and finding healthier alternatives for food coloring. keywords: allium assay, cytotoxicity, food dyes, mitodepressive. introduction in the technological process, especially after heat treatment, many food products lose or change their natural color. sometimes, the color changes during storage of the product, and these changes negatively affect the commercial appearance of food and reduce the sensory quality and consumer acceptance. in addition, foods, especially sweets, are more attractive to consumers, mainly children, if they are beautifully colored. dyes used in the food industry are chemical compounds responsible for the attractive colorful appearance of the food. their widespread use is determined by the increasing demand of buyers for the aesthetic qualities of the food on the market. synthetic dyes are also called artificial dyes. they do not exist as such in nature and are obtained by chemical synthesis. the solubility 144 elena bonciu et al. in water is due to the presence of an acid group (anionic dyes) and an amine group (cationic dyes), respectively (efsa). synthetic dyes are classified into: azodyes (–n = n–): (congo red, methyl yellow, sunset yellow; tartrazine); triarylmethane group: (brilliant blue, brilliant green) xanthenics: (erythrosine); quinoline: (quinoline yellow); indigo group: (indigotine, indigo). azodyes represent the most numerous class of food dyes, they account for over 50% of the world’s production of dyes, although so far no azoderivative has been found in nature. azodyes cover the entire spectrum of colors and satisfy practically the needs of coloring for any substrate, having representatives in all applicative classes. ultra-processed foods indeed often contain mixtures of additives. they represent about 330 authorized compounds in the european union (database on food additives). sunset yellow (e-110) is a water-soluble food dye, widely used in the confectionery industry. it has the chemical formula c16h10n2na2o7s2, molar mass 452.38 g/mol and the melting point is 300°c. brilliant blue (e-133) is a synthetic organic compound used mainly as a blue dye for processed foods, but also drugs, food supplements and cosmetics. its chemical formula is c37h34n2na2o9s3 and the molar mass is 792.85 g/mol (database on food additives). higher plants are suitable systems for a wide range of toxicological tests applicable to the assessment of risks to the environment, ecosystems (geras’kin et al. 2011) and, in some cases, to animals (arung et al. 2011). the bioassays with plants have been considered quite sensitive and simple in comparison to animal bioassays in the monitoring of the cytotoxic and genotoxic effects of chemical compounds (gomes et al. 2013; iganci et al. 2006). from this point of view, allium cepa l. (onion) has been indicated as an efficient test system for cytogenotoxicity assessment (mert and betül 2020; bonciu et al. 2018; samanta et al. 2012; metin and bürün 2008; evseeva et al. 2001). also, allium assay has advantages such as low costs and showing good correlation with mammalian test systems (özgün tuna-gülören et al. 2021; rosculete et al. 2019). with the mandatory mention of all ingredients on the food label, more and more consumers are wondering if the presence of different additives can affect their health. this study aimed to analyses the cytogenotoxic effect of sunset yellow (sy) and brilliant blue (bb) (two of the most used food dyes) in a. cepa root meristematic cells. materials and methods plant material clean and healthy onions bulbs were purchased from the craiova city central market. in order to promote root growth, bulbs were placed in small jars with discoid stem in contact with distilled water, and kept in laboratory, at room temperature (22 ± 2ºc) for 72 hours. the onion bulbs with freshly emerged roots were incubated in three different concentrations for each food dyes, for 24 hours, at room temperature. distilled water has been used as negative control. five onion bulbs were used for each experimental group. preparation of different concentrations of dyes for each food dye, three different concentrations prepared in distilled water were tested: c1 = 300 ppm, c2 = 150 ppm and c3 = 600 ppm for the testing of bb dye, and c1 = 100 ppm, c2 = 50 ppm and c3 = 200 ppm for the testing of sy dye, respectively. c1 represents the concentration recommended by the manufacturer on the package, and for the other variants we took into account the possibility of using lower doses (half of c1) or higher (twice as much as c1) than those recommended on the package. for the present study the both dyes were bought from one of the craiova city food store. microscopic preparations after 24 hours of treatment, the onion roots were carefully cut and processed for microscopic preparation. the biological material were fixed with a mixture of ethanol and glacial acetic acid (carnoy’s solution) in a volume ratio of 3:1 for 24 hours at 4°c in the refrigerator, followed by hydrolysis with 1n hydrochloric acid for 6 minutes at room temperature. then the onion roots were stained with 10% basic fuchsine solution (schiff ’s reagent). schiff reagent it was composed of basic fuchsine hydrochloride (5 g), hydrochloric acid (100 ml), sodium metabisulfite (10 g), distilled water (900 ml) and activated charcoal (5 g). its chemical formula is c19h21n3s2o7•4h2o. slides were prepared and cells were analyzed during the whole cell cycle for cellular and chromosomal aberrations totaling 5,000 cells for each tested dye concentration. the microscopic slides were prepared using the squash technique. for this purpose, after removing the root caps from the stained roots, they were immersed in a drop of 1% acetocarmine on a slide, squashed under a 145cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system cover slip and examined microscopically. five slides for each variant were analyzed for calculating the mitotic index (mi) and the cellular aberration frequency (two roots was used for each slide). the same slides were used to identify the cellular and chromosomal aberrations. all slides were examined using optika b-290tb microscope with digital camera (optika manufacturer, italy). statistical analyses the results have been interpreted statistically, using ms excel 2007. the analysis of variance (anova) was used to assess the significant differences between the control variant and each treatment. the differences between treatment means were compared using the least significant difference (lsd) test at a probability level of 0.05% subsequent to anova analysis. the mitotic index (mi) was calculated according to sehgal et al. (2006): mi (%) = × 100 the index of the cellular aberrations (ca) which comprising both chromosomal aberrations and nuclear anomalies were also calculated according to singh (2015): ca (%) = × 100 results table 1 presents the results of the influence of bb and sy food dyes on the mi and the number of cells in the different mitosis stages in a. cepa root tips. mi decreased with the increase concentration of food dyes solutions. thus, the intensity of mitotic activity was higher at the lowest concentration of both dyes namely at 150 ppm bb, when mi was 23.9% and at 50 ppm sy, when mi was 16.1%. however, these values are with 22%, respectively with 48% lower than the mi value recorded by the untreated control (30.8%). in the case of sy, a significant mitodepressive effect was recorded at all three tested doses, indicating the high cytotoxic potential of this food dye. the lowest mi value was observed at a concentration of 200 ppm sy (8.5%) i.e. 72.4% lower mitotic activity compared to negative control. it can be appreciated that the tested concentrations of bb and sy induced mitodepressive effect in meristematic root cells of a. cepa in a concentration-dependent manner. exposure of onion roots tips to both food dyes showed a large number of cells in prophase and low number of cells in anaphase, irrespective of the concentration applied. thus, compared to the control, the highest number of cells in prophase was registered at the variant bb 150 ppm mg, followed by the variant bb 300 ppm (508 cells) and sy 50 ppm (387 cells). on the other hand, the lowest number of cells in anaphase (43) was observed at sy 200 ppm. however, the exposure of the meristematic tissues of a. cepa to bb and sy also induced genotoxic effects, by increasing the number of cellular and chromosomal aberrations, in a concentration-dependent manner. thus, as observed in table 2 and figure 1, the main cellular aberrations identified were: irregular kinetics of chromosomes (ikc figure 1a-b); ring chromosomes (r figure 1c); laggard chromosomes (l figure 1d); sticky chromosomes (s figure 1e) and cells with micronuclei (mn figure 1f). regarding ikc, the highest values were registered to bb 600 ppm variant (11.20%) and sy 200 ppm varitable 1. total number of analysed cells and mitotic index (%) in root tips of allium cepa treated with different concentrations of brilliant blue and sunset yellow food dyes. dyes/ dose tci tcd mi± sem (%) cells in prophase cells in metaphase cells in anaphase cells in telophase control 3460 1540 30.8±0.79 510 302 383 345 bb 300 ppm 4090 910 18.2±0.48 508 156 114 132 bb 150 ppm 3804 1196 23.9±0.63 730 184 106 176 bb 600 ppm 4316 684 13.6±0.41* 345 112 68 159 sy 100 ppm 4265 735 14.7±0.52* 302 193 76 164 sy 50 ppm 4192 808 16.1±0.69* 387 205 92 124 sy 200 ppm 4575 425 8.5±0.38* 207 110 43 65 bb = brilliant blue; sy = sunset yellow; tci = total cells in interphase; tcd = total cells in division; mi = mitotic index; sem = standard error of mean; *significant at level 5% (p=0.05) for each treatment were analysed 5,000 cells. 146 elena bonciu et al. ant (11.08%), when compared to the negative control (1.10%). th e lowest values from this point of view can be observed at the bb 150 ppm variant (5.75%), respectively sy 50 ppm variant (7.25%). in the same vein, the appearance of cells with ring chromosomes was dependent by the increase of food dyes concentration. compared to the negative control, in which no aberrations were identifi ed, the highest values were registered to bb 600 ppm variant (5.16%) and sy 200 ppm variant (5.14%). th e lowest values can be observed at the bb 300 ppm variant (3.22%), respectively sy 50 ppm variant (3.20%). regarding laggard chromosomes, the highest values were registered to bb 600 ppm variant (6.05%) and sy 200 ppm variant (7.02%). th e lowest values from this point of view can be observed at the bb 150 ppm variant (3.20%), respectively sy 50 ppm variant (4.03%). th e identifi cation of other types of cell aberrations (s and mn) in the meristematic tissues of a. cepa suggests that their frequency it depends on the food dyes concentration. th us, the highest s values were registered to bb 600 ppm variant (11.14%) and sy 200 ppm variant (11.10%). th e lowest values from this point of view can be observed at the bb 150 ppm variant (4.28%), respectively sy 50 ppm variant (6.24%). on the other hand, the highest mn values were registered to bb 600 ppm variant (5.20%) and sy 200 ppm variant (6.35%). th e lowest values from this point of view was observed at the bb 150 ppm variant (3.52%), respectively sy 50 ppm variant (2.24%). th e highest frequency of total cell aberrations was recorded in the variants exposed to the highest concentration of food dyes, namely 38.75% (bb 600 ppm) and 40.69% (sy 200 ppm variant) respectively, this suggesting the genotoxic potential of the two types of food dyes when they are used in high concentrations, as can be seen in figure 2. discussion th e use of synthetic dyes is preferred because it off ers a uniform color intensity, they are stable, easily homogenized in the manufacturing processes and less expensive (pirvu et al. 2020; kanarek 2011). table 2. type and percentage of cellular aberrations induced by brilliant blue and sunset yellow food dyes on the meristematic roots of allium cepa. dyes/ dose ca (%) total aberrations (%)ikc r l s mn control 1.10 0 0 0 0 1.10 bb 300 ppm 7.15 3.22 3.92 8.12 4.28 26.69* bb 150 ppm 5.75 3.68 3.20 4.28 3.52 20.43 bb 600 ppm 11.20 5.16 6.05 11.14 5.20 38.75* sy 100 ppm 10.45 4.14 6.21 10.02 5.63 36.45* sy 50 ppm 7.25 3.20 4.03 6.24 2.24 22.96 sy 200 ppm 11.08 5.14 7.02 11.10 6.35 40.69* bb = brilliant blue; sy = sunset yellow; ca = cellular aberrations; ikc = irregular kinetics of chromosomes; r = ring chromosomes; l = laggard chromosomes; s = sticky chromosomes; mn = cells with micronuclei; *signifi cant at level 5% (p=0.05) for each treatment were analysed 5,000 cells. (a) (b) (c) (d) (e) (f) figure 1. some cellular aberrations identifi ed in meristematic cells of a. cepa exposed to brilliant blue and sunset yellow food dyes: irregular kinetics of chromosomes (a,b); ring chromosome (c); laggard chromosome (d); sticky chromosomes (e); cell with two micronuclei (f). figure 2. increased of cell aberrations and decreased of mitotic index in meristematic cells of a. cepa exposed to brilliant blue and sunset yellow food dyes. 147cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system maximum authorized levels of food additives are set by the european food safety authority (efsa) and aims to inform and warn consumers against the potential adverse effects of each individual substance in a given food product. nevertheless, the evaluation, recommendations and regulations has been based only on the currently available scientific evidence which is mainly derived from in vitro or in vivo experimental research. thus, essential information regarding the health impact of food additives in humans and the potential effects/ interactions is still missing yet urgently needed (chazelas et al. 2020). allium test has been indicated as an efficient test system for cytogenotoxicity evaluation (samanta et al. 2012; metin and bürün 2008; evseeva et al. 2001). also, the allium test is considered to be a standard procedure for quick testing and detection of toxicity and pollution levels in the environment (adesuyi et al. 2018). different parameters of allium cepa such as root shape, growth, mi, chromosomal aberrations etc. can be used to estimate the cytotoxicity and mutagenicity of environmental contaminants and pollutants (mert and betül 2020; adesuyi et al. 2018; bonciu et al. 2018; sutan et al. 2014). in our study, exposure of onion roots tips to both food dyes showed a large number of cells in prophase and low number of cells in anaphase at all concentrations applied. this might be due to the fact that dye may affect the tubulin and disturbed the mitotic spindle formation. as respects metaphase and anaphase, both mitotic stages showed a decrease in their frequency with increase of dyes concentration. similar results have been reported by bhattacharjee (2014) which evaluated the mitodepressive effect of sy using a. sativum assay; onyemaobi et al. (2012), who studied cytogenetic effects of food preservatives on a. cepa root tips and bhattacharjee and yadav (2005), while studying cytotoxicity of sy on vicia faba root tips. some authors reported that the mi in onion root tips was successively decreased with the increase in different dye concentrations and duration of treatments (gomes et al. 2013; kanarek 2011). these results are similar to those obtained in present study. our findings revealed that even at low concentration the both food dyes was cytotoxic for meristematic cells of a. cepa with a high significant effect on mitosis. the cytotoxicity level of a test compound can be determined based on the decrease in the mi (rosculete et al. 2019; adesuyi et al. 2018; singh 2015; liman et al. 2011). cytotoxicity is defined as a decrease in mi and as increase of frequency of cells with some cellular aberrations like c-mitosis, multipolar anaphase, sticky and laggards chromosomes (adesuyi et al. 2018; singh 2015). decrease of mi could be due to the inhibition of dna synthesis (sudhakar et al. 2001) or due to a block in the g2-phase of the cell cycle (marcano et al. 2004). cytogenetic abnormalities occur under biotic and abiotic stress conditions. several studies have been oriented to demonstrate the clastogenic effects of some food additives and pointed out their danger as carcinogens or mutagens (gultekin et al. 2015; andreatta et al. 2008; tsuda et al. 2001). on the other hand, some authors claim that chromosomal aberrations may have some benefits in plant breeding. e.g., chromosomal abnormality plants lead to not only gigantic effect, but also increase phytochemical compounds (ma et al. 2016; alam et al. 2015). many researches have given objects based on the outstanding benefits of chromosomal abnormality to plants. in some studies, the chromosomal abnormalities are regarded as a way to gain elite plant cultivars due to the fact that the increment in plant organs size derived from some of the most significant consequence of chromosomal abnormality (catalano et al. 2021; ruiz et al. 2020). as far as ecological perspectives are concerned, the cellular abnormalities enhance biotic and abiotic tolerance to adapt to climate change (ezquer et al. 2020). some chromosoma l aberration like stick iness observed in this study may occur as a result of physical adhesion of the proteins of the chromosomes, as ping et al. (2012) suggested. in april 2021, california’s office of environmental health hazards assessment released a ground-breaking, peer-reviewed report concluding that synthetic food dyes (such as red 40, red 3, yellow 5, yellow 6, blue 1, blue 2, and green 3) negatively affect children’s behavior. the final health effects assessment provides authoritative validation of what multiple independent reviews already concluded: that synthetic food dyes can cause or exacerbate behavior problems to some children (cspi, 2021). natural colors are gaining popularity because a natural food dye is a healthier and it does not cause health problems. usda has certified some natural food dyes that do not cause health problems to consumers (https:// sensientfoodcolors.com/en-us/color-solutions/certifiedorganic-colors/). most natural food dyes are obtained from fruits and vegetables and contain nutrients that are beneficial to health. there are simple and handy ways for anyone to easily extract color from some plants, fruits or vegetables, to color some food products. for example, the yellow color can be extracted from saffron soaked in water and then pressed; for different shades of red, purple or even blue, red cabbage or beetroot juice can be used mixed with different percentages of water, etc. finally, the decision to eat healthier remains with each of us. 148 elena bonciu et al. conclusions brilliant blue and sunset yellow (two of the most used food dyes) exerted mitodepressive and cytogenotoxic effects in meristemtic root cells of allium cepa l., in a concentration-dependent manner. results obtained in our study suggest caution in the consumption of foods that contain the two types of dyes and finding healthier alternatives for coloring of some food products. however, additional cytotoxicity studies are needed to add information to these and other previously obtained results in order to deepen the potential risks of these food dyes on a cellular level. acknowledgement n.a.ş. thanks the 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(poaceae) chandra bhanu singh1, vijay kumar singhal2, manish kapoor2,* genetic characterization of salicornia persica akhani (chenopodiaceae) assessed using random amplified polymorphic dna zhu lin1,*, hamed khodayari2 comparative chromosome mapping of repetitive dna in four minnow fishes (cyprinidae, cypriniformes) surachest aiumsumang1, patcharaporn chaiyasan2, kan khoomsab3, weerayuth supiwong4, alongklod tanomtong2 sumalee phimphan1,* classical chromosome features and microsatellites repeat in gekko petricolus (reptilia, gekkonidae) from thailand weera thongnetr1, surachest aiumsumang2, alongklod tanomtong3, sumalee phimphan2,* genotoxic and antigenotoxic potential of encapsulated enhalus acoroides (l. f.) royle leaves extract against nickel nitrate made pharmawati1,*, ni nyoman wirasiti1, luh putu wrasiati2 chromosomal description of three dixonius (squamata, gekkonidae) from thailand isara patawang1, suphat prasopsin2, chatmongkon suwannapoom3, alongklod tanomtong4, puntivar keawmad5, weera thongnetr6,* first report on classical and molecular cytogenetics of doi inthanon bent-toed gecko, cyrtodactylus inthanon kunya et al., 2015 (squamata: gekkonidae) in thailand suphat prasopsin1, nawarat muanglen2, sukhonthip ditcharoen3, chatmongkon suwannapoom4, alongklod tanomtong5, weera thongnetr6,* evaluation of genetic diversity and gene-pool of pistacia khinjuk stocks based on retrotransposon-based markers qin zhao1,*, zitong guo1, minxing gao1, wenbo wang1, lingling dou1, sahar h. rashid2 a statistical overview to the chromosome characteristics of some centaurea l. taxa distributed in the eastern anatolia (turkey) mikail açar1,*, neslihan taşar2 cytotoxicity of sunset yellow and brilliant blue food dyes in a plant test system elena bonciu1, mirela paraschivu1,*, nicoleta anca șuțan2, aurel liviu olaru1 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(4): 25-35, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1886 caryologia international journal of cytology, cytosystematics and cytogenetics citation: wendy ozols-narbona, josé imery-buiza (2022). morphological and cytogenetic characterization in experimental hybrid aloe jucunda reyn. x aloe vera (l.) burm. f. (asphodelaceae). caryologia 75(4): 25-35. doi: 10.36253/caryologia-1886 received: november 05, 2022 accepted: december 28, 2022 published: april 28, 2023 copyright: © 2022 wendy ozols-narbona, josé imery-buiza. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. morphological and cytogenetic characterization in experimental hybrid aloe jucunda reyn. x aloe vera (l.) burm. f. (asphodelaceae) wendy ozols-narbona*, josé imery-buiza departamento de biología, universidad de oriente, cumaná, 6101, venezuela *correspondign author. e-mail: wozolsnarbona@gmail.com abstract. aloe l. includes plants of economic interest worldwide for their medicinal properties and ornamental character. in this study, morphological and cytogenetic traits were evaluated in a hybrid obtained using aloe jucunda reyn. as pollen donor and a. vera (l.) burm. f. as female parent, to characterize it, determine its ornamental and agronomic potentialities and aspects related to its reproduction. conventional protocols for morphometric studies and cytogenetic analysis described for succulent plants were applied. progeny showed intermediate expressiveness in most of the characteristics, except in the colour of the leaves and flowers (hybrid = a. jucunda), as well as for the length of teeth, number, and area of leaf spots and angle between continuous leaves, where it surpassed the expression of both parents, giving it a high ornamental value. the length, width, and thickness of the leaves improved with respect to the paternal genome, so its potential for the exploitation of the gel and latex of its leaves cannot be ruled out. root tip cells showed a karyotype 2n = 2x = 14 = 8l + 6s = 1l(smsat) + 1l(sm) + 3l(st) + 3l(smsat) + 1s(m) + 5s(sm). microsporogenesis showed chromosomal abnormalities in 47.4% of the meiocytes, the most frequent being micronuclei in prophase-i, sticky chromosomes in metaphase-i, one or two dicentric bridges accompanied or not by acentric fragments in anaphase-i, -ii, and telophase-i, ii, as well as one or two additional microspores. these abnormalities reduce the fertility of their pollen grains and limit their sexual reproduction, providing a better explanation for their sterility. keywords: aloe, hybrid, morphological attributes, karyotype, microsporogenesis. introduction manual hybridization in plants has aroused interest in the genetic improvement of plants that represent crops of economic interest worldwide (marasek-ciolakowska et al. 2018). among these plants are those included within the genus aloe l., which comprises about 519 species with variable vegetative characteristics depending on their geographical location, temperature, fertility conditions, and availability of water in the soil (smith and vanwyk 2008; mbg 2022). these species are xerophytic and monocotyledonous plants that are characterized by being perennial herbs, shrubs or small trees 26 wendy ozols-narbona*, josé imery-buiza with thick roots and rosette-shaped leaves (carter 1994) with succulent tissues of economic importance for their ornamental attributes and therapeutic uses (rowley 1997, imery 2011). aloe vera (l.) burm. f. (=a. barbadensis mill.) is native to the arabian peninsula and now cultivated in several warm climatic zones of world including asia, america, and europe (grace et al. 2015, giannakoudakis et al. 2018). the a. vera industry has expanded throughout the world and the mucilaginous gel from the parenchymatous cells in the inner leaf pulp is used in many products, including fresh gel, juice, and multiple formulations for health, medicinal, and cosmetic purposes (saleem et al. 2022). its leaf extracts are rich in nutrients and contain over 200 active compounds including simple/complex polysaccharides, amino acids, proteins, enzymes, terpenoids, f lavonoids, saponins, minerals, vitamins, phenols, and other metabolites, allowing a broad spectrum of medicinal applications (saniasiaya et al. 2017). other industrial perspectives also include the use of a. vera derivatives as corrosion inhibitors (singh et al. 2016), obtaining biodiesel (silva et al. 2015), growth enhancer (el sherif 2017), germination accelerator and root development stimulant (tucuch-haas et al. 2022), post-harvest coating treatments (farina at al. 2020), improvement of the swelling capacity of commercial acrylic hydrogels (guancha-chalapud et al. 2022), nanobiotechnologies (arshad et al. 2022, song et al. 2022), decreased methane release in dairy cows (singh et al. 2021), bioremediation (giannakoudakis et al. 2018), among others. the global a. vera extracts market value is projected to increase from usd 2,454.5 million in 2022 to usd 5,153.71 million by 2032, showing opulent growth of 7.7% (fmi 2022). on the other hand, aloe jucunda reyn. evolved separately further south, in the somali desert, and it is considered an exclusively ornamental plant due to its small size, adaptability, and beauty of its bright green variegated leaves and pink pendant flowers (reynolds 1950). a. vera and a. jucunda only coexist in botanical gardens, nurseries, or research laboratories, both species present reproductive barriers such as protandry and self-incompatibility, which is why these plants propagate asexually (imery and cequea 2008). however, in reciprocal crosses trials, imery (2011) obtained viable progeny, using a. jucunda as the donor species for the pollen grains and a. vera as the female parent. the need to obtain information as a contribution to scientific knowledge about a completely unpublished genotype not yet described, led to the realization of this work, which aimed to evaluate morphological and cytogenetic traits that would allow characterizing this experimental hybrid. materials and methods vegetal material a. jucunda and a. vera adult plants (over eight years old) growing in the germplasm bank of succulent species of the biology department from universidad de oriente, located at 10°26’32’’ n and 64°09’14’’ w, in an area of very dry tropical forest in cumaná city (venezuela) were used. specimens of a. jucunda (p2) were originally acquired in local nurseries and those of a. vera (p1) came from península de araya naturalized population, located at 10°34’15’’ n and 64°12’08’’ w (albornoz and imery 2003). both species were identified considering the morphotaxonomic descriptions of jacobsen (1955), carter (1994) and van-wyk and smith (1996). five specimens of each of the evaluated genotypes were deposited in the irbr herbarium. other fresh specimens are preserved in the already identified germplasm bank. morphological evaluation fol low ing of t he mor phomet ric t ra its were determined in the progeny and their parents (figure 1): number, length, width, thickness, and volume (vh=π*lh*ah*eh/12) of the leaves (hernández-cruz et al. 2002), number and length of leaf teeth, number and area of leaf spots, leaf insertion angle, angle between continuous leaves, number of suckers, and flower colour, according to imery and cequea (2012). ten adult plants of each genotype (a. jucunda, a. vera, and experimental progeny) were characterized. quantitative variables were analysed using anova and lsd tests at p≤0.05 (sokal and rohlf 1979). cytogenetic evaluation mitotic chromosomes were studied from temporal slide prepared with meristems of root tips collected at 7:30-8:00 a.m., pre-treated with colchicine (0.05% m/v) for 2 h, fixed in carnoy ii solution (5:3:1 ethanol: glacial acetic acid: chloroform) for 30 min, hydrated in distilled water for 10 min, hydrolysed with hcl (1n) for 10 min and 24ºc, rehydrated in distilled water for 10 min, coloured with orcein ( 2% m/v) lactopropionic (45% v/v) for 4 min and gently squashed (fukui and nakayama 1996). chromosomes according to their size (stebbins 1971), length of the short arm (brandham 1971) and centromere position (levan et al. 1964) were classified. microsporogenesis was evaluated in flower buds between 3.7-4.3 mm in length, fixed in carnoy ii and 27morphological and cytogenetic characterization in experimental hybrid aloe jucunda reyn. x aloe vera (l.) burm. f. ca b h i ed f g figure 1. vegetative traits of the hybrid and its parents. (a) aloe jucunda, (b) hybrid, (c) a. vera, (d) cross section in leaves of a. jucunda (upper), hybrid (middle), and a. vera (lower), (e) leaf lengths in the three genotypes, (f ) contrasting details in the colour of the leaves, spots, and foliar teeth of the three genotypes evaluated, g) diff erences in the number of leaf spots between the adaxial face and the abaxial face in the leaves of the experimental hybrid, vegetative (h) and fl oral (i) details of the experimental hybrid. scale bars = 2 cm. 28 wendy ozols-narbona*, josé imery-buiza staining the content of an anther with lactopropionic orcein (alcorcés et al. 2012). at least five flower buds in meiosis for each genotype were analysed. all slides were systematically evaluated using a nikon labphot-2 microscope. photomicrographs at 400 and 1000 x with a sony 7.2 digital camera were captured and the images on a computer using the photoimpact and sigmascan pro 5 programs were examined. karyological data (chromosomal length, relative length, and long/short arm index) for anova between genotypes and ”t-student” tests between homologous chromosomes were used. viability of pollen grains fertility of the experimental hybrids was estimated by means of the in vitro germination of the pollen grains and pollen tube growth according to sunderland and roberts (1977) in culture medium with nutrient agar (6 g.l-1) and sucrose (0.125 mol.l-1), previously standardized for this genotype. culture medium was autoclaved at 15 psi for 15 min and five drops were added to ten slides. it was allowed to gel (10 min, 25ºc) and then the pollen grains of flowers kept in a humid chamber were dispersed until anthesis. observation was carried out in a nikon optical microscope, model labphot-2 at 100x, after 60 min in the 10 slides of the microcultures established and covered by a petri dish to avoid desiccation. for a better contrast, two drops of astra blue were added to each slide to colour the pollen tubes (danti et al. 2011). a viable pollen grain was expected when the length of the pollen tube was greater than or equal to the length of its polar axis (kalinganire et al. 2000). the percentage of in vitro germination of pollen grains was estimated using the relationship between the number of germinated pollen grains and the total number of pollen grains contained in each microscopic field. results and discussion genotypes evaluated (p1: a. vera, p2: a. jucunda, and h: hybrid) were significantly different (p≤0.05) in all the morphological variables studied. in most of the characteristics, the progeny was expressed in an intermediate way between its parents, except in the colour of the leaves, flowers and the presence of spots, which were inherited from the paternal genome of a. jucunda, as well as for the length of the teeth, angle between continuous leaves, number of spots on the adaxial side and number and area of leaf spots on both the adaxial and abaxial sides, in which the hybrid exceeded the expression of both parents (table 1). the bright green colour and variegated character of its leaves thanks to the presence of spots because of the contribution of the paternal genome of a. jucunda, give this hybrid a high ornamentable 1. morphological attributes evaluated in adult plants of the progeny and their parents aloe jucunda and a. vera, under nursery conditions in cumaná (venezuela). attribute/genotype a. vera (p1) a. jucunda (p2) hybrid (h) p1/p2 h/p1 h/p2 leaf colour grey-green pine-green pine-green number of leaves 24.90 ± 2.85a 18.90 ± 3.38c 23.40 ± 2.22b 1.32 0.94 1.24 leaf length (cm) 57.41 ± 5.24a 7.19 ± 0.28c 28.28 ± 3.27b 7.98 0.49 3.93 leaf width (mm) 74.79 ± 5.31a 15.74 ± 1.40c 35.61 ± 4.64b 4.75 0.48 2.26 leaf thickness (mm) 21.74 ± 2.34a 9.42 ± 0.83c 17.82 ± 1.63b 2.31 0.82 1.89 leaf volume (cm3) 244.39 ± 41.21a 2.81 ± 0.45c 47.47 ± 11.48b 86.97 0.19 16.89 leaf insertion angle (°) 31.71 ± 2.93c 79.06 ± 7.30a 38.44 ± 5.78b 0.40 1.21 0.49 angle between leaves (°) 80.90 ± 2.99c 105.37 ± 19.15b 124.72 ± 14.64a 0.77 1.54 1.18 number of leave teeth 35.08 ± 1.05a 20.30 ± 1.37c 23.43 ± 1.84b 1.73 0.66 1.15 teeth length (mm) 2.75 ± 0.22b 1.38 ± 0.20c 3.61 ± 0.47a 1.99 1.31 2.62 number of spots (adaxial) 0.00 ± 0.00c 51.13 ± 6.06b 59.27 ± 19.69a 0.00 ∞ 1.16 number of spots (abaxial) 0.00 ± 0.00c 225.37 ± 15.65a 194.37 ± 28.43b 0.00 ∞ 0.86 adaxial spot area (mm2) 0.00 ± 0.00c 1.82 ± 0.48b 16.35 ± 6.35a 0.00 ∞ 8.98 abaxial spot area (mm2) 0.00 ± 0.00c 0.93 ± 0.27b 15.56 ± 7.59a 0.00 ∞ 16.73 number of basal suckers 6.80 ± 2.04a 4.10 ± 0.99c 5.10 ± 3.03b 0.75 0.63 1.24 flower colour yellow orange-pink orange-pink values indicate mean ± standard deviation with n = 10 plants. numbers followed by same letter are not significantly different (lsd p≤0.05) between genotypes. 29morphological and cytogenetic characterization in experimental hybrid aloe jucunda reyn. x aloe vera (l.) burm. f. tal value. traits such as the length, width, thickness, and volume of the leaves were significantly improved in the progeny because of the contribution of the maternal genome (a. vera). in these cases, the magnitude of the improvements was between 1.24 to 16.89 times higher than the expression of the parent a. jucunda (smaller parent), so its possible agronomic potential for exploitation is not ruled out both of gel and latex of its leaves (figure 1). another attribute of interest is the increase in the dimensions of the foliar teeth, which gives this new genotype an advantage as a defence mechanism against some predators. watson et al. (2003) comment that the overexpression of some characteristic in hybrid descendants could be attributed to the accumulation of numerous loci in heterozygosis, propitiated by the interaction of two different genomes, in this case, that intervene in the organogenesis of the spines or biomass leaf to the edges. on the other hand, the prolific vegetative propagation guarantees the hybrid to perpetuate itself over time, compensating for its sterility, since it has not formed fruits and seeds through sexual reproduction, which, according to imery and cequea (2008), could be attributed to self-incompatibility mechanisms inherited from the maternal genome of a. vera. morphometric characterization of the progeny reveals a considerable ornamental value in this new genotype and the possibility of incorporating it as a model for studying the inheritance of traits of ornamental value and/or agronomic importance or for future crosses in the search for new genotypes, and complementary research. root tip meristematic cells presented bimodal karyotypes and chromosomal classifications (levan et al. 1964) described by the formulas 2n = 2x = 14 = 8l + 6s = 2l(smsat) + 4l(st) + 2l(stsat) + 2s(m) + 4s(sm) in a. vera with eight large chromosomes (l) measuring 13.4-15.5 µm and six small chromosomes (s) measuring 5.1-5.9 µm; 2n = 2x = 14 = 8l + 6s = 2l(sm) + 2l(st) + 4l(stsat) + 5s(sm) + 1s(m) in a. jucunda with eight l chromosomes (14.7-16.9 µm) and six s chromosomes (5.5-6.3 µm); and 2n = 2x = 14 = 8l + 6s = 1l(smsat) + 1l(sm) + 3l(st) + 3l(stsat) + 1s(m) + 5s(sm) in the hybrid with eight l chromosomes (13.8-15.8 µm) and six s chromosomes (5.2-6.2 µm) (figure 2). heteromorphisms between the homologues of chromosome pairs l2 and s1 were determined in the hybrids named vj6 and vj10, respectively (figure 2c,d). as the genotypes evaluated did not show significant differences in the length of each of their chromosomes, the possibility that heteromorphisms between homologues of the progeny are caused by chromosomal mutations such as deletions is ruled out. in this regard, brandham (1976) evaluated the karyotypes of 1543 diploid plants of the aloe, gasteria, and haworthia genera without finding evidence of deletion. however, in polyploid species of the genus aloe he found a frequency of 5.8%, indicating that structural mutations of this type have a lethal effect in diploid species. that is why heteromorphisms between homologous chromosomes are mainly attributed to the fact that their chromosomal complement comes from two different genomes. chromosomal abnormalities were found in 47.4% of the meiocytes evaluated. the most frequent meiotic aberrations were formation of micronuclei in prophasei, sticky chromosomes, and acentric fragments in metaphase-i and -ii, dicentric bridges accompanied or not with acentric or linked fragments in anaphase-i and -ii, occasionally persistent in prophase-ii, asynchrony between telophase-i and prophase-ii, bridges, fragments, and micronuclei in telophase-i and -ii, one or two additional microspores of variable size at the end of microsporogenesis (figure 4). although 31.6% of the pollen grains evaluated in the present investigation germinated under in vitro conditions (figure 5), the absence of fruits and seeds in this new genotype forces this plant to depend exclusively on vegetative propagation for its multiplication. reproductive barriers such as gametophytic and sporophytic self-incompatibility have been described for most species of the aloe genus (newton 2004), including a. vera (imery and cequea 2008) and a. jucunda (riley and majumdar 1979), limiting then its self-fertilization with those pollen grains not affected by microsporogenic irregularities. this leads to the deduction that the impossibility of sexual reproduction of the experimental hybrids is also related to incompatibility genes inherited from both parents. swamy and krishnamurthy (1980) and imery (2011), argue that many plant species are forced to propagate asexually due to the existence of chromosomal alterations (deletions, inversions, and translocations), transmitted from their parents, either because they were present in their genomes or because they originated during the formation of their sex cells. additional bridges, fragments, micronuclei, and microspores are frequent in aloe species with heterozygous paracentric inversions (riley and majumdar 1979, ahirwar and verma 2013). the pairing between the inverted chromosome and its pachytene homologue must involve the formation of a loop where crossovers occur between homologous chromatids that generate fine chromatin threads linked to two centromeres and totally unlinked fragments of these, causing the loss of 30 wendy ozols-narbona*, josé imery-buiza genes that reduce the fertility of gametes. chromosomal fragments present individually or linked to dicentric bridges during anaphase-i or telophase-i generally form additional micronuclei that increase the number of microspores at the end of meiosis and cause gene defi ciencies (ahirwar and verma 2013). th ese aberrations may be present in clones that were formed by vegetative propagation from carrier individuals (populations of a. vera from eastern venezuela) and may have been inherited by the progeny or may be generated during the formation of sexual cells (cequea et al. 2003, imery 2011). s1 s2 s3 s1 s2 s3 l1 l2 l3 l4 s1 s2 s3 l1 l2 l3 l4 a b l1 l2 l3 l4 l1 l2 l3 l4 c d s1 s2 s3 figure 2. mitotic chromosomes in root tip cells and bimodal karyograms (2n=2x=14=8l+6s) of a) aloe jucunda, b) a. vera, c, d) experimental hybrids with greater heteromorphisms between homologous chromosomes. typing of large (l) and small (s) chromosomes according to brandham (1971). scale bars = 5 µm. 31morphological and cytogenetic characterization in experimental hybrid aloe jucunda reyn. x aloe vera (l.) burm. f. on the other hand, baptista et al. (2000) mention that the high frequency of sticky chromosomes or agglutination could be due to a genetic-environmental interaction associated with high temperatures, causing chromatin instability mainly in metaphase-i. sapre (1975) suggests that the participation of neocentric activity in early displacement of smaller chromosomes is the main cause of dicentric bridges between large homologues; however, imery and cequea (2002) attributes this to early dissolution of the synaptonemal complex between small homologous chromosomes. other causes that have been mentioned to explain the presence of failures during meiosis are environmental conditions. palmer et al. (2000), obtained discrepancies in the percentage of abnormalities between glycine max plants that grew in diff erent environmental conditions. th ese authors argue that the plants analysed grew in two contrasting environments and that the high temperatures increased the frequency of meiotic aberrations. a cytogenetic study in abies sibirica (gymnosperm) conducted by bazhina et al. (2008) revealed the same trend, noting that temperature fl uctuations in the diff erent months of the year aff ected the frequency of abnormalities. in this case, the plants evaluated during the dry season of summer registered a greater number of anomalies with respect to those analysed in the cool season of spring. imery (2011) points out the possibility that the environmental conditions associated with high temperatures, solar radiation, and low humidity could promote the increase in the concentration of some substances typical of the plant (anthrones, anthraquinones) that alter the normal division of pollen mother cells in a. vera. it is possible, then, that the high environmental sensitivity and the existence of structural mutations already reported in a. vera were inherited to their sexual descendants a. jucunda x a. vera explain the origin of the abnormalities observed in this investigation. failures in the union of the kinetochores to the meiotic spindle could explain the presence of lagging or asynaptic chromosomes in metaphase and anaphasei and -ii, causing an independent behaviour of the rest of the chromosomes that make up the nucleus (ishii and akiyoshi 2022), and the depolymerization of spindle at diff erent times in each cell nucleus could be the reason on n mlk g g h i j edcba f figure 3. microsporogenesis and microspore mitosis of the experimental hybrid aloe jucunda x a. vera. a) prophase-i (leptotene), b) prophase-i (zygotene), c) prophase-i (pachytene), d) prophase-i (diplotene), e) prophase-i (diakinesis), f ) metaphase-i , g) anaphase-i, h) telophase-i, i) prophase-ii, j) metaphase-ii, k) anaphase-ii, l) telophaseii, m) tetrad, n) microspores initiating fi rst mitosis, o) microspore in metaphase showing haploid chromosomes (n=x=7=4l+3s). scale bars = 10 µm. 32 wendy ozols-narbona*, josé imery-buiza c d h k m n i ol g j a b e f figure 4. most frequent meiotic abnormalities in the hybrid aloe vera x a. jucunda. (a) micronucleus in prophase-i; (b-c) sticky chromosomes and early displacement of small chromosomes in metaphase-i; (d-h) acentric fragments in anaphase-i and telophase-i; (e-f ) one and two dicentric bridges in anaphase-i; (g) lagging chromosomes in anaphase-i; (i-k-n) bridge and fragment in telophase-i, -ii and prophaseii; (j) phase asynchrony between telophase-i and prophase-ii; (l) bridging and metaphase-ii fragment; (m) bridge and fragment in anaphase-ii and (o) additional microspore at the end of microsporogenesis. scale bars = 10 µm. ba * ** * * * * figure 5. pollen grains of the experimental hybrid aloe jucunda x a. vera. viability test of pollen cultured in vitro with agar-sucrose medium. arrows point to pollen tubes of germinated pollen grains considered viable, while the (*) indicate non-germinated or non-viable pollen grains. scale bars = 50 µm. 33morphological and cytogenetic characterization in experimental hybrid aloe jucunda reyn. x aloe vera (l.) burm. f. for the phase asynchrony between telophase-i/prophaseii and anaphase-i/telophase-ii (alcorcés et al. 2007). conclusions experimental hybrids of aloe vera x a. jucunda showed superiority of vegetative traits such as the length of the foliar teeth, angle between continuous leaves, number and area of the spots compared to their parents, conferring them a high ornamental value. traits such as the length, width, thickness, and volume of the leaves improved considerably with respect to the paternal genome, so its possible agronomic potential for the exploitation of the gel and latex of its leaves cannot be ruled out. root tip cells presented the expected bimodal karyotype and number of chromosomes for the species of this genus. the meiotic abnormalities present in the progeny decrease the fertility of the pollen grains and show the reasons for their limited sexual reproduction, providing a better explanation for the absence of fruits and seeds in all their flowering periods. acknowledgements the 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no correlation could be detected between the size or number of micronuclei/cell and the applied conditions. metaphase was the most sensitive stage to al stress compared to the other stages of mitosis; c-metaphase was the common abnormalities and it increased strongly when the exposure time was more than 6 h. under the influence of al stress for 24 h, the appearance c-metaphase in high frequency decreased the frequency of appearance of other forms of abnormalities during metaphase or ana-telophase. the previous cytological events created alteration either at or between the primer binding sites which could be detected by rapd and issr techniques. application of ten rapd primers resulted in amplification of 59 fragments including 20 monomorphic, seven unique and 32 polymorphic bands with polymorphism average of 60.09%. issr primers amplified 75 dna fragments including 18 monomorphic, eight unique and 45 polymorphic bands with polymorphism average of 72.90%. these data indicated that faba bean cultivar suffered from harmful effect of al on its genome when the duration of al treatment was more than 6 hr. issr was better than rapd to study genome stability of faba bean under abiotic stress agent. keywords: aluminum stress, chromosomal changes, molecular techniques, cytogenetic effect, vicia faba. introduction in several environments, soils are contained with metals in different concentrations. they resulted in beneficial or toxic effects on biological sys142 ahmed m. hassanein, ahmed h. mohamed, heba ahmed abd allah, hoida zaki tems that inhabit their environments; aluminum (al) is one of those widespread metals. in nature, the highest amount of al is present in un-soluble form as aluminum silicate. while soluble al form is present in a small amount, it finds a way to get from soil, water and air to biological systems (may and nordstrom 1991) leading to positive effects on cultivated plants (foy 1983; rout et al. 2001). on the other side, transfer of al from soil to plants in relatively high dose resulted in catastrophic effects and they vary depending on plant species especially under low ph (5.5) of the soil (rout et al. 2001). in root tips, aluminum intervenes with cell division, increases cell wall rigidity, increases the rigidity dna double helix and reduces dna replication (foy 1992). authors were also reported that al (0.2–1.0 mm) inhibits cell division due to severe inhibition of dna synthesis within 16 –24 h (minocha et al. 1992). while mitotic indices decreased, anaphase chromosome aberrations increased when faba bean root tips of faba ben exposed to different doses of al (0.01–10 mm) for 12 h (yi et al. 2010). under al stress, reduction in mitotic activity in several plant species was reported (rout et al. 2001; silva 2012). several studies indicated that al toxicity increased micronuclei formation, chromosomal abnormalities and sister chromatid exchanges (lima et al. 2007; yi et al. 2010). genotoxic effects of stress agents were associated with chromosome aberration, micronucleus formation and chromosomal recombination (achary and panda 2010). genotoxicity and dna destroy can be evaluated by cytogenetical or molecular techniques. rapd and issr used as reliable molecular tools to detect dna variation, damage and mutational events in cells of animals, microorganisms and plants (liu et al. 2005, 2009; salem and hassanein 2017; hassanein et al. 2018). they depend on the fact that the primer is joined with complementary dna sequence on opposite dna strands of the studied genome. under the influence of stress agent, primer binding sites vary under the influence of induced mutation and dna damage (gupta and sarin 2009; achary and panda 2010) leading to amplification of different dna fragments expressing different polymorphism values. the value of polymorphism gives a clear indication of the range to which the genome is affected by the factor in question. then, chromosomal destroy can be revealed by cytogenetical or molecular techniques. vicia faba belongs to fabaceae family and used as human food and animal feed. in addition, bean and other legumes are used to improve the fertility of soil through nitrogen fixation. aluminum has clear genotoxic and cytotoxic effects on cells of faba bean root tips (yi et al. 2010). consequently, faba bean root tips were used as a model plant to study the cytogenetic effects of dipterygium glaucum extracts (altwaty et al. 2016), herbicides and other materials on mitotic activity (el-rokiek et al. 2015; shafeek et al. 2016; prabhu et al. 2017). exposing the faba bean plant to pollutants in various concentrations is considered as a prominent test to determine the genotoxicity (foltête et al. 2011; prabhu et al. 2017). prominent study reported that application of al in low concentration induced adaptive response that led to genomic protection from genotoxic effects of al or other materials (achary and panda 2010). under the inf luence of al stress, inhibition the growth of plant roots was described as the most common symptoms exhibited by higher plants (rout et al. 2001; hassanein et al. 2020a). while several cytogenetical studies were explained the effects of al toxicity on root tips, clear view still needs further studies using model plants such as faba bean. in addition, molecular phenomenon that underlies cytogenetical events are still not fully understood. in this work, we described the effects of application of relatively high doses of al for different exposure periods on mitotic activity and dna integrity of faba bean root tips using cytogenetical and molecular approaches. material and methods plant materials seeds of vicia faba cultivar (misr 3) were obtained from the agriculture research and seeds center in qena, egypt. seeds were germinated in distilled h2o for two to three days to get roots with 2-3 cm long. the obtained roots were treated in solutions containing different concentrations of alcl3 (5, 15, 25 mm) for different periods (0, 6, 12 and 24 h). cytogenetical analysis procedure ten root tips of al treated seedlings were cut and placed 12 h in carnoy’s fixation solution containing ethanol and acetic acid, glacial (3:1 ratio). the cut seedling root tips were kept in 70% ethanol under dark condition at 4°c, hydrolyzed in 1 n hcl (darlington and la cour 1976) and subjected to the feulgen squash technique. total mitotic, mitotic indices, the frequency of mitotic stages and mitotic chromosomal abnormalities were determined according to the detailed formulas in hassanein et al. (2020b). 143chromosomal changes linked with the effect of high dose of aluminum on faba bean (vicia faba l.) root tips genomic dna extraction roots of 2-3 seedlings were treated with 5 mm al for different periods (0, 6, 12 and 24 h) and subjected for genome analysis to investigate how the genome of faba bean was influenced by al toxicity. genomic dna extraction was extracted from root tips and analyzed using rapd and issr techniques according to porebski et al. (1997). pcr conditions a total of ten rapd and nine issr primers (table 1) were used to detect induced genetic variation in faba bean roots under the influence of 5 mm al for different periods (6, 12 and 24 hr). genomic dna amplification was fulfilled in a dna thermal cycler (biometra tpersonal combi, biometra gmbh, germany). application of rapd and issr primers were carried out in a 25 µl pcr mixture solution containing 12.5 µl of go taq® green master mix (promega, madison, usa), 3 µl of primer 10 pmol, 6.5 µl of free nuclease water and 3 µl of 100 ng genomic dna templates. then, pcr run for dna amplification was started using initial denaturation cycle at 94°c for 5 min. then, 40 cycles were carried out using denaturation (94°c for 45 sec), annealing step (optimized for each primer), and elongation (72°c for 1 min) steps. extension step was used to finalize the amplification process at 72°c for 7 min. reproducibility reproducibility was taken in consideration to minimize personal errors. in this concern, each primer was used three times under the same pcr conditions. detection of pcr products the obtained amplification products of each pcr run were electrophoresed in a 1.5% or 2% agarose gel containing 0.5 µg/ml ethidium bromide in 1x tbe. run was carried out in run buffer at 70 volts. then, the amplified pcr products were visualized, photographed and analyzed. data analysis the experimental data were statistically analyzed by anova. data were compared using the least significant difference (lsd) test at 5% (*) and 1% (**) levels (snedecor and cochran 1980). also, dendrograms were generated for cluster analysis according to legendre and legendre (1983) using the community analysis package software program (cap) version 4.0 (richard and peter 2007). results under the inf luence of all alcl3 treatments, the decrease in mi and increase of total abnormalities were found to be statistically highly significant compared to that of control (table 2). irrespective the concentration of al, the value of mi was gradually decreased as the exposure time increased. roots treated with the highest concentration of alcl3 (25 mm) for 24 hr showed the highest inhibition of cell division and it was associated with the highest total abnormalities. in addition, the increase in interphase with increase of the concentrations of alcl3 and exposure time was appeared at all al treatments. table 1. the applied rapd and issr primers. primer type primer sequence (5’---------3’) primer type primer sequence (5’---------3’) rapd issr opa-02 tgccgagctg issr1 acacacacacacacacctg opa-05 aggggtcttg issr2 cacacacacacacacaaagct opa-07 gaaacgggtg issr3 acacacacacacacacaag opa-17 gaccgcttgt issr4 gagagagagagagagactg opat-08 tcctcgtggg issr5 gagagagagagagagactc opaw-10 ggtgtttgcc issr7 ctctctctctcta (ct)6a opd-1 accgcgaagg issr8 tcttcttcttctg opd-18 gagagccaac issr9 tgttgttgtgc opj-15 tgtagcaggg issr10 gtggtggtggc opp-13 ggagtgcctc 144 ahmed m. hassanein, ahmed h. mohamed, heba ahmed abd allah, hoida zaki the relative frequencies of different mitotic phases were affected by alcl3 treatments (table 2). variations in these frequencies appeared to be dependent on exposure time and concentrations of the applied alcl3 concentrations. prophase frequency and prophase abnormalities (fig. 1:4) usually increased as the concentration of alcl3 increased and the exposure time prolonged up to 15 mm alcl3. prophase minimum values of 36.2, 29.8, and 30.6% were observed when root tips were subjected to 25 mm alcl3 for 6, 12 and 24 h, respectively. metaphase frequency mostly increased as the concentration of alcl3 increased and exposure time was prolonged for more than 6 h; it was associated with increase in metaphase abnormalities. maximum value of metaphase frequency (69.4%) was detected when plant root tips were subjected to 25 mm alcl3 for 24 hr. when plant root tips were subjected to alcl3 toxicity, the registered values of ana-telophase frequency were generally lower than that of control. under the influence of 5 or 25 mm alcl3 for 24 h a complete inhibition in ana-telophase stage table 2. mitotic index (mi), % of total abnormalities, % of interphase and % of mitotic phases (prophase, metaphase and ana-telophase), include normal (total) and abnormal (abn) mitotic phases recorded for vicia faba (misr 3) root tips under the influence of different concentrations of alcl3 (5, 15 and 25 mm) and exposure times (6, 12 and 24 hr). treatment total mitosis mitotic index (mi) % total abnormal % interphase % prophase % metaphase % ana-telophase exposure time alcl3 conc. total abn. total abn. total abn. total abn. 6hr. 0 mm 355 7.10±0.36 4.09±0.42 92.9 0.4 37.6 0.4 24.7 1.4 42.2 5 mm 121 2.40±0.10** 83.93±6.41** 97.5 2.33 70.5 65.8 10.6 10.6 16.5 10.5 15 mm 99 1.95±0.15** 76.67±6.65** 98.0 1.37 66.6 62.8 7.7 7.7 25.6 7.7 25 mm 126 2.50±0.10** 65.15±11.05** 97.4 2.6 36.2 25.8 15.5 13.8 44.8 22.4 12hr. 5 mm 79 1.60±0.10** 92.60±1.30** 98.4 0.79 51.7 48.2 39.3 39.2 8.9 5.4 15 mm 64 1.26±0.05** 89.30±10.70** 98.7 2.5 56.5 43.4 30.4 30.4 13.04 13.1 25 mm 47 0.93±0.15** 95.70±0.10** 99.0 1.26 29.8 29.7 61.7 59.5 8.5 6.4 24hr. 5 mm 51 1.00±0.10** 88.13±8.15** 98.9 0.71 61.7 55.3 38.2 38.3 15 mm 86 1.70±0.00** 80.20±9.30** 98.2 0.76 76 50 26.0 26.0 8.0 2.0 25 mm 37 0.75±0.05** 97.90±2.10** 99.2 1.11 30.6 27.8 69.4 69.4 **. the mean difference is significant at the 0.05 level. table 3. types and frequency of chromosomal abnormalities [c-metaphase (c-m), sticky (stick), star, break, disturbed (dist), and diagonal (diag), bridge, free and c-anaphase (c-ana)] recorded for vicia faba (misr 3) root tips under the influence of different concentrations of alcl3 and exposure times (6, 12, and 24 hr). treatment interphase abnormalities metaphase abnormalities ana-telophase abnormalities exposure time alcl3 conc. micro bi nuclei c-m stick star break dist stick bridg dist diag free break star c-ana 6hr. 0 mm 1.3 5 mm 1.76 0.37 4.7 2.4 2.4 1.2 1.2 2.4 3.5 1.3 2.4 1.2 15 mm 0.79 0.28 2.6 1.3 3.8 1.3 1.3 2.6 1.3 25 mm 0.30 0.13 3.4 1.7 3.4 5.2 6.9 10.3 1.7 3.4 12hr. 5 mm 1.09 39.3 1.8 3.6 15 mm 2.53 26.1 4.3 8.7 4.3 4.2 25 mm 0.31 0.04 57.4 2.1 2.1 24hr. 5 mm 2.39 0.41 38.3 15 mm 1.22 0.45 18 2 25 mm 1.05 0.06 66.7 2.8 145chromosomal changes linked with the effect of high dose of aluminum on faba bean (vicia faba l.) root tips was detected. the detected abnormalities in each mitotic phase were higher than that of control. all the used concentrations of alcl3 enhanced micronuclei formation in the root tips (table 3 and fig. 1). the maximum value of micronuclei frequency (2.53%) was detected when root tips were subjected to 15 mm for 12 hr. under all the applied conditions, one (fig. 1: 1), or two (fig. 1: 2) micronuclei/cell were detected. there was no correlation between the number of micronuclei/ cell and the applied concentration of alcl3 or the exposure time. also, binucleated cells were detected under the applied conditions (fig. 1: 3). the type and frequency of chromosomal abnormalities that resulted from alcl3 treatments on faba bean (misr 3) roots were included in table 3 and illustrated in fig.1. metaphase was the most sensitive stage to al stress compared to the other stages of mitosis, where the abnormalities were detected at all treatments (table 3). c-metaphase (fig. 1: 5) was the common abnormalities and their appearance was independent on al concentration but it increased strongly when the exposure time was more than 6 h. in addition, during this stage, abnormalities including star shape (fig. 1: 7), chromosomes breaks (fig. 1: 5, 6) and disturbed configurations (fig. 1: 6) were detected. under the influence of the used high doses of al for the longest period of exposure (24 h), mi was drastically lowered but total abnormalities were increased and it was associated with drastic increase in c-metaphase. the concentration of most of the chromosomal abnormalities of the metaphase stage in one form of alteration (c-metaphase) made the appearance of other forms of abnormalities rare during metaphase or teloanaphase. ana-telophase chromosomal abnormalities including chromosomal bridges (fig. 1: 11), chromosomal breaks (fig. 1: 10), disturbed anaphase (fig. 1: 8) and sticky anaphase (fig. 1: 9) were detected. when plant root tips were subjected to the relatively high concentration of alcl3 (15 mm) for relatively long period (24 h), star shape chromosome was detected (fig. 1: 12) but other types of abnormalities were not registered during anatelophase stage. the same results were obtained when plant root tips were treated with 25 mm alcl3 for 12 hr. under different al treatments, star chromosome was figure 1. chromosomal aberrations of misr 3 cultivar root tips under the influence of alcl3 for different periods (6, 12, and 24 hr); (1, 2) micronuclei formation during interphase stage (arrows), (3) binucleated interphase, (4) irregular prophase, (5) c-metaphase with breaks (arrow), (6) disturbed metaphase, (7) star metaphase, (8) disturbed anaphase, (9) sticky anaphase, (10) breaks in anaphase (arrow), (11) bridge anaphase (arrow) and (12) star shape anaphase. figure 2. rapd profile generated by 9 primers using roots of different vicia faba misr 3 cultivar subjected for different periods in 5 mm alcl3. lanes1, 2, 3 and 4: 0.0, 6, 12 and 24 hr, respectively. m: dna ladder. 146 ahmed m. hassanein, ahmed h. mohamed, heba ahmed abd allah, hoida zaki the common type of chromosomal abnormalities during ana-telophase stage. root tips of misr 3 cultivar were subjected to molecular analysis to reveal genome variation which in consistent with chromosomal abnormalities under al toxicity (table 4). in this concern, ten rapd primers were used (fig. 2). misr 3 cultivar showed 32 polymorphic (54.24 %), 20 monomorphic bands (33.89 %) and seven unique bands (11.86 %) out of 59 fragments. the number of bands ranged from two using opat-08 primer to 13 bands using opa-17 primer. percentage of polymorphism ranged from 0 % when opd-18 was used to 92.31 % using opa-17 with an average of 60.09 %. primer opa-17 gave the highest number of polymorphic rapd markers. the average number of bands per polymorphic primers was 5.9 and the average number of polymorphic bands per polymorphic primers was 3.9. the highest number of bands was detected when opa-17 primer was used. the size of the obtained fragments using all the rapd primers ranged from 141 to 1811 bp. nine issr primers were used for amplification of genomic dna of v. faba misr 3 cultivar (table 5 & fig. 3). under the application of these nine primers 75 amplified fragments were detected. forty-five of them were polymorphic (60 %), 18 were monomorphic bands (24 %) and eight were unique bands (10.67 %). the number of bands per primers ranged from six using issr2 primer to ten bands using issr1, issr5 and issr10 primers. the size of the obtained fragments using all issr primers varied between 167-1557 bp. primers issr1 or issr9 table 4. ten rapd primers, their sequences, annealing temperature, size of amplified fragments (bp), total number of amplified fragments, number of polymorphic bands and unique bands identified per primer used to access genome stability of vicia faba misr 3 cultivar under the influence of al stress. primer sequence (5’→3’) annealing temperature (°c) polymorphic bands monomorphic bands unique bands total bands size range (bp) polymorphism (%) opa-02 tgccgagctg 32 4 2 1 7 141-753 71.43 opa-05 aggggtcttg 30 4 3 0 7 225-1102 57.14 opa-07 gaaacgggtg 30 3 2 0 5 497-1428 60.00 opa-17 gaccgcttgt 30 8 1 4 13 162-1811 92.31 opat-08 tcctcgtggg 32 1 1 0 2 218-958 50.00 opaw-10 ggtgtttgcc 30 4 1 0 5 313-912 80.00 opd-1 accgcgaagg 32 2 3 1 6 236-1364 50.00 opd-18 gagagccaac 30 0 4 0 4 292-1278 00.00 opj-15 tgtagcaggg 30 4 1 0 5 229-737 80.00 opp-13 ggagtgcctc 32 2 2 1 5 345-876 60.00 total 32 20 7 59 60.09 table 5. sequences and annealing temperature of nine issr primers were used to access genetic disorder of the root tip of vicia faba (misr 3 cultivar) as influenced by al concentration and exposure time. size of amplified fragments (bp), total number of amplified fragments, number of polymorphic bands, unique bands and specific bands identified per primer were included. primer sequence (5’→3’) annealing temperature (°c) polymorphic bands monomorphic bands unique bands total bands size range (bp) polymorphism (%) issr1 acacacacacacacacctg 56 7 3 0 10 175-1287 70.00 issr2 cacacacacacacacaaagct 60 5 1 0 6 239-703 83.33 issr3 acacacacacacacacaag 58 6 1 2 9 206-870 88.89 issr4 gagagagagagagagactg 50 4 2 2 8 167-985 75.00 issr5 gagagagagagagagactc 50 2 6 2 10 188-565 40.00 issr7 ctctctctctcta (ct)6a 38 6 1 1 8 804-1376 87.50 issr8 tcttcttcttctg 36 5 2 0 7 295-1489 71.42 issr9 tgttgttgtgc 32 7 0 0 7 169-828 100 issr10 gtggtggtggc 38 3 6 1 10 457-1557 40.00 total 45 18 8 75 72.90 147chromosomal changes linked with the effect of high dose of aluminum on faba bean (vicia faba l.) root tips gave the highest number of polymorphic issr fragments. the average number of bands per polymorphic primers was 8.33 and the average number of polymorphic bands per polymorphic primers was 5.9. percentage of polymorphism ranged from 40 % when issr5 and issr10 were used to 100 % using issr9 with an average of 72.90 %. when results of the application of issr were compared with others of rapd primers, valuable variations were detected. total bands per nine primers of issr (75) were higher than that of ten rapd (59) primers. in addition, the average polymorphism obtained from the application of issr primers (72.90 %) was higher than the average polymorphism obtained from applying of rapd primers (60.09 %). cluster tree based upon upgma analysis of ten rapd and nine issr primers of v. faba misr3 cultivar resulted in two main clusters (fig. 4 & 5). the first cluster included control and 6 h treated roots with alcl3. the second cluster included 12 and 24 h treated roots with alcl3. the obtained dendrogram as well as cytogenetical and polymorphism data showed that misr 3 cultivar suffered from harmful effect of al on the nuclear genome, especially when the duration of exposure time was more than 6 hr. discussion in this work, seedlings of faba bean were subjected to genotoxicity of high dose al (525 mm alcl3), the obtained nuclear genome variation was registered using cytogenetical and molecular approaches. cytogenetical effects of al on faba bean (yi et al. 2010; hassanein et al. 2020b) and other plant species (li et al. 2015; domingues et al. 2012; jaskowiak et al. 2018) were studied. in this work, irrespective the concentration of al, figure 5. upgma based cluster tree of vicia faba (misr 3) cultivar exposed to 5 mm alcl3 for (0.0, 6, 12 and 24hr) based on (10 rapd + 9 issr primers). figure 3. issr-pcr profiles generated by 9 primer using roots of different v. faba misr 3 cultivar subjected for different periods in 5 mm alcl3. lanes1, 2, 3 and 4 (0.0, 6, 12 and 24hr), respectively. m: dna ladder. figure 4. upgma based cluster tree of vicia faba (misr 3) cultivar exposed to 5 mm alcl3 for (0.0, 6, 12 and 24hr) based on (9 issr primers). 148 ahmed m. hassanein, ahmed h. mohamed, heba ahmed abd allah, hoida zaki the value of mi was gradually decreased as the exposure time increased as was also reported by other authors (yi et al. 2010; altwaty et al. 2016; hassanein et al. 2020b). variations in frequencies of different mitotic phases were registered and indicated that al induced cell cycle alterations in faba bean as was reported by yi et al. (2010). consequently, prophase frequency increased as the concentration of alcl3 increased and exposure time prolonged up to 15 mm alcl3. also, metaphase frequency mostly increased as the concentration of alcl3 increased except plant roots subjected al stress for 6 hr. increase of prophase and metaphase frequencies associated with increase in their abnormalities. in addition, increase in prophase frequency associated with decrease in ana-telophase frequency under al treatments and it showed complete inhibition when al stresses were applied for 24 h. micronuclei were detected in the root tips exposed to different concentrations of alcl3 for different periods and ranged from small to large size. there was no correlation between the size or number of micronuclei/cell and the applied conditions. binucleated cells are formed in al stressed misr 3 cultivar as the result of disturbed cytokinesis (alberts et al. 2002) and cell plate formation (gisselsson et al. 2001). micronuclei frequency is a good indicator of the cytogenetic effects of tested chemicals such as alcl3 in faba bean and other plant species (kanaya et al. 1994; gecheff 1996; grant and owens 1998; kundu and ray 2017). consequently, micronucleus assay was considered as the efficient, simplest and most effective tool to measure of chromosomal dna damages and analyze the mutagenic effect of different substances (auerbach 1962; el-azab et al. 2018; kursheed et al. 2018). under the used high doses of alcl3, metaphase was the most sensitive stage compared to the other stages of mitosis, where the abnormalities were detected at all treatments. in addition, c-metaphase was the common abnormalities, and their appearance was independent on al concentration, but it increased strongly when the exposure time was more than 6 hr. in addition, metaphase abnormalities including chromosomal stickiness, star shape chromosomes, chromosomal breaks and disturbed configurations were detected. during metaphase, failure of broken chromosome to recombine correctly due to the stickiness of chromosomes and their inability to arrive to the poles led to the appearance of chromosomal breaks under al or other stress agent (agarwal and ansari 2001). these abnormalities were also detected by other authors in faba bean (yi et al. 2010; hassanein et al. 2020b) and other plant species (de campos and viccini 2003; mohanty et al. 2004). concentration abnormalities in one form (c-metaphase) during metaphase made the appearance of other forms of abnormalities rare during metaphase or teloanaphase, especially under long exposure time (24 h). in addition, ana-telophase chromosomal abnormalities including chromosomal bridges, chromosomal breaks, disturbed anaphase, diagonal and star chromosomes, and c-anaphase were detected under al treatments. the registered stickiness in faba bean and other plant species gave an indication about the direct destructive effect of a toxic agent on chromosomes (renjana et al. 2013) or spindle disturbance (gaulden 1987). lagging chromosomes and chromosomal bridges were appeared which may be due to abnormal spindle fiber formation (badr et al. 2013). also, rieger et al. (1991) believed that the inhibition of cytoskeletal proteins leads to the formation of lagging chromosomes. data of our study indicated that al treatments in high dose induced chromosomal abnormalities and micronuclei formation. these two phenomenon may result from inhibition of dna synthesis (minocha et al. 1992), alteration of chemical or electrostatic properties of dna (unrau and laster 1952), elimination of genetic material (fernandes et al. 2007), induction of dna fragmentation (jaskowiak et al. 2018), formation of chromosomal bridges and chromosomal breaks (ignacimuthu and babu 1989), miss-repair of the broken dnas or fused of telomeres (souguir et al. 2018), stickiness of chromosomes (badr et al. 2014). in addition to point mutation, the previous events created alteration either at or between the primer binding sites, which could be detected by rapd and issr techniques (liu et al. 2007; gupta and sarin 2009). during pcr program, binding between primers and complemented loci resulted amplification of dna fragments with molecular weight of 100 to 2000 bp (ng and tan 2015). the amplified dna fragments were dependent on the extent of chromosomal changes under the toxic effect of al due to the previous events. these events created or abolished some primer binding sites leading to polymorphism. this means that the detected dna polymorphism may be due to mismatching at the primer site, appearance of a new primer site and/or change the distance between two opposite primers. using rapd primers, misr 3 cultivar showed 32 polymorphic out of 59 fragments primers (54.24 %), 20 monomorphic bands (33.89 %) and seven unique bands (11.86 %). percentage of polymorphism ranged from 0 % when opd18 was used to 92.31 % using opa-17 with an average of 60.09 %. under the application of issr primers, polymorphism average was 72.90 %. the average polymorphism obtained from the application of issr primers 149chromosomal changes linked with the effect of high dose of aluminum on faba bean (vicia faba l.) root tips (72.90 %) was higher than that of rapd primers (60.09 %). then, issr is an ideal molecular marker to study natural or induced genetic variation of faba bean cultivars as well as other plants (abdel-razzak et al. 2012; alghamdi et al. 2012; wang et al. 2012; salem and hassanein 2017; hassanein et al. 2018). cluster tree based on rapd and issr primers indicated that misr 3 cultivar suffered from harmful effect of al on its genome when plant root tips were exposed to al for more than 6 h where great cytogenetical events were happened and resulted in variation in primer binding sites leading to high polymorphism. in faba bean, yi et al. 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2012. genetic diversity and relationship of global faba bean (vicia faba l.) germplasm revealed by issr markers. theor appl genet. 124: 789–797. yi m, yi h, li h, wu l. 2010. aluminum induces chromosome aberrations, micronuclei, and cell cycle dysfunction in root cells of vicia faba. environ toxicol. 25: 124–129. caryologia. international journal of cytology, cytosystematics and cytogenetics 75(3): 109-122, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1628 caryologia international journal of cytology, cytosystematics and cytogenetics citation: inês da fonseca simão, hermenegildo ribeiro da costa, helena cristina correia de oliveira, maria helena abreu silva, paulo cardoso da silveira (2022). nuclear dna 2c-values for 16 species from timor-leste increases taxonomical representation in tropical ferns and lycophytes. caryologia 75(3): 109-122. doi: 10.36253/caryologia-1628 received: april 14, 2022 accepted: november 23, 2022 published: april 5, 2023 copyright: © 2022 inês da fonseca simão, hermenegildo ribeiro da costa, helena cristina correia de oliveira, maria helena abreu silva, paulo cardoso da silveira. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid is: 0000-0001-6548-8535 hc: 0000-0002-8059-2397 ho: 0000-0002-4673-0696 ms: 0000-0001-8060-2842 ps: 0000-0002-9253-5381 nuclear dna 2c-values for 16 species from timor-leste increases taxonomical representation in tropical ferns and lycophytes inês da fonseca simão1, hermenegildo ribeiro da costa1,2,3, helena cristina correia de oliveira1,2, maria helena abreu silva1,2, paulo cardoso da silveira1,2,* 1 department of biology, university of aveiro, 3810-193 aveiro, portugal 2 cesam-centre for environmental and marine studies, department of biology, university of aveiro, 3810-193 aveiro, portugal 3faculty of education, arts and humanities, national university timor lorosa’e (untl), avenida cidade de lisboa, dili, east timor * ccorresponding author. e-mail: psilveira@ua.pt abstract. knowledge regarding genome size allows us to infer relationships between taxa, address questions related to systematics and contribute to biodiversity studies. however, currently, less than 3% of the described pteridophyta species have genome size estimates reported in databases, and only around one third of these are tropical species, although the tropics are home of 86% of fern diversity. the region of timorleste, included in one of the 25 hotspots of biodiversity, is considered one of the richest areas of the world in terms of pteridophyte species. nonetheless, biodiversitydriven research focused on this territory’s biodiversity is scarce. this study presents novel 2c-values for 15 species of ferns collected in timor-leste, using flow cytometry. furthermore, one species of the lycophyte palhinhaea cernua (l.) vasc. & franco, was also studied and its estimated genome size compared to a previous report. estimates ranged from 10.45 pg in selliguea feei bory to 29.7 pg in microsorum punctatum (l.) copel, and are considered medium-size genomes. the data was compared with previous reports for closely related species. these are the first 2c-values for two families and seven genera of ferns, increasing the number of pteridophytes with reported c-values from 292 to 307. keywords: genome size, chromosome, cytogenetics, dna amount, nuclear dna content, malesia, geographical distribution. introduction information regarding genome size plays a fundamental role in understanding a species’ evolutionary history and is a tool that allows us to infer relationships between taxa, address questions related to cellular and developmental biology and systematics, among others, and contributes to biodiversity studies (leitch 2005; kumar et al. 2011). the considerable differences 110 inês da fonseca simão et al. in nuclear dna content across species can be related to adaptive features, which shows that genome size can be under selective pressure and its variations may be related to the evolutionary history of a given group (ohri 1998). currently, flow cytometry is the main technique used to obtain information related to species dna c-value (dolezel 2005). however, despite the importance of these studies, and the recent efforts concerning information about genome size in plants, there is still a substantial gap in knowledge, with only a very small portion of species studied, and more research is required. the majority of values reported in the plant dna c-value database (release 7.1, april 2019:https://cvalues. science.kew.org/) (leitch et al. 2019) belong to angiosperms. the 2c-value for 10.770 species of angiosperms is known, corresponding to 3.3% of their global diversity (antonelli et al. 2020). pteridophytes are even more under-represented, with only 292 species reported in the database. these numbers account for 2.45% of the 11,916 species of pteridophytes described (ppg 2016). in 2001, bennet & leitch set the goal of obtaining the c-value for 200 pteridophytes species by 2005, with a special focus on those that maximize systematic and geographic representation (bennet and leitch 2001). although this goal was met, further studies regarding this group are fundamental, since the pteridophytes represent an important evolutionary transition between bryophytes and spermatophytes and, as such, are critical to our understanding of how dna content has evolved across land plants (bainard et al. 2011). furthermore, since the laboratories adequately equipped to make 2c-values estimation are mostly located in temperate climate areas, with more difficult access to tropical fern species, we suspected such species would be underrepresented in the plant dna c-value database. yet, pteridophyte diversity in the tropics is significantly higher than in any other region of the globe. estimates point to the existence of 4500 species of ferns and lycophytes in southeast asia, more than twice the number of species of the entire holarctic kingdom (moran 2008). at the same time, the region of timor-leste, located in southeast asia, is included in the biogeographic region of malesia, which is considered one of the richest areas of the world in terms of tropical pteridophyte species diversity (ebihara and kuo 2012). additionally, timor-leste is included in wallacea, an area classified as one of the 25 hotspots of biodiversity identified by myers et al. (2000) as a priority of conservation at a global scale. despite the rich biological patrimony of timor-leste, research focused on the country’s biodiversity and genetic resources is lacking, mainly due to the military occupation of the territory that took place between 1975 and 1999 (bouma and kobryn 2004). in this sense, better coverage of pteridophy tes nuclear dna values data in this territory is crucial to understand the mechanisms behind genome size evolution and their relationship with geographic and ecological factors (dagher-kharrat et al. 2013). therefore, the aims of this paper are: 1. to check what percentage of genome size data from tropical pteridophytes has been estimated, comparing with other biogeographic regions; and 2. to expand knowledge about genome sizes of tropical fern species occurring in timor-leste. materials and methods plant material prior to the field work, a search was conducted in the plant dna c-value database to establish which pteridophytes species, known to occur in timor-leste, had already 2c-values estimations published, and which had not. from the latter list, those species with populations that could more easily be sampled were selected as target species for this study (table 1). leaves of 15 ferns and one lycophyte were collected from several field locations in timor-leste (table 1). these samples were kept fresh (at 0-5ºc) for a period no longer than a week and used for flow cytometry analysis. voucher specimens were prepared and kept in the herbaria of the university of aveiro (ave) and naturalis biodiversity center (l). duplicates were also kept at the national university of east timor (untl, díli, timor-leste). nuclear dna content estimation the nuclear dna content of fresh leaf samples was assessed using flow cytometry, currently the most used technique to estimate c/2c-value in plants for its simplicity, accuracy, convenience, and speed (galbraith et al. 1983, 2009). the methodology used followed loureiro et al. (2007), which included the preparation of nuclear suspensions by chopping 50 mg of leaf sample tissue and 50 mg of internal standard leaves, vicia faba “inovec” (2c= 26.90 pg; dolezel, sgorbati and lucretii 1992) or pisum sativum “ctirad” (2c= 9.09 pg; dolezel et al. 1992), with a razor blade in a glass petri dish containing 1 ml of wpb isolation buffer (200 mm tris.hcl, 4mm mgcl2.6h2o, 2 mm edta na2.2h2o, 86 mm nacl, 10 mm sodium metabisulfite, 1% pvp-10, 1% (v/v) triton x-100, ph 7.5; loureiro et al. 2007). the nuclear solution was then filtered through a nylon net of 50 µm, and 50 μg.ml-1 of propidium iodide (pi, sigma-aldrich, st. lou1112c-values for pteridophytes from timor is, mo, usa) and 50 μg.ml-1 of rnase (sigma-aldrich, st. louis, mo, usa) were added to the sample, to stain nuclear dna and prevent staining of double stranded rna, respectively. samples were analyzed within a 10 min period on an attune® acoustic focusing cytometer (termofisher scientific) equipped with a 488 nm laser. for each sample, at least 5,000 nuclei were analyzed. as a quality control, nuclear dna content estimates were only considered when the coefficient of variation of g0/g1 peaks (cvpeak) were below 5%. samples with higher cvpeak values were discarded and a new sample was prepared. for most of the taxa, three to five individuals were analyzed, but for selliguea feei and tectaria melanocaulos, only one individual for each of the species survived the time between sampling in timor and analysis in aveiro. the number of individuals measured for each population is provided in table 1. statistical analysis descriptive statistics were calculated for each taxa studied namely, mean, standard deviation (sd), coefficient of variation (cv), and minimum and maximum values of the holoploid genome size (2c, pg). table 1. scientific names and localities of samples collected for this study. voucher specimens are kept in the herbarium of the university of aveiro (ave) and of the naturalis biodiversity center (l). family circumscription according with ppg (2016). taxon family localities in timor-leste lycopodiophyta palhinhaea cernua (l.) vasc. & franco lycopodiaceae ainaro, roadside between maubisse and turiscai, [8°49’33” s, 125°38’10” e], costa et al. 254 (ave) pteridophyta calochlaena javanica (blume) m.d.turner & r.a.white dicksoniaceae ainaro, roadside from maubisse to turiscai, [8°49’22” s, 125°37’01” e], costa et al. 245 (ave, l.3959675) pityrogramma calomelanos (l.) link adiantum philippense l. pteris ensiformis burm. pteridaceae pteridaceae pteridaceae ainaro, roadside between maubisse and turiscai, [8°49’33” s, 125°38’10” e], costa et al. 253 (ave) liquiçá, roadside between tibar and faiten, [8°36’59” s, 125°29’09” e], costa et al. 8 (ave, l.3959700) manufahi, roadside of laclo, [8°51’28” s, 125°41’36” e], costa et al. 320 (ave) blechnopsis orientalis (l.) c.presl blechnaceae ainaro, roadside from maubisse to turiscai, [8°49’22” s, 125°37’01” e], costa et al. 244 (ave) diplazium esculentum (retz.) sw. athyriaceae aileu, from díli to aileu, after the crossroad to remexio and remexio, [8°37’05” s, 125°38’25” e], costa et al. 195 (ave, l.3959688) tectaria melanocaulos (blume) copel. tectariaceae aileu, asumau, [8°37’19” s, 125°38’37”], costa et al. 200 (ave, l.3959765) oleandra musifolia (blume) c.presl oleandraceae ainaro, roadside between maubisse and turiscai, [8°48’57” s, 125°38’39” e], costa et al. 258 (ave) goniophlebium subauriculatum (blume) c.presl microsorum punctatum (l.) copel. microsorum scolopendria (burm.f.) copel. platycerium bifurcatum subsp. willinckii (t.moore) hennipman & m.c.roos pyrrosia lanceolata (wall.) farw. pyrrosia longifolia (burm.f.) c.v.morton selliguea feei bory polypodiaceae polypodiaceae polypodiaceae polypodiaceae polypodiaceae polypodiaceae polypodiaceae viqueque, on the waibua forest at foothills of mundo perdido mountain, [8°43’59” s, 126°22’10” e], costa et al. 303 (ave) viqueque, on the waibua forest at the foothills of mundo perdido mountain, [8°43’59” s, 126°22’10” e], costa et al. 307 (ave) viqueque, on the waibua forest at foothills of mundo perdido mountain, [8°43’59” s, 126°22’10” e], costa et al. 301 (ave) díli, dare, [8°35’38” s, 125°34’07” e], costa et al. 84 (ave, l.3959789) aileu, roadside between aileu and maubisse, [8°48’16” s, 125°35’31” e], costa et al. 238 (ave) viqueque, roadside of urulita, [8°46’21” s, 126°22’11” e], costa et al. 290 (ave) ainaro, maubisse turiscai, at rita-uruho, [8°49’22” s, 125°37’01” e], costa et al. 243 (ave) 112 inês da fonseca simão et al. chromosome number the median of the chromosome numbers for 14 taxa was obtained from the online chromosome counts database (ccdb) (rice et al. 2015). floristic kingdoms versus 2c values analysis the floristic kingdom’s classification by takhtajan (1986) was applied to the pteridophyta taxa whose dna c-values are available in the plant dna c-value database. for that, global biodiversity information facility (gbif, at https://www.gbif.org/, january 2022) was consulted to establish each species’ main occurrence. finally, the distribution of species listed in the plant dna c-value database by each floristic kingdom was compared with the equivalent distribution of the total world number of pteridophyte species given by moran (2008). for this comparison, the paleotropical and the cape f loristic kingdoms had to be included in the same group, because moran (2008) gives a single total number for africa, without segregating the cape floristic kingdom. the same was not adopted for the holantarctic kingdom, because moran (2008), provides separate figures for new zealand, which allows some separation from other kingdoms. in south america no separation was possible between the holantarctic and the neotropical kingdoms, but since the number of neotropical species should be much greater than the holantarctic species present in the region, we assumed that the error would not be critical. results dna content estimates were obtained for the 16 samples, 15 of them representing taxa with no previous 2c-value reported. these estimates, as well as the chromosome median 2n value that are described in literature, are presented in table 2. the 2c dna content ranged from 10.45 pg in selliguea feei bory, with the vicia faba standard, to 29.7 pg in microsorum punctatum (l.) copel. with the pisum sativum standard. the average 2c-value for polypodiopsida was 20.62 pg, and for lycopodiophyta, represented only by one taxon, the 2c-value was 25.65 pg. the coefficients of variation (cvs) for the samples varied between 3.7% and 6.7%. the list of pteridophyte taxa for which nuclear dna 2c-values have been published in the plant dna c-value database (leitch 2019) is presented in the supplementary material 1, alongside with the tak htajan’s floristic kingdoms (takhtajan 1986) embraced by their geographical distributions ranges. this information is summarized in table 3, alongside with the total world estimated number, and percentage, of pteridophyte species for each f loristic kingdom, according with moran (2008). we can see in this table, that the paleotropical+cape kingdoms, together with the neotropical floristic kingdoms, with 45% and 42%, respectively, include the vast majority of the world’s pteridophyte diversity (87%). contrariwise, the most diverse group of pteridophytes whose nuclear dna 2c-values are known is the holarctic, with 44%, followed by the paleotropical+cape, with only 23% and the neotropical with 18%. with this study, the percentages of holarctic species is reduced to 42%, and the percentage of species from paleotropical+cape area increases to 25%. discussion in spite of the long journey between the field in timor-leste and the cytometry laboratory in aveiro, where the analysis was done, we succeed to analyze, at least, three individuals for 14 of the 16 species, and five/ six, for nine of the 16 species. the higher intraspecific variations detected are, most likely, related to difficulties associated to the flow cytometry technique, since the easiness of obtaining data differs between the taxa, as mentioned by obermayer, et al. (2002). following leitch, chase & bennet (1998) genome size classification, all taxa have “intermediate” genomes (7<2c≤28 pg). the median value established for genome size in ferns is 22.8 pg/2c and it has been related, partially, to variation in post-polyploidization processes such as additional chromosomes and dna arising from whole genome duplications-, since diploidization is not linked with genome downsizing in ferns in opposition to angiosperms, a group with smaller genomes (median= 3.4 pg/2c) (liu et al. 2019). regarding the lycophytes, the median 2c-value for the group is 0.26 pg (liu et al. 2019), corresponding to a very small genome (≤2.8 pg) (leitch et al. 1998). despite the 2c-value previously reported in the literature of 2.75 pg for palhinhaea cernua (l.) vasc. & franco (kuo et al. 2016), the 2c-value estimated for this species is 25.65 pg, corresponding to the “intermediate” category and to the highest genome size in the lycopodiaceae family reported until present, more than twice that of huperzia lucidula (michx.) trevis., which has 11.28 pg (bainard et al. 2011) and was the previous highest value reported. considering that the coefficient of variation for this estimate is 5.5%, it doesn’t seem likely that the 2c-value for p. cernua was 1132c-values for pteridophytes from timor ta bl e 2. m ea n 2c -v al ue e st im at es ( pg ) fo r 15 f er n sp ec ie s an d 1 ly co ph yt e co lle ct ed in e as tt im or , w ith s ta nd ar d de vi at io n (s d ), m in im um a nd m ax im um v al ue s, a ve ra ge c oe ffi ci en t of v ar ia ti on ( c v % ) fo r ea ch t ax on . f am ily c ir cu m sc ri pt io n ac co rd in g w ith p pg ( 20 16 ). e st im at es o bt ai ne d us in g th e v ic ia f ab a st an da rd ( 2c = 2 6. 90 ) ar e id en ti fie d w ith “ *” . th e re m ai ni ng m ea su re m en ts w er e ob ta in ed u si ng t he p is um s at iv um s ta nd ar d (2 c = 9. 09 p g) . r ep or te d ch ro m os om e nu m be r (m ed ia n n va lu e) f or t he t ax a av ai la bl e is a ls o pr es en te d, ac co rd in g to th e c c d b d at ab as e (r el ea se 1 .5 8, h tt p: // cc db .ta u. ac .il /) . ta xo n fa m ily m ed ia n 2n v al ue g en om e si ze ( 2c , p g) n. s am pl es m ea n ± sd m in . m ax . a ve ra ge c v ( % ) ly co po di op hy ta pa lh in ha ea c er nu a (l .) v as c. & f ra nc o ly co po di ac ea e 2 08 , 2 20 , 2 72 , 3 12 , 3 30 , 34 0, 4 16 25 .6 5 ± 0. 43 25 .3 2 26 .3 2 5. 52 5 p te ri do ph yt a c al oc hl ae na ja va ni ca ( bl um e) m .d .t ur ne r & r .a .w hi te d ic ks on ia ce ae ? 11 .4 1 ± 0. 11 11 .3 2 11 .4 3 4. 78 5 pi ty ro gr am m a ca lo m el an os ( l. ) li nk pt er id ac ea e 23 2, 2 40 26 .4 1 ± 0. 36 26 .1 2 26 .8 5 6. 72 4 a di an tu m p hi lip pe ns e l. 60 , 9 0 21 .9 2* ± 2 .3 18 .4 8 23 .2 9 4. 03 4 pt er is e ns ifo rm is b ur m . 58 , 8 788 , 1 16 , 1 68 , 1 85 19 .1 5* ± 0 .5 5 18 .7 1 19 .8 1 4. 71 5 bl ec hn op si s or ie nt al is ( l. ) c .p re sl bl ec hn ac ea e ? 13 .5 7* ± 0 .1 1 13 .4 3 13 .6 1 5. 91 5 d ip la zi um e sc ul en tu m ( r et z. ) sw . a th yr ia ce ae 82 22 .6 8 ± 0. 70 22 23 .5 7 5. 88 4 te ct ar ia m el an oc au lo s (b lu m e) c op el . te ct ar ia ce ae ? 24 .6 8 3. 69 1 o le an dr a m us ifo lia ( bl um e) c .p re sl o le an dr ac ea e 80 13 .6 5* ± 0 .0 8 13 .5 7 13 .7 5 6. 11 5 g on io ph le bi um s ub au ri cu la tu m ( bl um e) c .p re sl po ly po di ac ea e 72 21 .0 6* ± 0 .3 8 20 .5 9 21 .5 2 4. 98 5 m ic ro so ru m p un ct at um ( l. ) c op el . 72 29 .7 2 ± 0. 44 29 .4 3 30 .2 3 4. 56 3 m ic ro so ru m s co lo pe nd ri a (b ur m .f. ) c op el . 36 ** 24 .5 5 ± 1. 92 21 .1 4 25 .7 6 4. 53 5 pl at yc er iu m b ifu rc at um s ub sp . w ill in ck ii (t .m oo re ) h en ni pm an & m .c .r oo s 74 28 .4 7 ± 2. 88 23 .7 4 30 .8 3. 93 5 py rr os ia la nc eo la ta ( w al l.) f ar w . 74 23 .7 3 ± 0. 31 23 .4 7 24 .1 6 5. 34 4 py rr os ia lo ng ifo lia ( bu rm .f. ) c .v .m or to n 74 28 .7 9 ± 3. 58 26 .0 6 35 .7 4. 96 6 se lli gu ea fe ei b or y 74 10 .4 5* 5. 44 1 ** n v al ue p re se nt ed , n o 2n v al ue r ep or te d. 114 inês da fonseca simão et al. negatively influenced by artefacts such as the presence of interfering secondary metabolites (hanusová et al. 2014). this novel result shows that genome size within the lycopodiaceae family may be more variable than what was thought until now in fact, the chromosome numbers reported for this species varies from n=34 to 2n=208-416 (rice et al. 2015). comparing the 2c-value of diplazium esculentum (retz.) sw. (22.68 pg) with diplazium pycnocarpon (sprengel) m. broun (12.63 pg), the only other species of the same genus that has been screened for its genome size by bainard et al. (2011), the 2c-value differs by approx. 10 pg. this variation shows that even within the same genus, genome size may vary greatly, regardless of the two species’ chromosome number being very similar, with d. esculentum (2n=82) and d. pycnocarpon (2n=80). the same conclusion can be drawn when comparing our estimate for adiantum philippense l., (2c= 21.9 pg) with previous work on the genus: 2c-value estimates reported for adiantum pedantum l. are 10.16 pg (bainard et al. 2011) and for adiantum aleuticum (rupr.) c. a. paris are 11.42 pg (an approx. difference of 10.5 pg) (clark et al. 2016). the 2c-value discrepancy between adiantum species may be related, most probably, to differences in chromosome numbers between taxa, since both 2n=60 and 2n=90 have been reported for a. philippense in literature. although 2n=60 is similar to chromosome number for a. pedantum and a. aleuticum (2n=58), a 2n=90 could be a reason to explain this variation. the 2c-value discrepancy between adiantum species may also be related, in part, to the different geographical origin of the material. some evidence points towards the prevalence of smaller genomes in plant species that exist in harsher, drier, environments, with shorter growing seasons (knight, molinari and petrov 2005). but checking this would require investigations out of the scope of this paper. what we could contribute was towards improving the representation of the most diverse phytogeographical kingdoms for this group (table 3), following moran’s (2008) suggestion that this group of organisms shows a dominant pattern called “the latitudinal diversity gradient”, which means that species diversity in ferns increases from the pole towards the equator (moran 2008). despite this pattern, almost half of the studied species found in the plant dna c-value database (leitch et al. 2019) belong to the holarctic kingdom. therefore, an already understudied group of plants in terms of genome size lacks, to a great extent, estimates from species of the most representative phytogeographical kingdoms for this group, which we tried to counteract with the new data presented in this study (table 3). conclusions the present work includes novel data that contributes to the knowledge regarding genome size of 15 species of ferns and 1 species of lycophytes. our data increases the taxonomic representation of dna content in pteridophytes databases by two familiesblechnaceae and oleandraceae-, as well as seven genera (blechnopsis, goniophlebium, microsorum, palhinhaea, pityrogramma, pyrrosia and selliguea). furthermore, the representation of paleotropical fern species has increased by 2%. however, with almost 12.000 species of pteridophytes described to date, further work focused on the dna content of more lycophyte and fern species, especially from tropical regions, is crucial to expand taxonomic representation and fill in the phylogenetic gaps within the group. although we could not perform chromosome counts alongside with the 2c value estimations, this should be a future target, allowing to draw more complete conclutable 3. distribution of the number and percentage of species of pteridophytes recognized by each of takhtajan’s floristic kingdoms comparing with the same distribution in terms of species with published dna c-values including the contribution of this study. takhtajan’s floristic kingdoms no. (%) of species estimated* no. (%) of species with known dna c-values no. of species added in this study** current no. (%) of species with known dna c-values holartic 1470 (9.4) 190 (44) 188 (42) neotropical 6500 ((41.7) 76 (18) 4 80 (18) paleotropical + cape 6980 (44.7) 94 (23) 16 110 (25) australian 456 (2.9) 37 (8) 5 42 (9) holantarctic 193 (1.2) 29 (7) 29 (6) total 15599 (100) 429 (100) 25 454 (100) * numbers of species estimated to occur taken from moran (2008: 369); ** the numbers presented exceed the 16 species analyzed, because several of them are distributed among more than one floristic kingdom, as it was also adopted by moran (2008). 1152c-values for pteridophytes from timor sions about the genome of the studied species, namely, concerning ploidy levels. bearing this in mind, in spite of the relatively modest contribution in terms of species numbers (not so modest when we consider the number of new families and genera), this paper increases the representation of tropical pteridophy te diversity whose nuclear 2c-va lues are k nown, and highlights t hat f ur t her studies on genome size in ferns are crucial, especially in species from areas that are considered hotspots of tropica l fern biodiversit y, such as timor-leste. the lack of studies on the country’s biodiversity coupled with the human impact in the region, makes the execution of these studies even more important, since genome size data is basic information for an appropriate management and conservation of the plant genetic resources of the area. acknowledgements we are grateful to the timor-leste authorities, specially the minister of agriculture and fisheries and the quarentine, for allowing the collection and the export of samples by h.r.costa, and to u.n.t.l. 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chromosome numbers. new phytol. 206(1): 19-26. takhtajan a. 1986. floristic regions of the world. berkley and los angeles: university of california press (ca) wagner fs. 1992. cytological problems in lycopodium sens. lat. ann mo bot gard. 79(3): 718-729. doi: https://doi.org/10.2307/2399761 1172c-values for pteridophytes from timor supplementary material 1. distribution of pteridophyta taxa with studied dna c-value (from https://cvalues.science.kew.org/) among takhtajan’s floristic kingdoms (1986). genus species subspecies/variety phytogeographical region(s) acrostichum aureum neotropical, palaeotropical, australian adiantum aleuticum holarctic adiantum capillus-veneris holarctic,neotropical, palaeotropical, australian, holantarctic adiantum pedatum holarctic adiantum venustum holarctic alsophila spinulosa holarctic, palaeotropical amauropelta bergiana var. bergiana palaeotropical anemia collina neotropical anemia phyllitidis neotropical anemia rotundifolia neotropical anemia tomentosa neotropical angiopteris latipinna holarctic angiopteris lygodiifolia holarctic angiopteris pruinosa palaeotropical arthropteris orientalis palaeotropical asplenium achilleifolium neotropical asplenium adiantum-nigrum var. adiantum-nigrum holarctic, palaeotropical asplenium adulterinum holarctic asplenium aethiopicum subsp. tripinnatum palaeotropical asplenium aethiopicum subsp. dodecaploideum palaeotropical asplenium billotii holarctic asplenium boreale holarctic asplenium caucasicum holarctic asplenium ceterach holarctic asplenium cristatum neotropical asplenium cuneifolium holarctic asplenium dalhousiae holarctic asplenium daucifolium palaeotropical asplenium flabellifolium australian, holantarctic asplenium griffithianum holarctic, palaeotropical asplenium hallbergii neotropical asplenium hemionitis holarctic asplenium javorkeanum holarctic asplenium lividum palaeotropical asplenium marinum holarctic asplenium mauritiensis palaeotropical asplenium myriophyllum neotropical asplenium neolaserpitifolium palaeotropical asplenium nidus holarctic,neotropical, palaeotropical, australian asplenium obtusatum neotropical, palaeotropical, australian, holantarctic asplenium onopteris holarctic asplenium quadrivalens holarctic asplenium rhizophyllum holarctic asplenium richardii holantarctic asplenium ruta-muraria holarctic asplenium scolopendrium holarctic, holantarctic asplenium septentrionale holarctic asplenium subglandulosum australian, holantarctic asplenium tenerum compl palaeotropical 118 inês da fonseca simão et al. genus species subspecies/variety phytogeographical region(s) asplenium trichomanes holarctic, neotropical, palaeotropical, australian, holantarctic asplenium trichomanes subsp. quadrivalens holarctic, palaeotropical asplenium varians holarctic, palaeotropical asplenium víride holarctic asplenium viviparum palaeotropical asplenium xlolegnamense holarctic asplenium x-lucrosum holantarctic asplenium x-poscharskyanum holarctic athyrium filix-femina var. angustum holarctic azolla microphylla holarctic, neotropical blechnum microphyllum neotropical blechnum nudum australian, holantarctic blechnum spicant holarctic bolbitis heudelotii palaeotropical bolbitis singaporensis palaeotropical botrychium neolunaria holarctic botrychium alaskense holarctic botrychium boreale holarctic botrychium echo holarctic botrychium hesperium holarctic botrychium lanceolatum holarctic botrychium lunaria holarctic, australian botrychium matricariifolium holarctic botrychium michiganense holarctic botrychium minganense holarctic botrychium montanum holarctic botrychium pallidum holarctic botrychium pinnatum holarctic botrychium simplex holarctic botrychium spathulatum holarctic botrychium virginianum holarctic botrypus cf. virginianus holarctic, neotropical brainea insignis palaeotropical calochlaena dubia australian ceratopteris thalictroides holarctic, neotropical, palaeotropical, australian ceterach officinarum subsp. officinarum holarctic cheilanthes marantae holarctic cibotium barometz palaeotropical cibotium hawaiense palaeotropical cryptogramma crispa holarctic ctenitis sinii holarctic culcita macrocarpa holarctic cyathea crinita palaeotropical cyclosorus arbusculus palaeotropical cyclosorus asperum palaeotropical cyclosorus dentatus holarctic, palaeotropical cystopteris bulbifera holarctic cystopteris dickieana holarctic cystopteris fragilis agg. holarctic, neotropical, cape, holantarctic cystopteris tenuis holarctic danaea antillensis neotropical 1192c-values for pteridophytes from timor genus species subspecies/variety phytogeographical region(s) danaea kalevala neotropical danaea mazeana neotropical davallia denticulata var. denticulata palaeotropical, australian davallia tyermanii holarctic dendrolycopodium dendroideum holarctic dendrolycopodium obscurum holarctic dennstaedtia globulifera neotropical dennstaedtia wilfordii holarctic deparia acrostichoides holarctic deparia boryana holarctic, palaeotropical deparia japonica holarctic, palaeotropical dicksonia antarctica holarctic, australian dicranopteris linearis holarctic, neotropical, palaeotropical, australian, holantarctic diphasiastrum alpinum holarctic diphasiastrum digitatum holarctic diphasiastrum complanatum holarctic, neotropical, palaeotropical diphasiastrum tristachyum holarctic diplazium arborescens palaeotropical diplazium australe palaeotropical, australian, holantarctic diplazium proliferum palaeotropical, australian diplazium pycnocarpon holarctic diplopterygium bancroftii neotropical dipteris chinensis holarctic dracoglossum plantagineum neotropical drynaria heraclea palaeotropical dryopteris bernieri palaeotropical dryopteris carthusiana holarctic dryopteris clintoniana holarctic dryopteris cristata holarctic dryopteris cycadina holarctic, holantarctic dryopteris dilatata holarctic, holantarctic dryopteris filix-mas holarctic, neotropical, holantarctic dryopteris goldiana holarctic dryopteris intermedia holarctic dryopteris marginalis holarctic elaphoglossum aubertii palaeotropical elaphoglossum crinitum neotropical elaphoglossum hybridum neotropical, palaeotropical elaphoglossum lepervanchii palaeotropical equisetum arvense holarctic, holantarctic equisetum bogotense neotropical equisetum moorei holarctic equisetum fluviatile holarctic equisetum giganteum neotropical equisetum hyemale holarctic, neotropical, australian, holantarctic equisetum laevigatum holarctic, neotropical equisetum myriochaetum neotropical equisetum palustre holarctic equisetum pratense holarctic equisetum ramosissimum subsp. ramosissimum holarctic, palaeotropical equisetum scirpoides holarctic 120 inês da fonseca simão et al. genus species subspecies/variety phytogeographical region(s) equisetum sylvaticum holarctic equisetum variegatum holarctic gymnocarpium dryopteris holarctic gymnocarpium fedtschenkoanum holarctic gymnocarpium robertianum holarctic gymnosphaera podophylla holarctic, palaeotropical huperzia lucidula holarctic hymenophyllum badium cf holarctic, palaeotropical hymenophyllum sibthorpioides palaeotropical isoetes engelmannii holarctic isoetes lacustris holarctic lepisorus excavatus palaeotropical lindsaea ensifolia palaeotropical, australian llavea cordifolia neotropical lonchitis occidentalis palaeotropical loxsoma cunninghami holantarctic lycopodium annotinum holarctic lycopodium clavatum holarctic, neotropical, palaeotropical lycopodium dendroideum holarctic lycopodium obscurum holarctic lygodium japonicum holarctic, neotropical, palaeotropical, australian lygodium microphyllum holarctic, palaeotropical, australian lygodium volubile neotropical marattia purpurascens holarctic marsilea quadrifolia holarctic, neotropical, palaeotropical matteuccia struthiopteris var. pensylvanica holarctic megalastrum macrotheca neotropical mickelia nicotianifolia neotropical, palaeotropical microgramma percussa neotropical, palaeotropical microlepia speluncae neotropical, palaeotropical, australian microlepia strigosa holarctic, palaeotropical nephrolepis biserrata neotropical, palaeotropical, australian nephrolepis cordifolia ‘duffi’ holarctic, neotropical, palaeotropical, australian, holantarctic nephrolepis exaltata holarctic, neotropical, palaeotropical, australian oleandra neriiformis palaeotropical, australian onoclea orientalis holarctic onoclea sensibilis holarctic onychium lucidum holarctic, palaeotropical ophioglossum gramineum palaetropical, australian ophioglossum pendulum palaeotropical, australian ophioglossum petiolatum holarctic, palaetropical, holantarctic osmunda cinnamomea holarctic, neotropical osmunda claytoniana holarctic osmunda regalis var. spectabilis holarctic, neotropical paragymnopteris marantae holarctic paragymnopteris vestita holarctic pellaea atropurpurea holarctic, neotropical pellaea glabella subsp. glabella holarctic phegopteris connectilis holarctic phyllitis scolopendrium subsp. scolopendrium holarctic plagiogyria matsumureana holarctic 1212c-values for pteridophytes from timor genus species subspecies/variety phytogeographical region(s) platycerium coronarium palaeotropical pleopeltis macrocarpa neotropical, palaeotropical polyphlebium capillaceum neotropical polypodium australe holarctic polypodium cambricum holarctic polypodium glycyrrhiza holarctic polypodium interjectum holarctic polypodium scouleri holarctic polypodium virginianum holarctic polypodium vulgare holarctic, neotropical, cape, holantarctic polypodium vulgare x interjectum not defined polypodium x-font-queri holarctic polypodium x-mantoniae holarctic polypodium x-shivasiae holarctic polystichum acrostichoides holarctic psilotum nudum holarctic, palaeotropical, neotropical, australian, holantarctic pteridium aquilinum holarctic, neotropical, palaeotropical, australian pteridium revolutum palaeotropical pteridium subsp. caudatum var. arachnoideum neotropical pteridrys cnemidaria palaeotropical pteris croesus palaeotropical pteris linearis palaeotropical, neotropical pteris pseudolonchitis palaeotropical pteris vittata holarctic, neotropical, palaeotropical, holantarctic, australian ptisana salicina holantarctic, palaeotropical pyrrosia lingua holarctic, palaeotropical saccoloma domingense neotropical sadleria cyatheoides palaeotropical salvinia molesta holarctic, neotropical, palaeotropical, holantarctic, australian selaginella apoda holarctic, neotropical selaginella arenicola holarctic selaginella arizonica holarctic selaginella asprella holarctic selaginella bigelovii holarctic selaginella braunii holarctic selaginella cinerascens holarctic selaginella densa holarctic selaginella eremophila holarctic selaginella exaltata neotropical selaginella extensa neotropical selaginella flabellata neotropical selaginella hansenii holarctic selaginella helvetica holarctic selaginella involvens holarctic, palaeotropical selaginella kraussiana var. poulteri holarctic selaginella kraussiana holarctic, neotropical, holantarctic, palaeotropical, australian selaginella landii neotropical selaginella lepidophylla holarctic, neotropical selaginella leucobryoides holarctic selaginella martensii neotropical selaginella moellendorffii holarctic, holantarctic, palaeotropical 122 inês da fonseca simão et al. genus species subspecies/variety phytogeographical region(s) selaginella mutica holarctic selaginella oregana holarctic selaginella pallescens holarctic, neotropical selaginella peruviana neotropical selaginella pilifera neotropical selaginella pulcherrima neotropical selaginella rupestris holarctic selaginella rupincola holarctic selaginella selaginoides holarctic selaginella sellowii neotropical, holarctic selaginella tortipila holarctic selaginella uncinata holarctic, palaeotropical selaginella underwoodii holarctic selaginella vogelii palaeotropical selaginella wallacei holarctic selaginella watsonii holarctic selaginella weatherbiana holarctic selaginella willdenowii holarctic, neotropical, palaeotropical, australian selaginella wrightii holarctic, neotropical serpocaulon triseriale neotropical sphaeropteris lepifera neotropical, palaeotropical spinulum annotinum holarctic stenochlaena tenuifolia palaeotropical tectaria zeilanica palaeotropical thelypteris noveboracensis holarctic thelypteris palustris var. pubescens holarctic thyrsopteris elegans neotropical tmesipteris obliqua australian tmesipteris tannensis holantarctic todea barbara palaeotropical, australian, holantarctic trichomanes speciosum holarctic vandenboschia auriculata holarctic, palaeotropical vandenboschia davallioides palaeotropical vittaria lineata neotropical, palaeotropical woodsia alpina holartic woodsia ilvensis holartic woodsia pulchella holartic woodwardia fimbriata holartic woodwardia unigemata holarctic, palaeotropical caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 3 2022 firenze university press chromosome mapping of repetitive dnas in the picasso triggerfish (rhinecanthus aculeatus (linnaeus, 1758)) in family balistidae by classical and molecular cytogenetic techniques kamika sribenja1, alongklod tanomtong1, nuntaporn getlekha2,* chromosome number of some satureja species from turkey esra kavcı1, esra martin1, halil erhan eroğlu2,*, fatih serdar yıldırım3 l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations selma tabur1,*, nai̇me büyükkaya bayraktar2, serkan özmen1 characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques kristel ramírez-matadamas1, elva irene cortés-gutiérrez2, sergio moreno-limón2, catalina garcía-vielma1,* contributions of species rineloricaria pentamaculata (loricariidae:loricariinae) in a karyoevolutionary context a cius¹, ca lorscheider2, lm barbosa¹, ac prizon¹, ch zawadzki3, la borin-carvalho¹, fe porto4, alb portela-castro1,4 cadmium induced genotoxicity and antioxidative defense system in lentil (lens culinaris medik.) genotype durre shahwar1,2,*, zeba khan3, mohammad yunus khalil ansari1 biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts and evaluation of their biosafety potential denisa manolescu1,2, georgiana uță1,2,*, anca șuțan3, cătălin ducu1, alin din1, sorin moga1, denis negrea1, andrei biță4, ludovic bejenaru4, cornelia bejenaru5, speranța avram2 polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel (foeniculum vulgare mill.) egizia falistocco methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. sazada siddiqui*, sulaiman a. alrumman comparative study and genetic diversity in malva using srap molecular markers syamand ahmed qadir1, chnar hama noori meerza2, aryan mahmood faraj3, kawa khwarahm hamafaraj4, sherzad rasul abdalla tobakari5, sahar hussein hamarashid6,* nuclear dna 2c-values for 16 species from timor-leste increases taxonomical representation in tropical ferns and lycophytes inês da fonseca simão1, hermenegildo ribeiro da costa1,2,3, helena cristina correia de oliveira1,2, maria helena abreu silva1,2, paulo cardoso da silveira1,2,* nuclear dna content and comparative fish mapping of the 5s and 45s rdna in wild and cultivated populations of physalis peruviana l. marlon garcia paitan*, maricielo postillos-flores, luis rojas vasquez, maria siles vallejos, alberto lópez sotomayor identification of genetic regions associated with sex determination in date palm: a computational approach zahra noormohammadi1,*, masoud sheidai2, seyyed-samih marashi3, somayeh saboori1, neda moradi1, samaneh naftchi1, faezeh rostami1 comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(3): 13-18, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1660 caryologia international journal of cytology, cytosystematics and cytogenetics citation: esra kavcı, esra martin, halil erhan eroğlu, fatih serdar yıldırım (2022). chromosome number of some satureja species from turkey. caryologia 75(3): 13-18. doi: 10.36253/ caryologia-1660 received: may 17, 2022 accepted: february 12, 2022 published: april 5, 2023 copyright: © 2022 esra kavcı, esra martin, halil erhan eroğlu, fatih serdar yıldırım. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid ek: 0000-0002-5235-6979 em: 0000-0002-5484-0676 hee: 0000-0002-4509-4712 fsy: 0000-0003-4080-8488 chromosome number of some satureja species from turkey esra kavcı1, esra martin1, halil erhan eroğlu2,*, fatih serdar yıldırım3 1 necmettin erbakan university, faculty of science, department of biotechnology, konya, turkey 2 yozgat bozok university, faculty of science and arts, department of biology, yozgat, turkey 3 akdeniz university, faculty of education, department of science education, antalya, turkey *corresponding author: herhan.eroglu@bozok.edu.tr abstract. the genus satureja belonging to the lamiaceae family includes about 200 species, generally aromatic, distributed in the mediterranean basin. in genus satureja, the chromosomal data were reported from only 26 species. in this study, it is aimed to eliminate the deficiencies in the chromosomal data of satureja species, which are distributed in turkey, which is center of origin and diversity of the genus satureja. it was reported only one chromosome number (2n = 30), the first report for chromosome numbers of three taxa, the same chromosome count (excluding b-chromosomes) with previous report in only one species, and the new chromosome number in only one species. in conclusion, this study presented new data into the chromosomal records of genus satureja that might be useful for interpreting or understanding relationships among the species. in addition, dysploidy and polyploidy variations might probably have played an important role in speciation. in this regard, the results contributed to some missing data in satureja cytotaxonomy. keywords: satureja, chromosome, dysploidy, polyploidy, turkey. introduction the genus satureja l. belonging to the lamiaceae family includes about 200 species, generally aromatic, distributed in the mediterranean basin. satureja species are distributed in morocco, libya, saudi arabia, caucasus, iran, iraq and turkey, which are mostly mediterranean countries from europe, asia and north africa. turkey is center of origin and diversity of the genus satureja (harley et al. 2004; dirmenci et al. 2019). according to flora of turkey records, the genus satureja is represented by a total of 15 species, five of which are endemic. satureja species, popularly known as “pointed thyme” or “stone thyme”, have a wide range of uses in the food, pharmaceutical and cosmetic industries due to high amounts of thymol 14 esra kavcı, esra martin, halil erhan eroğlu, fatih serdar yıldırım and carvacrol (momtaz and abdollahi 2010; dirmenci et al. 2019). the dried leaves of satureja species are used as spice, food additive and herbal tea (babajafari et al. 2014). the genus satureja has been the focus of many studies due to its biological activities such as antimicrobial, antioxidant and anti-hiv-1. it has been stated that the rich essential oil (eg monoand sesquiterpenes) and phenolic content (eg phenolic acids, catechins, and flavonoids) of satureja species are responsible for these activities (eminağaoğlu et al. 2007; bektaş 2020). cytotaxonomy is a branch of taxonomy that uses karyological parameters to classify organisms. in cytota xonomy, chromosomal conf iguration is the most widely used parameter to understand the relationship between organisms. inference of species relationships is based on the assumption that closely related species share similar features in their chromosomal arrangement. by analyzing similarities and differences in chromosomes, karyotype evolution and species evolution can be reconstructed. the number, structure and behaviour of chromosomes are very valuable in taxonomy and the chromosome numbers (x, 2n) are the most commonly used characters (guerra 2008; eroğlu et al. 2020; martin et al. 2020; eroğlu et al. 2021). in genus satureja, the chromosomal data were reported from only 26 species. eighteen species were only diploid; however, they revealed two different basic numbers: x = 13 (2n = 26) and x = 15 (2n = 30). seven species were polyploid and revealed two different polyploidy levels: tetraploidy (2n = 4x = 24, 28, 44, and 60) and hexaploidy (2n = 6x = 48). the polyploid species revealed four different basic numbers such as x = 6, 7, 11, and 15. satureja sahendica bornm. had diploid and polyploid records. in addition, s. hortensis l. presented dysploidy, which is an alteration in basic number, generally by fusion, without the significant loss or gain of genetic material (shariat et al. 2013; irani et al. 2014; bordbar et al. 2021; chromosome counts database 2022; vozhdehnazari et al. 2022). in turkish satureja, the chromosomal data were reported from eight species, which were s. macrantha c.a. mey. (2n = 24), s. coerulea janka, s. cuneifolia ten., s. pilosa velen. s. thymbra l. (2n = 30), s. spinosa l. (2n = 30 + 2b), s. spicigera boiss. (2n = 44, 60), and s. hortensis (2n = 45-48) (shariat et al. 2013; irani et al. 2014; chromosome counts database 2022; vozhdehnazari et al. 2022). there was no record of the chromosome number of seven species, which were s. aintabensis p.h.davis, s. amani p.h.davis, s. boissieri hausskn. ex boiss., s. cilicica p.h.davis, s. icarica p.h.davis, s. parnassica heldr. & sartori ex boiss., and s. wiedemanniana lall. ex velen. therefore, the lack of some chromosomal reports from turkey, which is located in the mediterranean basin, may lead to some uncertainties in the cytotaxonomy. in this study, it is aimed to eliminate the deficiencies in the chromosomal data of satureja species, which are distributed in turkey, which is center of origin and diversity of the genus. materials and methods plant material within the scope of this study, s. aintabensis, s. boissieri, s. icarica, s. macrantha and s. spinosa distributed in different localities of turkey were examined by chromosome numbers. the examined plant samples were collected from their natural habitats and identified by prof. dr. tuncay dirmenci et al (figure 1). the collected plant samples were preserved in balıkesir university, necatibey faculty of education, department of biology education. the distribution regions and collection information are given in figure 2 and table 1. cytogenetic procedure the seeds of the collected plant samples were kept at -40°c for 1 month and then planted in petri dishes. the samples were kept in the dark at 4°c for 21 days, and at the end of 21 days, the samples placed in the climate cabinet were kept until the root tip tissue reached a few centimeters in length. germinated root tips were kept in α-monobromonaphthalene for 16 hours at 4°c for the first treatment. afterwards, root tips were fixed in 3:1 absolute alcohol:glacial acetic acid and stored in 70% alcohol in the refrigerator. the root tips were removed from the refrigerator, hydrolyzed in 1n hcl at room temperature for 10 minutes and stained with 2% aceto-orcein for 2 hours at room temperature. then, squash preparations were prepared with 45% acetic acid. after the preparations were frozen in liquid nitrogen, they were dried at room temperature and stabilized with depex medium (martin et al. 2018; eroğlu et al. 2021). ten metaphase plates were used for counting the somatic chromosomes of each species. after the mitotic chromosomes, which were well distributed, had good morphology, and were on the same plane, were detected, their photographs were taken at 1000× magnification with a camera attached to the microscope (olympus bx51). the chromosome photographs were analyzed using the image analysis system (bs200prop). 15chromosome number of some satureja species from turkey figure 1. habitat (1) and flowers (2) of satureja species. (a) s. aintabensis; (b) s. boissieri; (c) s. icarica; (d) s. macrantha; and (e) s. spinosa. scale bar, 10 cm for habitat (1) and 5 mm for flowers (2). figure 2. distribution map of the studied species in turkey. (a) s. aintabensis; (b) s. boissieri; (c) s. icarica; (d) s. macrantha; and (e) s. spinosa. 16 esra kavcı, esra martin, halil erhan eroğlu, fatih serdar yıldırım results figure 3 presented the mitotic metaphase chromosomes of five satureja species. the chromosome numbers of studied satureja species were given in table 2. in all species, the diploid number was 2n = 30, three of which were reported for the first time and the basic number was x = 15. in genus satureja, because the chromosomes were very small and the centromere region is unclear, detailed chromosomal measurements were not made. table 1. the collection information and localities of studied satureja species. species collection cite altitude date voucher number s. aintabensis gaziantep, samköy, behind dülükbaba promenade. 1000 m 03.09.2018 dirmenci 5210 & arabacı s. boissieri adıyaman, çelikhan, between yazıbaşı village and ulubaba mountain, 8th km. 1700 m 02.09.2018 dirmenci 5207 & arabacı s. icarica çanakkale, between gökçeada and aydıncık, 2nd-3rd km. 300 m 15.08.2018 dirmenci 5166 s. macrantha ardahan, between göle and şenkaya, 10th km. 1800 m 31.08.2018 dirmenci 5196 & arabacı s. spinosa muğla, fethiye, babadağ, above telpher. 1900 m 11.09.2018 dirmenci 5224 & yıldız figure 3. metaphase chromosomes of satureja species. (a) s. aintabensis; (b) s. boissieri; (c) s. icarica; (d) s. macrantha; and (e) s. spinosa. scale bar 10 µm. table 2. the chromosome numbers of studied satureja species. species x = basic number, 2n (ploidy level) s. aintabensis x = 15, 2n = 30 (diploid) s. boissieri x = 15, 2n = 30 (diploid) s. icarica x = 15, 2n = 30 (diploid) s. macrantha x = 15, 2n = 30 (diploid) s. spinosa x = 15, 2n = 30 (diploid) 17chromosome number of some satureja species from turkey discussion the genus satureja was represented by a total of 15 species in turkey. eleven turkish satureja whose chromosome numbers had been reported with present and previous studies were given in the table 3 for comparison. in the present study, the diploid number of all species was 2n = 30, three of which were reported for the first time: s. aintabensis, s. boissieri, and s. icarica. the chromosome number represented new cytotype in only one species, which was s. macrantha (2n = 30). vozhdehnazari et al. (2022) reported that the chromosome number of s. macrantha was 2n = 24. the chromosome number of s. spinosa agreed with the previous report excluding b-chromosomes. in turk ish satureja, f ive different chromosome numbers were recorded such as 2n = 24, 30, 44, 48, 60 and 2n = 30 was the most common diploid number. s. spinosa was the only species to have b-chromosomes. montmollin (1986) reported that the karyotype of s. spinosa was 2n = 30 + 2b, which were small supernumerary chromosomes other than a-chromosomes. b-chromosomes originated from the a-chromosomes and were a basic source of intraspecific variations of nuclear dna (heneida k et a l. 2019). although we obtained the same diploid number, we did not observe b-chromosomes. this was probably due to the locality difference. in table 3, satureja was a polybasic genus by x = 11, 12, 15 with ploidy levels of 2x and 4x. nine species were diploid with 2n = 2x = 30. s. hortensis and s. spinosa were polyploid, which revealed only one polyploidy level of tetraploidy (2n = 4x = 44, 48, and 60). a basic number of x = 15 dominated in reported satureja species (all species excluding s. hortensis in table 3), which were s. aintabensis, s. boissieri, s. coerulea, s. cuneifolia, s. icarica, s. macrantha (only in this study), s. pilosa, s. spicigera (only in this study), s. spinosa, and s. thymbra. in addition, the basic numbers of x = 11 and 12 were recorded. however, different basic numbers were reported, such as x = 6 for s. multiflora briq. and x = 7 for s. sahendica (krogulevich 1978; irani et al. 2014). in detecting karyotype evolution and speciation processes, basic chromosome number is one of the most important parameter. in genus satureja, basic number variations were probably caused by descending or ascending dysploidy and dibasic polyploidy. in table 3, nine species were diploid with 2x = 30 (81.82% of the species) and two species were polyploid with 4x = 44, 48, and 60 (18.18% of the species). polyploidy was probably one of the important mechanisms in the karyotype evolution of the genus, as it occurred at a non-negligible rate (chromosome counts database 2022) in the genus satureja. in the present study, it was reported only one chromosome number (2n = 30), the first report for chromosome numbers of three taxa, the same chromosome count (excluding b-chromosomes) with previous report in only one species, and the new chromosome number in only one species. in conclusion, this study presented new data into the chromosomal records of genus satureja that might be useful for interpreting or understanding relationships among the species. in addition, dysploidy and polyploidy variations might probably have played an important role in speciation. in this regard, the results contributed to some missing data in satureja cytotaxontable 3. the chromosome numbers of turkish satureja in present and previous studies. species (alphabetically) x = basic number, 2n (ploidy level) references observation s. aintabensis x = 15, 2n = 30 (diploid) present study first report s. boissieri x = 15, 2n = 30 (diploid) present study first report s. coerulea x = 15, 2n = 30 (diploid) chromosome count database 2022 previous report s. cuneifolia x = 15, 2n = 30 (diploid) chromosome count database 2022 previous report s. hortensis x = 12, 2n = 48 (tetraploid) 45-47 (probably dysploidy) chromosome count database 2022 previous report s. icarica x = 15, 2n = 30 (diploid) present study first report s. macrantha x = 15, 2n = 30 (diploid) x = 12, 2n = 24 (diploid) present study vozhdehnazari et al. 2022 new count s. pilosa x = 15, 2n = 30 (diploid) chromosome count database 2022 previous report s. spicigera x = 15, 2n = 60 (tetraploid) x = 11, 2n = 44 (tetraploid) shariat et al. 2013 irani et al. 2014 previous report s. spinosa x = 15, 2n = 30 (diploid) x = 15, 2n = 30 + 2b (diploid) present study chromosome count database 2022 b-chromosomes not observed s. thymbra x = 15, 2n = 30 (diploid) chromosome count database 2022 previous report 18 esra kavcı, esra martin, halil erhan eroğlu, fatih serdar yıldırım omy. however, all taxa should be investigated to elucidate the relationships between satureja species and the chromosomal data should be supported by molecular data. acknowledgments authors thank prof. dr. tuncay dirmenci (balıkesir university) for his contribution to the collection of plant specimens and mustafa koray şenova (hacettepe university) for his contribution to the germination of seeds. this work was supported by the [necmettin erbakan university research fund] under grant [number 191315001]. references babajafari s, nikaein f, mazloomi sm, zibaeenejad mj, zargaran a. 2014. a review of the benefits of satureja species on metabolic syndrome and their possible mechanisms of action. j evid based complementary altern med. 20:212–223. bektaş e. 2020. changes in essential oil composition, phenylalanine ammonia lyase gene expression and rosmarinic acid content during shoot organogenesis in cytokinin-treated satureja spicigera (c. koch) boiss. shoots. j plant biochem biotechnol. 29:450–460. bordbar f, mahani ze, mirtadzadini m. 2021. new chromosome counts in lamiaceae from flora of iran i. j appl biol sci. 15:310–317. chromosome counts database (ccdb, version 1.59). 2022. satureja [accessed 2022 march 4]. http://ccdb. tau.ac.il/ dirmenci t, yıldız b, öztekin m. 2019. türkiye florası için yeni bir tür kaydı: satureja metastasiantha rech. f. 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(convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(3): 3-11, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1731 caryologia international journal of cytology, cytosystematics and cytogenetics citation: kamika sribenja, alongklod tanomtong, nuntaporn getlekha (2022). chromosome mapping of repetitive dnas in the picasso triggerfish (rhinecanthus aculeatus (linnaeus, 1758)) in family balistidae by classical and molecular cytogenetic techniques. caryologia 75(3): 3-11. doi: 10.36253/ caryologia-1731 received: july 08, 2022 accepted: november 19, 2022 published: april 5, 2023 copyright: © 2022 kamika sribenja, alongklod tanomtong, nuntaporn getlekha. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. chromosome mapping of repetitive dnas in the picasso triggerfish (rhinecanthus aculeatus (linnaeus, 1758)) in family balistidae by classical and molecular cytogenetic techniques kamika sribenja1, alongklod tanomtong1, nuntaporn getlekha2,* 1 department of biology, faculty of science, khon kaen university, muang, khonkaen 40002, thailand 2 department of biology, faculty of science and technology, muban chombueng rajabhat university, chombueng, ratchaburi 70150, thailand *corresponding author. e-mail: nanthaphonket@mcru.ac.th abstract. this work presents the cytogenetic analysis conducted on the picasso triggerfish (rhinecanthus aculeatus (linnaeus, 1758)) from thailand. mitotic chromosomes were prepared from the anterior kidney. the cell suspensions were harvested by in vivo colchicine treatment. the present study includes the chromosomal investigation on r. aculeatus, using conventional (giemsa staining, ag-nor and c-banding) and molecular approaches (in situ mapping of five different repetitive dna classes including 18s rdna, 5s rdna, (ca)15, (ga)15 and (caa)10 as markers.) the results showed that r. aculeatus has karyotypes formed exclusively by telocentric chromosomes (44t; nf=44). the c-positive heterochromatic blocks are preferentially located in the centromeric and telomeric regions of some chromosomal pairs. the ag-nor sites occupy the interstitial position of the long arms of the largest telocentric pair (pair 1). the exclusive location of the major ribosomal sites in these pairs was confirmed by hybridization with 18s rdna probes. however, the 5s rdna genes are not located on 18s rdna-bearing chromosomes, but instead located exclusively in the subcentromeric region of pair 4. the mapping of (ca)15, (ga)15 and (caa)10 microsatellites are sparsely dispersed along all the chromosomes. the karyotype formula of r. aculeatus is 2n (44) = 44t. keywords: triggerfish, chromosome, repetitive sequences, fish cytogenetics. introduction the family balistidae belongs to order tetraodontiformes which includes the triggerfish of often brightly colored fish (nelson et al., 2016). around 40 species distributed in 12 genera are classified in this family (allen et al., 2017). triggerfish fishes are usually found in tropical and subtropical oceans throughout the world, with the greatest species richness in the indo-pacific. 4 kamika sribenja, alongklod tanomtong, nuntaporn getlekha the most abundance of species are found in relatively shallow, coastal habitats, especially at coral reefs (allen et al., 2017). in the present, several species from this family are popular in the marine aquarium trade. out of 40 described species of balistidae, only 15 species have been karyologically investigated: balistapus undulates, sufflamen fraenatus (takai and ojima, 1987), balistes capriscus (vitturi et al., 1988), balistes carolinensis (vitturi et al., 1992; thode et al., 1994), balistes vetula (gustavo and molina, 2005), balistoides conspicillus (takai and ojima, 1987; gustavo and molina, 2004), balistoides viridescens (takai and ojima, 1988), pseudobalistes flavimarginatus, rhinecanthus verrucosus, sufflamen chrysopterus (arai and nagaiwa, 1976), rhinecanthus aculeatus (arai and nagaiwa, 1976; kitayama and ojima, 1984), rhinecanthus echarpe (kitayama and ojima, 1984), melichthys niger (gustavo and molina, 2005) and melichthys vidua, odonus niger (kitayam and ojima, 1984). the members of the family balistidae have 2n ranging from 40 to 46, and most species have the karyotype present as acrocentric and telocentric chromosomes except b. viridescens and p. flavimarginatus, which are comprised of metacentric and submetacentric chromosomes (table 1). the study aims to investigate the evolutionary events associated with the chromosomal diversification in the picasso triggerfish (rhinecanthus aculeatus). the chromosomal investigation was conducted by obtaining the standard karyotype and idiogram using conventional (giemsa staining, ag-nor and c-banding) and molecular analyses to identify the chromosomal patterns and organization of five classes of repetitive dnas [18s rdna, 5s rdna, (ca)15, (ga)15, and (caa)10]. since there were only three previous cytogenetic studies of the genus rhinecanthus showing a diploid chromosome number of 2n=44 (arai and nagaiwa, 1976; kitayama and ojima, 1984), the results obtained from this study will increase our basic knowledge of the cytogenetics of r. aculeatus, which could form the basis for future research and support taxonomy of genus rhinecanthus. material and methods specimens collected and conventional methods cytogenetic analyses were conducted on the picasso triggerfish, rhinecanthus aculeatus (4 males and 4 females) from thailand gulf (figure 1). the specimens were caught using a hand-net, placed in sealed plastic bags containing oxygen and clean water and transported to the research station. the experiments followed ethical protocols and table 1. cytogenetic reviews of the family balistidae (8 genera). no. subfamily/species 2n nf nors formula references 1 balistapus undulates 42 42 2 42a/t takai and ojima (1987) 2 balistes capriscus 44 44 2 44t vitturi et al. (1988) 3 b. carolinensis 44 44 2 44t vitturi et al. (1992) 44 44 2 44t thode et al. (1994) 4 b. vetula 44 44 2 44t gustavo and molina (2005) 5 balistoides conspicillus 44 44 2 44t takai and ojima (1987) 44 44 2 44t gustavo and molina (2004) 6 b. viridescens 44 48 2 2m+2sm+40a/t takai and ojima (1988) 44 60 3 2m+14a+28t supiwong et al. (2013) 7 melichthys niger 40 40 2 40t gustavo and molina (2005) 40 40 2 40a/t de lima et al. (2011) 8 m. vidua 40 40 2 40a/t kitayama and ojima (1984) 9 odonus niger 42 – – 42a/t kitayama and ojima (1984) 10 pseudobalistes flavimarginatus 44 – – 2m+42a/t arai and nagaiwa (1976) 11 rhinecanthus aculeatus 44 44 2 44t arai and nagaiwa (1976) 44 44 2 44t kitayama and ojima (1984) 12 r. echarpe 44 – 2 44a/t kitayama and ojima (1984) 13 r. verrucosus 44 44 2 44t arai and nagaiwa (1976) 14 sufflamen chrysopterus 46 46 – 46a/t arai and nagaiwa (1976) 15 s. fraenatus 46 46 2 46a/t takai and ojima (1987) remarks: 2n = diploid chromosome number, nf = fundamental number (number of chromosome arm), m = metacentric, sm = submetacentric, a = acrocentric, t = telocentric chromosome, nors = nucleolar organizer regions and – = not available. 5chromosome mapping of repetitive dnas in the picasso triggerfish anesthesia with clove oil prior to sacrificing the animals to minimize suffering. the process was approved by the ethics committee of khon kaen university and by the rgj committee under no. phd/k0081/2556. mitotic chromosomes were obtained from cell suspensions of the anterior kidney, using the conventional air-drying method. the c-banding method was also employed to detect the distribution of c-positive heterochromatin and silver staining to detect the ag-nor location on chromosomes. the specimens were deposited in the fish collection of the cytogenetic laboratory, department of biology, faculty of science, khon kaen university. chromosome probes and fish experiments two tandemly arrayed dna sequences isolated from the genome of an erythrinidae fish species, hoplias malabaricus, were used as probes. the first probe contained a 5s rdna repeat and included 120 base pairs (bp) of the 5s rrna transcribed gene and 200 bp of the non-transcribed spacer (nts) sequence. the second probe contained a 1400 bp segment of the 18s rrna gene obtained via pcr from the nuclear dna. the 5s and 18s rdna probes were cloned into plasmid vectors and propagated in dh5a escherichia coli competent cells (invitrogen, san diego, ca, usa). the 5s and 18s rdna probes were labeled with spectrum green-dutp and spectrum orange-dutp, respectively, using nick translation according to the manufacturer’s recommendations (roche, mannheim, germany). the microsatellites (ca)15, (ga)15, and (caa)10 were synthesized. these sequences were directly labeled with cy3 at the 5’ terminus during synthesis by sigma (st. louis, mo, usa). fluorescence in situ hybridization (fish) was performed under high stringency conditions (yano et al., 2017). metaphase chromosome slides were incubated with rnase (40 μg/ml) for 1.5 h at 37°c. after the denaturation of the chromosomal dna in 70% formamide/2x ssc at 70°c for 4 min, 20 µl of the hybridization mixture (2.5 ng/μl probes, 2 μg/μl salmon sperm dna, 50% deionized formamide, 10% dextran sulphate) was dropped on the slides, and the hybridization was performed overnight at 37°c in a moist chamber containing 2x ssc. the first post-hybridization wash was performed with 2x ssc for 5 min at 65°c, and a final wash was performed at room temperature in 1x ssc for 5 min. finally, the slides were counterstained with dapi and mounted in an antifade solution (vectashield from vector laboratories). image processing approximately 20 metaphase spreads were analyzed to confirm the diploid chromosome number, karyotype structure and fish results. images were captured using an olympus bx50 microscope (olympus corporation, ishikawa, japan) with coolsnap and the image pro plus 4.1 software (media cybernetics, silver spring, md, usa). chromosomes were classified according to their arm ratios as metacentric (m), submetacentric (sm), acrocentric (a) or telocentric (t). results the picasso triggerfish (rhinecanthus aculeatus) have 2n=44. its karyotype is formed exclusively by telocentric chromosomes (44t) and a fundamental number (nf=44) (figure 2). the c-positive heterochromatic blocks are preferentially located in the centromeric regions, with some pairs exhibiting blocks in the telomeric ones (figure 2). the ag-nor sites are located in the interstitial region of the long arms of the largest telocentric pair (pair 1), the exclusive location of major ribosomal sites in these regions was confirmed by in situ hybridization with 18s rdna probes (figure 2). however, the 5s rdna genes are not located on 18s rdnabearing chromosomes, but instead located exclusively in the subcentromeric region of pair 4, while the 18s rdna sites are instead located on the interstitial position of the long arms of pair 1 (figure 2). the chromosomal mapping of all microsatellite sequences indicates a weak and dispersed distribution, without preferential accumulations in any of the chromosomal pairs (figure 3). the (ca)15 (ga)15 and (caa)10 figure 1. general characteristic of rhinecanthus aculeatus, its respective collection sites in the indian (andaman sea) and pacific oceans (gulf of thailand). 6 kamika sribenja, alongklod tanomtong, nuntaporn getlekha microsatellites are sparsely dispersed in most chromosomes though they can still form conspicuous clusters. they however exhibit less defined clusters in some chromosome pairs (figure 3). these clusters occupy the centromeric regions of chromosomes but at rather low frequency. for all the chromosomal markers, no differential hybridization patterns were detected between males and females. figure 2. mataphase and karyotypes of rhinecanthus aculeatus arranged from conventionally giemsa-stained, ag-stained (highlighted in the boxes), c-banded and after fluorescence in situ hybridization with 5s and 18s rdna probes. bar 5 μm. 7chromosome mapping of repetitive dnas in the picasso triggerfish the idiogram of r. aculeatus represents gradually declining length of the chromosomes (figure 4). the karyotype is notably attributed solely to telocentric chromosomes. the karyotype formula of r. aculeatus is as follow: 2n (44) = 44t figure 3. chromosomal mapping of diand tri-nucleotide microsatellites in the chromosomes of rhinecanthus aculeatus by fluorescence in situ hybridization. the general distribution pattern of (ca)15, (ga)15 and (caa)10 microsatellites is mainly diffuse, with the occurrence of few conspicuous clusters in some centromeric and chromosome arm regions. bar = 5 μm. 8 kamika sribenja, alongklod tanomtong, nuntaporn getlekha discussions karyotype uniformity among rhinecanthus species cytogenetic analyses were conducted on rhinecanthus aculeatus from the gulf of thailand (indo-pacific). the 2n of r. aculeatus is 44 chromosomes in all specimens, with karyotypes predominantly formed by telocentric chromosomes (figure 2, 3 and table 2). however, considerable cytogenetic research has been conducted on a number of species in the family balistidae (table 1). the karyotype of rhinecanthus aculeatus is 44t; this finding is consistent with that of other species in the genera rhinecanthus, balistes, and balistoides, particularly balistes capriscus, b. carolinensis, b. vetula, balistoides conspicillus, rhinecanthus aculeatus, r. echarpe, and r. verrucosus, which still have 2n=44t. it suggests that even after speciation, their karyotypes remain conserved. although b. viridescens and pseudobalistes flavimarginatus have the same number of chromosomes (2n=44) with above species but exhibit an asymmetrical karyotype due to both species are the high variability of chromosomal rearrangements and their higher adaptive divergence. moreover, like all other species in the family balistidae, in which the morphologically differentiated sex chromosome could not be observed (arai and nagaiwa, 1976; kitayama and ojima, 1984). in addition, rhinecanthus species exhibited karyotypes which are broadly similar in structural patterns, with all of them displaying 2n = 44 and a high number of telocentric chromosomes. these characteristics present in all of the rhinecanthus species analyzed so far (arai and nagaiwa, 1976; kitayama and ojima, 1984; montanari et al. 2016; present study). chromosome markers of r. aculeatus the only one pair which bear ag-nor/18s rdna sites are useful chromosomal markers shared among the rhinecanthus species. the result here is also similar to the chromosome bearing nucleolar organizer region in previous studies (table 1) except balistoides viridescens that found tree nors (supiwong et al., 2013) this suggests that this event may be related to chromosomal change during evolution. furthermore, the interstitial region of the largest telocentric chromosome pair 1 of r. aculeatus showed clearly observable nucleolar organizer regions. this is quite consistent with the report by de lima et al. (2011) on the karyotype of melichthys niger in the same family. their study reported the presence of a conspicuous secondary constriction in the interstitial position on the long arm of the chromosome pair no. 2 which was, corresponding to the nucleolar organizer regions, identified by ag-nor sites and by in situ hybridization with an 18s rdna ribosomal probe. normally, most fishes have only one pair of nors on chromosomes. only some fishes have more than two nors, which may be caused by the translocation between some parts of the chromosomes that have nor and another chromosome (sharma et al., 2002). the present study shows that the species analyzed presents nor site on a single chromosome pair. this is considered a simple isomorphic condition in fish (almeida-toledo, 1985). another peculiar cytogenetic aspect of tetraodontiformes is the small quantity of heterochromatic regions, localized in telomeric or centromeric positions on most of the chromosome pairs (supiwong et al., 2013). organization of repetitive dnas in the chromosomes of r. aculeatus the c-positive heterochromatins in the chromosomes of r. aculeatus are distributed in centromeric and telomeric positions in most of the chromosomes (figure 2). this recurring distribution pattern is similar to those reported for species of other balistidae genera, such as melichthys (de lima et al., 2011). the 18s rdna sites are equally located on the interstitial position of pair 1, whereas 5s rdna sites occur in the subcentromeric region of pair 4. the non-syntenic organization of these genes is frequent and it could be a plesiomorphic condition in balistidae. this is the first report of the presence of microsatellite sequences in the heterochromatin of r. aculeatus which show recognizable organizational patterns. figure 4. standardized idiogram showing lengths and shapes of chromosomes of rhinecanthus aculeatus (2n=44) by conventional staining and ag-nor banding techniques. the arrow indicates nucleolarnucleolar organizer regions. 9chromosome mapping of repetitive dnas in the picasso triggerfish the (ca)15 (ga)15 and (caa)10 microsatellites present a weak and diffuse distribution on all chromosomes, but they also present a small number of conspicuous clusters characterized by intense signs in some parts of chromosomes (figure 3). thus, this data is useful for comparing the phylogenetic proximity of this genus that may share the same distribution pattern of the microsatellite sequences which points to independent evolutionary pathways, constituting homoplastic chromosomal characters. however, since these sequences are subject to high rates of change, their distribution may show marked evolutionary differentiation (cioffi et al., 2011; molina et al., 2014a; 2014b). in fact, the organization of microsatellite sequences demonstrates the particular arrangements that repetitive dnas can be achieved in different species. chromosome evolution of the family balistidae chromosomal rearrangements represent the main cause of karyotype diversification among several perciformes species (arai, 2011; molina and galetti, 2002). the different balistidae species underwent an extremely diversified karyotype evolution, considering the numerical and structural aspects of their complements, with diploid chromosome number varying from 2n=40 to 46, and marked differences in the nf that varied from 40 to 60, possibly due to the occurrence of pericentric inversions (getlekha et al., 2018). analyses performed highlight the combined importance of the different chromosome rearrangements in the evolutionary modelling of their karyotypes, such as centric fission fusion, and especially pericentric inversions (getlekha et al., 2016a; 2016b). the family balistidae has 2n values lower than 2n=48 with most of their representatives presenting acrocentric and telocentric chromosomes. this karyotypic pattern was also observed in the present study in r. aculeatus (2n=44). the origin of the reduced diploid chromosome numbers in these species seems to be centric fissions but chromosome lost in tandem, which seems to be common in other species of the family (arai and nagaiwa 1976; marques et al., 2016). table 2. mean length of short arm chromosome (ls), length long arm chromosome (ll), length total arm chromosome (lt), relative length (rl), centromeric index (ci) and standard deviation (sd) of rl, ci from 20 metaphase cells of the male and female the picasso triggerfish (rhinecanthus aculeatus), 2n=44. chromosome pair ls ll lt rl±sd ci±sd chromosome type 1* 0.00 2.60 2.60 0.070±0.009 1.000±0.000 telocentric 2 0.00 2.20 2.20 0.059±0.004 1.000±0.000 telocentric 3 0.00 2.06 2.06 0.056±0.005 1.000±0.000 telocentric 4 0.00 2.04 2.04 0.055±0.004 1.000±0.000 telocentric 5 0.00 1.97 1.97 0.052±0.003 1.000±0.000 telocentric 6 0.00 1.93 1.93 0.051±0.003 1.000±0.000 telocentric 7 0.00 1.90 1.90 0.051±0.005 1.000±0.000 telocentric 8 0.00 1.86 1.86 0.049±0.003 1.000±0.000 telocentric 9 0.00 1.75 1.75 0.047±0.003 1.000±0.000 telocentric 10 0.00 1.74 1.74 0.047±0.004 1.000±0.000 telocentric 11 0.00 1.72 1.72 0.046±0.005 1.000±0.000 telocentric 12 0.00 1.71 1.71 0.045±0.003 1.000±0.000 telocentric 13 0.00 1.68 1.68 0.044±0.003 1.000±0.000 telocentric 14 0.00 1.61 1.61 0.043±0.003 1.000±0.000 telocentric 15 0.00 1.59 1.59 0.042±0.003 1.000±0.000 telocentric 16 0.00 1.55 1.55 0.041±0.003 1.000±0.000 telocentric 17 0.00 1.45 1.45 0.038±0.003 1.000±0.000 telocentric 18 0.00 1.40 1.40 0.036±0.005 1.000±0.000 telocentric 19 0.00 1.40 1.40 0.036±0.006 1.000±0.000 telocentric 20 0.00 1.30 1.30 0.034±0.006 1.000±0.000 telocentric 21 0.00 1.19 1.19 0.030±0.007 1.000±0.000 telocentric 22 0.00 1.10 1.10 0.028±0.007 1.000±0.000 telocentric remark: * nor-bearing chromosome. 10 kamika sribenja, alongklod tanomtong, nuntaporn getlekha conclusion based on the chromosome study of the picasso triggerfish (r. aculeatus) using conventional analyses (giemsa staining, ag-nor and c-banding) and molecular analysis (in situ mapping of five different repetitive dna classes including 18s rdna, 5s rdna, (ca)15, (ga)15 and (caa)10 as markers), this research can verif y diploid chromosome, fundamental number and distribution patterns of microsatellites on the chromosomes. the results show that r. aculeatus has 2n=44 with predominantly telocentric chromosome. the fundamental number (nf) was 44. the c-positive heterochromatic blocks are preferentially located in the centromeric and telomeric regions of some chromosomal pairs. the ag-nors sites were located on the interstitial region of long arms of the telocentric chromosome pair 1. the exclusive location of the major ribosomal sites in these pairs was confirmed by in situ hybridization with 18s rdna probes. however, the 5s rdna genes are not located on 18s rdna-bearing chromosomes, but instead located exclusively in the subcentromeric region of pair 4. the mapping of (ca)15, (ga)15 and (caa)10 microsatellites are sparsely dispersed along all the chromosomes. the idiogram of r. aculeatus represents gradually declining length of the chromosomes. the karyotype is notably attributed solely to telocentric chromosomes like in common ancestor of marine fish, the high ability of distribution results in high gene flow and low chromosome evolution. the karyotype formula of r. aculeatus is 2n (44) = 44t. up to the present, there are 3 species of the genus rhinecanthus that were cytogenetically analyzed. rhinecanthus species provides remarkable karyotype features for chromosomal and genetic conservatism discussion. further studies of other species as well as additional information from molecular chromosome analyses are expected to explain the karyotype pattern and chromosome evolution in these fishes. references allen, g.r., erdmann, m.v. & purtiwi, p.d. 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(ed) springer, berlin, pp. 429–444. caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 3 2022 firenze university press chromosome mapping of repetitive dnas in the picasso triggerfish (rhinecanthus aculeatus (linnaeus, 1758)) in family balistidae by classical and molecular cytogenetic techniques kamika sribenja1, alongklod tanomtong1, nuntaporn getlekha2,* chromosome number of some satureja species from turkey esra kavcı1, esra martin1, halil erhan eroğlu2,*, fatih serdar yıldırım3 l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations selma tabur1,*, nai̇me büyükkaya bayraktar2, serkan özmen1 characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques kristel ramírez-matadamas1, elva irene cortés-gutiérrez2, sergio moreno-limón2, catalina garcía-vielma1,* contributions of species rineloricaria pentamaculata (loricariidae:loricariinae) in a karyoevolutionary context a cius¹, ca lorscheider2, lm barbosa¹, ac prizon¹, ch zawadzki3, la borin-carvalho¹, fe porto4, alb portela-castro1,4 cadmium induced genotoxicity and antioxidative defense system in lentil (lens culinaris medik.) genotype durre shahwar1,2,*, zeba khan3, mohammad yunus khalil ansari1 biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts and evaluation of their biosafety potential denisa manolescu1,2, georgiana uță1,2,*, anca șuțan3, cătălin ducu1, alin din1, sorin moga1, denis negrea1, andrei biță4, ludovic bejenaru4, cornelia bejenaru5, speranța avram2 polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel (foeniculum vulgare mill.) egizia falistocco methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. sazada siddiqui*, sulaiman a. alrumman comparative study and genetic diversity in malva using srap molecular markers syamand ahmed qadir1, chnar hama noori meerza2, aryan mahmood faraj3, kawa khwarahm hamafaraj4, sherzad rasul abdalla tobakari5, sahar hussein hamarashid6,* nuclear dna 2c-values for 16 species from timor-leste increases taxonomical representation in tropical ferns and lycophytes inês da fonseca simão1, hermenegildo ribeiro da costa1,2,3, helena cristina correia de oliveira1,2, maria helena abreu silva1,2, paulo cardoso da silveira1,2,* nuclear dna content and comparative fish mapping of the 5s and 45s rdna in wild and cultivated populations of physalis peruviana l. marlon garcia paitan*, maricielo postillos-flores, luis rojas vasquez, maria siles vallejos, alberto lópez sotomayor identification of genetic regions associated with sex determination in date palm: a computational approach zahra noormohammadi1,*, masoud sheidai2, seyyed-samih marashi3, somayeh saboori1, neda moradi1, samaneh naftchi1, faezeh rostami1 comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(4): 15-24, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1922 caryologia international journal of cytology, cytosystematics and cytogenetics citation: basoz sadiq muhealdin, sahar hussein hamarashid, fairuz ibrahim ali, nakhshin omer abdulla, syamand ahmad qadir (2022). studying some morphological responses of stevia (stevia rebaudiana bertoni) to some elicitors under water deficiency. caryologia 75(4): 15-24. doi: 10.36253/ caryologia-1922 received: november 08, 2022 accepted: december 14, 2022 published: april 28, 2023 copyright: © 2022 basoz sadiq muhealdin, sahar hussein hamarashid, fairuz ibrahim ali, nakhshin omer abdulla, syamand ahmad qadir. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. studying some morphological responses of stevia (stevia rebaudiana bertoni) to some elicitors under water deficiency basoz sadiq muhealdin1, sahar hussein hamarashid1,*, fairuz ibrahim ali1, nakhshin omer abdulla1, syamand ahmad qadir2 1 agricultural project management department, technical college of applied science halabja, sulaimani polytechnic university, iraq 2 medical laboratory techniques department/halabja technical institute, research center/ sulaimani polytechnic university, sulaymaniyah, iraq *corresponding author. e-mail: sahar.rashid@spu.edu.iq abstract. this research evaluated the effect of foliar spraying of different elicitors on modulating the effect of water stress on the stevia. the experimental design was the split-plot based on a randomized complete block design with three repetitions. experimental treatments included different irrigation regimes (90% fc, 65% fc, and 40% fc) and foliar application of different elicitors (control, chitosan, salicylic acid, and melatonin). in this study, the content of chlorophyll a and b was reduced by intensifying the water deficit stress. also, the highest content of the two pigments was allocated to the treatment of melatonin application. in the present study, melatonin foliar application under 90% fc irrigation conditions had the highest plant height, leaf area index, biomass, and carotenoid content, moreover, the highest content of proline, phenol, dpph, rebaudioside a, and steviosid was assigned to melatonin foliar application treatment under 40% fc irrigation conditions. results revealed, although water stress reduced plant height, leaf area index, and plant biomass, the application of melatonin and salicylic acid under different irrigation conditions moderated the effect of water stress on these traits. application of melatonin and salicylic acid under water deficit stress conditions also increased the content of proline, phenol, dpph, rebaudioside, and steviosid. keywords: drought stress, enzyme activities, foliar spraying, pigment. introduction about 230-220 species have been recorded for stevia, one of the most economically important species is s. rebaudian (al hassan et al 2017). the distinguishing feature of s. rebaudiana (stevia) from other species is the relatively high production of non-toxic and non-nutritive diterpenoid anticorn glycosides in the leaves of this species, the sweetness of which is 300 times of sucrose (aghighi shaverdi et al., 2018). consumers’ desire for a natural zero-calorie sweetener has made the cultivation, production, and extraction of glycosides in this plant attractive (aghighi shaverdi et al., 2018). among the glycosides in 16 basoz sadiq muhealdin et al. stevia leaves and tissues, the most abundant glycosides are rebaudioside a (reb-a) and steviosid (arnon 1949) drought is known as one of the most destructive stresses that affect the growth, development, and reproduction of plants (bi et al 2021). a typical effect of water deficit is to cause oxidative damage through the widespread accumulation of reactive oxygen species (ros) (duan et al., 2022). ros overproduction leads to lipid peroxidation, enzyme inactivation, impaired protein structure / function, and nucleic acid damage (duncan 1955). plants, in turn, use enzymatic and non-enzymatic antioxidant systems to prevent the accumulation of ros (duncan 1955). some stimulants of biological origin or eustress or with appropriate dosage and length, not only activate chemical defense in plants, but also increase plant productivity (elizabeth abreu and munné-bosch 2008). one of the purposes of plant physiologists is to identify and introduce substances that improve the resistance of plants to biotic and abiotic stresses. salicylic acid is one of these substances. salicylic acid treatment with appropriate dose has increased resistance to environmental stresses in different species. regulation of tougher pressure and activation of the antioxidant system are the mechanisms of salicylic acid in improving water shortage stress resistance (eraslan et al., 2007). another substance that plays a role in modulating environmental stresses is chitosan. it is a natural polymer derived from chitin, which is used as a biological elicitor in agriculture (efsa 2010). chitosan treatment enhances photosynthesis and stomatal closure by aba synthesis (guo et al., 2022; 2021). this substance increases antioxidant enzymes through the h2o2 and no signaling pathways and induces the produces sugars, amino acids, organic acids, and other metabolites needed to signal stress, osmotic balance, and energy metabolism under unfavorable environmental conditions (gao et al., 2016). another compound that increases plant resistance to biological and non-biological stresses is melatonin (n-acetyl-5-methoxytryptamine) (janda et al., 1999; meng et al., 2014; ma et al., 2018; porra et al., 1989; peng et al., 2021; su et al., 2019; sun et al., 2021). a wide range of physiological reactions has been attributed to melatonin, which can moderate the adverse effects of environmental stresses such as drought and salinity on plants. melatonin has an antioxidant activity and can detoxify ros in cells (karimi et al., 2015). melatonin can increase photosynthetic capacity by increasing the absorption of water and nutrients and increasing the expression of genes associated with mitogen-activated protein kinases (li et al., 2021). it has been reported that the use of melatonin under drought stress conditions prevents the accumulation of abscisic acid in the cell and has a synergistic effect with cytokinin (lehmann et al., 2010; liang et al., 2019; mahajanab et al., 2021 ). therefore, in the present study, we investigate the role of chitosan, salicylic acid, and melatonin on the physiological and biochemical responses of stevia under water deficit stress conditions. materials and methods this experiment was carried out in the two cropping years of 2019 and 2020 as a split-plot based on a randomized complete block design with three replications in a research farm in the city of sulaymaniyah located in the north of iraq. the experimental area was located at 45°11’ n; 45°58’ e longitude and altitude, respectively, 690 m above sea level. the average annual rainfall in the experimental area was about 250 mm and the average maximum and minimum temperatures were 22 and 9 degrees, respectively. the soil properties of the test site are listed in table 1. experimental treatments included different irrigation regimes (90% fc, 65% fc, and 40% fc) and foliar application of different elicitor (control, chitosan (2 gl-1), salicylic acid (100 mg l-1), and melatonin (100 μm), which were assigned to main and sub-plots, respectively. table 1. soil physical and chemical properties of the experimental site. physical properties value unit chemical properties value unit sand 133.6 g kg-1 organic mater 20 g kg-1 silt 244.3 g kg-1 ph 7.57 clay 622.1 g kg-1 ece 1.4 ds m-1 soil texture clay total nitrogen 20 mg kg-1 bulk density 1200 kg m-3 phosphors (soluble) 19 mg kg-1 field capacity (33 k pa) 320 g kg-1 potassium (soluble) 13.7 meq kg-1 wilting point (1500 k pa) 188 g kg-1 calcium (soluble) 7.5 meq kg-1 17studying some morphological responses of stevia (stevia rebaudiana bertoni) to some elicitors under water deficiency the seedlings obtained by tissue culture were initially cultivated in peat moss medium to select the wellestablished plantlets. after three weeks, the uniform seedlings were trans-planted into soil in may each year. in this experiment, the dimensions of each plot were considered 2×2.5 m. each plot also included four rows of crops. in this study, the distance between the rows was 50 and the distance between the plants was 30 cm. the first two weeks the soil moisture was maintained within the range of field capacity and, then, the irrigation treatments were applied as 90, 65, and 40% of field capacity (fc). the soil moisture content was measured using the gravimetric method. irrigations for each plat were conducted to replenish 100% of soil field capacity. chitosan, salicylic acid, and melatonin were sprayed three times after 30, 40, and 50 days from transplanting in both years. harvesting was done 62 days after transplanting the seedlings to the field. in the first stage, the leaves and stems were separated and then weighed. evaluating morphological characteristics at the time of harvest, the plants in each plot were carefully removed from the soil and the roots of each sample were washed to remove soil residues. stem length, leaf area index, and biological yield of each sample were measured. leaf area index was calculated according to formula (1), in which la leaf area and lg land area were occupied lai = la/lg photosynthetic pigments 80% of acetone extract with adsorption readings at 663, 646, and 440 nm was used to measure the concentration of photosynthetic pigments according to arnon’s (18) method. in addition to quantify the measurements, porra et al. (19) method was used chlorophyll a = 12.25×a663–2.55×a646 chlorophyll b = 20.31×a646–4.91×a663 carotenoids = 4.69×a440–0.267× (a663×a646) pigment content was expressed as mg/gdw. proline content to determined the proline content. 50 mg of leaf dry matter was homogenized in 5 ml of ethanol: water mixture (60:40) and the homogenized solution was incubated for 24 h at +4 °c. after centrifugation at 10,000 rpm, the supernatant was collected. in the next step, 100 ml of ninhydrin acid 1% was mixed with 500 μl of supernatant of the extracted solution. the samples (reaction mixture) were placed in a water bath at 95 °c for 20 min and, then, cooled to the room temperature. after cooling, the reaction absorbance was measured at 520 nm and using proline as standard, the proline content of the samples was measured. the proline content of the samples was expressed based on mg proline / g fresh weight zheng et al. (2018). secondary metabolite analysis to extract phenol, plant samples were dried and, then, macerated in 80% methanol ((hplc grade). in the next step, the samples were incubated for 24 h at 4 °c. the incubated samples were centrifuged at 2000 rpm for 15 min and the supernatants of the extracts were collected for further analysis. finally, wolf et al.’s (20) method was used to analyze the phenol content. in this method, folin-ciocalteu reagent and gallic acid were used as the standard. the total antioxidant capacity was determined by the dpph (2,2-diphenyl-1-picrylhydrazyl) assay according to karimi et al. (21). analysis of steviol glycosides to estimate the glycoside content of the leaves, at the time of harvest, the leaves in each plot were randomly selected and dried at 40 °c in an air oven. then, 100 g of the powdered leaf sample was placed in a 25 ml flask containing 10 ml of methanol and filtered for 24 hours. the reduced pressure filter was vacuum dried and, then, defatted with n-hexane. afterwards, the moving phase was used to solve the samples (hplc grade-acetonitrile: water in the ratio 1:1). filters with pores of 0.22 μm were used to re-filter the samples. filtered samples were used to determine the content of stevoside, reb-a by liquid chromatography-mass spectrometry (lcms-shimadzu, 2020 system) (22). standard curves with the samples were used to quantify glycoside content; these curves are used for standard stevoside and reb-a samples (22). statistical analysis analysis of variance was performed using sas software, also the comparison of the mean of treatments with the duncan (1955) method was performed at a level of 5% probability. results results showed the effect of irrigation regimes on all the traits was significant at the level of 1% probability. 18 basoz sadiq muhealdin et al. a significant difference was detected among the different treatments in terms of the effect on plant height, leaf area index, biological yield, proline, chlorophyll a, chlorophyll b, phenol, rebaudioside a, and steviosid at the probability level of 1% and in terms of carotenoid content at the level of 5% probability. there was a significant interaction effect of irrigation and allicator treatments on plant height, biological yield, proline, phenol content, dpph, rebaudioside a, and steviosid at 1% probability and on leaf area index and carotenoid content at 5% probability level was observed (table 2). plant height in this study, the highest plant height with the average of 37.17 cm was allocated to normal irrigation conditions (90% fc) and the foliar application of salicylic acid. with the intensification of water stress, the plant height significantly decreased; but, in treatment of the 65% fc, application of salicylic acid could significantly increase plant height compared to the control treatment. there was no significant difference between the control treatment and other treatments using growth stimulants (table 4). the results also showed that under irrigation conditions of the 40% fc, there was no considerable difference among the control treatment and the application of elicitors. results revealed, the lowest plant height was allocated to irrigation treatment of the 40% fc in all the four growth elicitor treatments (table 4). leaf area index the results of mean comparisons showed that melatonin application under normal irrigation conditions (90% fc) had the highest leaf area index, while the control and chitosan treatment in the treatment of 40% fc showed the lowest amount. in this study, although in the irrigation treatment of 65% fc, there was no considerable difference between the control treatment and the other elicitor treatments, in the irrigation treatment of 40% fc, the use of salicylic acid increased the leaf area index significantly compared with the control and other treatments (table 4). biological yield among the interactions of irrigation and plant growth stimulant treatments, the use of melatonin in 90% fc treatment with the average of 49.34 g/plant showed the maximum biological yield. the lowest biological yield with the average of 12.14 and 10.97 g/plant was allocated to the control treatment and application of chitosan under irrigation conditions of 40% fc. results showed that although biological yield decreased with exacerbation of water deficit, the melatonin spraying was able to improve this trait in the treatment of the 65% fc by 45.61% compared with the control treatment, in addition there was no remarkable difference among control treatment and elicitors application treatments in the irrigation treatment of the 40% fc (table 4). table 2. combine analysis of variance of irrigation and spraying elicitors on studied traits in stevia (stevia rebaudiana bertoni). sov df mean square plant height lai biomass proline chlorophyll a chlorophyll b carotenoid phenol dpph rebaudioside a steviosid year (y) 1 39.01ns 1.25ns 0.60ns 0.0004ns 0.0004ns 0.008ns 0.00021ns 0.027ns 0.056ns 0.50ns 2.72ns year (replication) 4 13.08 0.85 0.21 0.0005 0.0002 0.004 0.0006 0.018 0.021 0.64 1.24 irrigation levels (i) 2 1606.57** 305.92** 4182.82** 0.0029** 0.0057** 28.32** 0.0031** 0.065** 42.81** 27.73** 98.38** y× i 2 7.76ns 17.36ns 10.06ns 0.0001ns 0.0004ns 0.0024ns 0.00007ns 0.017ns 0.18ns 5.50ns 4.05ns error 1 8 13.56 5.88 60.04 0.0006 0.0007 4.33 0.000012 0.011 3.71 5.91 11.73 elicitors (e) 3 37.29** 31.62** 289.38** 0.0075** 0.0147** 29.94** 0.0013* 0.040** 1.95ns 13.47** 216.91** y×e 3 19.94ns 0.45ns 0.06ns 0.0074ns 0.0004ns 0.052ns 0.000025ns 0.012ns 0.092ns 0.47ns 7.16ns i × e 6 41.33** 8.88* 189.68** 0.0011** 0.0005ns 5.54ns 0.00040* 0.332** 3.98** 8.39** 46.95* y×e×i 6 10.36ns 1.54ns 11.16ns 0.0004ns 0.0004ns 0.0011ns 0.000032ns 0.031** 0.05ns 0.46ns 6.27ns e2 36 11.12 3.41 48.21 0.0003 0.0006 2.12 0.00013 0.011 1.05 0.98 14.34 cv% 12.56 14.20 24.78 15.16 19.01 16.96 20.57 10.52 9.67 13.51 15.55 ns, *, and ** were significant at levels 1 and 5% respectively. 19studying some morphological responses of stevia (stevia rebaudiana bertoni) to some elicitors under water deficiency proline the results revealed that the leaf proline content increased with exacerbation of drought, so that the irrigation conditions of the 40% fc with spraying of melatonin with the average of 0.173 (mg/gfw) had the maximum proline content. results demonstrated that spraying of melatonin in both 65% fc and 40% fc conditions was able to improve the proline content compared with the control by 41.62 and 55.85%, respectively. the lowest proline content was assigned to 90% fc treatment with all four eliminator treatments (table 4). photosynthetic pigments the results showed that irrigation conditions of 40% fc and 65% fc reduced the chlorophyll content by 10.85 and 20.91 percent, respectively, compared to 90% fc irrigation conditions (table 3). in the present study, the use of melatonin with the average of 0.163 (mg/gfw) had the highest leaf chlorophyll a content and increased the amount of this trait compared to the use of chitosan, salicylic acid, and control by 15.60, 15.60, and 72.30 percent, respectively. in this study, the control treatment with the average of 0.094 (mg/gfw) had the lowest chlorophyll a content (table 3). table 3. mean comparison of main effects of irrigation and spraying elicitors treatments of on studied traits in stevia (stevia rebaudiana bertoni). irrigation plant height (cm) lai biomass (g plant-1) proline (mg gfw1) chlorophyll a (mg gfw-1) chlorophyll b (mg gfw-1) carotenoid (mg gfw-1) phenol mggae gdw-1 dpph (%) rebaudioside a (%) steviosid (%) 90% fc 34.50a 16.71a 41.13a 0.108b 0.149a 0.096a 0.069a 0.961b 9.36c 6.48b 22.34b 65% fc 26.99b 12.75b 28.19b 0.120a 0.133b 0.086b 0.052b 1.026a 10.45b 7.03b 24.36ab 40% fc 18.16c 9.58c 14.73c 0.130a 0.118c 0.074c 0.047b 1.067a 12.00a 8.55a 26.39a elicitors control 24.41b 11.49c 23.08b 0.148a 0.094c 0.068c 0.046c 0.822d 10.49a 6.63b 21.63b chitosan 27.42a 12.64bc 27.56ab 0.119b 0.141b 0.087b 0.056b 1.174a 10.52a 6.43b 21.09b salicylic acid 26.99ab 14.68a 28.57ab 0.108bc 0.136b 0.089ab 0.055b 0.993c 10.31a 8.11a 27.33a melatonin 27.38ab 13.23ab 32.85a 0.102c 0.163a 0.098a 0.067a 1.087b 11.07a 8.24a 27.39a data in columns followed by different letters are significantly different (p ≤ 0.01) by duncan’s multiple range test. table 4. mean comparison of irrigation and spraying elicitors interaction treatments of on studied traits in stevia (stevia rebaudiana bertoni). irrigation elicitors plant height (cm) lai biomass (g plant-1) proline (mg gfw-1) carotenoid (mg gfw-1) chlorophyll a (mg gfw-1) chlorophyll b (mg gfw-1) phenol mggae gdw-1 dpph (%) rebaudioside a (%) steviosid (%) 90% fc control 30.35bcd 14.73bcd 32.88bcd 0.0937d 0.062bc 0.109a 0.081a 1.1197bc 8.11f 5.887ghi 18.76f chitosan 33.95abc 15.94abc 38.84abc 0.0957cd 0.058b-e 0.147a 0.091a 1.1017bc 9.29ef 6.65f-h 20.81ef salicylic acid 37.17a 17.65ab 43.45ab 0.1207bcd 0.07b 0.164a 0.102a 0.6317d 10.03de 5.302i 26.65bc melatonin 36.53ab 18.51a 49.34a 0.123bcd 0.087a 0.176a 0.068a 0.674d 9.94de 8.088cd 23.15c-f 65% fc control 23.08ef 11.36def 20.61def 0.1033cd 0.047de 0.086a 0.111a 0.6747d 11.12bcd 6.328f-i 22.49c-f chitosan 26.57de 13.69cd 28.26cde 0.1237bcd 0.056cde 0.149a 0.082a 1.1567ab 10.37de 5.649h-i 20.40ef salicylic acid 30.02cd 14.03bcd 26.82cde 0.1063cd 0.045e 0.134a 0.089a 1.1683ab 9.92de 8.883bc 29.10b melatonin 28.89cde 11.9def 37.07abc 0.1498ab 0.059bcd 0.164a 0.105a 1.108bc 10.4de 7.277def 25.43bcd 40% fc control 19.8f 8.39f 12.14f 0.1113cd 0.031f 0.088a 0.055a 1.0033c 12.24ab 7.7de 23.66cde chitosan 18.9f 8.3f 10.97f 0.1057cd 0.055cde 0.126a 0.094a 1.2647a 11.91abc 7.018d-g 22.06def salicylic acid 17.22f 12.36cde 20.05def 0.1313bc 0.048cde 0.11a 0.07a 1.18ab 10.98cd 10.15a 26.25bcd melatonin 16.71f 9.28ef 15.75ef 0.1733a 0.055cde 0.148a 0.079a 1.151ab 12.88a 9.362ab 33.60a data in columns followed by different letters are significantly different (p ≤ 0.01) by duncan’s multiple range test. 20 basoz sadiq muhealdin et al. chlorophyll b content decreased due to water shortage so that the supply of the 90% fc and 40% fc had the maximum and minimum chlorophyll b contents, respectively (table 3). results revealed that melatonin application had the highest chlorophyll b content. the difference between melatonin and salicylic acid was not significant. the lowest chlorophyll content was recorded for control treatment of foliar application (table 3). in our experiment spraying of melatonin in irrigation treatments of 90% fc improved carotenoid content compared with the control treatment by 40.32 percent and had the maximum carotenoid content (table 4). in our study, although in irrigation treatment of 60% fc, there was no notable difference among the control treatment and elicitors, in the irrigation treatment of 40% fc, foliar spraying of chitosan, salicylic acid, and melatonin increased the carotenoid content by 77.41, 54.88, and 77.10 percent compared to the control. phenol content based on the results, with the intensification of water deficit stress, the phenol content was increased, so that the foliar spraying of chitosan and salicylic acid in irrigation treatment of 60% fc and the use of chitosan, salicylic acid, and melatonin in irrigation treatment of 40% fc had the highest phenol content and enhanced the amount of this trait remarkable compared with the control. the lowest phenol content was allocated to irrigation treatment of 90% fc and the foliar spraying of salicylic acid and melatonin (table 4). dpph in our study water stress rose the amount of dpph, so that the treatment of melatonin, chitosan, and control under irrigation treatment of 40% fc had the highest amount of dpph activity. the minimum dpph content was recorded in the control treatment under 90% fc irrigation conditions (table 4). in our experiment, water shortage and foliar application of elicitors had a synergistic effect on dpph activity. rebaudioside a results revealed that water deficit stress and foliar spraying of salicylic acid and melatonin had a synergistic effect on rebaudioside a content. irrigation treatment with 40% fc with foliar spraying of salicylic acid and melatonin had the highest amount of glycoside rebaudioside a. it should be noted that the application of these two treatments in the irrigation regime of 65% fc significantly increased the amount of rebaudioside a in comparison to the control and foliar spraying chitosan treatments (table 4). the lowest amount of rebaudioside a was recorded under normal irrigation (90% fc) and salicylic acid application. steviosid in our study irrigation regime of 40% fc along with melatonin foliar spraying had the highest steviosid glycoside content, furthermore foliar spraying of salicylic acid under 90% fc irrigation conditions, foliar spraying of salicylic acid and melatonin under 65% fc irrigation conditions, and application of melatonin under 40% fc irrigation conditions could significantly improved steviosid glycoside content compared with control and other treatments (table 4). discussion our research findings showed water deficit diminished plant height, but spraying of salicylic acid, especially in irrigation treatment of the 65% fc, was able to moderate the negative effect of water deficit on plant height. under drought stress, cell elongation and cell differentiation are reduced due to decreased total water potential inside the plant (xu et al., 2021). it seems that the application of salicylic acid can mitigate the adverse effect of drought stress on plant growth by preventing a reduction in cell divisions and cell size (yan et al., 2018). in the study by karimi et al. (tardieu et al., 2000) on stevia, drought stress decreased plant height, while the use of external svglys could not affect this trait. in this study, foliar application of melatonin under normal irrigation conditions produced the maximum leaf area index. leaf area index decreased with the intensification of water shortage. results revealed the use of salicylic acid in the irrigation treatment of 40% fc, increased the leaf area index significantly compared to the control and other treatments. probability salicylic acid can improve nutrient uptake, especially under stress, which in turn can increase growth (25). it seems that salicylic acid can increase photosynthesis and, thus, increase growth by increasing the amount of chlorophyll in the leaves that are at the beginning of the aging process (26). karimi et al. (21) showed that drought stress decreased leaf growth in stevia, but the external application of svglys reduced the leaf losses. 21studying some morphological responses of stevia (stevia rebaudiana bertoni) to some elicitors under water deficiency in our research, the use of melatonin under normal irrigation had the highest biological yield, this trait was reduced by reducing the available water of the plant. the results also revealed that foliar application of melatonin under water shortage conditions increased biological yield compared to the non-foliar spraying treatment. increased biological yield in the present study can be due to the positive effect of foliar application on leaf area index and photosynthetic pigments under different irrigation, leaf area development and photosynthetic pigments increased the rate of photosynthesis and accelerate plant vegetative growth. one of the adverse effects of drought stress is accelerating the production of reactive oxygen species (ros) (ucar et al., 2016), which leads to cell damage, reduced growth and biomass production, and ultimately cell death. the result showed that under irrigation conditions of the 40% fc with the use of melatonin had the highest proline content. results also revealed that the use of melatonin in both 65% fc and 40% fc conditions was able to raise the proline content compared to the control treatment. proline acts as an osmolytic / sprotactant factor under water deficit stress (zheng et al., 2018). the results of our study showed a significant increase in proline content under water deficit stress treatments. it has been reported that the use of melatonin under water deficit conditions induces drought resistance in plants by increasing proline biosynthesis (zhang et al., 2015). consistent with the results of the present study, liang et al. (zhao et al., 2017) in actinidia cinnis, campos et al. (2019) in brassica napus, and li et al. (2019) in cophea arabica have found that melatonin application significantly increases leaf proline content in these plants. in the study by ghanbari et al., the use of chitosan and salicylic acid in milk thistle (silybum marianum l.) remarkably raised the proline content under drought stress. water deficit causes the ros accumulation, resulting in the degradation of the molecular structure of chlorophyll, and finally declining plant photosynthesis. under moisture shortage conditions, a decrease in chl content was considered a typical sign of oxidative stress. the decrease in chl content under drought stress is mainly due to degradation of chl as the result of ros activity (zhang, et al ,2022). reduction of chlorophyll content due to water deficiency, in chickpea, maize , and basil , has been documented in previous investigations. in the present study, the use of melatonin had the highest leaf chlorophyll a and b content. similar to other photosynthetic pigments, water deficit decreases carotenoid content, but the use of all three elicitors increased carotenoid compared with non-foliar spraying treatment (control) under irrigation treatment of 40% fc. under water deficit conditions stress, the use of elicitors can prevent the degradation of pigments molecules. decreased pigments molecule degradation after melatonin treatment may be due to decreased down-regulation of genes of chlorophyll-degrading enzymes such as chlase, pph, and chl-prx. the results showed that induction of water deficit stress and foliar application of elicitors increased leaf phenol content so that the use of chitosan and salicylic acid under irrigation treatment of 60% fc and the use of chitosan, salicylic acid, and melatonin in irrigation treatment of 40% fc showed the highest amount of phenol content. as mentioned, drought stress increases the accumulation of ros in cells that can damage cellular structures. the use of elicitors can increase the activity of antioxidant enzymes to strengthen the plant’s defense system and prevent accumulation of ros in cells. increased phenol content in response to melatonin treatment under drought stress had been reported in the study by sharma et al. (2017) on grafted (carya cathayensis). in the study by karimi et al. (2017), the highest values of antioxidant capacity in stevia were allocated to water deficit conditions and plants treated with svglys, while the lowest values were reported under normal irrigation conditions and no use of svglys. application of salicylic acid by regulating the activity of antioxidant enzymes can improve plant tolerance to adverse conditions (li, et al, 2021; sun, et al. 2021; xu, et al, 2021; zhang, et al. 2022). the results showed the spraying of melatonin and chitosan under irrigation treatment of 40% fc showed the maximum amount of dpph activity. consistent with our results, the use of melatonin had a protective role against water shortage in corn and oats . in the study by ahmed et al. (42), water deficit increased rebaudioside a and stevioside content in steva. compatible solutes such as soluble sugars under melatonin treatment increase, these substances are responsible for maintaining the turgor and osmotic pressure of plant cells under water deficit conditions. in the study by jalal et al. (2018), salicylic acid treatment increased sugar content on plant in both drought stress and control treatments. salicylic acid increases plant resistance to drought stress by stimulating sugar production in the cell. karimi et al. (2019) showed that drought stress increased rebaudioside a content in stevia (bi, et al., 2021; duan, et al., 2022; guo, et al, 2021; guo, et al, 2022). the results showed that application of salicylic acid under 90% and 65% fc irrigation conditions and application of melatonin under 40% fc irrigation had a positive effect on increasing steviosid glycoside content, 22 basoz sadiq muhealdin et al. therefore, the content of this substance increased in all irrigation conditions in response to different elicitors. in a study on milk thistle (silybum marianum l.), the highest soluble sugars was reported in the application of chitosan and salicylic acid under water stress conditions. the positive effect of melatonin on increasing the amount of soluble sugars has also been reported in the studies by liang et al. (2011), campos et al. (2012), and li et al. 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(caryophyllaceae) grown under different salt stress conditions. physiol mol biol plants. 24(2):231–238. doi:  10.1007/s12298017-0496-x zhang m, jin zq, zhao j, zhang g, wu f. 2015. physiological and biochemical responses to drought stress in cultivated and tibetan wild barley. plant growth regul. 75(2): 567-574. 24 basoz sadiq muhealdin et al. zhao p, lu gh, yang yh. 2017. salicylic acid signaling and its role in responses to stresses in plants. in: pandey g (ed) mechanism of plant hormone signaling under stress, 1st edn, vol 11. zhang, j., m. khayatnezhad, and n. ghadimi, 2022. optimal model evaluation of the proton-exchange membrane fuel cells based on deep learning and modified african vulture optimization algorithm. energy sources, part a: recovery, utilization, and environmental effects, 44(1):287-305. caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 4 2022 firenze university press avicennia genus molecular phylogeny and barcoding: a multiple approach laleh malekmohammadi1, masoud sheidai1,*, farrokh ghahremaninejad2, afshin danehkar3, fahimeh koohdar1 studying some morphological responses of stevia (stevia rebaudiana bertoni) to some elicitors under water deficiency basoz sadiq muhealdin1, sahar hussein hamarashid1,*, fairuz ibrahim ali1, nakhshin omer abdulla1, syamand ahmad qadir2 morphological and cytogenetic characterization in experimental hybrid aloe jucunda reyn. x aloe vera (l.) burm. f. (asphodelaceae) wendy ozols-narbona*, josé imery-buiza assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions seyedeh mahsa hosseini1, sepideh kalatejari1, mohsen kafi2,*, babak motesharezadeh3 assessment of protein and dna polymorphisms in corn (zea mays) under the effect of non-ionizing electromagnetic radiation ekram m. abdelhaliem1,*, hanan m.abdalla1, ahmed a. bolbol1, rania s. shehata1,2 chromosome counts of some species of wetland plants from northwest iran saeedeh sadat mirzadeh vaghefi*, adel jalili delimiting species using dna and morphological variation in some alcea (malvaceae) species based on srap markers chnar hama noori meerza1, basoz sadiq muhealdin2, sahar hussein hamarashid2,*, syamand ahmad qadir3, yusef juan4 mapping cap-a satellite dnas by fish in sapajus cay paraguay and s. macrocephalus (platyrrhini, primates) simona ceraulo, francesca dumas* determination of genome size variation among varieties of ilex cornuta (aquifoliaceae) by fow cytometry peng zhou1, jiao li2, jing huang1, fei li1, qiang zhang2,*, min zhang1,* first report of chromosome and karyological analysis of gekko nutaphandi (gekkonidae, squamata) from thailand: neo-diploid chromosome number in genus gekko weera thongnetr1, suphat prasopsin2, surachest aiumsumang3,*, sukhonthip ditcharoen4, alongklod tanomtong5, prayoon wongchantra6, wutthisak bunnaen7, sumalee phimphan3 intraspecific karyomorphological and genome size variations of in vitro embryo derived iranian endemic asafoetida (ferula assa-foetida l., apiaceae) narges firoozi, ghasem karimzadeh*, mohammad sadegh sabet, vahid sayadi cytogenetic studies in the centaurea aucheri group (sect. phaeopappus) seyed mahmood ghaffari¹,*, seyed mohsen hesamzadeh hejazi² karyomorphology, genome size, and variation of antioxidant in twelve berry species from iran saeed mohammadpour1, ghasem karimzadeh1,*, seyed mahmood ghaffari2 caryologia. international journal of cytology, cytosystematics and cytogenetics 75(4): 37-48, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1827 caryologia international journal of cytology, cytosystematics and cytogenetics citation: seyedeh mahsa hosseini, sepideh kalatejari, mohsen kafi, babak motesharezadeh (2022). assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions. caryologia 75(4): 37-48. doi: 10.36253/caryologia-1827 received: april 18, 2022 accepted: december 04, 2022 published: april 28, 2023 copyright: © 2022 seyedeh mahsa hosseini, sepideh kalatejari, mohsen kafi, babak motesharezadeh. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. the data that suppor t the findings of this study are openly available in [repository name e.g “zenodo”] at http://doi.org/[10.5281/zenodo.6344758]. competing interests: the author(s) declare(s) no conflict of interest. assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions seyedeh mahsa hosseini1, sepideh kalatejari1, mohsen kafi2,*, babak motesharezadeh3 1 department of horticultural science and agronomy, science and research branch, islamic azad university, tehran, iran 2 department of horticultural science, university college of agriculture & natural resource, university of tehran, karaj, iran 3 department of soil science, faculty of agricultural engineering & technology, university college of agriculture & natural resources, university of tehran, karaj, iran corresponding author. e-mail: mkafi@ut.ac.ir abstract. heavy metals pollution is an important challenge that was cussed by human activity, this stress decreases under salinity. the aim of this study was to investigate the ability of japanese raisin in the absorption of nitrate (0, 30, and 60 mgl-1) and lead (0, 300, and 600 mgl-1) under salinity stress (0 as control and 3 and 6 dsm-1). results showed that the studied plant continued to uptake nitrate and potassium under stress conditions of pb and salinity. although na and cl uptake were observed as a defense mechanism in the plant, the k/na ratio, and k content increased from 1 to 6 and from 1.8 to 5%, respectively. also, the most appropriate physiological responses were observed at treatments under contamination level of 300 mg pb and salinity level of 3 dsm-1, so that the synthesis of malondialdehyde (mda) and enzymatic activity increased at these levels of hms and salinity. based on the results, the studied species were able to uptake moderate concentrations of pb (34.1-71mg kg-1) under experimental conditions. hence, its potential for the clean-up of some contaminants in the environment can be considered by researchers for further research. novelty statement. this study investigated the cleaning up of some heavy metals and nitrate from the environment and plants’ physiological responses under stressful conditions. the plant species (japanese raisin) characteristics and the results have enough novelty and will be published for the first time. most hardwood trees such as walnut, oak, beech, poplar, etc. have a slow growth rate. but japanese raisin tree a new and unknown plant in iran has very important features. this tree is one of the few trees that in addition to having hardwood, has a very high growth rate. which can be useful in creating artificial forests, landscapes, as well as in industrial applications, buildings, and furniture industries. so far, no special research has been done on the phytoremediation characteristics of this plant and the selection of this plant in the present study and the study of the ability of this plant to absorb nitrate and lead under salinity stress is a completely new and innovative topic. keywords: antioxidant enzymes, heavy metal, phytoremediation, proline, malondialdehyde, salinity. 38 seyedeh mahsa hosseini et al. 1. introduction metal pollution is harmful for human health and the environment. human activities have been considered an important factor in the contamination of the soil with heavy metals (hms) (akinci and guven 2018; motesharezadeh et al, 2016). the presence of hms reduces soil fertility, crop yield, and soil microbial activity (pinto et al, 2004; sumiahadi and acar, 2018). lead (pb), a hm pollutant in industrial ecosystems, is important in plant life because of its easily absorption by the plant roots, which is induced by its high accumulation in the surface area of the soil (mosaferi et al, 2008; wang et al, 2019). in addition to natural processes, pb is also produced through the artificial sources (exhaust fumes from automobiles, factories, battery tanks, and pesticides). after pb is absorbed by roots, it causes changes in metabolic activities of plants, disrupting their growth and development (sharma and dubey, 2005; oguntade et al, 2018). presence of pb, leads to a disruption of membrane carriers’ activity of the root cells, depleting nutrients such as magnesium, calcium, and iron. as a result of an experiment, deficiency symptoms of these nutrients were reported in pb-treated plants (sharma and dubey, 2005). moreover, the overuse of nitrate fertilizers in agriculture fields leads to nitrate pollution of ground and surface waters (castro‐ rodríguez et al, 2016). climate change and water deficit is the important challenge in agriculture activities all over the world and soil salinity is the one of most important problem that cause by these challenges (isayenkov and maathuis, 2019). there are many studies have reported that salinity stress induced by nacl restricts agriculture and crop yield (isayenkov and maathuis, 2019). plant resistance to salinity depends on some mechanisms such as antioxidants activity, ion homeostasis, biosynthesis of osmolytes, and gene expression. phytoremediation is a useful technic based on the living plant’s ability to absorb ionic compound by their roots or leaves and clean up soil, air and water contamination (berti and cunningham, 2000). there are many reports on phytoremediation, such as phytoremediation of high levels of nitrate with poplar trees (castro‐rodriguez et al, 2016), zinc (zn) and pb nitrates with sunflowers (adesodun et al, 2010), nitrate with salvinia molesta (ng and chan, 2017) and zn, cd, and pb with typha angustifolia and eichhornia crassipes (sricoth et al, 2018). generally, stresses such as salinity and heavy metals in which salinity increases the uptake of heav y metals, occur simultaneously in the environment. the results of a study indicated that the presence of cd along with nacl in the root environment of four barley cultivars significantly reduced soil cd concentration and increased its uptake by plants (huang et al, 2007). similarly, abbasi et al. (2013) investigated the effect of irrigation water salinity on the rate of heavy metals uptake in potamogeton berchtoldi and reported that the concentration of heavy metals (pb and cd) in plant increased with the increasing salinity up to 4 and 6 dsm-1, respectively. in general, based on the results of the numerous studies, it can be concluded that under conditions of hms (such as pb and cd) and salinity stresses, the plant’s nutritional needs for nutrients such as n, p and k will increase (khoshgoftarmanesh, 2010; yan et al, 2020). in fact, the recommendation for more application of these nutrients under stress conditions, is a strategic management to prevent reducing plant dry matter. it should be mentioned that nitrate, in principle, increases the resistance to salinity, which has been similarly reported in several studies (bai et al, 2021). however, there are limited reports on the phytoremediation ability of japanese raisin or its ability to grow in the contaminated soils. based on this background, this study was aimed to assess the potential use of japanese raisin for the phytoremediation of a nitrate/pb polluted soil under conditions of salt stress. 2. material and methods 2.1. plant material and growth condition one-year old seedlings of japanese raisin (hovenia dulcis l.) were prepared from hirkania greenhouse (nowshahr, mazandaran, iran). seedlings were grown in plastic bags (25×30 cm), and fertilized with npk fertilizer and a hoagland based solution during the test period (motesharezadeh et al, 2016), also the average temperature and the humidity were 25 °c and 70%, respectively (ramesh et al, 2006). the seedlings were kept for five months and then harvested. due to the stress induced by moving seedlings from a distant place in the north of the country, it was necessary to apply some treatments to plants reinforcement (table s11). therefore, to improve the seedlings growth and before the application of experimental treatments, complete fertilizer and hoagland nutrient solution were used and also leaching was considered. 1 supplementary data. 39assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions 2.2. experimental design to execute the experiment, a neutral culture media (a combination of 70% non-enriched cocopeat and 30% perlite) was used. in order to investigate the capability of japanese raisin for the phytoremediation of a no3/ pb polluted soil under conditions of nacl stress, a factorial greenhouse trial was arranged in a completely randomized design with four replications. the treatments consisted of (1) nacl salinity as the primary factor at three levels of 0 (control), 3, and 6 dsm-1, according to previous reports (salimi et al, 2012); (2) nitrate derived from potassium nitrate, as the secondary factor, was applied at three levels (0, 30, and 60 mgl-1) (gheshlaghi et al, 2015); and (3) pb form the source of lead nitrate (0, 300, and 600 mgl-1) (shabani et al, 2015) as the third factor. to have only the effect of nitrate, equivalent of potassium added in the treatments (from the source of potassium nitrate), potassium sulfate was added to other pots. in order to avoid the negative effect of possible stress on plant, potassium nitrate was applied during the holding period every two weeks via irrigation water. 2.3. biochemical measurements 2.3.a lead content measurement dry ashing method was used to analyze plant samples and measure pb (wallis, 1996). during the method, to measure metals, plants organic matter is destroyed with controlled heat. based on the method, one gram of dried and powdered sample of the plant was poured into a crucible and placed in an electric oven at 550 degrees for 4 hours. after leaving the crucibles out of the furnace and reaching ambient temperature, samples were transferred to small beakers using 10 ml of 2 m hydrochloric acid. then, the beakers were placed on an electric stove until the first white vapors appeared. next, after reaching the ambient temperature, the contents of beakers were filtered through a filter paper inside a 100 ml volumetric flask and made up to volume with distilled water. finally, pb concentrations were reported in samples extract by use of icp-oes (inductively coupled plasma atomic spectroscopy). pb uptake was measured and reported by multiplying its concentration by the plant dry matter (sharma et al, 2012). 2.3.b total nitrogen and nitrate content measurement total concentration of nitrogen (n) in plant samples was measured by kjeldahl method (horneck and miller,1998). in this method ground plant material were digested to h2so4 at high temperatures by the help of metal catalyst. total nitrogen of plant was changed to ammonium (nh4+), which is then by titration the concentration of n was quantified. to measure the nitrate of the plant samples, ion-selective electrode method was used (miller, 1988). 2.3.c potassium/sodium ratio and chlore content measurements sodium and potassium concentrations were determined based on the method of wet digestion with hydrochloric acid using flame photometer elea (ryan et al, 2002). in order to measure the chlore (cl) content in the plant material (liu, 1998), after extraction, the cl in the filtrate was analyzed using the colorimetric method on the traacs 800tm auto-analyzer. in this method, the sample is mixed with the color reagent and dialyzed into the color reagent again. the procedure is based on the release of thiocyanate ions from mercuric thiocyanate by cl ions in the sample. the liberated thiocyanate reacts with ferric iron to form a red color complex of ferric thiocyanate. the color of the resulting solution is stable and directly proportional to the original cl concentration. the color complex is measured at 480 nm using a 10-mm flow cell. nitrite (no2), sulfide, cyanide, thiocyanate, bromide, and iodine ions cause interferences when present in sufficient amounts. 2.4. antioxidant enzymes activity measurement in order to evaluate the effect of salinity, nitrate and lead contamination stresses on plant physiological responses, the changes in enzymatic activity were measured. among plant enzymes, catalase (cat) and superoxide dismutase (sod) are considered as sensitive enzymes indicating plant resistance mechanisms under stress conditions (khadem moghadam et al, 2016). hence, these two enzymes were selected for this goal of the present study. total protein content was determined following the method described by bradford (1976). protein  content was determined using spectrophotometry  at a wavelength of  595 nm. also, the modified method of chance and maehly (1955) was used to measure the activity of peroxidase (pod) enzyme. the activity of superoxide dismutase, the basis of which is its ability to inhibit the photochemical reduction of nitro blue tetrazolium (nbt), was determined according to the method described by dhindsa et al. (1981). 40 seyedeh mahsa hosseini et al. 2.5. soluble carbohydrate content measurement the method of irigoyen et al. (1992), was used to measure soluble carbohydrates. for this purpose, leave sample were ground in liquid nitrogen, 100 mg of them were blended with 5 ml of 70 % ethanol (wv-1) for 5 min, then centrifuged at 3500 rpm for 10 min at 4 °c. after that 200 ml of the supernatant were added to 1 ml of an anthrone solution, then the absorbance was read by uv/vis spectrophotometer at 625 nm. 2.6. malondialdehyde level assessment malondialdehyde (mda) concentration was measured by thiobarbituric acid method by spectrophotometry. its concentration was calculated using the extinction coefficient mda-tba for the complex (dandekar et al., 2002). 2.7. proline content measurement to measure proline level, the procedure of bate et al. (1973) were used. first, 10 ml of acid sulfuric were added to 100 mg of fresh leaf, they were then passed through filter paper. after 2ml ninhydrin and 2ml acid acetic glacial were added to 2 ml of extract and kept in benmary counter for 1 h, then toluene was added to them. after 2 h the supernatant was extracted. the absorbance at 520 nm was recorded. 2.8. lipid peroxidization measurement to quantify the amount of this enzyme, the modified chance and maehly (1955) method was used. in this method, 1 ml of potassium phosphate buffer (ph = 6.7) was poured into the cuvette and 17.6 μl of hydrogen peroxide and 17.6 μl of leaf extract were added to it. the resulting solution was immediately read in a spectrophotometer at a wavelength of 240 nm for 2 minutes at intervals of 15 seconds to calculate the activity of this enzyme according to the amount of light absorption. 2.9. statical analysis the present study was executed based on a factorial trial arranged in a completely randomized design (crd) with four replications. data were analyzed using sas 9.2 and mstatc software. differences between treatments were determined following duncan’s multiple range test (dmrt), (p ≤ 0.05). figures were drawn using excel 2010 software. 3. results 3.1. biochemical content measurements 3.1.a. plants lead level according to the variance analysis results, the interactions of salinity, no3-, and pb significantly affected pb concentration (table s2). based on the means comparison results, salinity at levels of 3 and 6 dsm-1 reduced pb content by 13% and 36%, respectively; nitrate at levels of 30 and 60 mgl-1 decreased pb content by 11% and 12%, respectively; and pb at levels of 300 and 600 mgl-1 increased pb content by 45% and 53%, respectively. also, the highest pb content (69 mg kg.-1) belonged to the treatments of s1n0pb2 and s0n0pb2, and the lowest one was observed at treatments with no added pb (fig. 1). to better understand the ability of the studied plant how cope with stress and phytoremediation, the uptake rate was calculated for different treatments (fig. 2). results showed that in high salt concentration the high uptake of lead was observed in high nitrate concentration. accordingly, pb uptake and accumulation can be considered as a reliable indicator for phytoremediation under salinity stress conditions. 3.1.b. plants total nitrogen and nitrate level regarding nitrogen content, it is understandable that there was a significant (p≤0.01) interaction between salinity, no3-, and pb. additionally, no3significantly affected shoot nitrogen content (p≤0.01) (table s2). the application of 30 and 60 mgl-1 no3increased n content by 10% and 18%, respectively. the highest n content (7.3%) was m c a m e b m ef c m d a m g ef m g f m i jk m k h m ij i 0 10 20 30 40 50 60 70 80 p b0 p b2 p b1 p b0 p b2 p b1 p b0 p b2 p b1 p b0 p b2 p b1 p b0 p b2 n0 n1 n2 n0 n1 n2 n0 n1 n2 control s1 s2 p b (m g. kg -1 ) figure 1. lead (pb) content in japanese raisin in response to salinity (s0: control, s1: 3 and s2: 6 dsm-1), nitrate (n0: 0, n1: 30 and n2: 60 mgl-1), and pb (pb0: 0, pb1: 300 and pb2: 600 mgl-1). values in each group followed by the same letter are not significantly different according to dmrt at p≤0.05 41assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions observed in s0n2pb0 and s2n1pb2 treatments, while the lowest n content (3.3%) was recorded for s1n0pb2. based on the results, the interaction of salinity, no3-, and pb, significantly affected shoot no3content (table s2). no3content reduced by 11% and 21% with the application of 3 and 6 dsm-1 salinity. however, application of 30 and 60 mgl-1 of no3increased no3content by 24% and 36%, respectively. the interaction between all three treatments showed that the highest no3content (0.61%) was recorded for the treatment of s0n1pb0, while the lowest no3content (0.05%) was observed at the treatment of s2n0pb2. 3.3.c plants sodium, potassium and chlore level in accordance with the obtained results, the interactions of salinity, no3-, and pb significantly (p≤0.01) affected shoot potassium content (table s2). the lowest values of shoot k concentration were observed at salinity treatments of 3 and 6 dsm-1, which were reported to be 1.85% and 1.95%, respectively; while no3application at levels of 30 and 60 mgl-1 increased k by 4% and 22%, respectively. the interaction between the treatments indicated that the highest shoot k concentration (5.05%) belonged to the treatment of s0n2pb0, that was almost 3 times more than the lowest one at treatment of s1n1pb0, s1n0pb0 and s2n0pb2 (1.85%). as results showed, the interactions of salinity, no3-, and pb significantly (p≤0.01) affected shoot na concentration (table s2). considering to the data, it can be found that shoot na concentration increased up to the 39% by salinity application of 3 or 6 dsm-1, while no3application at levels of 30 and 60 mgl-1 decreased shoot na content by 26% and 25%, respectively. based on the interactions of studied factors, the highest shoot na concentration )2.77% (belonged to the treatments of s2n0pb0, s2n1pb0 that was fivefold of the s2n2pb2 treatment as the lowest one. considering to the k/na ratio shown in table 2, it can be found that the accumulation of k effectively controlled salinity stress. the highest ratio was recorded in the sin0pb1treatment that was six times more than s2n0pb2 treatment as lowest one. according to variance analysis results it is clear that the interactive effects of salinity, no3-, and pb, significantly affected cl concentration (table s2). also, the results of means comparison of indicated that salinity at levels of 3 and 6 dsm-1 increased cl content by 17% and 30%, respectively; nitrate at levels of 30 and 60 mgl1 increased cl content by 9% and 2%, respectively; and pb at levels of 300 and 600 mgl-1 increased cl content by 4.9% and 6.8%, respectively. the highest (4.7%) and the lowest (1.4%) values of cl content were recorded for the treatments of s2n1pb2 and s0n0pb0, respectively. high cl concentration was observed in the treatments with high salinity and nitrate concentration. 3.2. antioxidant enzymes activity results showed salinity significantly affected the activities of antioxidant enzymes (table s3), so that the levels of 3 and 6 dsm-1 increased the activity of pod by 11% and 6%, respectively, while they reduced the activities of sod by 2 and 15% and cat by 17 and 63% (table 1), respectively. additionally, no3at levels of 30 and 60 mgl-1 significantly decreased the activities of pod by 12% and 20%, sod by 9% and 24%, and cat by 20% and 15% (table 1), respectively. furthermore, pb at levels of 300 and 600 mgl-1 significantly increased the activities of pod by 12% and 11% and cat by 27% and 34%, respectively, while they reduced the activity of sod by 39% and 50%, respectively (table 1). considering to the results, it can be found that the highest enzymatic activity of sod, pod and cat, which are the best indicators of assessing stress conditions, were observed at treatments of 3 dsm1 salinity + 30 mgl-1 nitrate (without pb), 3 dsm-1 salinity + 600 mgl-1 pb (without nitrate) and 600 mgl-1 pb + 30 mgl-1 nitrate application (without salinity), respectively. (table 1). in other words, with increasing the studied stresses levels including pb contamination up to 600 mg, salinity up to 6 dsm-1 and nitrate up to 60 mgl-1, the enzymatic activity reduced indicating the reduction of plant defense mechanisms under severe stress conditions. generally, the relationships between biochemical traits and nutrients status can provide a clear underh h h h c d h d a h h h h e e h f e h h h h b g h d f 0 0,5 1 1,5 2 2,5 3 pb0 pb2 pb1 pb0 pb2 pb1 pb0 pb2 pb1 pb0 pb2 pb1 pb0 pb2 n0 n1 n2 n0 n1 n2 n0 n1 n2 control s1 s2 )1sh oo t l ea d up ta ke (m g po t figure 2. shoot lead (pb) uptake in japanese raisin in response to salinity (s0: control, s1: 3 and s2: 6 dsm-1), nitrate (n0: 0, n1: 30 and n2: 60 mgl-1), and pb (pb0: 0, pb1: 300 and pb2: 600 mgl-1). values in each group followed by the same letter are not significantly different according to dmrt at p≤0.05 42 seyedeh mahsa hosseini et al. standing of positive and negative correlations among whole studied parameters. for this purpose, the correlation between traits were calculated. the results showed the high positive correlation between proline and mda by r2=0.879, n and k by r2= 0.718, soluble carbohydrate and mda by r2= 0.612 and lipid peroxidase and mda by r2= 0.574 (table 2). 3.3. soluble carbohydrates content to evaluate the biochemical and physiological responses of plant against studied stresses, soluble carbohydrates were measured. based on the results, the most values of soluble carbohydrates were reported at treatments of s1n0pb2 and s0n0pb2, indicating that hms stress had more effect on this trait in comparison with salinity. the highest content of soluble carbohydrate was recorded in s0n0pb2 that was 3 time more than s2n2pb2 as lowest treatment. 3.4. malondialdehyde level of plants also, there was a significant difference in proline concentration among different studied treatments compared to control. malondialdehyde was measured as an important indicator of plant response to abiotic stresses. results showed, the most values of this parameter were observed at moderate levels of pb application (without salinity) and also the synthesis of this biochemical product significantly reduced by the expansion of hms, salinity and nitrate stress. lowest value of mda was recorded in s2n2pb2 treatment that show this parameter were deceased in high salt, nitrate and lead concentration. 3.5. proline level of plants the most values of proline were observed at moderate levels of pb application (without salinity) and also the synthesis of this biochemical product significantly reduced by the expansion of hms, salinity and nitrate stress. the highest value of proline was recorded in the s0n0 treatment that was twofold higher than s2n2 treatment as last one. 3.6. lipid peroxidation the increasing trend was observed for lipid peroxidation and the most values of this parameter belonged to the treatments with low levels of stress (without salinity and without nitrate). highest content was observed in the s0n0 treatment, while the treatment with high level of nitrogen and lead ranked last treatment. 4. discussion 4.1. biochemical traits affected by different level of nitrate and lead under salinity stress 4.1.a lead uptake and concentration were affected under different nitrate level based on the results, pb pollution caused more salinity (na) uptake. in other words, salinity can increase hms stress, which means the intensification of the stress induced by salinity and also has been table 1. activities of antioxidant enzyme in response to treatments. salinity (dsm-1) nitrate (mgl-1) pb (mgl-1) pod sod cat control 0 0 0.52d* 161.9c 5kl 300 0.29lmn 82.15ijk 56.37cd 600 0.36hij 87.87ij 21.83gh 30 0 0.19p 68.63lmn 3.01l 300 0.35 h-k 93.02i 54.08cd 600 0.38ghi 128.4ef 86.26a 60 0 0.45ef 120.7fg 30.8f 300 0.56cd 140.1d 58.77c 600 0.58c 117.5g 67.41b 3 0 0 0.34ijk 67.05mn 72.25b 300 0.34ijk 132.9de 50.19d 600 0.8a 175.5b 55.34cd 30 0 0.54cd 188.5a 43.41e 300 0.69b 154.3c 11.05jk 600 0.31klm 59.99no 9.76jkl 60 0 0.23op 52.44o 53cd 300 0.27mno 74.59klm 10.55jkl 600 0.33jkl 86.29ij 14.98hij 6 0 0 0.48e 115.2gh 3.52jl 300 0.57c 132.3de 25.93fg 600 0.36hij 105.6h 27.79fg 30 0 0.42fg 106.3h 14.84hij 300 0.36hij 79.4jkl 12.66ij 600 0.38ghi 89.69ij 20.79gh 60 0 0.23op 49.88o 8jkl 300 0.39gh 114.4gh 18.7hi 600 0.26no 58.77no 10.07jkl * means within a column followed by the same letters are not significantly different at p ≤ 0.05 according to duncan’s multiple range test. 43assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions considered by many researchers. generally, when the stress inhibits plant growth and reduces transpiration, the increase in contaminant uptake will be stopped or reduced. accordingly, in the present experiment, the salinity stress without hms contamination, led to reduce contaminant (pb) uptake. the critical level of pb contamination in soil, considered by researchers, is 50 mg kg-1 soil (prasad, 2004). also, the normal range of pb in plant tissues is between 0.2 to 20 mg kg-1, but its critical and contamination levels in plant is more than 20 mg kg-1, reducing the yield and plants dry matter (alloway, 1990). accordingly, high concentrations (34.6-71.6 mg kg -1) of pb accumulated in treatments of pb contamination indicating the ability of the studied species for hms phytoremediation. in fact, the studied plant has an appropriate potential for phytoremediation under conditions of simultaneous stresses. 4.1.b n and nitrate level improved the plant resistance under salinity stress the percentage of n and no3 and the accumulation of k, na and cl represent the intensity of plant response to the studied stresses (hms and salinity). results indicated that salinity or na content reduced by supplying nitrate. it should be mentioned that nitrate increases plants resistance to salinity, which also has been reported in numerous researches (kafkafi et al, 1992). some researchers believe that reduction of nitrate concentration is because of the negative interaction between cl and nitrate and antagonistic effect of cl on nitrate uptake. while, others attribute it to the plant’s response under saline conditions reducing water uptake (lauter and munns, 1986). the results of a study showed that phytoremediation of hvs (co, cu, cr, ni, and pb) pollution by aquatic hyacinth was only effective at high concentrations of nitrate and by decreasing nitrate concentration the phytoremediation efficiency decreased (tangahu et al, 2011; bai et al, 2021). loska and wiechula (2003) reported that the presence of any type of contaminants in water and soil resources led to pollutants accumulation in plant organs, changing enzymatic activities. similarly, the results of the present study are consistent with those of recent studies (dayani et al, 2009; husejnovic et al, 2018). 4.1.c high potassium to sodium ratio improve phytoextraction in contract to chlore khoshgoftar et al. (2004) reported that hms uptake from the soil solution increased with the increase of nacl level, while no such effect was observed for nano3. it is assumed that chloride ion positively affected hms (pb and cd) solubility in soil and their uptake by plants. generally, chloride ion increases the dynamics and adsorption capacity of the metals. having high salinity tolerance, is another important strategy of plants for resistance against salinity. for example, grasses and atriplex/salicornia are capable to grow at the salinity levels up to 1.2-1.7 dsm-1 and 21-28 dsm-1, respectively. table 2. pearson correlation coefficients between characteristics. n no3 k na k/na cl pb con. pb uptake sol. carb proline mda lipid pox sod cat n 1 no30.495** 1 k 0.768** 0.575** 1 na -0.093ns 0.071ns 0.049ns 1 k/na 0.468** 0.359** 0.520** -0.721** 1 cl 0.497** 0.409** 0.412** 0.104ns 0.249* 1 pb con. 0.340** 0.158ns 0.344** -0.331** 0.259* 0.145ns 1 pb uptake -0.209ns -0.217ns -0.198ns -0.174ns 0.086ns 0.060ns -0.149ns 1 sol. carb -0.084ns 0.083ns -0.065ns 0.215ns -0.140ns 0.022ns -0.484** 0.008ns 1 proline -0.006ns 0.179ns 0.189ns 0.366** -0.180ns -0.094ns -0.249* -0.222* 0.504** 1 mda -0.126ns 0.064ns -0.054ns 0.009ns 0.016ns -0.136ns -0.418** 0.042ns 0.818** 0.612** 1 lipid 0.056ns 0.215ns 0.195ns 0.302ns -0.067ns -0.071ns -0.234* -0.288** 0.475** 0.879** 0.574** 1 pox -0.221* -0.146ns -0.032ns 0.259* -0.293** 0.131ns -0.205ns 0.165ns 0.256* 0.106ns 0.122ns 0.004ns 1 sod -0.113ns -0.140ns -0.085ns 0.037ns 0.067ns -0.285** 0.218ns 0.073ns 0.328** 0.158ns 0.363** 0.206ns 0.386** 1 cat -0.213ns -0.270* -0.099ns -0.299** 0.163ns -0.240* -0.050ns -0.219* 0.202ns 0.302** 0.373** 0.276* -0.081ns 0.222* 1 ** represent significant difference at p ≤ 0.01, * represent significant difference at p ≤ 0.05, n.s represent no significant difference 44 seyedeh mahsa hosseini et al. regarding to the results of the present study, it seems that the japanese raisin (hovenia dulcis) can be considered as a relatively tolerant species at moderate salinity levels due to its appropriate responses to salinity levels of 3 and 6 dsm-1, the accumulation of k and na and also the high ratio of k/na in different stress treatments. in addition to salinity stress, pb contamination also has a specified critical level based on soil and plant studies. potassium accumulation is probably a defense mechanism and increasing the ratio of k/na is a strategic way for resistance to salinity stress (kibria and hoque, 2015). generally, these results indicated the intensity of the effect of stress treatments (hms and salinity) on the one hand and the plant resistance responses to pb and salinity. also, kibria and hoque (2015) conducted a field experiment to investigate the effect of the mitigation of soil salinity on rice by application of k and zn fertilizers. the results demonstrated that k+/na+ ratio in the grains significantly affected by the application of k. therefore, it may be induced by the fact that the application of higher doses of k and zn fertilizers could alleviate the adverse effects of salinity in rice via increasing nutrient uptake and maintaining a higher k+/na+ ratio. boudaghi malidareh et al. (2014) reported a significant relationship between the amount of k fertilizer and hms pollution (cadmium concentration) in the soil. furthermore, the results of a study demonstrated that soil salinity can be improved by the application of nitrogen and potassium fertilizers (shanker, 2005). azari et al. (2005) investigated the role of potassium on nitrate and cd contamination in potato and onion. they found that the concentrations of nitrate and cadmium in potato and onion tubers significantly decreased following the application of potassium and zinc fertilizers, and the highest nitrate and cd contamination was recorded for the treatment with the unbalanced fertilizer use. under salinity stress, the concentrations of potassium and phosphorus in the stem significantly decreased, while the concentration of sodium in the leaves increased. the similar results were also reported by khalilpoor and jafarinia (2017) and yousefinia and ghasemiyan (2016). 4.2. antioxidant enzymes activity was affected by different levels of hm and salt stress it seems that the increase in enzymatic activity is one of the main strategies tolerating hms contamination and salinity stresses (malar et al, 2016). accordingly, the changes in activity of sod enzyme are considered as the appropriate indicators of stress management. the results of the present study showed that the increased sod activity under stress conditions was due to the plant’s survival on the one hand and contaminants purification on the other hand. however, the simultaneous increase in the stresses of hms contamination, salinity and nitrate, up to the maximum levels, reduced the activity of all three studied enzymes. malar et al. (2016) reported that plants use different mechanisms to cope with hms toxicity. alizadeh (2012) reported that the contamination lead and cadmium disrupted the growth of two poplar species and plant biomass significantly reduced at high levels of pollutants. furthermore, the reduced vegetative growth caused by hms stress in plants may be because of the suppressed activities of catalase and superoxide dismutase (schutzendubel and polle, 2002). jabeen and ahmad (2012) reported that salinity increased the activity of peroxidase but decreased that of catalase. similar results were also reported on canola (abili and zare, 2014) and maize (abdelgawad et al, 2016). michalak (2006) has found that antioxidant enzymes can scavenge reactive oxygen species (ros) when the plant grows under hms stress. also, verma and dubey (2003) observed that pb toxicity changed the activity of antioxidant enzymes in rice plants. barandeh and kavousi (2017) reported that the activities of antioxidant enzymes, including superoxide dismutase, catalase, and ascorbate peroxidase significantly increased in lentil seedlings with the increasing in cd concentration. similarly, hendry et al. (1992) illustrated that hms are also a reason for oxidative stress via the production of free radicals of reactive oxygen which can react with lipids and finally lead to the lipid peroxidation, membrane damage, and enzymatic inactivation (dixi et al, 2001). similar results were reported by verma and dubey (2003) and abdelqawad et al. (2016). generally, it has been reported that stress increases the activities of antioxidant enzymes (meloni et al, 2003) but at an intolerable intensity obviously reduces their activities (amiriyan mojarad et al, 2018). the results of the present study in agreement with previous reports showed the increment of sod, pod and cat by increasing the hm and salt stresses level. 4.3. soluble carbohydrate was affected by different levels of hm and salt stress soluble carbohydrate content was affected by different nitrate level and stress condition. the results of current study showed the decreasing trend by increasing salt and lead level. weisany et al. (2014) reported that at all three growth stages (pre-flowering, post-flowering, and seed filling), salinity stress decreased shoot fresh and dry weights, plant yield, root and leaf also soluble carbohydrate content of these tissue in soybean, but the 45assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions application of zinc fertilizer alleviated these negative effects. decreased biomass production induced by hms stress may be because of a disturbance in uptake and transmission of nutrients and water into the aerial parts of plants (sudova and vosatka, 2007). 4.4. malondialdehyde synthesis were changed under hm and salt stress the synthesis of biochemical compounds such as malondialdehyde has been considered as another important mechanism to withstand stress conditions. the stress-adapted plants seem to be more capable to synthesis these metabolites. there are numerous studies confirming the increased synthesis of malondialdehyde and some plant biochemical/enzymatic compounds as a response to stress conditions (juknys et al, 2012; aljahali and alhassan, 2020). based on the results of the present study, there was a significant and negative correlation between shoot pb concentration with mda synthesis and also between the activity of pox with the k/na ratio. the similar results have been reported by aljahali and alhassan (2020). 4.5. proline level affected under stress condition proline is a non-enzymatic antioxidant known as bio-marker that showed plants response to the stress (petrovic et al, 2020). in the water caltrop plant, they suggested proline accumulation as a good biomarker of hms stress (petrovic et al, 2020; bi, et al., 2021; duan, et al., 2022; guo, et al, 2021; guo, et al, 2022). the results of proline accumulation, showed the decreasing trend by increasing the nitrate concentration under high level of lead. these results indicated the positive effect of nitrate to deceasing the side effect of hms and salt stress as mentioned before. similarly, bai et al, (2021) suggested that phytoremediation of hvs pollution by sweet sorghum was only effective at high concentrations of nitrate and by decreasing nitrate concentration the phytoremediation efficiency decreased. 4.6. lipid peroxidation were affected under stress condition heavy metals pollution, causes changes in some plant processes such as lipid peroxidation (ashraf et al, 2017). the results of this study showed the high lipid peroxidation under control treatment. similar results were reported on rice, that by increasing lead level the lipid peroxidation was decreased (ashraf et al, 2017; li, et al, 2021; sun, et al. 2021; xu, et al, 2021; zhang, et al. 2022 ). 5. conclusions based on the obtained results of the present study, it can be concluded that with the increasing in salinity stress from 0 to 3 dsm-1, the content of n (from 5.69% to 5.26%), k (from 4% to 3.21%) and nitrate (from 0.36 to 0.28 mg kg-1) significantly reduced. also, results showed that with the increasing in salinity stress (from 3 to 6 dsm-1) and nitrate level (from 30 to 60 mgl-1) in the soil, plant pb concentration significantly decreased. additionally, under conditions of pb stress, the uptake of nutrients (especially macronutrients) significantly improved with the increasing in the nitrate level from 30 to 60 mgl-1. it appears that stress conditions increased plant’s nitrate requirement, which could be considered as a strategy for the improvement of plant tolerance under hms stress. on the other hand, the increase in k ranged from 1.8 to 5% and also k/na ratio ranged from 1 to 6 can be considered as a resistance mechanism of plants under salinity stress. in addition, the synthesis of mda and other biochemical compounds in japanese raisin, grown under pb contamination stress, has been reported for the first-time providing  ideas for future studies. it should be mentioned that the studied species absorbed the moderate concentrations of pb (34.1-71 mgkg-1) indicating its potential for hms phytoremediation. generally, based on the results of the present study described before, japanese raisin can be considered as a relatively susceptible to salt stress. references abbasi sm, chorom n, zamir e. 2013. the effect of salinity and heavy metals (cd and pb) on phytoremediation of potamogeton berchtoldi. 2nd national conference on sustainable agriculture and health environment. 12 september. hamedan, iran. abdelgawa h, zinta g, hegab mm, pandey r, asard h, abuelsoud w. 2016. high salinity induces different oxidative stress and antioxidant responses in maize seedlings organs. journal of frontiers in plant science. 7: 276. https:/ doi:org/10.3389/fpls.2016.00276. abili j, zare s. 2014. evaluation of antioxidant enzymes activity in canola under salt stress. journal of advances in agriculture. 2(2), 88-92. adesodun jk, atayese mo, agbaje ta, osadiaye ba, mafe of, soretire aa. 2010. phytoremediation potentials of sunflowers (tithonia diversifolia and helianthus annuus) for metals in soils contaminated with zinc and lead nitrates. journal of water, air, and soil pollution. 207(1-4),195-201. 46 seyedeh mahsa hosseini et al. bi, d., c. dan, m. khayatnezhad, z. sayyah hashjin, z. y. ma 2021. molecular identification and genetic diversity in hypericum l.: a high value medicinal plant using rapd markers markers. genetika 53(1): 393-405. duan, f., fei song, sainan chen, majid khayatnezhad, noradin ghadimi, 2022. model parameters identification of the pemfcs using an improved design of crow search algorithm. international journal of hydrogen energy, 47(79): 33839-33849 gholamin, r. & khayatnezhad, m. 2020a. assessment of the correlation between chlorophyll content and drought resistance in corn cultivars (zea mays). helix, 10, 93-97. gholamin, r. & khayatnezhad, m. 2020b. the effect of dry season stretch on chlorophyll content and rwc of wheat genotypes (triticum durum l.). bioscience biotechnology research communications, 13, 1833-1829. gholamin, r. & khayatnezhad, m. 2020c. study of bread wheat genotype physiological and biochemical responses to drought stress. helix, 10, 87-92. gholamin, r. & khayatnezhad, m. 2020d. the study of path analysis for durum wheat (triticum durum desf.) yield components. bioscience biotechnology research communications, 13, 2139-2144. gholamin, r. & khayatnezhad, m. 2021. impacts of peg-6000-induced drought stress on chlorophyll content, relative water content (rwc), and rna content of peanut (arachis hypogaea l.) roots and leaves. bioscience research, 18, 393-402. gheshlaghi z, khorasani r, haghnia gh, kafi m. 2015. effect of nitrate and harvest time on yield and concentration of iron, zinc and copper in lettuce. journal of crop production and processing. isfahan university of technology. 5 (16), 315-330. https://doi: 10.18869/acadpub.jcpp.5.16.315. 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international journal of agriculture and crop sciences. vol, 4 (7), 398-403. zhang, j., m. khayatnezhad, and n. ghadimi, 2022. optimal model evaluation of the proton-exchange membrane fuel cells based on deep learning and modified african vulture optimization algorithm. energy sources, part a: recovery, utilization, and environmental effects, 44(1):287-305. caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 3 2022 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 73(3): 97-102, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-218 caryologia international journal of cytology, cytosystematics and cytogenetics citation: y.-c. tang, p.-p. yang, m.-h. yang, g.-r. he, y.-w. cao, l.-f. xu, j. ming (2020) karyotype analysis of lilium lancifolium and four related cultivars. caryologia 73(3): 97-102. doi: 10.13128/ caryologia-218 received: april 13, 2019 accepted: june 19, 2020 published: december 31, 2020 copyright: © 2020 y.-c. tang, p.-p. yang, m.-h. yang, g.-r. he, y.-w. cao, l.-f. xu, j. ming. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyotype analysis of lilium lancifolium and four related cultivars yu-chao tang1,#, pan-pan yang1,#, mei-hua yang2, guo-ren he1, yu-wei cao1, lei-feng xu1,*, jun ming1,* 1 the institute of vegetables and flowers, chinese academy of agricultural sciences, beijing 100081, china 2 school of landscape architecture, beijing university of agriculture, beijing 100081, china *corresponding authors. e-mail: xuleifeng@caas.cn (leifeng xu); mingjun@caas.cn (jun ming) # these authors contribute equally to the article. abstract. lilium lancifolium is one of the most important species of genus lilium. besides ornamental value, it also has highly edible and medicinal properties. we investigated the karyotypes of l. lancifolium and four related cultivars. the results indicated that the ploidies of four cultivars varied from diploid to tetraploid. both ‘flore pleno’ and ‘red velvet’ were triploid (2n=3x=36), consistenting with the wild species l. lancifolium. ‘sweet surrender’ was diploid (2n=2x=24), and ‘red life’ was tetraploid. all karyotypes of candidates belonged to 3b type except ‘flore pleno’, which belonged to 3a type. karyotype symmetry analysis revealed that the wild species l. lancifolium had a middle value of a1, a2, and tf%, which meant that the cultivars related to l. lancifolium had different tendencies to symmetry compared to l. lancifolium, but whether they were higher or lower was unclear. keywords: lilium lancifolium, cultivars, karyotype, chromosome. introduction genus lilium includes approximately 100-115 wild species (liang and tamura, 2000). lilium lancifolium, the tiger lily, one of the most important species of genus lilium, is widely distributed in northern and eastern asia, including korea, japan, china and sakhalin (feldmaier and mcrae, 1982). besides ornamental value, l. lancifolium is also extensively used as both food and a traditional chinese medicine for many centuries in china due to its health-promoting properties and treatments of bronchitis, pneumonia, chronic gastritis (chau and wu, 2006; luo et al., 2012; joung et al., 2007; kwon et al., 2010; gao et al., 2015). in addition, l. lancifolium has been used as a modal plant to study the mechanism of bulbil formation in lilies (yang et al., 2017, 2018; he et al, 2020), because it is one of the four lilium species which can bear bulbils in leaf axils (mcrae, 1998; liang and tamura, 2000; bach and sochacki, 2012). 98 yu-chao tang et al. karyomorphological investigations are very important for the views of ta xonomica l, ecologica l and cy tological studies. although the karyotype features are generally constant in a group of species and even a genus, the variations in structure and/or number can change the number, size, and position of the centromere on chromosomes, causing genetic variation (ahn et al., 2017). in addition, chromosome number can also complement information of polyploidy and other highly significant genome changes which are invisible by morphological and molecular methods. for more than 40 years, karyotype analysis of many wild lilium species including l. lancifolium (gill et al., 1974; vosa et al., 1976; gao et al., 2011) and hybrids (khan et al., 2009; liu et al., 2011) has been carried out. that provides a lot of significant theoretical basis for cytological and taxonomic studies of lilium. it is interesting that all lilium species are diploids (2n=2x=24) except l. lancifolium, which consists of ploidy complex with diploids, narrowly distributed, and triploids (2n=3x=36), widely distributed in inlands of china, korea, japan and russia, in nature (noda 1978, 1986; kim et al., 2006; hwang et al., 2011). because l. lancifolium distributed in china is natural triploid (2n=3x=36) (noda, 1986), it is highly sterile in cross breeding, neither as male or female parent, which seriously stunts the upgrading of its varieties. recent years, several varieties related to l. lancifolium have emerged on market, but the karyotype analysis of l. lancifolium related cultivars have never been reported. here, we collected four cultivars related to l. lancifolium and investigated the karyotypes of l. lancifolium and four related cultivars, providing cytological and genetic foundation for the breeding of l. lancifolium. materials and methods plant materials l. lancifolium and four related cultivars (‘red life’, ‘sweet surrender’, ‘red velvet’, ‘flore pleno’) were used as materials in this study (figure 1). l. lancifolium bulbs were harvested from our farm (beijing, china: 116.58°e; 40.07°n) in october 2017. bulbs of l. lancifolium related cultivars were purchased from licai garden co., ltd (zhejiang, china). all bulbs were planted in the matrix of peat, vermiculite, perlite with volume ratio of 5:3:1, in a greenhouse at the institute of vegetables and flowers, chinese academy of agricultural sciences, beijing, china in march 2018. root tips were obtained from each population for squashing. chromsome preparation and observation actively growing root tips of each populations for the length around 0.5 cm were taken between 9 am and 11 am in a clear day, and pretreated with 0.7% cycloheximide solution at 4°c for 10 h, then fixed in carnoy’s i fluid (methanol: glacial acetic acid = 3:1) for 24 h at room temperature, and finally kept in 70% alcohal until use. the roots were hydrolyzed in 1 mol/l hcl at 60°c for 10 min, then the chopped root tips were stained in carbol fuchsin stain for 5~6 min at room temperature. the roots must be rinsed in distilled water for 3 to 5 times before each step. the observations of the best metaphase plates were made using an olympus cx31 (olympus light microscope, tokyo, japan) equipped with a 100 × /1.25 oil objective and a mounted canon 550d digital camera (canon, japan). figure 1. the five materials in this study. a: l. lancifolium, b-e: l. lancifolium cultivars: 'flore pleno', 'sweet surrender', 'red life', 'red velvet', respectively. 99karyotype analysis of lilium lancifolium and four related cultivars karyological analyses the measurement of basic parameters such as long arm (la), short arm (sa) related to every chromosome was performed utilizing photoshop v.7. based on which we calculated total length of genome (tlg), ar (arm ratio), ci (centromeric index), la% (long arm percentage), sa% (short arm percentage), tf% (total form percentage), vrc (value of relative chromatin), a1 (intrachromosome asymmetry index), a2 (interchromosome asymmetry index) and di (dispersion index). the karyotype formula was calculated according to the definition of metacentric (m), submetacentric (sm), telocentric (t), and sbtelocentric (st), proposed by levan et al. (1964). the karyotype classification was defined by using the method of stebbins (1971). finally, the data analysis was conducted in excel. results this study revealed detailed pictures of mitotic chromosome plates, related karyotypes and karyograms in l. lancifolium and its four cultivars (figure 2). the related parameters and karyotypic formula were summarized in table 1 and table 2. the results showed that, l. lancifolium (figure 2-a) is triploid, which had a chromosome number of 2n=3x=36; while in the four cultivars, only ‘flore pleno’ (figure 2-b) and ‘red velvet’ (figure 2-e) were triploid, with the same chromosome number as l. lancifolium, 2n=3x=36; ‘sweet surrender’ (figure 2-c) was diploid, 2n=2x=24; and ‘red life’ (figure 2-d) was tetraploid, 2n=4x=48. in five materials, only l. lancifolium had satellites attached to the first two pairs of chromosomes, and ‘flore pleno’had one group of chromosomes attached with satellites, the other three cultivars had no satellites. according to the karyograms in figure 2, besides ‘red velvet’ had two groups of telocentric chromosome, l. lancifolium, ‘flore pleno’ and ‘sweet surrender’ had one set telocentric chromosome, and ‘red life’ has no telocentric chromosome (figure 2). the length of shortest chromosome in l. lancifolium (table 1-a) is 10.27 μm, which was obviously longer than its four cultivars’. the total genome length ranged from 117.44 to 191.05 μm between tetraploid ‘red life’ (table 1-d) and wild species l. lancifolium in all populations. even the karyotype formulas of five materials were complicated, at least four different chromosometypes were contained in each species, most of the five materials had the same karyoty pe 3b except ‘flore pleno’ (table 1-b), whose belongs to 3a (table 1). the table 1. karyotype analysis of l. lancifolium and four related cultivars. sample chromosome number chromosome size range (μm) total genome length (μm) karyotype karyotypic formula a 2n=3x=36 10.27-21.15 191.05 3b 3m(2sat)+3sm(3sat)+12st+15t+3t b 2n=3x=36 7.57-14.80 127.37 3a 3sm(3sat)+18st+12t+3t c 2n=2x=24 8.68-19.83 155.29 3b 2m+4sm+4st+12t+2t d 2n=4x=48 6.52-14.15 117.44 3b 4m+8sm+28st+8t e 2n=3x=36 7.25-16.54 153.44 3b 3m+9sm+18st+6t notes: a: l. lancifolium, b-e: l. lancifolium cultivars: 'flore pleno', 'sweet surrender', 'red life', 'red velvet', respectively. table 2. mean of parameters of chromosomes analysis of l. lancifolium and four related cultivars. species tl la sa ar ci la% sa% tf% vrc a1 a2 di a 15.64 12.98 2.66 4.88 0.17 6.79 1.39 16.70 15.92 0.79 0.20 2.47 b 10.45 8.92 1.53 5.83 0.15 7.00 1.20 14.45 10.61 0.81 0.21 3.01 c 12.94 10.49 2.45 4.28 0.19 6.67 1.58 18.95 12.94 0.77 0.26 3.09 d 9.78 7.58 2.20 3.45 0.22 6.45 1.87 22.50 9.79 0.71 0.22 3.44 e 12.79 10.06 2.73 3.68 0.21 6.56 1.78 21.35 12.79 0.70 0.19 3.43 notes: a: l. lancifolium, b-e: l. lancifolium cultivars: 'flore pleno', 'sweet surrender', 'red life', 'red velvet', respectively. tl: total length of chromosome, la: long arm, sa: short arm, ar: arm ratio, ci: centromeric index, la%: long arm percentage, sa%: short arm percentage, tf%: total form percentage, vrc: value of relative chromatin, a1: intrachromosome asymmetry index, a2: interchromosome asymmetry index, di: dispersion index. 100 yu-chao tang et al. mean value of the chromosome long arm varied from 7.58 μm to 12.98 μm in ‘red life’ (table 2-d) and l. lancifolium (table 2-a), respectively. the average of short arm lengths ranged between 1.53 μm and 2.66 μm in ‘flore pleno’ (table 2-b) and l. lancifolium. and the average total length of chromosomes varied from 9.78 μm to 15.64 μm in ‘red life’ and l. lancifolium (table 2). figure 2. mitotic chromosome plates, related karyotypes and karyograms of l. lancifolium and four related cultivars. a: l. lancifolium, b-e: l. lancifolium cultivars: 'flore pleno', 'sweet surrender', 'red life', 'red velvet', respectively. 101karyotype analysis of lilium lancifolium and four related cultivars to evaluate the symmetry of karyotype, we calculated ar (arm ratio), ci (centromeric index), la% (long arm percentage), sa%(short arm percentage), tf% (total form percentage), vrc (value of relative chromatin), a1 (intrachromosome asymmetry index), a2 (interchromosome asymmetry index) and di (dispersion index) respectively based on tl (total length of chromosome), la (long arm) and sa (short arm) (table 2). the results showed that, among the studied populations, the highest tf% value (22.50) was estimated in ‘red life’ and the lowest tf% value (14.45) was estimated in ‘flore pleno’, the tf% valve (16.70) of l. lancifolium was the second lowest. the analysis of the intra-chromosome asymmetry (a1) and inter-chromosome asymmetry (a2) revealed that, ‘red velvet’ (with mean value of a1=0.70, a2=0.19) presented the smallest asymmetry. in this study, the wild species l. lancifolium had a lowest di value (2.47), and the di values four cultivars were much higher (3.01~3.44) (table 2). discussion the karyomorphological investigations of lilium are very important for the views of taxonomical and ecological studies (ahn et al., 2017). in this study, the results showed that, l. lancifolium (a) was triploid, which had a chromosome number of 2n=3x=36, coinciding with previous report (gao et al., 2011). while the ploidy of the four cultivars related to l. lancifolium (a) varied from diploid to tetraploid. since the chromosome numbers of f1 hybrids of triploid l. lancifolium × diploid l. leichtlinii range from 24 to 34 (suzuki and yamagishi, 2016), and the chromosome numbers of f1 hybrids of triploid l. lancifolium × tetraploid ‘brunello’ can reach to 50 (ma, 2017), these four cultivars we studied might be obtained by hybridization between triploid l. lancifolium and other tetraploid lilies. according to previous studies, all the karyotypes of lilium belong to 3b and 3a type (stebbins, 1971; gao et al., 2011). in our study, only ‘flore pleno’ belonged to 3a, the other four belonged to 3b. that corroborate that the karyotype of genus lilium is stable. both the karyograms and the value of tf% indicate that the karyotypes of five materials are very asymmetric. by using the values of a1, a2 (zarco, 1986) and tf% (huziwara, 1962), we can evaluate the symmetry of karyotypes among close classes. the present study revealed that the wild species l. lancifolium had a middle value whether of the a1, a2, or tf%, which meant that the cultivars related to l. lancifolium had different tendencies to symmetry compared to l. lancifolium, but whether they were higher or lower was uncertain. di index plays an important role in arranging the species within the same class of karyotype asymmetry in an advancing order of specialization by permitting further gradations, as depicted by species arrangement within sections (lavania and srivastava, 1992). we found that the wild species l. lancifolium had the lowest di value compared to its cultivars. this might indicate that the hybrid progenies of 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frontiers in plant science, 8: 1508. yang pp, xu lf, xu h, he gr, feng yy, cao yw, ming j. 2018. morphological and anatomical observation during the formation of bulbils in lilium lancifolium. caryologia, 71(2): 146-149. zarco cr. 1986. a new method for estimating karyotype asymmetry. taxon, 526-530. zhang t, gao j, jin zy, xu xm, chen hq. 2014. protective effects of polysaccharides from lilium lancifolium on streptozotocin-induced diabetic mice. international journal of biological macromolecules, 65: 436440. caryologia. international journal of cytology, cytosystematics and cytogenetics 75(3): 19-29, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1791 caryologia international journal of cytology, cytosystematics and cytogenetics citation: selma tabur, nai ṁe büyükkaya bayraktar, serkan özmen (2022). l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations. caryologia 75(3): 19-29. doi: 10.36253/caryologia-1791 received: august 22, 2022 accepted: november 02, 2022 published: april 5, 2023 copyright: © 2022 selma tabur, nai ṁe büyükkaya bayraktar, serkan özmen. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations selma tabur1,*, nai̇me büyükkaya bayraktar2, serkan özmen1 1 department of biology, faculty of arts and science, süleyman demirel university, 32260 isparta, turkey 2 süleyman demirel education complex, 32260 isparta, turkey *corresponding author. e-mail: taburs@gmail.com abstract. the objective of the present study was to with all details explain of the efficiency of l-ascorbic acid (l-asa) also known as vitamin c on cytotoxicity and genotoxicity induced by salt stress in the barley apical meristems. as a result of the statistical analysis salt stress caused a significant (p ≤ 0.05) decrease in mitotic index of barley seeds depending on concentration increase, while the frequency of chromosomal aberration (ca) increased. in addition, it was determined that mitotic index value was decreased by 46% with 1 μm l-asa supplementation as compared to control and chromosomal abnormalities were increased by 8.96% as well as. however, in the case of simultaneously application of 1 μm l-asa and different salt concentrations, the high salt concentrations exhibited an excellent success according to low salt concentrations in alleviating the mitodepressive effect of salt stress. moreover, the frequency of chromosomal aberrations in the root meristem cells of those seeds with 1 μm l-asa supplementation germinated at different salt concentrations was substantially reduced compared to own control group (alone 1 μm l-asa pretreatment). the 1 μm l-asa pretreatment at the highest salt concentration (at 0.40 m) was showed an excellent success by reducing the frequency of the chromosomal aberrations by approximately 90 %. different salt concentrations and/or 1 μm l-asa supplementation caused micronuclei and granulation as well as various chromosomal aberrations in prophase, metaphase, anaphase and telophase. keywords: cytotoxicity, genotoxicity, hordeum vulgare l., mitotic index, ascorbic acid, salinity. introduction together the global climate change, which is starting to make its presence felt more and more, plants are becoming more frequently subjected to adverse abiotic stresses, such as extreme temperatures, cold, high salinity, and drought, which limiting plant growth and crop productivity. salinity is one of the major environmental factors that reduce plant productivity 20 selma tabur, nai̇me büyükkaya bayraktar, serkan özmen (tobe et al. 2003; sabagh et al., 2019). nearly 20% of the world’s cultivated land and also five-hundred thousand hectares of irrigation area in turkey are threatened by salinity (fao 2016). salt stress inhibits or delay growth and development of plants by negatively affects plant growth via oxidative stress, especially ion toxicity, nutritional and hormonal imbalance, and osmotic stress (parida and das 2005; ashraf, 2009; elsheery et al. 2020a). moreover, the retardant effects of salinity stress on growth, physiological aspects, productivity and cellular activity were also recorded on other different many plants species (bargaz et al. 2016; nassar et al. 2016; elsheery 2020b; tabur et al. 2021). however, plants develop highly complex mechanisms for tolerate salinity. tolerance to salt stress of plants is of three types: osmotic stress tolerance, na+ or clexclusion, and the tolerance of tissue to accumulated na+ or cl(munns and tester 2008; zvanarou et al. 2020). since the mechanisms behind salinity are quite complex and difficult to understand, impact of salinity on plants, type and causes of salinity, and salt tolerance strategies of plants are still discussed in level cellular and molecular (zhu et al. 2016). the common view of many researchers in combating salinity is the development of high salt-tolerant plant varieties. however, this method, which is one of the economical ways to eliminate the negative effects of salinity on plants, shows inconsistency between different crops. therefore, there is a great scientific burden on researchers to cope with this important environmental stress that also limit crop productivity. for all these reasons, most of the researchers contributed to overcome the disadvantages of salt stress and to develop salt tolerant varieties by using various hormones, plant growth regulators, leaf extracts, vitamins biofertilizer and amino acids (tabur and demir 2010 a,b; mohsen et al. 2014; çavuşoğlu et al. 2016 a,b; naser et al. 2016; mahfouz and rayan 2017; farheen et al. 2018; özmen and tabur 2020; tabur et al. 2021). in recent studies, it has been reported that some vitamins may be effective to alleviate the negative effects of salinity by increase resistance to salt stress (shalata and neumann 2001), plant growth and yield quality (el-bassiouny et al. 2005; bassuony et al. 2008), seed germination, seedling growth (emam and helal, 2008), mitotic activity (özmen and tabur 2020) some metabolic changes. ascorbic acid (asa) is a naturalist product that acts as an antioxidant and enzyme and also improves cofactor. it acts as an essential substrate in the cyclic pathway of enzymatic detoxification of hydrogen peroxide. there are various isomers of ascorbic acid or vitamin c (l-asa, d-asa, d-izoasa). d-asa and d-isoasa do not have vitamin c function. therefore, when ascorbic acid is mentioned, l-ascorbic acid (3-keto-l-gulofuranolaktan) comes to mind from these isomers because of the only isomer with biological activity (dizlek and gül, 2007). the stimulatory roles of l-asa, a minor, water-soluble antioxidant, in plant growth and other developmental processes are well documented (gallie 2013; hossain et al. 2017; gaafar et al. 2020). in plants l-asa serves as a major redox buffer and regulates various physiological processes controlling growth, development, and stress tolerance. being a major component of the ascorbate-glutathione (asa-gsh) cycle, l-asa helps to modulate oxidative stress in plants by controlling ros detoxification alone and in co-operation with glutathione. any fluctuations, increases or decreases, in cellular l-asa levels can have profound effects on plant growth and development, as l-asa is associated with the regulation of the cell cycle, redox signaling, enzyme function and defense gene expression (hossain et al. 2017). the ascorbic acid concentration increases in plant cells exposed to stress conditions and plays a role in providing tolerance against oxidative stress by playing a role in the direct clearance of o-2 and oh-. as a result of enzyme and gene expression analyzes carried out under different abiotic conditions in many plants, it was determined that ascorbic acid-related gene expression levels increased and these increases were given as a defense response against stress. on account of this, it is emphasized that higher l-asa levels are important to minimize oxidative stress and regulate plant metabolic processes. (athar et al. 2008, 2009; akram et al. 2017). the cellular asa pool size in plants can be regulated by the coordinated action of many related enzymes. numerous recent studies have confirmed that asa level increases the tolerance and adaptation of crops to many abiotic stresses such as cold, drought, salinity, heavy metal toxicity and ozone stresses (xie et al. 2009; çavuşoğlu and bilir 2015; akram et al. 2017; xu 2017; sabagh et al. 2019; gaafar et al. 2020; nunes et al. 2020; wang et al., 2020; chen et al. 2021). as mentioned above, there are many studies on the effects of asa on seed germination, seedling growth, plant resistance, plant growth and yield quality, antioxidant enzyme activity, and some biochemical and metabolic changes under various abiotic stress conditions. in addition, it has been known for few decades that asa plays an important role in plant growth and development by regulating cell division (smirnoff 1996; gallie 2013). however, a rather limited number of studies have been found on the response of ascorbic acid to cytotoxicity and genotoxicity caused by various abiotic stresses, including salinity (barakat 2003; yu et al. 2014; el-araby 21l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells et al. 2020). for this reason, this work was designed to comprehensively test of the efficiency level of exogenous l-asa against effects cytotoxic and genotoxic in caused by salt stress in barley meristem cells and to contribute to the gap in the literature. namely, it is aimed at clarifying to what extent exogenous l-asa is able to tolerate salt stress, whether it encourages cells to enter the mitosis division, and whether it causes any changes in the structure and behavior of chromosomes. materials and methods the barley cultivar (hordeum vulgare cv. ‘bülbül 89’) used in this study was requested from the field crops research institute, ankara, turkey. nacl and ascorbic acid (l-asa) used in the experiments were obtained from merk and sigma-aldrich, respectively. primarily, to prevent fungal contamination, the barley seeds were surface sterilized by immersion in 1% (w/v) naclo solution for 10 min, rinsed thoroughly five times with sterile distilled water and dried on filter papers at room temperature prior to experimental procedure. the sterilized seeds were divided into two groups and soaked in constant volumes (50 ml) of distilled water (control, c) and l-asa (1 µm, micromolar) for 24 h at 20 ± 1oc. the solutions were filtered at the end of this pretreatment session and 20-25 barley seeds which uniform sized were placed in petri dishes covered with two sheets filter papers moistened with 7 ml of distilled water or three different nacl (0.32, 0.35 and 0.40 m, molar) concentrations. after, petri dishes were transferred to incubators at constant temperature (20 ± 1 °c) for germination for several days. these salt levels hindering germination of seeds on a large scale and the most proper concentration of l-asa level in alleviation of the salt inhibition at the germination were determined in a preliminary investigation conducted by us. to cytogenetic analyses after 3 or 4 days, the root tips reached to 0.5-1 cm were excised, pretreated with a saturated solution of paradichlorobenzene for 4 h at 20ºc, fixed with carnoy’s fluid i (absolute ethanol: glacial acetic acid, 3:1, v/v) for 24 h, and stored in 70% ethanol at 4ºc until required. then, root tips were hydrolyzed in 1 n hcl at 60ºc for 15-18 min, stained for 1-1.5 h in accordance with the standard procedure for feulgen staining, and squashed in 45 % acetic acid (sharma and gupta 1982; elçi and sancak 2013). after one day, microscopic slides were made permanent in by mounting canada balsam by alcohol vapor exchange method. the best mitosis phases and aberrances were observed in permanent slides and photographed (100x) with a digital camera (olympus c-5060) mounted on an olympus cx41 microscope. the prepared slides were examined under the microscope at 100x magnification, and mitotic index, i.e. percentage of dividing cells were accounted by counting approximately 15000 cells (three repeat, 5000 per slide) for all per-application. the mitotic index (mi) was calculated using the following the equation: in addition, chromosomal aberrations (ca) occurring at all stages of mitosis during microscopic observation of the slides were calculated according to the following the equation for each per-application as the percentage of 350 dividing cells counted. all experiments were repeated three times. statistical evaluations of obtained data were actualized using the spss 14.0 program and duncan’s multiple range test (duncan, 1955). results the mitotic index (mi) data obtained from the cytological analysis of barley root tips treated with different nacl levels and 1 μm l-asa (vitamin c) are summarized in table 1. based on these data, mi gradually drastic reduced with parallel to increasing nacl levels as compared to the control group. at the highest salt level (at 0.40 m, molar), the mitotic index was reached to the lowest value by reducing from 7.0 ± 1.5 (control, in distilled water) to 1.6 ± 0.07 (77%). in the root meristem cells exposed to 1 μm l-asa alone, a mitotic index reduction of about 46% was recorded according to control group. when samples with l-asa treated germinated at different salt levels were compared with their selves control group (l-asa alone), it was determined that the mitotic index increased statistically a little except for 0.32 m nacl. 0.35 and 0.40 m nacl levels exhibited major successful compared as each other the mitotic index values of l-asa pre-treated and untreated samples at the same salt concentrations (table 1). especially, it was recorded that at the highest salt concentration (0.40 m nacl) the mitotic index value was increases approximately two and a half times (from 1.6 ± 0.07 in control group to 4.0 ± 0.3 in 1 μm asa). 22 selma tabur, nai̇me büyükkaya bayraktar, serkan özmen the chromosomal aberration frequencies data obtained from barley root tips germinated both distilled water and different nacl levels in the absence or presence of 1 μm l-asa are summarized in table 1. in parallel with the increasing salt concentrations, a very high rate of chromosomal aberrations observed in the root meristem cells of barley seeds. that is, while the chromosomal aberration frequency was 0.00±0.0 in the control seeds germinated in distilled water medium, it was recorded as 2.30±1.0 at 0.32 m salinity, 8.96±2.5 at 0.35 m salinity, and 21.2±2.5 at 0.40 m salinity. on the other hand, the frequency of chromosomal aberrations in seeds germinated in salt stress-free medium after 1 μm l-asa supplementation alone was remarkably higher than that in the control group (distilled water, 0.00 m nacl) and was also statistically significant. however, the frequency of chromosomal aberrations of seeds germinated at different salt concentrations after 1 μm l-asa supplementation has exhibited a statistically significant decrease compared to the percentage of seeds treated with 1 μm l-asa alone. when these values are compared with the frequencies of seeds germinated only at different salt concentrations, although1 μm l-asa supplementation partially increased the chromosome aberration rate at the lowest salt level studied, it significantly reduced the negative effect of salt stress on this parameter, especially at high salt levels (at 0.35 and 0.40 m salinity). in other words, while the chromosomal aberration rate was 8.96 % at 0.35 m salinity and 21.2 % at 0.40 m salinity, the application of 1 μm l-asa showed an excellent success, reducing these aberration rates to 1.70% and 2.00%, respectively (table 1). as a result of scans in mitosis slides, no abnormality was found in the meristem cells of the control group barley seeds germinated in distilled water and at 20°c, and all stages of mitosis were observed normally (figure 1). microscopic images of a wide range of chromosome aberrances observed in the preparations prepared with table 1. mitotic index scores and frequency of chromosome aberrations in meristem cells of h. vulgare l. exposed to different nacl concentrations after 1 μm l-asa supplementation nacl (m, mol/l) and l-asa (μm) concentrations mitotic index (%) chromosome aberrations (%) control (0.00, distilled water) *7.0±1.5c *0.00±0.0a 1 μm l-asa 3.8±0.4b 8.96±2.5b 0.32 m nacl 6.3±0.4c 2.30±1.0a 0.32 m nacl + 1 μm l-asa 3.3±0.7b 2.50±2.0a 0.35 m nacl 2.8±0.1b 8.96±2.5b 0.35 m nacl + 1 μm l-asa 4.0±0.2b 1.70±0.6a 0.40 m nacl 1.6±0.07a 21.2±2.5c 0.40 m nacl + 1 μm l-asa 4.0±0.3b 2.00±2.0a *values with insignificant difference (p ≤ 0.05) for each column are indicated with same letters (± standard deviation). as test solution, 1 µm ascorbic acid (l-asa) was used. concentrations of nacl were 0.32, 0.35, 0.40 m (mol/l). the pretreatment process of seeds was performed by soaking 24 h in constant volumes of distilled water (control) or l-asa. different concentrations of salt were added to germination medium. all data were evaluated as three replicates figure 1. normal mitosis stages in meristem cells of h. vulgare l. germinated in distilled water (control). aprophase bmetaphase (2n = 14) canaphase dtelophase. scale bar = 10µm. figure 2. aberrations observed in pre-prophase and prophase stage in meristem cells of h. vulgare l. germinated at different nacl concentrations after 1 µm l-asa supplementation for 24 h. a-bmicronuclei, c-dchromatin granulation in interphase, e-fdisorderly prophase. scale bar = 10µm. 23l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells root tips belonging to all other application groups are shown in figure 2-5. the most common chromosome abnormalities observed in all application were micronucleus, disorderly prophase and anaphase, uncoiling chromosome, sticky chromosome, bridges in anaphase, and false polarization in anaphase and telophase. the abnormalities such as alignment anaphase and vagrant chromosomes were observed in the minimal level. discussion in the present work, effect cytotoxic and genotoxic in the apical meristem cells of barley seeds exposed salt stress of exogenous 1 μm l-asa supplementation were investigated comprehensive. a lt hough its mechanism has not been f u lly explained yet, the effects of salinity stress one of the most important abiotic stresses have been known for a long time by many researchers at cellular and chromosomal levels (lutsenko et al. 2005; tabur and demir 2010 a,b; pekol et al. 2016; kiełkowska et al. 2017; elaraby et al. 2020). all these researchers agree that salt stress causes chromotoxic actions and total inhibition of mitotic processes on meristematic cells, just as in our study. in addition to, zvanarou et al. (2020) reported figure 5. aberrations observed in telophase stage in meristem cells of h. vulgare l. germinated at different nacl concentrations after 1 µm l-asa supplementation for 24 h. a-bfalse polarization in telophase, c-dfalse polarization in telophase and vagrant chromosomes (arrows). scale bar = 10µm. figure 3. aberrations observed in metaphase stage in meristem cells of h. vulgare l. germinated at different nacl concentrations after 1 µm l-asa supplementation for 24 h. a-buncoiling chromosomes, c-dsticky chromosomes. scale bar = 10µm. figure 4. aberrations observed in anaphase stage in meristem cells of h. vulgare l. germinated at different nacl concentrations after 1 µm l-asa supplementation for 24 h. adisorderly anaphase, b-dbridges in anaphase (arrows), elaggard chromosome (arrow), f-galignment anaphase, h-ıfalse polarization in anaphase. scale bar = 10µm. 24 selma tabur, nai̇me büyükkaya bayraktar, serkan özmen that dividing root meristem cells are more sensitive to nacl than other tissues since remains in direct contact with abiotic stress factors. however, salt damage extent depends upon plant species, stages of plant development, genotype, salinity concentration, and exposure time (vicente et al. 2004; tabur et al. 2021). to date, many studies have been conducted on the effect of ascorbic acid on morpho-physiological, biochemical and metabolic changes under both normal and various stress conditions using various plant species (khan et al. 2006; dolatabadian ve jouneghani 2009; fatemi 2014; mohsen et al. 2014; gaafar et al., 2020; nunes et al. 2020; chen et al. 2021). however, studies on the protective role of exogenous l-asa supplementation against the cytotoxic effects of various abiotic stresses and its effect on mitotic activity and chromosomal abnormalities, especially against salt stress, are quite insufficient (barakat 2003; yu et al. 2014; el-araby et al. 2020). therefore, first of all, it was found appropriate to compare the effects of l-asa during germination in distilled water at 20°c before proceeding to its effects on these parameters under salt stress conditions. as mentioned in the research findings section, the mitotic index value of barley seeds that were not pretreated with l-asa (0.00 control, c) was 7.0±1.5, while this value was 3.8±0.4 in seeds that were pretreated. in other words, l-asa supplementation alone caused a decrease of approximately 46% on the mitotic index compared to the control group (see table 1). mitotic index, as known is one of the most important indicators reliably identified the presence of cytotoxicity (fiskesjö 1985). the decrease of the mitotic index value below 50% compared to the control variant leads to a sublethal effect, while below 22% it can cause lethal effects on test organisms (mesi and kopliku 2013). undoubted, in this case 1µm l-asa supplementation alone has a potential for sublethal effects. in addition, l-asa application alone increased the rate of chromosomal aberrations by 8.96% compared to distilled water (see table 1). as a result of this study, it was revealed that 1µm l-asa supplementation alone reduced the mitotic index value and had a negative effect on chromosomal aberrations in barley seeds germinated in distilled water environment. our findings regarding mitotic index and chromosomal abnormalities are in agreement with the study reported in allium cepa by asita et al, (2017). however, cenanovic and durakovic (2016) reported that ascorbic acid treatment at different concentrations (250, 500 and1000 μg/ml) increased the mitotic index in allium cepa root meristems. it is thought that this difference may have occurred depending on the plant species studied and/or the dose and application time of the ascorbic acid used. as for the effect of l-asa application on the mitotic index and chromosomal aberrations of barley seeds germinated in saline conditions, the data obtained from our study on the mentioned parameters will be presented for the first time for barley plant. as a result of our literature research, only three previously reported studies were found that were more or less close to the subject. firstly, barakat (2003) reported that high salt concentrations significantly reduced mitotic activity and increased chromosomal aberrations in allium cepa l. however, the researcer has determined that the ascorbic acid supplementation significantly increased the mitotic index and reduced chromosomal aberrations by reducing inhibitory effect of salt. secondly, yu et al. (2014) emphasized that the application of the asa (0, 0.5, 1, 2, 4 mm) decreased markedly chromosome aberrations frequency, and increased mitotic index on vicia faba roots exposed to different concentration of pb (no3)2. finally, el-araby et al. (2020) has been researched the effects of two concentrations of asa (50 and 100 ppm) on the cytological parameters of pea seedlings under salinity stress. they reported that asa (100 ppm) treatments significantly reduced the damaging effect of salinity stress on mitotic index and chromosomal abnormalities percentage. similarly, also in our study, 1µm l-asa showed an excellent performance on the mitotic index of barley seeds under high salt stress conditions. for example, 1µm l-asa supplementation has increased mitotic index by approximately two and a half times at the highest salt stress condition (at 0.40 m salinity), (see table 1). in addition, l-asa supplementation under especially high salt stress conditions showed statistically positive effects on chromosomal aberrations in root meristems of barley seeds too. although the application of 1µm l-asa alone caused a significant increase of chromosomal aberrations in root meristem cells of seeds germinated in distilled water, in parallel with the increasing of the salt concentrations, the detrimental effect of supplementation 1µm l-asa has seriously reduced, from 8.96 ± 2.5% abnormal cells (at distilled water, control) to 2.00 ± 2.0% (at 0.40 m). moreover, while ratio of the chromosomal aberrations in the highest salt level studied (at 0.40 m) was 21.2 ± 2.5%, it was reduced to 2.00 ± 2.0% with the application of 1µm l-asa. in other words, 1µm asa application at 0.40 m salinity has shown an excellent success by almost zeroing the detrimental effect of salt stress (see table 1). that is, we can say that l-asa application may be more successful in high salt levels than in low salt levels in alleviating the detrimental effect of salt stress on chromosome structure and behaviors. from here, it can be concluded that effective in including the adaptive response to genotoxic stress since l-asa at 25l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells high salt concentrations significantly reduces the clastogenic effects induced by salinity. also, it is important to point out that l-asa, known as vitamin c, have the ability to reduce the toxic effect of various genotropic toxicants if used in appropriate doses and at the convenient stage of growth and development. in unstressed conditions, administration of l-asa alone might have been function as a stimulator, slowing down the mitotic cycle by suppressing the synthesis of proteins required for normal cell division (tabur et al. 2021). the slowdown in the mitotic cycle might have triggered mitodepressive effects during cell division, thus causing a significant increase (8.96%) of chromosomal aberrations. it has been known for a long time that external stimulatory growth regulator applications are useless and even harmful under normal conditions without stress (tabur ve demir 2010a). therefore, it is not surprising that l-asa application alone in distilled water reduces the mitotic index and increases chromosome aberrations. then, we can say that l-asa supplementation under stress conditions, especially at high salt concentrations (at 0.35 m and 0.40 m salinity), may have accelerated mitotic activity and consequently reduced chromosomal aberrations caused by stress. undoubtedly, these results supported that exogenous l-asa may play a protective role against the harmful effect of salt stress on chromosomes by eliminating the mitodepressive effects that occur under stress conditions. chromosomal abnormalities that occur spontaneously or as a result of exposure to environmental stresses are indicate the harmful effect of a toxic agent on plant cells (nag et al. 2013). many biotic and abiotic toxic agents can promote the occurrence of chromosome aberrations by different mechanisms, including aneugenic (changes in total chromosome number) and clastogenic (changes in chromosome structure) actions. feretti et al. (2007) sugessted that if toxic ajans cause damage to plant cell chromosomes, they may also be potentially harmful for mammalian cell chromosomes. micronucleus (mn) assay is accepted as the most effective endpoint to analyze the mutagenic effect of the toxic agents. the large mn in the cell indicates aneugenic effect resulting from chromosome loss while small mn indicates clastogenic effect due to chromosome breaks (kontek et al. 2007). briand and kapoor (1989) have reported that the micronuclei (figure 2 a, b) are probably the result of vagrant chromosomes and fragments. dane and dalgıç (2005) reported that chromatin granulation is related to the inhibition of enzymes and histone proteins. it emphasized by many researchers that several chromatin regulation-related factors, such as histone modification enzymes, linker histone h1, hmg proteins and atp-dependent chromatin remodeling factors have been functioned in plant abiotic stress responses (kim et al. 2010; asensi-fabado et al. 2017). chromatin granulation at interphase (figure 2 c, d), most likely caused to deformation of the nuclear material by toxic agents, might be a consequence of all these reasons and abnormal chromatin condensation and indicative of many abnormalities that may occur in future mitosis phases. uncoiling chromosomes (figure 3 a, b) and disorderly prophase (figure 2 e, f ) may be the result of a weak mitotic effect and irregular chromosome contractions (tabur et al. 2021). sticky chromosomes (figure 3 c, d) could be originated from abnormal dna condensation, abnormal chromosomal wrapping and inactivation of the axes (asita and mokhobo 2013). at the same time it has been asserted that such aberrations may be a result of improper folding of the chromatin fibers (klášterská et al. 1976). according to some researchers, sticky chromosomes are a marker of high toxic effect on chromatin and irreversibility of the change (fiskesjö and levan 1993; türkoğlu 2007). in the current study, all of mitoclassic impacts in anaphase and telophase (figure 4-5) that form an important portion of chromosomal abnormalities might have been largely resulted from spindle dysfunction. fiskesjö (1997) have informed that bridges (figure 4 b-d) are clastogenic effects, both resulting from chromosome and chromatid breaks. according to tabur and demir (2010 b) the bridges in anaphase and telophase might have been the result of inversions. moreover, bonciu et al. (2018) have asserted that nucleoplasmic bridges originate from dicentric chromosomes or occur as a result of as faulty longitudinal break of sister chromatids during anaphase. the disorganizations in mitosis such as disorderly anaphase (figure 4 a), fault polarization at ana-telophases (figure 4 h, ı; figure 5 a-d), alignment anaphase (figure 4 f, g) and bridges may be mainly the result of faulty kinetochore attachment or of spindle dysfunction (rieder and salmon 1998). such irregularities constitute a significant portion of chromosomal aberrations. vagrant (figure 5 c, d) and lagging chromosomes (figure 4 e) occurs during the anaphase where one or more chromatids gets detached from the rest of the chromatids and is incapable of moving towards the poles. patil and bhat (1992) have suggested that laggard chromosomes could be originate from the failure of spindle apparatus to organize in normal way. also, the laggard of chromosomes may have occurred due to a weak mitotic impress. it known that salt stress, particularly nacl caused too many c-mitotic reactions (fiskesjö 1997). therefore, increasing salt concentrations may have been reason to the formation of laggard chromosomes at high rates. briefly, l-asa alone and/or dif26 selma tabur, nai̇me büyükkaya bayraktar, serkan özmen ferent salt levels used in our study may have been caused to all these abnormalities mentioned above by triggering the stimulation/ inhibition of enzymes and proteins necessary for the normal cell division, by disturbing the spindle mechanism. conclusion in the present work, it has been compared the interactions between the mitotic index and chromosome behaviors of l-asa under normal and salt stress using barley seeds. as known, the mechanisms by which salinity affections plant growth and development are rather complex and also controversial since a long time. unfortunately, although the causes of salinity have been characterized, our understanding of the mechanisms by which salinity prevents plant growth is still rather poor. in summary, it was determined that l-asa supplementation alone significantly reduced mitotic activity (46%) and caused a very high (8.96%) abnormality on chromosome behaviors in this study. in this case, l-asa supplementation alone can create various types of mutations over time. however, this study supports that exogenous l-asa pretreatment, especially at high salt concentrations, can eliminate the negative effects of salinity on the mentioned parameters in barley plant. the obtained results in our work may provide new conceptual tools for designing the hypotheses of different salt tolerance in plants and to brighten many contradictions particularly in relation to effects of l-asa and high salt stress on mitotic activity and chromosomal abnormalities. surely! further investigation is needed to confirm these findings. consequently, surveying the effects of l-asa on principal metabolic events, which can be directly or indirectly effective on 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repetitive dnas in the picasso triggerfish (rhinecanthus aculeatus (linnaeus, 1758)) in family balistidae by classical and molecular cytogenetic techniques kamika sribenja1, alongklod tanomtong1, nuntaporn getlekha2,* chromosome number of some satureja species from turkey esra kavcı1, esra martin1, halil erhan eroğlu2,*, fatih serdar yıldırım3 l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations selma tabur1,*, nai̇me büyükkaya bayraktar2, serkan özmen1 characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques kristel ramírez-matadamas1, elva irene cortés-gutiérrez2, sergio moreno-limón2, catalina garcía-vielma1,* contributions of species rineloricaria pentamaculata (loricariidae:loricariinae) in a karyoevolutionary context a cius¹, ca lorscheider2, lm barbosa¹, ac prizon¹, ch zawadzki3, la borin-carvalho¹, fe porto4, alb portela-castro1,4 cadmium induced genotoxicity and antioxidative defense system in lentil (lens culinaris medik.) genotype durre shahwar1,2,*, zeba khan3, mohammad yunus khalil ansari1 biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts and evaluation of their biosafety potential denisa manolescu1,2, georgiana uță1,2,*, anca șuțan3, cătălin ducu1, alin din1, sorin moga1, denis negrea1, andrei biță4, ludovic bejenaru4, cornelia bejenaru5, speranța avram2 polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel (foeniculum vulgare mill.) egizia falistocco methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. sazada siddiqui*, sulaiman a. alrumman comparative study and genetic diversity in malva using srap molecular markers syamand ahmed qadir1, chnar hama noori meerza2, aryan mahmood faraj3, kawa khwarahm hamafaraj4, sherzad rasul abdalla tobakari5, sahar hussein hamarashid6,* nuclear dna 2c-values for 16 species from timor-leste increases taxonomical representation in tropical ferns and lycophytes inês da fonseca simão1, hermenegildo ribeiro da costa1,2,3, helena cristina correia de oliveira1,2, maria helena abreu silva1,2, paulo cardoso da silveira1,2,* nuclear dna content and comparative fish mapping of the 5s and 45s rdna in wild and cultivated populations of physalis peruviana l. marlon garcia paitan*, maricielo postillos-flores, luis rojas vasquez, maria siles vallejos, alberto lópez sotomayor identification of genetic regions associated with sex determination in date palm: a computational approach zahra noormohammadi1,*, masoud sheidai2, seyyed-samih marashi3, somayeh saboori1, neda moradi1, samaneh naftchi1, faezeh rostami1 comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 caryologia. international journal of cytology, cytosystematics and cytogenetics 72(4): 69-78, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-192 citation: s. heneidak, e. martin, f. altinordu, a. badr, h.e. eroğlu (2019) chromosome counts and karyotype analysis of species of family apocynaceae from egypt. caryologia 72(4): 69-78. doi: 10.13128/cayologia-192 published: december 23, 2019 copyright: © 2019 s. heneidak, e. martin, f. altinordu, a. badr, h.e. eroğlu. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. chromosome counts and karyotype analysis of species of family apocynaceae from egypt samia heneidak1,*, esra martin2, fahim altinordu2, abdelfattah badr3, halil erhan eroğlu4 1 department of botany, faculty of science, suez university, suez, egypt 2 department of biotechnology, faculty of science, necmettin erbakan university, konya, turkey 3 department of botany and microbiology, faculty of science, helwan university, egypt 4 department of biology, faculty of science and art, bozok university, yozgat, turkey *correspondence: sheneidak2000@yahoo.com abstract. the chromosome counts of 13 species of family apocynaceae in the flora of egypt have been reported; one species from subfamily periplocoideae and the other 12 species from subfamily asclepiadoideae. the chromosome numbers are 2n = 22 for periploca angustifolia, glossonema boveanum, pentatropis nivalis, cynanchum acutum, calotropis procera, gomphocarpus sinaicus, pergularia daemia and pergularia tomentosa; 2n = 24 for leptadenia arborea and solenostemma arghel; 2n = 22, 44 for caudanthera edulis, caudanthera sinaica and desmidorchis acutangulus. the chromosome numbers and karyotype analyses were firstly reported in leptadenia arborea (2n = 24). the polyploid nature was demonstrated by the prevalence of cells with 2n = 4x = 44 chromosomes in caudanthera edulis, caudanthera sinaica and desmidorchis acutangulus. the chromosomes are median and submedian as most species in the apocynaceae. the intrachromosomal asymmetry and interchromosomal asymmetry were estimated with mca and cvcl values. in intrachromosomal asymmetry, desmidorchis acutangulus is the most symmetrical karyotype, while pergularia tomentosa is the most asymmetrical karyotype. in interchromosomal asymmetry, glossonema boveanum is the most symmetrical karyotype, while cynanchum acutum is the most asymmetrical karyotype. keywords. apocynaceae, chromosome number, egyptian flora, karyotype asymmetry. introduction the family apocynaceae comprises 366 genera and ca. 5100 species (meve, 2002; endress et al., 2014). this family is currently divided into five subfamilies; periplocoideae, asclepiadoideae, apocynoideae, rauvolfioideae, secamonoideae (endress and bruyns, 2000; endress et al., 2014). the majority of species represented in the egyptian flora are classified in the two subfamilies periplocoideae and asclepiadoideae. the subfamily periplocoideae is a small group of species comprising only ca. 195 species in 33 genera (heneidak and naidoo, 2015). on the other hand, asclepiadoideae is the largest 70 samia heneidak et al. subfamily of the apocynaceae and contains about 3000 species in 164 genera of five tribes. the tribes are divided into 15 subtribes (meve, 2002; endress et al., 2014). chromosome data have been constantly used for systematic purposes but chromosome number alone is not sufficient to exactly trace the evolutionary history of taxonomic groups. however, comparative karyotype analysis of related species has traditionally been used to describe patterns and directions of chromosomal evolution within plant groups and to infer the evolutionary role of chromosomal changes in plant evolution (stebbins, 1971; badr et al, 1997; 2009; eroğlu et al., 2013; kamel et al. 2014). more detailed information about the karyotype has been found necessary in order to provide diagnostics criteria for the systematics and phylogeny of plants (altay et al., 2017). in fact, karyological features are evaluated as important taxonomic characters only when provide additional information and allow conclusions about evolutionary events in the group of interest (badr and elkington, 1977; peruzzi and eroğlu, 2013). survey of chromosome counts in the apocynaceae in chromosome count reports, particularly the index to plant chromosome numbers of the missouri botanical garden (http://www.tropicos.org/project/ipcn) and the chromosome counts database (ccdb) (http://ccdb. tau.ac.il) which is a community resource of plant chromosome numbers (new phytol. 206(1): 19-26) as well as the old counts reported in federov (1969) as well as the chromosome count reports that was frequenty published in the journal taxon indicated thatseveral authors have reported chromosome numbers of many species of the apocynaceae. several authors have reported chromosome numbers of many species of the apocynaceae (francini, 1927; mitra and datta, 1967; fedorov, 1969; arrigoni and mori, 1976; albers and delfs, 1983; albers and austmann, 1987; khatoon and ali 1993; liede 1996; albers et al., 1993; albers and meve, 2001; kamel et al., 2014). these studies showed that the family is karyologically almost entirely homogenous, especially subfamilies asclepiadoideae and periplocoideae, with nearly 96% of the taxa investigated so far having chromosome complements in multiples of a basic number of x = 11, with a few deviating numbers. deviating chromosome numbers were reported with 2n = 18, 24 in funastrum clausum (jacq.) schulr. and funastrum cynanchoides (decne.) schulr. (tribe asclepiadeae) (albers et al., 1993), 2n = 20 in microloma incanum decne. microloma calycinum e. mey., microloma sagittatum (l.) r. br. and microloma tenuifolium (l.) k. schum. (tribe asclepiadeae) (albers et al., 1993) and 2n = 24 in periploca graeca l. (subfamily periplocoideae) (pesci, 1971). in literature, x = 9 was only reported in cynanchum acutum l. and pergularia tomentosa l. (fedorov, 1969). the deviating chromosome numbers, i.e. 2n = 24 and x = 9 that were found previously and in the present work were reported a deviating base chromosome numbers in the genera cynanchum, microloma, and sarcostemma (albers et al. 1993). these authors gave an account of previously published deviating chromosome numbers in the asclepiadaceae. in subfamily asclepiadoideae, the polyploidy rate is approximately 6%. the polyploid species are mostly tetraploid (85%) with 2n = 44 and only a few are hexaploid with 2n = 66 (albers and meve, 2001). albers (1983) reported the polyploid taxa in most of the genera of tribe ceropegieae. albers and meve (1991) observed that the proportion of polyploid cells in the meristems of adventives roots is significantly higher than in the meristems of primary and secondary roots in genera duvalia haw., hoodia sweet ex decne., orbea haw., pectinaria haw., stapelia, trichocaulon n.e.br. and tridentea haw. high ploidy levels were recorded in tylophora anomala n. e. br.; for example the decatetraploid (2n = 132-154) and the hexaploid (2n = 66) (meve, 1999). in apocynaceae, the counting and measuring of small size of the chromosomes is difficult. the chromosomes form a graded series with only very slight differences in morphology (albers, 1983). within a single karyotype the chromosomes are comparatively similar in size. the heterogeneous karyotypes were only found where chromosome sizes varied considerably in the subfamilies periplocoideae, asclepiadoideae and secamonoide (albers and meve, 2001). in the present study, 13 species of the family apocynaceae were investigated karyologically to determine the chromosome numbers and to compare with earlier results. in addition, the karyotype of the examined species growing in egypt has been analysed using a number of chromosome characterizing parameters such as varaitions in length, arm ration and centromeric asymmetry indices in order to gather more information that might help a better understanding of the taxonomic treatment of the species of apocynaceae in the egyptian flora. materials and methods plant materials seeds of 13 species of apocynaceae were collected from mature flowers from sites in their natural habitats as given in table 1 and mapped as in figure 1. voucher specimens of the examined species are kept at suez university herbarium. in the two succulent species (caudanthera edulis and desmidorchis acutangulus), 71chromosome counts and karyotype analysis of species of family apocynaceae from egypt the root tips of adventitious roots were collected from plants, except caudanthera sinaica from seedlings. cytogenetic procedure for cy tological preparations, seeds were germinated on moist whatman paper and actively–growing root tips were pre-treated in saturated aqueous α-bromonaphthalene at 4ºc for 24 hours, or in a solution of 0.002 m 8-hydroxyquinoline at 18ºc for 5-6 hours. they were fixed in absolute ethanol:acetic acid (3:1) for at least one hour, hydrolysed in 1n hcl at 60ºc for 8 minutes and stained in feulgen staining solution. the slides were mounted in euparal for long-term storage (martin et al., 2011). photographs of chromosome spreads were taken using a carl zeiss axiostar plus microscope fitted with a canon (pc 1200 power shoot a641) digital camera. the number of somatic chromosomes was carefully counted in five slides for each species. karyotype analyses were made by using bs200pro image analysis software. homologous pairs of somatic chromosomes were determined according to their total and relative lengths for each species. the following parameters were used to characterize the chromosomes: long arm (la), short arm (sa), total length (tl = la + sa) and arm ratio (la / sa). total haploid lengths and mean haploid lengths were calculated. for the karyotype formula, chromosomes were classified using the nomenclature of levan et al. (1964). several karyotype symmetry indices have been applied to express the asymmetry of the karyotype. karyotype asymmetries were estimated by mean centromeric asymmetry (mca) (peruzzi and eroğlu, 2013) and coefficient of variation of chromosome length (cvcl) (paszko, 2006). the intrachromosomal asymmetry was table 1. list of species examined and the localities from which plants used for chromosome counts were collected and date of collection. taxa date locality 1. periploca angustifolia labillardiere 12.06.2009 el-salûm: wadi salufa, 31º37’24’’n–25º09’00’’ e, morsy et al. s.n. 2. caudanthera edulis (edgew) meve & liede 27.01.2009 gebel elba: wadi yahameib, 22º25’18’’n–36º18’33’’e, morsy et al. s.n. 3. caudanthera sinaica (decne.) plowes 10.05.2009 north sinai: gidda pass, 30º13’06’’n–33º03’04’’e, heneidak s.n. 4. desmidorchis acutangulus decne. 23.08.2009 gebel elba: wadi aideib, 22º15’00’’n–36º26’12’’e, morsy et al. s.n. 5. leptadenia arborea (forssk.) schweinf. 17.02.2009 aswan: 24º05’00’’n–32º54’18’’e, heneidak s.n. 6. glossonema boveanum (decne.) decne. 09.04. 2009 sharm el sheikh: nabq protectorate, south sinai, 28º07’00’’n– 34º25’00’’e, heneidak s.n. 7. solenostemma arghel (delile) hayne 25.11. 2009 dahab: south sinai, 28º29’05’’n–34º31’18’’e, heneidak s.n. 8. pentatropis nivalis (j. f. gmel.) d. v. field & j. r. i. wood 30.05.2009 gebel elba: abu ramad, 22º20’00’’n–36º34’00’’e, morsy et al. s.n. 9. cynanchum acutum l. 07.10. 2009 suez: shalufa, 30º07’03’’n–32º32’27’’e, heneidak s.n. 10. calotropis procera (willd.) r. br. 06.10. 2009 ismailia: 30º64’00’’n–32º27’00’’e, 06.10.2009, heneidak s.n. 11. gomphocarpus sinaicus boiss. 15.04. 2009 saint catherine: wadi el arbeen, south sinai, 28º32’12’’n– 33º95’00’’e, heneidak s.n. 12. pergularia daemia (forssk.) chiov. 25.10. 2009 gebel elba: wadi acaw, 22º15’31’’n–36º21’00’’e, morsy et al. s.n. 13. pergularia tomentosa l. 13.12. 2009 ismailia: suez desert road, 29º38’33’’n–32º16’32’’e, heneidak s.n. figure 1. the distribution map of the studied species in egypt. periploca angustifolia (■); caudanthera edulis, desmidorchis acutangulus, pentatropis nivalis, pergularia daemia (♦); caudanthera sinaica (▲); leptadenia arborea (●); glossonema boveanum, solenostemma arghel, gomphocarpus sinaicus (¶); cynanchum acutum (▼); calotropis procera, pergularia tomentosa (▬). 72 samia heneidak et al. calculated with mca = [mean (l – s) / (l + s)] × 100. the formula contains the length of long arm (l) and short arm (s) of each chromosome. the interchromosomal asymmetry was calculated with cvcl = [standard deviation / mean chromosome length] × 100. finally, a scatter diagram between intrachromosomal asymmetry (mca) and interchromosomal asymmetry (cvcl) was drawn. results and discussion the subfamily periplocoideae is represented with one species in tribe periploceae. the subfamily asclepiadoideae is represented with 12 species in tribe ceropegieae and asclepiadeae. the photographs illustrating the chromosomes of the studied species are shown in figures 2 and 3. the ideograms are given in figure 4. the gametic and somatic chromosome counts of the investigated species in present and previous studies are given in table 2. detailed chromosomal data are given in table 3. chromosome numbers table 2 summarizes the chromosome number and the previous counts for the studied species of apocynaceae. eleven of the 13 species examined here have 2n = 22, based on a basic number of x = 11. these results confirmed previous records for other species, and therefore, it is clear that the dominance of a basic number of x = 11 and a majority of 2n = 22 is the base in the subfamilies periplocoideae and asclepiadoideae. it is the first time to count the chromosomes of leptadenia arborea (2n = 24); (figure 2f). both diploid chromosome number (2n = 22) and tetraploid chromosome number (2n = 44) cells were scored in the three succulent species, which belong to tribe ceropegieae; i.e. caudanthera sinaica, c. edulis (figures 2c, 2d) and desmidorchis acutangulus. diploid number (2n = 22) is reported also in caudanthera edulis by albers and meve (2001), in caudanthera sinaica by albers and meve (2001), kamel et al. (2014), and in desmidorchis acutangulus by albers and delfs (1983), albers and meve (2001). however, tetraploid number (2n = 44) is recorded also in caudanthera edulis by albers and austmann (1987), in desmidorchis acutangulus by kamel et al. (2014), while in caudanthera sinaica recorded in the present study only). polyploidy is known to occur in 11 genera of subfamily asclepiadoideae with eight genera belonging to tribe ceropegieae (albers and meve, 2001). there are different patterns (mixoploidy) in terms of the number of chromosomes. this is probably the state of the endopolyploidy that is the result of enderoduplication. no odd-number polyploidy was figure 2. photomicrographs of somatic metaphase chromosomes in root tip cells: periploca angustifolia (a), caudanthera sinaica (b), diploid caudanthera edulis (c), tetraploid caudanthera edulis (d), desmidorchis acutangulus (e), leptadenia arborea (f). scale bar = 10 µm. 73chromosome counts and karyotype analysis of species of family apocynaceae from egypt found. the diploid count supports the findings of albers and meve (2001); while the tetraploid count in this study supports the findings of albers and austmann (1987). albers and meve (1991) reported that the frequency of tetraploid cells in the adventitious roots is higher than in the primary and the secondary roots. this phenomenon may lead to a complete polyploidization of adventitious roots, and can be ascribed to ecological rather than morphological or genetic factors (albers and meve, 1991). diploid chromosome number (2n = 22) is recorded in periploca angustifolia in this study and by arrigoni and mori (1976). deviations from this number are absent in this species as reported before in subfamily periplocoideae by albers and meve (2001). in the current study, three species; glossonema boveanum, pentatropis nivalis and gomphocarpus sinaicus was also found to have a diploid chromosome number of 2n = 22 as scored by albers and meve (2001), kamel et al. (2014) and other four gomphocarpus species examined by albers and meve (2001). the same for cynanchum acutum was also found to have a diploid chromosome number of 2x = 22 as recorded by kamel et al. (2014) and in other 25 cynanchum species examined by albers and meve (2001). the old records of earlier numbers of n = 9 and 2n = 18 in cynanchum acutum quoted in francini (1927) and federov (1969) as well as the count of 2n = 24 in cynanchum virens (albers et al., 1993) may be regarded as deviating numbers as argued by albers et al. (1993). calotropis procera was also found to have a diploid chromosome number of 2n = 22 as recorded by fedorov (1969), albers and meve (2001) and kamel et al. (2014). the other number of 2n = 26 recorded for this species by bramwell et al. (1972) may be regarded as deviating number as argued by albers et al. (1993). the two pergularia species were also found to have a diploid chromosome number of 2n = 22 as recorded by albers and meve (2001) and kamel et al. (2014) in pergularia daemia or by albers and meve (2001) in pergularia tomentosa. the old records of earlier numbers of n = 9 in pergularia tomentosa quoted in federov (1969) as well as the count of 2n = 24 in pergularia daemia (mitra and datta, 967) may be regarded as deviating numbers as argued by albers et al. (1993). chromosome number of leptadenia arborea was 2n = 24 in this report, while albers and meve (2001) found 2n = 22 in two leptadenia species (l. pyrotechnica decne. and l. hastata (pers.) decne.). this may be regarded as deviating number as argued by albers et al. (1993). the same for solenostemma arghel was also found to have a diploid chromosome number of 2n = 24 in this study, while kamel et al. (2014) found 2n = 22 in this species. karyotype analyses the chromosomes of the examined species are all small with slight morphological differences among the complements of the studied samples. when compared the chromosome morphology among the species, the smallest mean chromosome length (2.60 µm) figure 3. photomicrographs of somatic metaphase chromosomes in root tip cells: pentatropis nivalis (a), glossonema boveanum (b), solenostemma arghel (c), cynanchum acutum (d), pergularia tomentosa (e), pergularia daemia (f), gomphocarpus sinaicus (g), calotropis procera (h). scale bar = 10 µm. 74 samia heneidak et al. figure 4. idiograms of studied species: periploca angustifolia (a), caudanthera sinaica (b), caudanthera edulis (c), desmidorchis acutangulus (d), leptadenia arborea (e), pentatropis nivalis (f), glossonema boveanum (g), solenostemma arghel (h), cynanchum acutum (i), pergularia tomentosa (j), pergularia daemia (k), gomphocarpus sinaicus (l), calotropis procera (m). 75chromosome counts and karyotype analysis of species of family apocynaceae from egypt was observed in cynanchum acutum of tribe asclepiadeae. in contrast the largest mean length (6.51µm) was observed in gomphocarpus sinaicus of tribe asclepiadeae. albers and meve (2001) concluded that the average karyotype size diminished from rather large chromosomes in the periplocoideae to the smallest karyotype length in the presumed most advanced tribe of the asclepidoideae, the asclepiadeae. the mean chromosomes length in leptadenia arborea is 2.61µm, whereas albers and meve (2001) noticed an average length of 0.72 µm in two leptadenia species (l. pyrotechnica and l. hastate). in this study, mean chromosome lengths in caudanthera sinaica, desmidorchis acutangulus and caudanthera edulis were relatively larger (7.25, 6.14 and 5.38 µm, respectively). these three species also express evolutionarily basic morphological characters (albers and meve, 2001). meve and heneidak (2005) reported that the average mean chromosome length is (1.06-1.38 μm) in apteranthes europaea of tribe ceropegieae. the chromosomes of the three polyploid species studied here are usually smaller than those of diploid ones as reported before in polyploidy taxa by albers and meve (2001). a general tendency of size reduction can be seen starting with the presumably most primitive subfamily periplocoideae to the more evolved asclepiadoideae, and within the latter subfamily starting table 2. the gametic and somatic chromosome counts of the investigated species in present and previous studies. subfamily, tribe, subtribe species previous results reference present counts 2n explanation n 2n subfamily periplocoideae tribe periploceae periploca angustifolia — 22 arrigoni and mori (1976) 22 detailed measurements subfamily asclepiadoideae tribe ceropegieae subtribe stapeliinae caudanthera edulis — — 22 44 albers and meve (2001) albers and austmann (1987) 22 44 detailed measurements caudanthera sinaica — 22 albers and meve (2001), kamel et al. (2014) 22 & 44 new count & detailed measurements desmidorchis acutangulus — — 22 44 albers and delfs (1983), albers and meve (2001) kamel et al. (2014) 22 44 detailed measurements subtribe leptadeniinae leptadenia arborea — — — 24 first report tribe asclepiadeae subtribe asclepiadinae calotropis procera 11 — — — — 22 26 44 fedorov (1969) albers and meve (2001), kamel et al. (2014) bramwell et al. (1972) kamel et al. (2014) 22 detailed measurements gomphocarpus sinaicus — 22 kamel et al. (2014) 22 detailed measurements pergularia daemia — — 22 24 albers and meve (2001), kamel et al. (2014) mitra and datta (1967) 22 detailed measurements pergularia tomentosa 9 — — — 22 44 fedorov (1969) albers and meve (2001) kamel et al. (2014) 22 detailed measurements solenostemma arghel — 22 kamel et al. (2014) 24 new count subtribe cynanchinae cynanchum acutum 9 — — — 18 22 fedorov (1969) francini (1927) kamel et al. (2014) 22 detailed measurements glossonema boveanum — 22 albers and meve (2001), kamel et al. (2014) 22 detailed measurements subtribe tylophorinae pentatropis nivalis — 22 albers and meve (2001), kamel et al. (2014) 22 detailed measurements 76 samia heneidak et al. with the most primitive fockeeae to the most advanced asclepiadeae, a decrease in chromosome size has taken place (albers and meve, 2001). the chromosomes of most karyotypes are comparatively similar in size. only rarely were heterogeneous karyotypes found where chromosome size varied considerably (albers and meve, 2001). the smallest arm ratio was observed in desmidorchis acutangulus (1.06) and the highest one was observed in leptadenia arborea (2.12). cynanchum acutum has the smallest chromosome length as 1.67 μm and the biggest chromosome length is measured in gomphocarpus sinaicus as 9.20 μm. liede et al. (2002) also found that the chromosomes are generally short and varying in length, one pair of the large sized chromosomes in glossonema boveanum. in apocynaceae, chromosomes are typically submetacentric, rarely acrocentric with one pair of chromosomes possessing secondary constrictions with satellites (albers, 1983; albers and meve, 2001). albers and meve (2001) found the smaller chromosomes in tribe asclepiadeae, in particular the subtribes asclepiadinae, astephaninae and metastelminae where mean length ranges from 0.70 to 1.15 µm. in tribe ceropegieae, the mca values indicated that desmidorchis acutangulus is the most symmetrical karyotype, while caudanthera edulis is the most asymmetrical karyotype. whereas, the cvcl values indicated that the most homogeneous centromere position is observed in caudanthera sinaica. on the other hand the most heterogeneous centromere position is observed in desmidorchis acutangulus. in tribe asclepiadeae, the mca values indicated that glossonema boveanum is the most symmetrical karyotype, while pergularia tomentosa is the most asymmetrical karyotype. the cvcl values indicated that the most homogeneous centromere position is observed in glossonema boveanum. on the other hand the most heterogeneous centromere position is observed in cynanchum acutum. in all tribe, the symmetrical and asymmetrical karyotypes are quite different. in parallel, a weak positive correlation is determined between mca and cvcl (r = 0.120) (figure 5). in figure 5, three tribes of family apocynaceae have different karyotypes in terms of asymmetry degrees: tribe asclepiadeae with higher intrachromosomal asymmetry and interchromosomal asymmetry, tribe ceropegieae with lower intrachromosomal asymmetry and interchromosomal asymmetry, one species of tribe periploceae with relatively average intrachromosomal and interchromosomal asymmetry. on the other hand the results need to be supported by data from more species, because the species number investigated (per tribe) is much too low. the possible origin of deviating chromosome numbers called numerical aneuploidy are defects in cell division as anaphase lagging, nondisjunction or presence of b-chromosomes. b-chromosomes, which are also known as supernumerary chromosomes, are a major source of intraspecific variation in nuclear dna (jones et al., 2008). the general consideration is that b-chromosomes are derived from the a-chromosomes. probably, a b-chromosome may have originated from paracentrotable 3. the measurement data of the studied apocynaceae species. species kf sc (μm) lc (μm) rl (%) sc-lc thl (μm) mcl (μm) cvcl mca periploca angustifolia 20m + 2sm 3.08 6.68 5.52-11.99 55.73 5.06 19.87 15.37 caudanthera edulis 20m + 2sm 2.84 5.38 6.37-12.07 44.55 4.05 19.34 16.45 caudanthera sinaica 22m 4.08 7.25 6.95-12.37 58.61 5.33 17.35 13.01 desmidorchis acutangulus 22m 2.59 6.14 5.31-12.59 48.74 4,43 25.05 10.81 leptadenia arborea 20m + 4sm 1.88 3.80 5.99-12.11 31.35 2.61 23.20 16.29 glossonema boveanum 22m 2.70 4.88 6.59-11.92 40.97 3.72 16.63 11.20 solenostemma arghel 24m 2.04 4.54 5.11-11.34 40.00 3.33 22.57 17.71 pentatropis nivalis 22m 2.52 5.32 6.13-12.93 41.17 3.74 21.85 12.07 cynanchum acutum 18m + 4sm 1.67 4.10 5.82-14.31 28.63 2.60 29.16 16.98 calotropis procera 22m 2.08 5.37 5.53-14.26 37.67 3.42 26.27 15.19 gomphocarpus sinaicus 22m 4.20 9.20 5.86-12.83 71.71 6.51 23.94 14.42 pergularia daemia 22m 3.30 6.19 6.31-11.83 52.36 4.76 18.65 12.82 pergularia tomentosa 22m 2.88 5.12 6.71-11.92 42.94 3.90 17.72 19.94 abbreviations: karyotype formula (kf), shortest chromosome length (sc), longest chromosome length (lc), relative length (rl), total haploid chromosome length (thl), mean chromosome length (mcl). 77chromosome counts and karyotype analysis of species of family apocynaceae from egypt meric region amplication of a fragmented a chromosome or from a chromosome fusions. conclusion with this study, new chromosome data were given for 13 taxa of family apocynaceae. more karyological data are needed to understand the phylogeny of apocynaceae. in conclusion, some intrageneric relationships within apocynaceae will clarify with comparative chromosomal analysis. also, additional comparative high-resolution molecular cytogenetic studies will be necessary to clarify phylogenetic relationships between genera or species. acknowledgements we would like to thank prof. dr. ahmed mursi ahmed for collecting caudanthera edulis, desmidorchis acutangulus, pentatropis nivalis and pergularia daemia from gebel elba, in south east area of egypt. references albers f (1983). cytotaxonomic studies in african asclepiadaceae. bothalia 14: 795-798. albers f, austmann m (1987). chromosome number reports xcv. taxon 36: 494-496. albers f, delfs w (1983). in iopb chromosome number reports lxxxi. taxon 32: 667-668. albers f, liede s, meve u (1993). deviating chromosome numbers in asclepiadaceae. nord j bot 13: 37-39. albers f, meve u (1991). mixoploidy and cytotypes: a study of 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karyotype asymmetry indices. plant syst evol 258: 39-48. peruzzi l, eroğlu he (2013). karyotype asymmetry: again, how to measure and what to measure? comp cytogen 7: 1-9. pesci g (1971). in numeri cromosomici per la flora italiana. inf bot italiano 3: 124-157. stebbins gl (1971). chromosomal evolution in higher plants. london: edward arnold ltd. substantia an international journal of the history of chemistry vol. 2, n. 1 march 2018 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 72(2): 63-68, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-194 citation: a.-m. dutrillaux, b. dutrillaux (2019) telomeric heterochromatin and meiotic recombination in three species of coleoptera (dorcadion olympicum ganglebauer, stephanorrhina princeps oberthür and macraspis tristis laporte). caryologia 72(2): 63-68. doi: 10.13128/cayologia-194 published: december 5, 2019 copyright: © 2019 a.-m. dutrillaux, b. dutrillaux. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. telomeric heterochromatin and meiotic recombination in three species of coleoptera (dorcadion olympicum ganglebauer, stephanorrhina princeps oberthür and macraspis tristis laporte) anne-marie dutrillaux, bernard dutrillaux* systématique, évolution, biodiversité, isyeb umr 7205 – cnrs mnhn upmc ephe, muséum national d’histoire naturelle, sorbonne universités, 57 rue cuvier cp50 f-75005, paris, france *corresponding author: bdutrill@mnhn.fr abstract. centromeres are generally embedded in heterochromatin, which is assumed to have a negative impact on meiotic recombination in adjacent regions, a condition required for the correct segregation of chromosomes at anaphase i. at difference, telomeric and interstitial regions rarely harbour large heterochromatic fragments. we observed the presence at the heterozygote status of heterochromatin in telomere region of some chromosomes in 3 species of coleoptera: dorcadion olympicum; stephanorrhina princeps and macraspis tristis. this provided us with the opportunity to study the relationship between heterochromatin, chiasma location and meiotic recombination independently from the proximity of centromeres in this order of insects. in acrocentric chromosomes, the presence of heterochromatin in telomere region of the long arm displaces recombination near the centromere. in sub-metacentrics, recombination is almost always restricted to the other arm. this at distance effect of heterochromatin may deeply influence genetic drift. keywords. c-banded, telomeric heterochromatin, meiosis, recombination, coleoptera. introduction in almost all living organisms, centromeres are surrounded by heterochromatin, which harbours repetitive dna (nakaseko et al. 1986), whose function is not yet completely understood. in cells in mitotic growth, heterochromatin represses transcription and expression of genes located into it (grewal and jia 2007). during meiosis of most living organisms, recombination is necessary for the correct chromosome segregation at anaphase i, but it does not occur in heterochromatin. consequently, recombination is repressed in centromeric regions, which harbour heterochromatin. in schizosaccharomyces pombe, it was found to be approximately 200 times less in het64 anne-marie dutrillaux, bernard dutrillaux erochromatin than in the genome-wide average (ellermeier et al. 2010). a current interpretation is that recombination, thus chiasma formation in centromeric region, would lead to abnormal chromosome segregation (lynn et al. 2004). thus, a function of centromeric heterochromatin would be to displace recombination far from the centromeres and allow the correct chromosome segregation. large and variable amounts of heterochromatin are present in the karyotype of many animals belonging to various taxonomic groups, but their position is not at random: frequently juxta-centromeric and rarely interstitial or terminal. in mammals, a wellknown example of terminal heterochromatin is that of the hedgehog (insectivora) in the karyotype of which 2 to 4 chromosome pairs are involved. however, the heterochromatic blocks are not strictly terminal because nors (nucleolar organizer regions) are located at their extremity (mandhal 1979). it was noticed that no meiotic recombination occurred in and at proximity of heterochromatin (natarajan and gropp 1971). the same particularities were observed in the primate cebus capucinus (dutrillaux 1979) but such examples remain rare. in insects, terminal heterochromatin was observed in acrocentric chromosomes of several species of grasshoppers (john and king 1982, 1985, torre et al. 1985). these authors attributed the displacement of chiasmata to proximal position to the presence of terminal heterochromatin. in coleoptera, large heterochromatic fragments are commonly seen in most families (juan and petitpierre, 1989, correa et al., 2008, dutrillaux and dutrillaux, 2016), but almost always in the centromere region, as in other taxonomic groups. this preferential location may result from the amplification of dna repeated sequence surrounding centromeres, as shown for α satellite sequences (rudd et al. 2006, shepelev et al. 2009). as it will be discussed, the correct segregation of chromosomes at meiosis may also depend on the embedding of centromeres in heterochromatin. displacement of heterochromatin from centromere regions to intercalary or terminal regions would necessitate secondary events, but terminal heterochromatin may have other origins. it will be also discussed that the presence of heterochromatin in telomeric position may not confer a selective advantage, by imposing meiotic constraints. among hundreds of species of coleoptera we studied, karyotypes with large amounts of heterochromatin in terminal position were rarely observed. we confirm that heterochromatin terminally located on the long arm of acrocentrics may not decrease recombination, but simply displaces it near to the centromere, usually considered as a cold region (mahtani and willard 1998). in addition, we show that in nonacrocentric chromosomes, heterochromatin terminally located on one arm displaces recombination to the other arm. thus, heterochromatin can suppress recombination on a whole arm and influence meiotic recombination at much larger distance than it was generally thought. material and methods three examples belonging to three different families or sub-families were found among about 400 species of polyphagan beetles: dorcadion olympicum ganglebauer 1882 (cerambycidae: lamiinae: dorcadionini). two specimens were captured in may 2014 in eastern greece, near alexandroupolis (40° 50’57”n and 25°52’46”e). stephanorrhina princeps oberthür 1880 (scarabaeidae: cetoninae: goliathini). two specimens of african origin (malawi) were obtained in september2007 from a private breeding. macraspis tristis laporte 1840 (scarabaeidae: rutelinae: rutelini). eight adult specimenswere obtained in march 2008 from grubs captured in guadeloupe (basseterre, near deshayes 16°18’00”n and 61°47’00”w) in december 2006. chromosome preparations of cells at various stages of meiosis were obtained as described (dutrillaux and dutrillaux 2009, dutrillaux et al. 2010). proliferating cells obtained from either eggs, testes or mid gut were processed as described. chromosomes were giemsa stained and further silver stained for localization of the nucleolus organizer region (nor) and/or c-banded for localization of heterochromatin. image capture and karyotyping were performed using ikaros software (metasystems, germany). chromosome nomenclature: to avoid ambiguous interpretations, we will call acrocentric all chromosomes with a single euchromatic arm, whatever the size of the heterochromatin (generally c-banded) forming the other arm. chromosomes with euchromatin (not c-banded) on both arms are either metacentric or sub-metacentric. we will focus on chromosomes with large heterochromatic blocks distally attached to euchromatic arms. results dorcadion olympicum the mitotic male karyotype is composed of 24 chromosomes. pairs 3 and 5 are sub-metacentric and all other autosomes are acrocentric. the x chromosome is sub-metacentric and the y is punctiform (24,xy). in one of the two specimens studied, the length of one chromo65telomeric heterochromatin and meiotic recombination in three species of coleoptera some 11 is enlarged by the addition of a large amount of c-banded heterochomatin at the telomeric region of the long arm. (fig. 1a). following a simple giemsa staining, the chromatids of this fragment look hyper-cohesive, thin and pale. centromeric regions are faintly c-banded, as it frequently occurs in the genus dorcadion (personal data). at diakinesis/metaphase i of meiosis, the sex bivalent has the parachute configuration, usually found in polyphagan coleoptera. the compaction of the additional heterochromatin of bivalent 11 is much variable: quite elongated at early diakinesis, it becomes highly compacted at late metaphase i. bivalent 11 remains easily identified by c-banding (fig. 1b). the euchromatic component of bivalent 11 has the same aspect in the 131 analysed metaphases i: a cross with very uneven branches. the block of heterochromatin is always located at the tip of the longest branch. this indicates that chiasmata are systematically located near the centromere. in 90/95 spermatocytes ii at metaphases, the heterochromatic block is located on a single chromatid of chromosome 11, demonstrating that one crossing-over had occurred in its long arm (fig. 1c). in 5 instances only, the heterochromatin is present on both chromatids, which may be interpreted as either a lack of recombination in the long arm or the result of a double recombination between the centromere and the heterochromatin. interestingly, in these chromosomes, the heterochromatin blocks remain cohesive, while euchromatic arms are well separated, as usual at this stage. this gives it a ring appearance (fig.i d). finally, in 4 additional pairs of sister spermatocytes ii (or diploid ones), the heterochromatin carrier chromosomes remain close to each other suggesting heterochromatin remained associated at anaphase, inducing chromosome lagging. stephanorrhina princeps the mitotic karyotype is composed of 18 metaor sub-metacentric autosomes, one large x, and one punctiform y (20, xy). large and variable fragments of heterochromatin are c-banded on one chromosome of pairs n°5, 6, 8 and on the x. in pair n°5, the heterochromatin is distally located on the short arm (fig. 2a). we will focus on the meiotic behaviour of the heterozygote chromosomes 5. as in the previous species, the chromatids are hyper-cohesive in their heterochromatic portion. at diakinesis/metaphase i, the heterochromatin is fuzzy and hardly detectable without c-banding. in 37/40 instances, the heterochromatin is located at one extremity of bivalent 5, which looks asymmetrical after c-banding (fig. 2b). in 3 instances, heterochromatin is in the centre of the bivalent, which has a symmetrical appearance. amongst 23 spermatocytes ii at metaphase, chromosomes 5 have a c-band on either both chromatids (12 times), or a single chromatid (twice, fig. fig. 1. chromosomes of dorcadion olympicum : a) giemsa-stained karyotype and c-banded chromosomes 11 exhibiting additional heterochromatin in one homologue. b) giemsa stained (left) and c-banded (right) spermatocyte i at diakinesis/metaphase with bivalent 11 carrying heterochromatin at one side (arrow). x and y form a parachute bivalent (xyp). barr= 10 mm, as in other figures. c) group of 3 spermatocytes ii at metaphase, sequentially giemsa stained (g) and c-banded (c). as in most other metaphases ii, centromeric heterochromatin is poorly c-banded and chromosome 11 is asymmetrical, with compacted heterochromatin at the tip of a single chromatid (arrows). d) 12,y spermatocyte ii in which the 2 heterochromatin carrier chromatids of chromosome 11 remain cohesive, whereas all other chromosomes have clearly non-cohesive chromatids. fig. 2. sequentially giemsa stained (g) and c-banded (c) chromosomes of stephanorrhina princeps a) giemsa stained (center) and c-banded karyotype of a spermatogonium exhibiting additional heterochromatin on one chromosome of pairs 5, 6, 8 and on the x. only heterochromatin of chromosome 5 is clearly separated from the centromere region. b) diakinesis/metaphase i: heterochromatin (arrows) remains opposite to chiasmata. c) exceptionally, in each of these 2 brother spermatocytes ii, chromosome 5 is asymmetrically carrier of heterochromatin. d) spermatocyte i at pachynema: synapsis defect (sd) of the proximal (euchromatic) part of the short arm of bivalent 5. 66 anne-marie dutrillaux, bernard dutrillaux 2c), or had no c-band (9 times). thus, recombination rarely occurred in the heterochromatin carrier arm. at pachynema, the bivalents with enlarged heterochromatin tend to remain close to each other, in spite of the drastic hypotonic shock to which they had been submitted. a synapsis defect of the euchromatic fragment comprised between the telomeric and centromeric heterochromatin was recurrently observed (fig. 2d). macraspis tristis the karyotype, composed of 18 chromosomes, is characterized by the frequent presence of large and variable heterochromatic fragments at telomeric regions of one arm of 3 pairs of sub-metacentrics (n°6, 7 and 8) and on the x (fig. 3a). in all diakineses/metaphases i examined from all the specimens, the heterochromatin carrier bivalents have the same configuration: chiasma or terminal association in the euchromatic arms and opposite position of the heterochromatin (fig.3b). this suggests that no recombination occurred in the heterochromatin carrier arms. in the specimen considered here, pairs n°7 and 8 were heterozygote for the presence or absence of a large heterochromatin fragment. among 11/12 spermatocytes ii, all carrier chromosomes had similarly heterochromatin in both chromatids (fig. 3c). in a single one, one chromosome was asymmetrical, with heterochromatin on a single arm, indicating that recombination took place between the centromere and the heterochromatin (fig. 3d). in a second specimen, only pair n° 8 was heterozygote for the presence of heterochromatin. no asymmetry was observed on chromosomes from 28 spermatocytes ii. finally, in a third specimen, both pairs n° 7 and 8 were heterozygote, and no asymmetry was detected among 8 spermatocytes ii. thus, in 65 analysed sub-metacentrics, recombination was almost always suppressed between the distally located heterochromatin and the centromere, and occurred in the other arm (64/65 times). as in s. princeps, the bivalents with enlarged heterochromatin tended to associate at pachynema. discussion recombination and heterochromatin only few data exist on meiotic recombination and chromosome segregation in beetles, and most of them were obtain before the heterochromatin detection was possible (smith and virkki 1978). in both literature and our own data, a general observation is that most bivalents exhibit a single chiasma in a fairly distal, if not terminal, position at diakinesis/metaphase i. this is in agreement with the findings, in other organisms, that repression of recombination occurs not only in juxta-centromeric heterochromatin, but also in adjacent regions. thus, recombination hot spots are rarely located near to the centromeres, but most frequently in intercalary and near telomeric regions (lichten and goldman, 1995). the presence of large heterochromatin fragments in chromosomes is not exceptional. they generally occur in centromeric regions, where recombination rarely occurs (ellermeier et al., 2010). their more exceptional occurrence at telomeric regions offers the possibility to look for the possible influence of heterochromatin on meiotic recombination and chromosome segregation, independently from the proximity of the centromere and associated repetitive dna. such analysis was already performed in grasshoppers, in which some species have terminal heterochromatin in acrocentric chromosomes. it showed the displacement of chiasmata to a proximal position in heterochromatin carrier, compared to other chromosomes (john and king, 1982, 1985, de la torre et al., 1986). our observations in d. olympicum confirm these findings: at metaphase i, the heterochromatic block of chromosome 11 is almost always at distance from the chiasma. in addition, we could quantify recombination through the analysis of 95 spermatocytes at metaphase ii: 90/95 chromosomes 11 carry heterochromatin on a single chromatid, which formally demonstrates an almost systematic occurrence of crossing over between the centromere and the heterochromatic block. thus, there was fig. 3. giemsa (g) stained and c-banded (c) chromosomes of macraspis tristis. a) c-banded karyotype of a spermatogonium exhibiting heterozygosity for heterochromatin of pairs 7 and 8 and homozygosity for pairs 5 and 6. b) sequentially giemsa stained and c-banded spermatocyte i at metaphase with asymmetric bivalents 7 and 8. heterochromatin of bivalents 5, 6, 7 and 8 is always external, opposite to chiasmata or terminal association. c) spermatocyte ii: all heterochromatin fragments (arrows)are symmetrically distributed on both chromatids. d) unique spermatocyte ii with asymmetrical chromosome 7. 67telomeric heterochromatin and meiotic recombination in three species of coleoptera no crossing-over suppression but a displacement towards proximal regions. the 5 metaphases ii with symmetrical chromosomes 11 cannot be interpreted univocally: either 2 or no crossing-over occurred or crossing-over occurred in the short (heterochromatic) arm. as discussed below, these few cells provide us with interesting information about chromosome cohesion and segregation. in m. tristis and s. princeps, all autosomes are submetacentric, and the analysis of spermatocytes i at metaphase indicates that most bivalents form a single chiasma. for chromosomes with one heterochromatin carrier arm, chiasmata are almost systematically located on the other arm. this absence of recombination in the heterochromatin carrier arm is confirmed by the analysis of spermatocytes ii in metaphase: at difference with the acrocentric of dorcadion, heterochromatin is almost always either present or absent on both arms. this suggests a “choice” between the two arms, by suppression of recombination in the whole heterochromatin carrier arm. as observed in a proportion of pachytene cells of s. princeps, there is an asynapsis of the whole euchromatic fragment located between centromeric and telomeric heterochromatin, which may be related to the lack of recombination. these 3 examples show that large telomeric heterochromatin fragments can drastically influence meiotic recombination in euchromatin, with an effect at a long distance. it may create a hot spot of recombination fairly close to the centromere, which is quite unusual, as in d. olympicum. it may also generate a cold and a hot arm, as in the two other species. this effect is probably not due to the heterozygote status, because when both arms are carrier, they are generally not involved in chiasmata at diakinesis. by altering meiotic recombination, these occasional heterochromatic fragments alter the gene linkage between all the genes of the chromosome and influence genetic drift. cohesion, compaction and heterochromatin at metaphase i, sister chromatids are maintained together by cohesins, a ring-shaped protein complex formed by 4 sub-units: scc1 (rec8), scc3, smc1 and smc3. at anaphase i, cohesins are cleaved by separase all along chromatids, except in centromere regions where the sub-unit rec8 is protected from cleavage by the protein complex shugoshin/protein phosphatase pp2a, which counteracts its phosphorylation (riedel et al. 2006). this permits the resolution of chiasmata, which occur in euchromatic fragments. at the following metaphase ii, cohesins are no more efficient. chromatids are well separated and chromosome cohesion is maintained at the centromere regions only. finally, after inactivation of pp2a, centromere cleavage occurs at anaphase ii, enabling the segregation of monochromatidic chromosomes. why and how shugoshin/pp2a complex is located in centromere regions remains unknown. our observation may provide some information. in spermatocytes ii, all chromosomes have well separated chromatid, with the exception of chromosomes 11 of d. olympicum, which are ring shaped when they are symmetrically carrier of terminal heterochomatin. it means that cohesin was not cleaved both at centromere and telomere, the two heterochromatic regions. thus, rec8 seems to be protected from cleavage by heterochromatin. this protection, necessary for the correct chromosome segregation at anaphase i, may be one of the reasons why centromeres are systematically embedded in heterochromatin. it is commonly observed that in metaphases of mitotic cells also, heterochromatic fragments are more cohesive than euchromatin. at the molecular level, the search of a particular relationship between the protein complexes involved in cohesion and heterochromatin components could be of interest. the dna methylation status may play a role in this context. in human cells, although juxta-centromeric heterochromatin of chromosomes 1, 9 and 16 is not g-c rich, it is strongly labelled by antibodies to 5-mdc (5-methyldeocycytidine), indicating its strong methylation status (miller et al., 1974; montpellier et al., 1994). in mouse germ cells, large variations of dna methylation were reported during the progression of gametogenesis (coffigny et al. 1999; bernardino-sgherri et al. 2002; marchal et al. 2004). drastic changes from hypoto hyper-methylation occur in heterochromatin and euchromatin in an opposite way. for example, in early spermatogonia, hypo-methylated and elongated centromeric heterochromatin displays premature cleavage, while chromosomes remain cohesive at hyper-methylated chromatids. on the opposite, euchromatic chromatids are cleaved in spermatocytes ii, when they are poorly methylated while chromosomes remain cohesive at their methylated and compacted centromere regions. unfortunately, we could not study the methylation status of beetle chromosomes during gametogenesis, but the high similitude of variations of chromosome compaction and cohesion suggests they might correlate with dna methylation 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cytosystematics and cytogenetics volume 72, issue 2 2019 firenze university press karyotype analysis of a natural lycoris double-flowered hybrid jin-xia wang1, yuan-jin cao1, yu-chun han1, shou-biao zhou1,2, kun liu1,* insights on cytogenetic of the only strict african representative of genus prunus (p. africana): first genome size assessment, heterochromatin and rdna chromosome pattern justine germo nzweundji1, marie florence sandrine ngo ngwe2, sonja siljak-yakovlev3,* assessment of cytotoxicity and mutagenicity of insecticide demond ec25 in allium cepa and ames test arzu özkara cytogenetic effects of fulvic acid on allium cepa l. root tip meristem cells özlem sultan aslantürk evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of allium test ioan sarac1, elena bonciu2,*, monica butnariu1, irina petrescu1, emilian madosa1 fluorescence in situ hybridisation study of micronuclei in c3a cells following exposure to elf-magnetic fields luc verschaeve1,2,*, roel antonissen1, ans baeyens3, anne vral3, annemarie maes1 phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities nicoleta anca şuţan1,*, irina fierăscu2, radu fierăscu2, deliu ionica1, liliana cristina soare1 telomeric heterochromatin and meiotic recombination in three species of coleoptera (dorcadion olympicum ganglebauer, stephanorrhina princeps oberthür and macraspis tristis laporte) anne-marie dutrillaux, bernard dutrillaux* a whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of populus trichocarpa l. subjected to different stresses alberto vangelisti#, gabriele usai#, flavia mascagni#, lucia natali, tommaso giordani*, andrea cavallini differences in c-band patterns between the japanese house mice (mus musculus) in hokkaido and eastern honshu hikari myoshu, masahiro a. iwasa* karyotypic description and repetitive dna chromosome mapping of melipona interrupta latreille, 1811 (hymenoptera: meliponini) natália martins travenzoli1, ingrid cândido de oliveira barbosa2, gislene almeida carvalho-zilse2, tânia maria fernandes salomão3, denilce meneses lopes1,* caryologia. international journal of cytology, cytosystematics and cytogenetics 75(4): 93-101, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1902 caryologia international journal of cytology, cytosystematics and cytogenetics citation: peng zhou, jiao li, jing huang, fei li, qiang zhang, min zhang (2022). determination of genome size variation among varieties of ilex cornuta (aquifoliaceae) by fow cytometry. caryologia 75(4): 93-101. doi: 10.36253/caryologia-1902 received: november 11, 2022 accepted: december 15, 2022 published: april 28, 2023 copyright: © 2022 peng zhou, jiao li, jing huang, fei li, qiang zhang, min zhang. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid qz: 0000-0002-2025-9576 determination of genome size variation among varieties of ilex cornuta (aquifoliaceae) by fow cytometry peng zhou1, jiao li2, jing huang1, fei li1, qiang zhang2,*, min zhang1,* 1 jiangsu academy of forestry, nanjing 211153, jiangsu, china 2 co‑innovation center for sustainable forestry in southern china, key laboratory of state forestry and grassland administration on subtropical forest biodiversity conser‑ vation, college of biology and the environment, nanjing forestry university, nanjing 210037, jiangsu, china *corresponding author. e-mail: zhangqiang@njfu.edu.cn; e-mail: nmzhang@163.com abstract. ilex cornuta lindl. & paxton is a commercially important horticultural species worldwide, and extensive cultivation and hybridization have produced many varieties. despite the considerable breeding, selection, widespread cultivation and domestication, which may have a significant role in the composition of genomes, there are no other previous reports of intraspecific genome size variation in the different cultivars or hybrids of this species. in the present work, genome size of 12 varieties of i. cornuta was assessed and analyzed through high-resolution flow cytometry (fcm). nuclear dna was analyzed using nuclei isolated from young leaves, which used propidium iodide (pi) staining, with rice (oryza sativa cv. nipponbare) as internal reference. as a result, statistically significant differences in genome size were detected among all diploid i. cornuta varieties considered. the estimated genome size (2c value) of i. cornuta varieties ranged from 1.47 to 1.80 pg, with 1.22-fold variation and an average size of 1.65 pg. the domestication and interspecific hybridization induced variation of genome size in i. cornuta, and the genome size of hybrids exhibited a wider range of variation compared with that of cultivars. in summary, flow cytometry is a useful tool to analyze the genome size of i. cornuta. the first report of the genome sizes of varieties of this species would provide useful data for further research on i. cornuta, and enrich the c value database of ilex l. what’s more, our findings could be the foundation in the future of i. cornuta genome sequencing and breeding programs. keywords: ilex cornuta, genome size, dna content, flow cytometry, hybrids, cultivars. introduction the ilex l. (holly) is the only living woody dioecious angiosperm genus, accounting for approximately 600 species with a broad distribution from tropics to temperate regions within the monogeneric family of aquifoliaceae (loizeau et al. 2016). the ilex species are prized for their glossy evergreen 94 peng zhou et al. foliage and abundant showy fruits that can bloom from autumn to early spring, when many other plants in the landscape are dormant (yao et al. 2021). ilex cornuta lindl. & paxton, one of very important native landscape woody materials and distributed only in eastern china and korea (hu 1949), has been utilized as a horticultural species because its leaves are distinctive rectangular foliage (one or two spines per side) and its fruits are red berries (park et al. 2019). i. cornuta, the most speciose and commercially significant specie of the diverse genus ilex, has a long and complex horticultural history. together with i. aquiforlium usually distributed in europe, it is a typical species for christmas tree inside home. in addition, it has been utilized as medical plant in china so that it contains several useful compounds (zhang et al. 2012; kang et al. 2014). extensive cultivation and hybridization have produced many varieties of i. cornuta, including commercially important horticultural species such as cultivated tea and iconic flowering shrubs (hodges et al. 2001; park et al. 2019). genome size (c value/haploid nuclear dna content) is an important attributes of living organisms, which is correlated with size of nucleus/cell, cellular process including dna synthesis rate and ecological traits, etc. genome size has fundamental significance in a wide range of applications including molecular biology, ecology, systematic biology, cytology, evolutionary biology and genomics (jatt et al. 2019). genome sizes of more than 7500 plant species have been estimated (bennett and leitch 2012), but the genome sizes of higher species are still poorly understood (bennett and leitch 2011; doležel and bartoš 2005). flow cytometric analysis in plants has proved to be useful to determine dna content and ploidy level in different species and accessions (sliwinska 2018; pellicer et al. 2014; d’hondt et al. 2011). the genus ilex is one of the largest plant genera, but of which only 6 species of genome size have been determined (bennett and leitch 2012). as the most speciose member of this genus, i. cornuta has become widely cultivated throughout asia, europe and america. despite the considerable breeding, selection, widespread cultivation and domestication of i. cornuta, which may have a significant role in the composition of genomes, except for i. cornuta (zhang et al. 2013), there are no other previous reports of intraspecific genome size variation in the different cultivars or hybrids of this species. improved knowledge of genome size of key cultivars and complex hybrids would be a valuable resource for further breeding and improvement of i. cornuta. therefore, in this study, the genome size of cultivars and hybrids of i. cornuta were identified and analyzed by fcm. the genome size variation of different i. cornuta varieties were explored to provide a basis for the development and utilization of i. cornuta germplasm resources. materials and methods plant materials the sampling site was located in the national holly germplasm resources repository of the jiangsu academy of forestry, which is located at jiangning district, nanjing city, jiangsu province, china. the tested materials were 12 i. cornuta varieties in consideration of its commodity value; the age of trees is 5-7a, each was healthy and without pests and diseases. diploid rice, oryza sativa subsp. japonica cv. nipponbare (1c = 389 mb, gc = 43.6 %; international rice genome sequence project 2005), were used as an internal standard, which was provided by nanjing agricultural university and germinated in petri dishes in the laboratory. leaves were collected from 12 healthy i. cornuta varieties and rice. flow cytometry analysis experimental design prior to fcm measurement, flow cytometer parameters were determined, based on external analyses of sample and primary standard. subsequently, internal fcm procedure was performed. preparation of plant nuclei suspensions for f low cytometry (1) sampling: 0.05 g of young leaves of i. cornuta and 0.05 g of young leaves of rice was collected, washed in distilled and deionized water successively to remove surface dirt, and dried on filter paper. (2) dissociation: 1 ml of pre-cooled tris dissociation solution was added to a precooled culture dish, and the cut leaf tissues were immersed in this solution and then chopped quickly with a razor blade. after chopping, 1 ml tris dissociation solution was added, well mixed and allowed to stand for 1-3 min at 4 °c. (3) filtration: the liquid mixture was drawn from the culture dish, filtered once through a 400 mesh membrane and placed into a centrifuge tube. the mixture was incubated at 4 °c for 5 min. (4) centrifugation: the cell nuclear suspension was obtained by centrifugation at 4 °c at 1000 r/min for 5 min. (5) dyeing: the supernatant was discarded. the nuclei were stained with propidium iodide (pi), and rnase 95determination of genome size variation among varieties of ilex cornuta (aquifoliaceae) by fow cytometry was added to a final concentration of 50 μg/ml. the mixture was dyed at 4 °c for 5-10 min in the dark environment before being analyzed. settings of flow cytometer and calculations samples were run on a bd influxtm cell sorter (bd, piscataway, nj, usa) with an argon laser exciting at 488 nm. pulse area was detected using 670 mean/30 bandwidth detector, as well as with side (ssc) and forward (fsc) scatters. prior to analysis, the instrument was checked for linearity and the amplification was adjusted so that the peak corresponding to rice was positioned approximately at channel 10000. the voltage was maintained at a constant high level throughout each experiment. each plant was measured at least three times by the same operator to eliminate potential artefacts. if the difference among the three measurements exceeded 2%, the most deviating value was discarded and the sample was re-analyzed. coefficient of variation values (cv) was used to evaluate the results. nuclear genome size was calculated as a linear relation between the ratio of g0/g1 peak of the samples and the standard according to the following formula (dolezel and bartos 2005): sample genome size = [(sample g0/g1 peak mean)/(standard g0/g1 peak mean)] × standard genome size. genome size data are presented in absolute terms in pg (1c and 2c value) and mbp (1 pg dna = 978 mbp; doležel 2003). statistical analyses of genome size fcm detection results were edited and analyzed by bd facs sortware1.0.0.650, and a flow histogram was obtained. variance analysis was carried out using excel 2003 and spss 13.0 with convective detection parameters. a one-way anova (analysis of variance) was used to compare genome sizes among individuals of the same varieties and among 12 sampled varieties respectively. fisher’s least significant difference (lsd) test (p<0.05) was used for the multiple comparison. a t test was used to compare genome size values for four cultivars and eight hybrids to determine whether differences were significant between two groups. genome size data were log10 transformed prior to analyses. results optimization of flow cytometry for i. cornuta based on recommendations from specialized fcm bibliography and small genome size values reported in aquifoliaceae (bennett and leitch 2012), we chose rice as primary standard by internal standardization to lower the bias (noirot et al. 2003; lysák et al. 2000). the fluorescence intensity range of standard and sample was determined by observing the position of peak in the flow cytometric histogram, when they were analyzed separately on the machine. as shown in figure 1 and figure 2, the debris peak and nuclei peak were effectively separated, and the sample peak had good linearity, indicating that the nuclear dissociation solution was suitable. the g0/g1 peak of rice was tuned to fluorescence channel 10000 (figure 1b), and followed the same protocol, the g0/g1 peak of i. cornuta sample was positioned at channel number around 18000 (figure 2b). when rice and i. cornuta being chopped simultaneously, the ssc (figure 3a) showed that the particles of the target species and the internal standard are clearly concentrated with good discrimination, and it was easy to distinguish two dominant g0/g1 peaks in histogram (figure 3b). nuclear dna content was calculated as a linear relationship between the ratio of g0/g1 peaks of the sample and standard, indicating that the i. cornuta nuclei contained more dna than rice nuclei. therefore, in the flow cytometry histograms of mixed samples, the peak reflecting the nuclei isolated from rice should be positioned at the left side of the histogram, while the peak reflecting the nuclei isolated from i. cornuta sample should be positioned at the right side of the histogram (figure 3b). table 1. variance analysis of fcm detection parameters of different varieties in i. cornuta. index variation source sum of squares degree of freedom mean square f value significance level 2c value between varieties 0.390 11 0.035 17.631 0.000 within varieties 0.058 29 0.002 total variation 0.448 40 1c value between varieties within varieties 0.058 29 0.002 total variation 0.448 40 96 peng zhou et al. fcm histograms of i. cornuta varieties the peak histograms of different classified i. cor‑ nuta varieties obtained with fcm were shown in figure 4. mean fluorescence values from 12 i. cornuta varieties showed g0/g1 nuclei peaks in a fluorescence range from 17240 to 23090. flow cytometry analyses produced high-resolution histograms with cv values for the internal standard and sample peaks varying between 2.77% and 5.10% (mean 4.15%) and between 1.82% and 5.15% (mean 3.95%), respectively, which suggested that the resolutions of the high quality histograms were appropriate for genome size analysis (table 2). assessment of genome size of i. cornuta varieties the mean 2c value was determined for each varieties of i. cornuta by comparing the relative g0/g1 nuclei pi-fluorescence peak of rice (primary standard) to that of each i. cornuta sample. analysis of variance (anofigure 1. fcm detection analysis result for rice that was separately processed. (a) scatterplot on side scatter (ssc) versus pi fluorescence with manually drawn polygon gate; (b) histogram of relative fluorescence intensity derived from nuclei isolated from rice only. peak 4 represent g0/g1 nuclei of rice. figure 2. fcm detection analysis result for i. cornuta sample that was separately processed. (a) scatterplot on side scatter (ssc) versus pi fluorescence with manually drawn polygon gate; (b) histogram of relative fluorescence intensity derived from nuclei isolated from i. cornuta only. peak 4 represent g0/g1 nuclei of i. cornuta sample. figure 3. fcm detection analysis result for mixed samples of rice and i. cornuta sample that were simultaneously processed (cochopped). (a) scatterplot on side scatter (ssc) versus pi fluorescence with manually drawn polygon gate, (b) histogram of relative fluorescence intensity derived from nuclei isolated from rice and i. cornuta processed simultaneously. peak 4 represent g0/g1 leaf nuclei of rice, peak 5 represent g0/g1 nuclei of i. cornuta sample. figure 4. fcm histograms obtained after analyses of propidium iodine-stained nuclei isolated i. cornuta varieties ’burfordii’ (a), ’dwarf burford’ (b), ’luteocarpa’ (c), ’’o’spring’ (d), ’emily bruner’ (e), ’james swan’ (f ), (g) ’ilex dabieshanensis no.1’; ’mary nell’(h), ’nellie r. stevens’ (i) , ’edward j. stevens’ (j), ’golden nellie r stevens’ (k) and ’china girl’(l) with internal standard rice. peak 4 represent g0/g1 nuclei of rice, peak 5 represent g0/g1 nuclei of i. cor‑ nuta varieties. 97determination of genome size variation among varieties of ilex cornuta (aquifoliaceae) by fow cytometry va) of genome sizes variation in different varieties of i. cornuta was significant (table 1). the 2c genome sizes (2c value) varied 1.22-fold among all i. cornuta varieties, ranging from 1.47±0.0217 to 1.80±0.0148 pg, thus with 1c genome size estimates for i. cornuta ranging from 717 to 881 mb (0.733–0.901 pg) with an average of 805 mb (0.823 pg). ‘emily bruner’ had the smallest genome size, whereas ’nellie r. stevens’ had the largest genome size (table 2). comparison of genome sizes between cultivars and hybrids the i. cornuta varieties measured were distinguished into two groups based on genetic origin, which were significantly different (p < 0.05). the first represents four i. cornuta-related cultivars, whereas the second includes eight interspecific hybrids developed based on i. cornuta. the 2c value of genome size was estimated between 1.61±0.0048 and 1.77±0.0263 pg for cultivar family and 1.47±0.0217 and 1.80±0.0148 pg for hybrids family (table 2). box and whisker plot showing differences in the 2c values assessed with different germplasm sources (figure 5). the median and average 2c value of genome sizes were 1.688 pg and 1.678 pg for cultivars and 1.634 pg and 1.627 pg for hybrids, respectively. the variation in genome size of hybrids (1.22-fold) was somewhat larger than that of cultivars (1.10-fold), possibly due to those hybrid nature. discussion genome size estimation of i. cornuta in the present work, the genome sizes of 12 i. cor‑ nuta varieties were measured, according to their global commodity value in different countries. results of flow cytometric analysis showed that the mean 2c genome size estimated of i. cornuta in this study ranged from 1.47 pg to 1.80 pg (1.63 pg on average), corresponding to the 1c genome size of 805 mb or 0.823 pg, which indicated that genome size estimates of i. cornuta varieties were very small (1c≤1.4 pg; leitch et al. 1998; soltis et al. 2003) and considerable variations were found among all varieties. therefore, caution should be taken when cultivars/genotypes are selected for genome sequencing and other genome-based studies. nevertheless, these new data on the genome sizes of both cultivars and complex hybrids are larger than the previous result of i. cornuta (2c=1.31 pg; zhang et al. 2013). the possible reasons for this difference might be due to the use of different origins of plant materials, reference plants, methods for lysates, staining protocols and instruments (wang et al. 2019; jatt et al. 2019; kolano et al. 2012). given the large variation in genome sizes of i. cornuta germplasm resources, it is essential to investigate a large number of varieties before a more accurate estimation of their average genome sizes can be achieved. optimization of internal reference to estimate genome size accurately due to its indirect nature, one of the important steps in fcm analysis was to choose the reference plant species used as an internal standard, which can reveal significant differences in dna contents among the same cultivar or plant species (ortega-ortega et al. 2019; jatt et al. 2019; doležel et al. 2003). to be useful as a primary standard, a plant must have similar, but not identical, genome size to the analyzed plant, and the g0/g1 peaks of the standard should not overlap to the peaks of the sample and is located relatively at a distance from the samples that can help to decrease the errors in measuring dna content (doležel and greilhuber 2010; jatt et al. 2019). ideally, the 2c peak of the target species should be located between the 2c and 4c peaks of the internal reference standard and the genome size of the target and internal standards should not differ more than fourfold (suda and leitch 2010). in addition, the standard must be easy to use, genetically stable, nuclei must be obtained in enough amounts for analysis (ortega-ortega figure 5. boxplot of genome sizes in cultivars and hybrids. the horizontal black line within each bar represents the median value of the genome size, while a black dot within the bar denotes the average value of genome size. 98 peng zhou et al. et al. 2019). rice has all these characteristics. initially in this study, rice was selected as internal standard. the g0/ g1 peak of i. cornuta was about twice that of the diploid cultivated rice and they don’t overlap each other (figure 3), which proved the rice was a advisable standard for i. cornuta flow cytometric analysis. performance of flow cytometry for i. cornuta the cv has been considered an important fcm parameter, indicating the quality of nuclei suspensions (favoreto et al. 2012). cv within 9% indicated that test results were relatively reliable (georgiev et al. 2009); cv below 5% indicated the highest accuracy for fcm assessments in plants (doležel and bartoš 2005). in the present work, for ‘o spring’ and ‘golden nellie r stevens’, it was very difficult to obtain cv values at the level of below 5%, which mainly due to the high amount of autofluorescence and phenolics in the gold leaves hampering the dyeing and analysis of the nuclei (choudhury et al. 2014). except for these two varieties, the fcm procedure used here provided fluorescence peaks of g0/g1 nuclei showing cv are all below 5%, which indicated that the extraction and staining procedure using tris dissociation solution combined with a centrifugation step can result in the accepted histograms. thus, the fcm procedure in this work is adequate for determination of genome size for i. cornuta and can be applied in other fcm studies of ilex. intraspecific variations in genome size statistical analysis can help assess the extent of genome size variation among varieties of related species or cultivars. in the case of i. cornuta it is important to determine the genetic variability between different cultivars (including genome size) since most of them are not biologically defined species, but rather the result of somatic mutations and artificial hybridization (hodges et al. 2001). thus, fcm analysis applied to estimate total nuclear dna content in i. cornuta cultivars can help to identify those cultivars with higher possibilities of havtable 2. genome size of the varieties of i. cornuta analyzed in this work. no varieties genome size cv (%) of samples cv (%) of standard 2c (pg) 1c (pg) mean 1c (mpb) meanmean ± sd min. max. significance* ilex cornuta -related cultivars 1 ‘burfordii’ 1.77±0.0263 1.72 1.81 ab 0.883 863 4.55 4.40 2 ‘dwarf burford’ 1.61±0.0048 1.60 1.62 def 0.805 787 3.50 2.77 3 ‘luteocarpa’ 1.71±0.0351 1.63 1.81 bc 0.855 836 3.49 4.09 4 ‘o spring’ 1.63±0.0282 1.57 1.67 de 0.813 794 5.15 4.94 average 1.68 0.839 820 interspecific hybrids ilex (cornuta x latifolia) 5 ‘emily bruner’ 1.47±0.0217 1.42 1.53 g 0.733 717 4.56 4.81 6 ‘james swan’ 1.55±0.0294 1.50 1.60 ef 0.774 757 4.85 4.90 ilex 7 ‘dabieshanensis no.1’ 1.56±0.0355 1.50 1.62 ef 0.781 763 2.81 3.76 ilex [(cornuta x pernyi) x latifolia] 8 ‘mary nell’ 1.71±0.0048 1.70 1.72 bc 0.856 837 4.57 4.97 ilex (aquifoilium x cornuta) 9 ‘nellie r. stevens’ 1.80±0.0148 1.78 1.83 a 0.901 881 1.82 3.60 10 ‘edward j. stevens’ 1.54±0.0039 1.54 1.55 f 0.772 754 3.98 4.26 11 ‘golden nellie r stevens’ 1.72±0.0146 1.69 1.76 bc 0.859 839 5.08 3.58 ilex (rugosa x cornuta) 12 ‘china girl’ 1.66±0.0093 1.64 1.68 cd 0.831 812 3.09 3.78 average 1.63 0.813 795 overall average 1.65±0.0165 1.42 1.83 0.823 805 3.95 4.15 99determination of genome size variation among varieties of ilex cornuta (aquifoliaceae) by fow cytometry ing sexual compatibility and therefore hybridization via conventional breeding. among angiosperms, there is a great variation in genome sizes, ranging from 0.065 pg/1c of dna in genlisea margaretae hutch. to 152.23 pg/1c in paris japonica franch (kolano et al. 2012). also, numerous studies revealed the existence of considerable variation in genome size at the interspecific level, e.g. coffea arabica (ortega-ortega et al. 2019; noirot et al. 2003), phoenix dactylifera (jatt et al. 2019), chenopodium qui‑ noa (kolano et al. 2012), several pisum species (baranyi et al. 1996), three saccharum species (zhang et al. 2012), agave tequilana (palomino et al. 2003) and arabidop‑ sis thaliana (schmuths et al. 2004). amongst the cultivars of i. cornuta there was a 1.16-fold range of variation, and although some of this might be attributable to methodological variation, it is possible that not all can be explained in this way. murray (2005) has suggested that intraspecific variation in c-value may be indicative of taxonomic heterogeneity and there is no doubt that i. cornuta is a highly variable species that exhibits a wide range of morphological variation. in general, these cultivars, resulting from spontaneous mutations and being thus related, have remarkably similar genome sizes and a different dna content relative to their progenitors. this is the case of the i. cornuta cultivar that gave rise to ‘burfordii’, ‘dwarf burford’, ‘luteocarpa’ and ‘o’spring’. in contrast, a wider range of variation in the genome size were exhibited among the hybrids, one possible explanation for which was that varieties of hybrid origin have undergone substantial genome size changes as compared with natural mutations (ortega-ortega et al. 2019). other varieties have different genetic origin and yet possess similar dna content, i.e., ‘luteocarpa’ and ‘golden nellie r stevens’. it is known that variations in genome size has been primarily attributed to fluctuation within highly repetitive dna, variation in chromosome number, amplification/deletion of dna sequences (wang et al. 2017; kolano et al. 2012; sharma et al. 2019). mechanisms underlying intraspecific and interspecific genome size variation in plants still remain controversial; thus, more research is fairly required in this regard (ortega-ortega et al. 2019; huang et al. 2013). conclusion in conclusion, genome size of 12 commercially important i. cornuta varieties was analyzed in picograms by flow cytometry technique for the first time. this study can fill a gap in the literature by providing information about the genome size of i. cornuta varieties and enrich the c value database of ilex l. it also provides a valuable reference for other ilex l. species to determine genome size by flow cytometry. this information can be helpful for i. cornuta breeding programs, give the paucity of genome size studies with fcm in different important i. cornuta cultivars. additionally, these results may be relevant for genomic analysis as well as for a better understanding of i. cornuta evolutionary relationships, diversification, hybridization, and polyploidy. acknowledgments thanks go to yanwei zhou and yiping zou, who assisted with the material collection and feng lin for the technical support with fcm. author contributions conceptualization, p.z. and m.z.; formal analysis, q.z.; investigation, j.l. and p.z.; data curation, j.h.; writing—original draft preparation, p.z.; writing— review and editing, q.z. and m.z.; visualization, f.l. and j.h.; funding acquisition, m.z. and p.z. all authors have read and agreed to the published version of the manuscript. funding this research was funded by the jiangsu academy of forestry youth foundation [jaf-2022-03], the jiangsu province innovation and extension project of forestry science and technology [lykj[2020]02], the jiangsu province innovation and extension project of forestry science and technology [lykj[2021]07], the jiangsu province innovation and extension project of agricultural science and technology [2021-sj-008] and independent research projects of jiangsu academy of forestry [zzky202105]. references baranyi m, greilhuber j, swięcicki wk. 1996. genome size in wild pisum species. theor appl genet. 93:717721. bennett md, leitch ij. 2012. plant dna c-values database (release 6.0, december 2012). see http://www. kew. org/cvalues. 100 peng zhou et al. bennett md, leitch ij. 2011. nuclear dna amounts in angiosperms: targets, trends and tomorrow. ann bot. 107: 467-590. choudhury rr, basak s, ramesh am, rangan l. 2014. 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l, cao b, bai c. 2013. new reports of nuclear dna content for 66 traditional chinese medicinal plant taxa in china. caryologia. 66: 375-383. zhang sx, yao zr, li j. tu pf. 2012. flavonoids from the leaves of ilex cornuta. chinese j nat med. 10: 84-87. caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 3 2022 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 75(4): 49-66, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1716 caryologia international journal of cytology, cytosystematics and cytogenetics citation: ekram abdelhaliem, hanan m.abdalla, ahmed a. bolbol, rania s. shehata (2022). assessment of protein and dna polymorphisms in corn (zea mays) under the effect of non-ionizing electromagnetic radiation. caryologia 75(4): 49-66. doi: 10.36253/caryologia-1716 received: june 27, 2022 accepted: november 23, 2022 published: april 28, 2023 copyright: © 2022 ekram abdelhaliem, hanan m.abdalla, ahmed a. bolbol, rania s. shehata. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. assessment of protein and dna polymorphisms in corn (zea mays) under the effect of nonionizing electromagnetic radiation ekram m. abdelhaliem1,*, hanan m.abdalla1, ahmed a. bolbol1, rania s. shehata1,2 1 department of botany and microbiology, faculty of science, zagazig university, 44519, egypt 2 biology department, faculty of science, jazan university, 45142, saudi arabia *corresponding author. e-mail: ekram.esa@gmail.com abstract. many reports highlight biological responses of crop plants after nonionizing electromagnetic radiation (emr) exposure based on the phenotypic and physiological levels. so, this study aimed to estimate genetic alterations in proteins, isozymes, and dna banding patterns as well as the extent of nuclear dna damage of economic corn (zea mays) under the stress of emr using accurate and reliable bioassays like sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page), isozymes (leucineaminopeptidase, esterases, peroxidase, and catalases), random amplified polymorphic dnapolymerase chain reaction (rapd-pcr), and comet assay, respectively. sds-page analysis showed distinct polymorphisms (96.66%) between emr exposed and non-exposed corn seedlings depending on the number and type of bands, their intensities as well as molecular weight which ranged from (60.27 to 192.35 kda), gain, and loss of bands. the four isozymes generated varies isozymatic polymorphisms based on relative front, zymogram number, and optical intensities. rapd analysis generated 85 amplified dna products with high polymorphism values ranged from 90.91 to 100% based on primers, band type, dna sizes which ranged from 153 to 1008-bp, lose, gain, and intensity of dna bands. comet assay scored highest extent of loosed dna from nuclei (dna damage) reached the value of (tailed ratio 20%) at emr exposed corn nuclei for 5 days compared to nonexposed nuclei which reached the value of (tailed ratio 3%). this study concluded that each emr exposure time had unique interaction with proteins, isozymes, and dna of corn cells exhibiting wide range of genotoxic stress and subsequently, adversely effect on growth and yield of this sensitive crop plants. keywords: electromagnetic radiation, zea mays, sds-page, isozymes, rapd-pcr, single cell gel electrophoresis. introduction nowadays, non-ionizing electromagnetic radiation (emr) arise from both natural and wide human made sources such as variety of electronic devices (rio & rio, 2013) can influence the growth, yield, and quality of 50 ekram abdelhaliem et al. plants based on flux of emr and exposure time (nyakane et al., 2019). important components of cells are proteins that classified into varies classes according to their functions. the stress proteins generated from abiotic stressor like emr consider a new class from these proteins with functions related to protection of cell ( iderawumi and friday 2020). when emr interact with the dna can stimulate the synthesis of this stress protein, causing dna strand breaks which increase by increasing of emr energy (blank & goodman, 2012, shabrangi et al., 2011). field parameters and characteristics (frequency, intensity, and wave-shape), cell type, and exposure duration all influence emr genetic effects. the gene expression changes (for example, genes implicated in cell cycle arrest, apoptosis and stress responses, and heat-shock proteins) are consistent with the results that emr causes genetic damage (lai, h 2021). the study of ruiz-gómez and martínez-morillo (2009) reported that a major concern of the genotoxic effects of non-ionizing electromagnetic field (emf) is overproduction of ros in cells and inducing oxidative stress on protein and dna because of emf exposure can induce dna strand breaks and acts as a co-inductor of dna damages rather than as a genotoxic agent. the genetic mechanisms by which emr interact with protein and dna are radical pair recombination led to increasing the concentration, activity, and lifetime of reactive oxygen species (ros), which might cause changes in cell cycle, genetic mutation, damage to dna which can lead to changes in cellular functions and cell death, modification of protein expression and oxidation of proteins, inhibition of enzymes (kıvrak et al., 2017). higher plant species differ in their sensitivity and response to environmental stresses because they have a variety of stress perception, signaling, and response skills (ahanger et al., 2017). corn (zea mays) is one of the world’s major cereals and food crops for humans, it belonging to family gramineae and genus zea. several physical abiotic stresses affect the total production of maize due to damage in its dna, like emf (yan, et al., 2011). it provides a promising genetic bio-monitors model to detect genotoxicity of environmental stress and dna lesions induced by abiotic stress (grant & owens, 2006; erturk, et al., 2014). many studies focused on the effect of emf on plant growth and its development (ortiz, et al., 2015) but rarely concerned with their effects on banding patterns and genetic polymorphisms of proteins, isozymes and dna. this attracted the attention of this study to explore the interaction of low frequency emfs (60 hz+) with proteins ( enzymatic and nonenzymatic) using sds-page and isozymatic technique, respectively and dna using rapdpcr and single cell gel electrophoresis bioassays. identification of the biochemical and molecular mechanisms for plant tolerance like maize to environmental stress is important. detection of proteins and isozymes alterations at the gene product level is carried out by biochemical markers which measuring allele frequencies for specific genes (hailu&, alatawi 2014). meanwhile, molecular markers monitored differences (polymorphisms) within nucleotide sequences to indicate alterations at the dna level, such as nucleotide changes: deletion, duplication, inversion, and/or insertion (qi et al., 2014). sds-page (sodium dodecyl sulphate polyacrylamide gel electrophoresis) is a biochemical bioassay that is used to detect genetic differences in polypeptide banding patterns and to profile proteins induced by abiotic stress due to changes in the dna coding sequences and active structural genes leading to modifications of structure protein, protein interaction, and stressed oxidative proteins (karaca, 2013). on the other hand, isozyme analysis is a sophisticated biochemical approach that has a wide range of applications in detecting genetic alterations in plant cells (hailu et al., 2014). isozymes are enzymatic proteins, arise from multiple gene loci coding for distinct structural polypeptide chains, and their electric charge depends on the amino acids they contain (hailu et al., 2014). they have different molecular forms showing the same substrate specificity due to changes in the nucleotide sequence of the dna that codes for the protein, they differ in size, molecular weight, electrophoretic mobility, electric charge, and amino acid content (karaca, 2013). native polyacrylamide gel electrophoresis (native page) is used to differentiate protein variants in isozyme analysis, and an enzyme-specific staining combination, which contains a substrate, co-factor, and oxidized salt, is used to visualize them. (karaca, 2013). recently mutations induced by genotoxic stress, damage and fragmentation of dna can be estimated using molecular cytogenetic techniques such as rapdpcr and the single cell gel electrophoresis (comet assay) which can determine the direct genotoxic effect of exogenous factors on plant genotypes at the nuclear dna level (cenkci et al., 2009; santos, pourrut, ferreira de oliveira, 2015). rapd (random amplified polymorphic dna) is a pcr-based technology that uses short randomized nucleotide sequence primers to amplify random dna segments of genomic dna without the need for prior genomic dna knowledge. it can be used to detect genotoxicity, nucleotide sequence polymorphism, and alteration in rapd profiles induced by environmental genotoxic stresses which lead to changes in the structure 51assessment of protein and dna polymorphisms in corn (zea mays) of dna in living organisms, such as point mutations, tiny insertions and deletions of dna, and rearrangement of nucleotide sequences, all of which cause dna damage and lesions (atienzar & jha, 2006). comet assay, also known as single cell gel electrophoresis, is one of the most modern procedures for analyzing dna damages brought to agricultural sciences and genetic toxicology in recent years, reflected as single and double-strand breaks, oxidative-induced base damage and dna-dna/dna protein cross linking induced by oxidative and genotoxic environmental factors (nandhakumar et al., 2011). comet-like shape of nuclei (with a head, the nuclear region and a tail which contains dna fragments) is formed after electrophoresis and staining with a fluorescent dye and observed by a fluorescence microscopy (dikilitas et al., 2009). the aim of this study is to investigate biochemical and molecular mechanisms of nonionizing electromagnetic radiation (emr) on proteins and dna of corn (zea mays) based on sds-page, isozymatic analyses, rapd-pcr, and comet assay to estimate genetic polymorphism in economic corn crop under the stress of emr as well as understand how this plant was adapted. material and methods plant material the bio-monitor plant material in this investigation was maize grains (hybrid-323) supplied from the (agronomy research department, field crops institute, agriculture research center, giza, egypt). grains were checked for viability and homogeneity size before being divided into two groups: non-exposed and exposed to electromagnetic radiation (emr). 30 grains of each group were sterilized and germinated in earthenware pot 60 cm in diameter containing soil obtained from the agriculture field until reached seedlings after thirty-days-old. emr exposure facility electromagnetic radiations (emr) are created when electric current flows: the greater the current, the stronger the magnetic intensity. so, (emr) have magnetic and electrical properties that surround plant samples within that field. emr generator system was designed a locally and presented at biophysics department, faculty of science, zagazig university, egypt. this system consists of two coils, each formed by 1,000 turns of 1 mm copper wire, with a mean diameter of 260 mm and 25 cm length. emr were generated by a handmade cylindrical shaped coil that was connected to a 220v ac power supply (ed-345bm, china), to generate electrical current of 60 hz. emr intensities were measured through use of magnetic flux meter type 4048 with probe t4048, 001, manufactured by usa. to keep the temperature from rising, a standard fan was used. the temperature was measured with a thermometer to be 22+1°c. thus, a vertical sinusoidal magnetic field of 10 mt was generated in the central zone of the coils system when a 60 hz sinusoidal electric current passed through the coils. corn seedlings were put in a vessel (a glass jar with diameter of 7 cm and height of 12 cm) by placing a glass jar daily in the center zone of the coils system, and then subjected to strengths of emr (10 mt) for four different durations of exposure 1, 3, and 5 days, termed as (ex-1, ex-3, and ex-5 days) while emr non-exposed seedlings termed as (ex-0). leaves of ten corn seedlings were collected from emr exposed, and non-exposed, and thoroughly cleaned with distilled water, for removal of any debris and then completely dried in air conditions and then subjected to biochemical and molecular cytogenetic analyses. biochemical and molecular cytogenetic bioassays dried leaves of emr exposed, and non-exposed corn seedlings were defatted and processed into leaf powder according to the methods outlined by hojillaevangelista & evangelista, (2006) and used for sdspage, isozymatic, rapd-pcr, single cell gel electrophoresis analyses. biochemical bioassays protein extraction and sds-page analysis protein extraction from leaves was carried out as stated in the work of abdelhaliem and al-huqail (2016) with some modifications. 0.2 g of powdered and defatted leaves was added to extraction buffer (0.5 m trishcl, ph 6.8, 2.5% sds, 5% urea, and 5% 2-merkaptoethanol) in an eppendorf tube and mixed thoroughly by overtaxing. extraction buffer was boiled for 5 min before centrifugation at 10,000 g for 5 min at 4°c and the supernatant was used. to visualize the mobility of protein on the gel, bromophenol blue was added to the supernatant as a tracking dye. sds-page was used to examine proteins using 10% sds-polyacrylamide gels, as described by laemmli (1970). the protein bands were observed after electrophoresis using coomassie brilliant blue g-250 staining. marker proteins (fermentas) were 52 ekram abdelhaliem et al. used as references.the molecular weights (kda) of the polypeptide bands formed in the electropherogram were compared to the standard pharmacia protein marker. to capture the image and determine band intensities, gels were digitally photographed and analyzed using the gel doc viller lourmat system. data analysis of polypeptide banding patterns the bio-rad video densitometer, model gel doc 2000, was used to determine the number, concentration, and band density of polypeptide bands on each gel lane. electropherograms of each germplasm of emr exposed and non-exposed corn plants were evaluated for the presence (1) or absence (0) of protein bands to assess variance in the protein banding pattern. protein polymorphisms were evaluated based on previous polypeptide banding pattern differences. isozymes extraction and isozymatic analysis identification of isozymatic variations induced in emr exposed, and non-exposed corn seedlings were performed using the sodium dodecyl sulphate-native polyacrylamide gel electrophoresis (page) according to the methods of majumder, hassan , rahim, kabir (2012). four isozymes, leucine-aminopeptidase (lap. e.c. 3.4.1 1. i), esterases (est, e.c. 3.1.1.1), peroxidase, (prx e.c. 1.11.1.7), catalases (cat, 1.11. 1.6), were used in this study. isozymes of each sample was extracted according to method of majumder et al. (2012) that described briefly in the study of abdelhaliem & alhuqail, (2014). the staining of gel of lap and cat isozymes was performed according to protocol of pasteur, pasteur, bouhomme, catalan, davidian (1988) while est and prx isozymes followed the protocol of tiwari & bakshi (2015) for. the vilber lourmat gel documentation system was used to photograph the gels. the most common allele at each locus for each isozyme was assigned as relative front mobility (rf) value. the value rf was calculated in equation (1). rf = distance of zymograms migrated / distance of marker dye migrated (eq. 1) page and data analyses zymograms of each enzyme were observed and studied against an intense fluorescent light. after the staining of lap, est, pex, and cat isozymes, the isozymatic data were collected and immediately only consistent and clear zymograms were scored. the isozymatic banding patterns were compared among of emr non-exposed, and exposed corn seedlings based on their relative front (rf) values on gel electrophoresis, zymogram number, their intensities, and the percentages of polymorphic, unique and non-unique loci. different isozymatic patterns were scored as discrete variables, the presence “1” or absence “0” of zymogram. alterations of isozymatic patterns and zymograms at each locus were calculated using the popgene 32 version 1.31 software based on the computer program (labate, 2000). molecular cytogenetic bioassays genomic dna isolation and rapd-pcr analysis rapd analysis was performed to analyze the genotoxic effects of emr exposed, and nonexposed corn dna. genomic dna of powdered and defatted leaves were isolated following a modified hexadecyl trimethyl ammonium bromide (ctab) buffer protocol kit & chandran (2010) as described briefly in the study of abdelhaliem & al-huqail, (2013). the absorbance of diluted dna solution at 260 nm and 280 nm was used to measure the purity and concentration of dna. the dna quality was evaluated using ethidium bromidestained agarose gel electrophoresis. dna amplification process by pcr reactions of dna amplification by pcr, analysis of amplification products by agarose gel electrophoresis were conducted following the protocol of williams williams, kubelik, livak, rafalski, tingey (1990) with some modifications. the mixture of pcr amplification reaction as described briefly in the study of abdelhaliem, & al-huqail (2016) was consisted of 2.5 µl 10x buffer with 15 mm mgcl2 (fermentas, vinius, lithuania), with 0.25 mm each of datp, dctp, dgtp and dttp (sigma, st. louis, mo, usa), 0.5 u taq dna polymerase (sigma), 0.3 mm primer and 50 ng template dna. the pcr was performed in a palm thermal cycler apparatus (corbett research) was programmed using the following method: initial denaturation of 4 min at 95°c followed by 45 cycles of 1 min at 95°c, 1 min at 38°c, and 2 min at 72°c with final extension at 72°c for 10 min and a hold temperature of 4°c. only five primers (p-02, 06, 08, 10, and 14) effectively generated reproducible amplified products after a total of 20 random dna oligonucleotide primers 53assessment of protein and dna polymorphisms in corn (zea mays) (10 mer) were employed in the pcr (university of british columbia, canada). amplification dna products were analyzed by electrophoresed on 1.5% agarose gel (sigma) in tae buffer (0.04 m tris-acetate, 1 mm edta, ph 8). the run lasted one hour at a constant voltage of 100 volts. for 15 minutes, gels were stained with 0.2 mg/ml ethidium bromide. a uv light transilluminator was used to examine the pcr products. to determine band sizes, a 100-bp dna ladder (gibco-brl, grand island, ny, usa) was put into the first lane of each gel. a gel documentation system was used to photograph the gels under uv light (bio-rad, hercules, ca, usa). analyses of dna banding patterns visualization of amplified dna products on agarose gel electrophoresis was carried out using photo print (vilber lourmat, france) imaging system. banding patterns generated by rapd were analyzed using one-dimensional software (advanced american biotechnology and imaging, fullerton ca 92831, usa) while dna polymorphisms in rapd profiles included disappearance of a band and appearance of a new band with respect to the non-exposed profile, were calculated using several amplified dna parameters such as losses of normal bands, appearance of new bands, the number of polymorphic (unique and non-unique dna bands), and monomorphic dna bands, and the molecular sizes of dna bands as well as intensity of bands for each emr exposed sample compared to non-exposed one. amplified dna products were scored based on the presence (1) or absence (0) of dna bands for each primer and dna band intensity estimated by using image analysis software. polymorphic dna bands (unique and non unique) and monomorphic bands were also scored. estimation of genetic polymorphisms polymorphism (p, in %) of protein or isozyme or dna were estimated according to gjorgieva et al., (2013) based on the lost bands (non-unique) and the appearance of a new band (unique bands) as well as the monomorphic bands (bands with the same loci at all samples) as in equation (2). polymorphism % =[a+b/c] x 100 (eq. 2) where a is the number polymorphic bands (a is new bands unique, b is the number of lost nonunique bands and c is the total number of scored bands (polymorphic and monomorphic bands). isolation of nuclei and comet assay (single cell gel electrophoresis) technique isolation of nuclei and slide preparation the nuclei of emr exposed, and non-exposed corn leaves were isolated following the protocol of juchimiuk et al., (2006). five hundred mg of leaves were rinsed in distilled water twice, dried with a paper towel and then placed in a glass petri dishes containing 200 μl of cold tris-hcl buffer, ph 7.5 (on ice). under yellow light, leaves were carefully sliced into a ”fringe” with a new razor blade to release nuclei into the buffer. this approach of nuclei isolation found to be the most effective in obtaining low dna lesions in non-exposed samples. each slide was covered with a mixture of 55 l nuclear suspension and 55 l lmp agarose (1% generated with phosphate-buffered saline) and cover slipped at 40°c after being coated with 11% nmp agarose and dried. after putting the slide on ice for at least 5 min., the coverslip was removed. the coverslip was then replaced after 110 l of lmp agarose (0.5%) was poured on the slide. the coverslip was removed after 5 minutes on ice. single cell gel electrophoresis technique comet assay slides were prepared as described by juchimiuk et al. (2006). the corn nuclei slides were horizontally put in a gel electrophoresis tank with freshly prepared cold electrophoresis buffer (300 mm naoh, 1 mm edta, ph > 13) and incubated for 15 min. at 4°c, electrophoresis was carried out at 16 v, 300 ma for 30 min. the gel was then neutralized by washing three times in 400 mm tris-hcl, ph 7.5, and then stained for five minutes with ethidium bromide (20 g/ml). the gels were immediately dipped in ice-cold distilled water after staining and examined. comet imaging and software analysis the level of dna damage in 50 randomly chosen nuclei was examined in each slide using a computerized image analysis system or a fluorescence microscope with an excitation filter of 546 nm and a barrier filter of 590 nm (komet version 3.1. kinetic imaging, liverpool, uk). tail dna (td percent, relative proportion of dna in the comet tail) and tail moment (tm, integrated value of density multiplied by dna migration distance from nuclei) were used as dna damage parameters to quantify nuclear dna damages (juchimiuk et al., 2006). the 54 ekram abdelhaliem et al. percentage of nuclei having tails was also estimated, as well as relative tail length. results biochemical genetic bioassays sds-page bioassay the electrophoretic profiling of polypeptide banding patterns based on molecular weight (kda), band number, band intensity, fractionation of bands, appearance of new bands and the loss of some bands as parameters of polypeptide banding showed variations between emr exposed and non-exposed corn seedlings by sds-page analysis (table 1 and figure 1). there were 39 polypeptide bands with molecular weights ranging from 60.27 to 192.35 kda, 29 of which were polymorphic with a value of 74.36%, according to the data obtained, (24 unique bands with a value of 61.54%, plus 5 non-unique bands with value of 12.82%) in addition to one monomorphic band value of 2.56%.sds-page analysis indicated distinctive polymorphism value of 96.66% based previous banding variations. when comparing emr exposure samples to non-exposed samples, there were noticeable differences in the number of polypeptide bands and molecular weight. the highest number of polypeptides bands was 12, with a value 30.77% scored at corn seedlings exposed to emf for 5 days while the lowest number was 8, with a value 20.51% for non-exposed samples (control). unique polypeptide bands obtained from sds-page were varied in molecular weight (kda), and in number and intensity of bands among emr exposed and nonexposed samples (table 1). as a result, unique bands can be employed as a tool for the appearance new characteristic polypeptide bands that are specific for each emr exposure time. the highest number of unique polypeptides bands was 9, with a value of 37.50% for samples exposed to emr for 5 days while the lowest number (4 unique bands), and with a value of 16.67 scored at samples exposed to emr for one a day (table 1). isozymatic bioassay lap, est, prx, and cat isozymes used in this study revealed clear isozymatic polymorphisms among emr exposed and non-exposed zea mays reached the values of 91.66 % for lap and est, 88.89% for cat, and 80.00% for pex based on zymograms number, loci, rf values, and optical densities generated by each isozyme, separately (tables 2, 3, 4 and 5; figures 2 and 3 a and b). a total of 72 different electrophoretic zymograms produced by the four isozymes showed varied relative front (rf ) values, varying from 0.01 to 1.20 and varied values of optical densities (od). of these zymograms, 38 with a value of 52.77% were polymorphic (27 unique zymograms with a value of 37.5% plus 11 nonunique zymograms with a value of 15.28%). the higher number of zymograms was (19) generated by est and prx while lap and cat isozymes generated 17 zymograms. the four enzymes scored maximum number of zymograms (21) at corn seedling exposed to emf for 5 days compared with 13 zymograms scored for non-exposed samples. on the other hand, lap , est, pr x, and cat isozymes generated unique zymograms varied in rf values and optical densities. the highest number of unique zymograms produced by four isozymes was 9; with a value of 33.33% recorded for samples exposed to emr for 1 and 5 days, in comparison to 5 unique zymograms; with a value of 18.51 % for non-exposed samples (tables 2, 3, 4 and 5). molecular cytogenetic bioassays rapd-pcr bioassay profiles and banding patterns of amplified dna bands generated by rapd exhibited clear variations among corn seedlings exposed to emr compared to non-exposed one (table 6 and figure 4). only five of the 20 random decamer primers examined revealed distinct alterations in the amplified dna banding patterns and provided specific and reliable results and consistent bands. 85 dna bands were produced by rapd analysis, the sizes of these bands ranged from 153 and 1008 bp in length. the rapd analysis, on the other hand, identified three types of amplified dna bands (polymorphic, monomorphic, and polymorphic), which differed quantitatively and qualitatively in band number, size (bp), and intensity on an agarose gel (table 6). there were, 59 bands were polymorphic (unique and non-unique bands) with a value of 69.41% (36 unique bands with a value of 42.35% and 23 non-unique bands with a value of 27.06%) and 5 monomorphic bands with a value of 5.88%. an average of 17 bands per primer was scored. furthermore, table 6 shows the total polymorphisms produced by the five primers, which reached a value of 97.01%. the primers differed with respect to the value of polymorphisms detected. the highest level of polymorphism (100%) was revealed by primers (p-14) because of it do not detect any monomorphic bands, followed by primers (p-02 and p-10) which recorded polymorphism value of 90.91%, p-06 recorded polymorphism value of (88.89%) 55assessment of protein and dna polymorphisms in corn (zea mays) table 1. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) analysis of proteins of emf exposed and non-exposed zea mays seedlings for days of exposure times (ex-0, ex-1, ex-3,and ex-5) using the documentation system gel doc bio rad system 2000. lanes 1–4 represented the days of exposure times (ex-0, ex-1, ex-3,and ex-5), respectively. lanes rows molecular weight (kda) polypeptide bands in each lane lane1 lane 2 lane 3 lane 4 band types kda % kda % kda % kda % 1 192.35 0 0 1 10.90 0 u 2 184.13 1 2.27 0 0 0 u 3 180.15 0 1 5.67 0 0 u 4 158.02 0 0 0 1 6.67 u 5 154.61 0 0 1 5.98 0 u 6 148.00 0 1 4.08 0 0 u 7 145.31 1 10.40 0 0 0 u 8 138.81 0 0 1 5.31 1 1.55 non-u 9 130.19 0 1 1.17 0 0 u 10 126.66 0 0 1 2.23 0 u 11 118.79 1 11.10 0 0 0 u 12 115.57 0 1 17.40 1 13.00 0 nonu 13 112.44 0 0 0 1 17.60 u 14 105.45 1 10.40 1 5.51 0 0 non-u 15 104.49 0 0 1 6.53 0 u 16 89.84 1 28.30 1 27.60 1 21.50 1 24.60 m 17 80.24 0 0 0 1 8.28 u 18 76.16 1 2.70 0 0 0 u 19 75.50 0 0 0 1 1.43 u 20 71.66 0 0 0 1 4.24 u 21 71.04 0 1 15.20 0 0 u 22 70.42 0 0 1 13.40 0 u 23 67.43 0 0 0 1 2.07 u 24 66.27 1 13.40 0 0 0 u 25 63.66 0 0 0 1 2.07 u 26 62.00 0 1 16.90 1 13.90 1 1.00 non-u 27 62.22 0 0 0 1 10.30 u 28 61.45 1 21.50 0 0 0 u 29 60.91 0 1 6.46 1 7.12 0 non-u 30 60.27 0 0 0 1 21.20 u no. of polypeptide bands 8 9 10 12 total polypeptide bands 39 % of total bands 20.51 23.07 25.64 30.77 unique (u) bands (non-u) bands polymorphic bands monomorphic bands polymorphisms % no % no % no % no % frequency of polypeptide bands and polymorphisms 24 61.54 5 12.82 29 74.36 1 2.56 96.66 lane1 lane2 lane 3 lane 4 no. kda no. kda no. kda no. kda no.and mw(kda) of unique polypeptide bands 6 184.13–145.3176.16–66.27–61.45 4 180.1501–48.00– 130.19–71.04 5 192.35–154.61 126.66– 104.49–70.42 9 158.02–112.44–80.24– 75.50–71.66–67.43– 63.66–62.22-60.27 total unique bands 24 % of unique bands 25 16.67 20.83 37.50 56 ekram abdelhaliem et al. while primer (p-08) scored the lowest level of polymorphism value of (81.83%). these genetic dna polymorphisms based on the gain and/or loss of dna bands in emr exposed samples compared to the non-exposed one (control). besides, the number of rapd amplified dna bands varied among emr exposed corn seedlings compared to control and correlated positively with increasing exposure time of emr. the highest number of amplified dna bands was 26, with a value of 30.59%, which was produced by five primers and was detected in emr exposed sample for 5 days exposure time compared to 21 dna bands, with a value of 24.71% which recorded at non-exposed samples. unique amplified dna bands created by rapd analysis were distinctive loci specific for one exposure time based on their number, their molecular sizes, and optical intensities. the highest number of unique dna bands produced by five primers was 13, with a value of 36.11% recorded in emr exposed samples for 5 days exposure time compared to 10 dna unique bands, with a value of 27.78% which recorded at non-exposed samples, while the lowest number of unique dna bands was six, with a value of 16.67% for emr exposed samples for one days exposure time (table 6). single cell gel electrophoresis technique bioassay the comet assay or single cell gel electrophoresis assay (scge) is one of the very widely used assays to microscopically detect dna damage at the level of a single cell. cells containing damaged dna have the appearance of a comet with a bright head and tail. the scge or comet test was employed in this investigation to identify nuclear dna (ndna) damage caused by an electromagnetic radiation stressor in corn seedlings for different exposure times (table 7 and figure 5). the extent of dna migration from nuclei (tailed ratio), tail length μm, % of tailed dna (td percent), and tail moments(tm) were utilized to evaluate the level of dna damage caused by the comet assay. the recent findings revealed that each emr exposure time led to inconsistent differences in the level of dna damage in corn nuclei. emr exposed samples for 5 days exposure time (ex-5) detected the highest dna migration from corn nuclei (tailed ratio 20%) with tail length (2.88 μm), td% (2.79%), and tm (8.04); this demonstrated that this emr exposure time had clastogenic and genotoxic stress increased ndna damage of corn cells in comparison to non-exposed nuclei (ex-0) which detected the lowest dna migration (tailed ratio 3%) with tail length (0.99 μm), td% (1.05%), and tm (1.73). discussion the current study used sds-page and isozymatic, rapd-pcr, and scge as accurate, reliable, to detect genetic effect of 60 hz emr on proteins, isozymes, and dna, respectively. sds page and isozymatic analyses are biochemical bioassays generated genetic polymorphisms at the level of gene product such as alterations in non-enzymatic and enzymatic proteins (storage proteins and isozymes, respectively) and amino acids. sdspage analysis revealed varied polypeptide banding patterns and high level of protein polymorphisms among emr exposed corn seedlings and non-exposed samples depend on number of bands, their molecular weights, and band intensity, the gain of new protein bands (unique bands) and the loss of normal bands (nonunique bands). the banding pattern of electrophoretic polypeptide may be due to interaction of emr with the transcriptional events occurring during the expression of genes under emr stressor leading to different mutations in sequencing of mrna and changes in amino acids of proteins as end products and consequently, polypeptide banding patterns of proteins on electrophoretic gel of sds-page (sadia et al., 2009). on the other hands, the high levels of polymorphisms based on the gain of polypeptide bands or loss of others which generated by sds-page analysis may be resulted from conformational changes in the amino acid figure 1. polypeptide banding patterns analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) technique of non-exposed and emr exposed seedlings of zea mays for days of exposure times (ex-0, ex-1, ex-3,and ex-5) based on relative front (rf) values and optical densities (od). lanes 1–4 represented the days of exposure times (ex-0, ex-1, ex3,and ex-5), respectively. 57assessment of protein and dna polymorphisms in corn (zea mays) sequences of proteins, or may result from gene duplication followed by a point mutation (insertion or addition of nitrogenous base sequences) that encodes the fractionated polypeptide bands led to appearing (gain) of new bands (unique bands) or may be result from deletion of sequences or loss of genes and consequently, deficiency of amino acids between mutated sites of polypeptide chain of emr-exposed samples led to loss of protein bands (non-unique) (galani et al., 2011). moreover, variation in the number of polypeptide bands and band intensities observed in emr-exposed samples in comparison to the control may be resulted from changes in nitrogenous bases of dna, in protein sites or amino acid sequences or frameshift mutations led to changes in bands number while band intensity may be resulted from duplication of gen or point mutation which led to manufacturing of longer and shorter of polypeptide chains (shikazono et al., 2005). additionally, emrexposed corn seedlings for 5 days caused alteration in profile and banding patterns of proteins as evident in increasing bands number more than non-exposed ones. on native-page , lap, est, pex, and cat isozymes employed in this work displayed numerous zymograms at various loci. these variations in electrophoretic zymogramatic patterns and isozymatic polymorphisms based on rf values and zymograms intensitable 2. distribution of leucine-aminopeptidase (lap) zymograms of emf non-exposed and exposed zea mays seedlings for days of exposure times (ex-0, ex-1, ex-3, and ex-5) based on relative front (rf) values and optical densities (od). lanes 1–4 represented the days of exposure times (ex-0, ex-1, ex3, and ex-5), respectively. rows rf values leucine-aminopeptidase (lap) zymograms zymogram types lane 1 lane 2 lane 3 lane 4 rf od rf od rf od rf od 1 0.19 √ 38.4 u 2 0.25 √ 34.9 u 3 0.35 √ 5.15 u 4 0.40 √ 10.8 u 5 0.43 √ 28.3 u 6 0.46 √ 32.4 u 7 0.66 √ 35.1 √ 33.6 √ 25.9 √ 31.3 m 8 0.85 √ 30.4 u 9 0.88 √ 29.9 u 10 0.90 √ 41.7 u 11 0.95 √ 38.1 u 12 1.20 √ 7.23 √ 4.34 √ 3.05 non-u no. zymograms 3 5 4 5 total zymograms 17 % of zymograms 17.64 29.41 23.53 29.41 lane 1 lane 2 lane 3 lane 4 no. rf no. rf no. rf no. rf no. and rf of unique loci 2 0.25–0.88 3 0.35–0.43– 0.95 2 0.46–0.90 3 0.19–0.40– 0.85 % of unique loci 20.00 30.00 20.00 30.00 unique (u) (non-u) polymorphic monomorphic (m) polymorphisms no. % no. % no. % no. % % frequency of isozymetic bands and polymorphisms 10 58.82 1 5.88 11 64.71 1 5.88 91.67 58 ekram abdelhaliem et al. ties among emrexposed corn seedlings for different exposure times compared to non-exposed ones. the alterations in zymogramatic patterns may be due to mutation or changes in the dna nucleotide sequence that codes for the protein leading to the substitution of one to several amino acids or changes in amino acids compositions that result in a change in the net charge of a proteins consisted isozyme (karaca, 2013). they might also be attributable to a gene’s interaction with oxidative stress caused by emr exposure times or to changes in enzyme conformation which altering the rate of proteins migration on page and their relative front mobility as well as efficiency and stability of the isozyme (kumar, gupta, misra, modi, pandey, 2009). on the other hand, alteration in the electrophoretic zymograms mobility may be due to changes in the sequences of encoding dna or from the shapes and different sizes of the affected isozyme (karaca, 2013). recently, rapd-pcr and single-cell gel electrophoresis (scge) are molecular and cytogenetic bioassays used in this study at dna level to detect reliable and accurate genetic polymorphisms in banding patterns and dna damages induced by emr-oxidative stress on nuclear dna of corn seedlings. rapd bioassay is used in this study to provide information about nucleotide sequence polymorphisms that might have occurred across coding and noncoding regions of the entire genome (elsh & mcclelland, 1991). the data obtained table 3. distribution of esterase (est) zymograms of emf non-exposed and exposed zea mays seedlings for days of exposure times (ex-0, ex-1, ex-3, and ex-5) based on relative front (rf) values and optical densities (od). lanes 1–4 represented the days of exposure times (ex0, ex-1, ex-3, and ex-5), respectively. rows rf values esterase (est) zymograms lane 1 lane 2 lane 3 lane 4 zymogram typesrf od rf od rf od rf od 1 0.15 √ 33.8 √ 42.2 non-u 2 0.16 √ 34.3 u 3 0.29 √ 57.6 u 4 0.36 √ 15.6 u 5 0.38 √ 10.2 u 6 0.39 √ 13.5 u 7 0.57 √ 25.8 √ 22.9 non-u 8 0.58 √ 19.7 √ 20.0 non-u 9 0.69 √ 3.62 u 10 0.78 √ 26.3 √ 36.3 √ 19.4 √ 8.54 m 11 0.97 √ 1.50 √ 21.40 non-u 12 1.10 √ 2.52 u no. zymograms 5 5 4 5 total zymograms 19 % of zymograms 26.31 26.31 21.05 26.31 lane 1 lane 2 lane 3 lane 4 no. rf no. rf no. rf no. rf no. and rf of unique loci 3 0.16–0.39– 0.77 3 0.38–0.79– 1.10 1 0.36 2 0.29–0.69 % of unique loci 42.86 42.86 14.29 28.57 unique (u) (non-u) polymorphic monomorphic (m) polymorphisms no. % no. % no. % no. % % frequency of zymograms and polymorphisms 7 36.84 4 21.05 11 57.90 1 5.26 91.66 59assessment of protein and dna polymorphisms in corn (zea mays) scored variations in dna polymorphisms and banding patterns among emr-exposed zea mays compared to the non-exposed ones based on primers used, alterations in the bands number of dna, their sizes, intensities, the gain of new amplified dna bands (unique), and disappearance of normal bands (non-unique). additionally, this study found that these variations was dependent on emr exposure times. the number of amplified dna bands may be related to the number of nucleotides and their directions within genomic dna sequences that are complementary to the sequence of the related primer (abdelhaliem & alhuqail, 2016). dna polymorphisms generated by rapd analysis may be due to alterations in dna nucleotide sequences during duplication of dna or gene expression under the emr stressor such as additions of the amplified dna bands, insertions of new nitrogenous bases, and transpositions of genes within genomic dna that led to appearance of new dna bands ( unique dna bands) ( atienzar & jha, 2006).or due to the deletion of existing genes present on genomic dna or transpositions of genes from their own dna to another dna or due to the hybridization site of a primer in one gene that is altered at a single nucleotide in second amplified gene that can eliminate of a specific amplified nucleotide sequences from second gene amplified resulting of disappearance of amplified dna genes (non-unique bands) (welsh & mcclelland, 1991). therefore, the unique bands can be assumed as a characteristic bioassay specific for each corn germplasm affected by emr. the comet assay (single-cell gel electrophoresis) is a simple method for measuring dna strand breaks ( dna damage) in eukaryotic nuclei. cells containing damaged dna have the appearance of a comet with a bright table 4. distribution of peroxidase (pex) zymograms of emf non-exposed and exposed zea mays seedlings for days of exposure times (ex-0, ex-1, ex-3, and ex-5) based on relative front (rf) values and optical densities (od). lanes 1–4 represented the days of exposure times (ex-0, ex-1, ex-3, and ex-5), respectively. rows rf values peroxidase (per) zymograms lane 1 lane 2 lane 3 lane 4 zymogram rf od rf od rf od rf od types 1 0.01 √ 2.5 √ 1.5 √ 1.0 non-u 2 0.10 √ 24.6 √ 35.7 √ 32.4 √ 44.6 m 3 0.38 √ 57.9 √ 37.0 √ 24.9 √ 43.4 m 4 0.59 √ 23.4 u 5 0.62 √ 2.00 u 6 0.73 √ 17.5 √ 2.50 non-u 7 0.88 √ 12.00 u 8 0.89 √ 27.4 u 9 0.92 √ 19.2 u 10 1.20 √ 2.00 u no. zymograms 3 5 5 6 total zymograms 19 % of zymograms 15.78 26.31 26.31 31.57 lane 1 lane 2 lane 3 lane 4 no. rf no. rf no. rf no. rf no. and rf of unique loci 0 – 2 0.62–0.89 2 0.59–0.92 2 0.88–1.20 % of unique loci 0.00 33.33 33.33 33.33 unique (u) (non-u) polymorphic monomorphic (m) polymorphisms no. % no. % no. % no. % % frequency of zymograms and polymorphism 6 31.57 2 10.52 8 42.10 2 10.52 80.00 60 ekram abdelhaliem et al. head and tail. in contrast, undamaged dna appears as an intact nucleus with no tail. in this study, it used to determine the degree of dna damage induced by emr in corn seedlings. scge data illustrated notable alterations in the degree of dna damage in corn nuclei exposed to emr for one, three-, and five-days dependent on exposing time. this may be due to interaction of emr with the dna can stimulate the synthesis of this stress protein, causing dna strand breaks which increase by increasing of emr energy (blank & goodman, 2012). this nuclear dna ( ndna) damages led to increase in migration of dna fragments (tail) from the nucleus (head). this revealed that the increased emrexposure of corn seedlings induced dna lesions in their cells. the dna lesions induced by emr may be directly due to energy deposition in cells or indirectly due to reactive oxygen species (ros) and oxidative dna damage (kıvrak et al., 2017). they showed that a major concern of the genotoxic effects of non-ionizing electromagnetic radiation (emr) is overproduction of ros in cells and inducing oxidative stress on protein and dna because of emr exposure can induce dna strand breaks and acts as a co-inductor of dna damages rather than as a genotoxic agent. they also interpreted the genetic mechanisms by which emr interact with protein and dna are radical pair recombination led to increasing the concentration, activity, and lifetime of reactive oxygen species (ros), which might cause changes in cell cycle, genetic mutation, damage to dna leading to changes in cellular functions and cell death, modification of protein expression and oxidation of proteins, and inhibition of enzymes. table 5. distribution of catalase (cat) zymograms of emf non-exposed and exposed zea mays seedlings for days of exposure times (ex-0, ex-1, ex-3, and ex-5) based on relative front (rf) values and optical densities (od). lanes 1–4 represented the days of exposure times (ex0, ex1, ex-3, and ex-5), respectively. rows rf values esterase (est) zymograms zymogram types lane 1 lane 2 lane 3 lane 4 rf od rf od rf od rf od 1 0.02 √ 2.00 √ 2.34 √ 1.0 non-u 2 0.09 √ 32.4 √ 32.9 √ 25.9 √ 21.1 m 3 0.22 √ 15.4 u 4 0.33 √ 17.2 √ 27.9 non-u 5 0.34 √ 32.9 √ 21.5 non-u 6 0.69 √ 57.6 u 7 0.71 √ 34.7 √ 46.2 non-u 8 0.85 √ 49.9 u 9 0.94 √ 3.34 u no. zymograms 4 4 4 5 total zymograms 17 % of zymograms 23.53 23.53 23.53 29.41 lane 1 lane 2 lane 3 lane 4 no. rf no. rf no. rf no. rf no. and rf of unique loci 0 – 1 0.85 1 0.36 2 0.29–0.69 % of unique loci 0.00 25.00 25.00 50.00 unique (u) (non-u) polymorphic monomorphic (m) polymorphisms no. % no. % no. % no. % % frequency of zymograms and polymorphism 4 23.53 4 23.53 8 47.06 1 5.88 88.89 61assessment of protein and dna polymorphisms in corn (zea mays) conclusion the data obtained in current study observed that the longer emr exposing time could induce notable alterations in banding patterns profile generated by sdspage, isozymatic, and rapd bioassays in addition to distinct extent of dna damages as estimated by comet assay. therefore, this study concluded that each emr exposing time had unique interaction with proteins, isozymes, and dna in corn cells exhibiting wide range of genetic and oxidative stress on these macromolecules. due to these distinct alteration, it might be asserted that the exposure of economic crop plants to emr may change gene expression and subsequently, will affect their growth and grain yield. also, it concluded that bioassays used should be augmented for accurate and precise estimation of alterations of protein and dna profiles after emr exposure of crop plants and for providing excellent results and understanding how this plant was adapted. author contribution e. m. a. and h. m.a. designed and performed the experiments. e. m. a. analyzed the data and discussed the results. e. m. a. and r. s. s. wrote the manuscript in consultation with a. a. b., and h. m.a. all of the authors read and approved the manuscript. figure 2. (a) isozymatic patterns and (b) schematic distribution of leucine-aminopeptidase (lap) and esterase (est) zymograms (rf values) of non-exposed and emr exposed seedlings of zea mays for days of exposure times (ex-0, ex-1, ex-3, and ex-5) based on relative front (rf) values and optical densities (od). lanes 1–4 represented the days of exposure times (ex-0, ex1, ex-3, and ex-5), respectively. 62 ekram abdelhaliem et al. acknowledgment the authors would like to express their deep thanks to professor magda hanafy, biophysics department, science college, zagazig university, for her effort in performing electromagnetic radiation (emr). references abdel haliem e, al-huqail aa. 2013. oxidative damage and mutagenic potency of fast neutron and uv-b radiation in pollen mother cells and seed yield of vicia faba l. biomed research international 2013:1:12 abdelhaliem e, al-huqail aa. 2016. detection of protein and dna damage induced by elevated carbon dioxide and ozone in triticum aestivum l. using biomarker and comet assay. genetics and molecular research 15(2):1-19. ahanger ma, akram na, ashraf m, alyemeni mn, wijaya l, ahmad p. 2017. plant responses to environmental stresses—from gene to biotechnology. aob plants 9(4): 1-17. atienzar fa, jha an. 2006. the random amplified polymorphic dna (rapd) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review. mutation research/ reviews in mutation research 613(2-3):76-102. figure 3. (a) isozymatic patterns and (b) schematic distribution of peroxidase (pex) and catalases (cat) zymograms (rf values) of nonexposed and emr exposed seedlings of zea mays for days of exposure times (ex-0, ex-1, ex-3,and ex-5) based on relative front (rf) values and optical densities (od). lanes 1–4 represented the days of exposure times (ex-0, ex-1, ex3,and ex-5), respectively. 63assessment of protein and dna polymorphisms in corn (zea mays) blank m, goodman r. 2011. dna is a fractal antenna in electromagnetic fields. international journal of radiation biology 87(4): 409-415. blank m, goodman r m. 2012. electromagnetic fields and health: dna-based dosimetry. electromagnetic biology and medicine 31(4): 243-249. cenkci s, yıldız m, ciğerci i̇h, konuk m, bozdağ a. (2009). toxic chemicals-induced genotoxicity detected by random amplified polymorphic dna (rapd) in bean (phaseolus vulgaris l.) seedlings. chemosphere 76 (7): 900-906. dikilitas m, kocyigit a, yigit f. (2009). a molecularbased fast method to determine the extent of dna damages in higher plants and fungi. african journal of biotechnology, 8(14). elsh j, mcclelland m. (1991). genomic fingerprinting using arbitrarily primed pcr and a matrix of pairwise combinations of primers. nucleic acids research 19:5275–5279. erturk fa, agar g, arslan e, nardemir g, sahin, z. 2014. determination of genomic instability and dna methylation effects of cr on maize (zea mays l.) using rapd and cred-ra analysis. acta physiologiae plantarum 36(6):1529-1537. galani s, naz f, soomro f, jamil i, azhar a, ashraf a. 2011. seed storage protein polymorphism in ten elite rice (oryza sativa l.) genotypes of sindh. african journal of biotechnology 10(7): 1106-1111 gjorgieva d, kadifkova panovska t, ruskovska t, bačeva k, stafilov t. 2013. influence of heavy metal stress on antioxidant status and dna damage in urtica dioica. biomed research international 2013:1-6. table 6. amplification banding patterns and genomic template stability (gts) of nuclear dna analyzed by rapd-pcr isolated from emf non-exposed and exposed zea mays seedlings for days of exposure times (ex-0, ex-1, ex-3,and ex-5) and analyzed by bio-one d++ software (vilber lourmat, france). lanes 1–4 represented the days of exposure times (ex-0, ex-1, ex-3,and ex-5), respectively. primer code primers sequences (5´→3´) amplicon sizes (bp) number of scorable bands in each lane types of dna bands polymorphisms polymorphic monomorphictotal bands unique (u) non -u total lane1 lane2 lane3 lane4 no % no % no % no % no % % p-02 gga ccc aac c 1008-170 3 3 4 5 15 17.65 5 5.88 3 3.53 8 9.41 1 1.18 90.91 p-06 acc tga acg g 938-709 4 3 3 5 15 17.65 4 4.71 6 7.06 10 11.76 1 1.18 88.89 p-08 gtg tgc ccc a 877-438 3 3 4 5 15 17.65 6 7.06 3 3.53 9 10.59 2 2.35 81.83 p-10 ggt cta cac c 774-214 5 5 3 7 20 23.53 9 10.59 5 5.88 14 16.47 1 1.18 90.91 p-14 ctt ccc caa g 846-153 5 5 5 4 20 23.53 12 14.12 6 7.06 18 21.18 0 – 100.00 overall total bands in each lane 21 19 19 26 85 100 36 42.35 23 27.06 59 69.41 5 5.88 92.19 sum 85 % of total bands in each lane 24.71 22.35 22.35 30.59 primer code no. and sizes (bp) of unique amplified dna bands lane 1 lane 2 lane 3 lane 4 no. sizes no. sizes no. sizes no. sizes p-02 1 985 1 979 1 1000 2 1008–450 p-06 2 688–595 0 – 0 – 2 780–640 p-08 0 – 1 706 2 650–500 3 780–520 p-10 2 600–200 2 550–418 1 366 4 690–640–493– 297 p-14 5 721–652–594– 528–153 2 371–153 3 797–483–406 2 846–700 total unique bands 10 6 7 13 sum of unique bands 36 % of unique bands 27.78 16.67 19.44 36.11 64 ekram abdelhaliem et al. hailu hw, kristiyanto dh, alatawi ara, raqib sm. 2014. isozyme electrophoresis and morphometric comparison of reed (imperata cylindrical) adaptation to different altitudes. international journal of innovative research in science, engineering and technology 3(5):12387-12394. hojilla‐evangelista mp, & evangelista rl. 2006. effects of cooking and screw‐pressing on functional properties of cuphea psr23 seed proteins. journal of the american oil chemists’ society 83(8):713-718. iderawumi am, friday ce. 2020. effects of magnetic field on pre-treatment of seedlings and germination. journal of agriculture and research 6 (9):1-8. juchimiuk j, gnys a, maluszynska j. 2006. dna damage induced by mutagens in plant and human cell nuclei in acellular comet assay. folia histochemica et cytobiologica, 44(2):127-131. karaca m. 2013. isozymes as biochemical markers in plant genetics. international journal of agriscience 3(11):851-861. kit ys, chandran s. 2010. a simple, rapid, and efficient method of isolating dna from chokanan mango (mangifera indica l.). african journal of biotechnology 9(36): 58055808. figure 4. amplified dna banding patterns of genomic dna isolated from non-exposed and emr exposed seedlings of zea mays for days of exposure times (ex-1, ex-3,and ex-5) and analyzed by rapd-pcr dna using five random primers (p-02, 06, 08, 10, and 14). lanes 1–4 represented the days of exposure times (ex-0, ex-1, ex-3,and ex-5), respectively. 65assessment of protein and dna polymorphisms in corn (zea mays) kıvrak eg, yurt kk, kaplan aa, alkan i, altun, g. 2017. effects of electromagnetic fields exposure on the antioxidant defense system. journal of microscopy and ultrastructure 5(4):167-176. kumar p, gupta vk, misra ak, modi dr, pandey bk. 2009. potential of molecular markers in plant biotechnology. plant omics 2(4): 141-162. labate ja. 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(asphodelaceae) wendy ozols-narbona*, josé imery-buiza assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions seyedeh mahsa hosseini1, sepideh kalatejari1, mohsen kafi2,*, babak motesharezadeh3 assessment of protein and dna polymorphisms in corn (zea mays) under the effect of non-ionizing electromagnetic radiation ekram m. abdelhaliem1,*, hanan m.abdalla1, ahmed a. bolbol1, rania s. shehata1,2 chromosome counts of some species of wetland plants from northwest iran saeedeh sadat mirzadeh vaghefi*, adel jalili delimiting species using dna and morphological variation in some alcea (malvaceae) species based on srap markers chnar hama noori meerza1, basoz sadiq muhealdin2, sahar hussein hamarashid2,*, syamand ahmad qadir3, yusef juan4 mapping cap-a satellite dnas by fish in sapajus cay paraguay and s. macrocephalus (platyrrhini, primates) simona ceraulo, francesca dumas* determination of genome size variation among varieties of ilex cornuta (aquifoliaceae) by fow cytometry peng zhou1, jiao li2, jing huang1, fei li1, qiang zhang2,*, min zhang1,* first report of chromosome and karyological analysis of gekko nutaphandi (gekkonidae, squamata) from thailand: neo-diploid chromosome number in genus gekko weera thongnetr1, suphat prasopsin2, surachest aiumsumang3,*, sukhonthip ditcharoen4, alongklod tanomtong5, prayoon wongchantra6, wutthisak bunnaen7, sumalee phimphan3 intraspecific karyomorphological and genome size variations of in vitro embryo derived iranian endemic asafoetida (ferula assa-foetida l., apiaceae) narges firoozi, ghasem karimzadeh*, mohammad sadegh sabet, vahid sayadi cytogenetic studies in the centaurea aucheri group (sect. phaeopappus) seyed mahmood ghaffari¹,*, seyed mohsen hesamzadeh hejazi² karyomorphology, genome size, and variation of antioxidant in twelve berry species from iran saeed mohammadpour1, ghasem karimzadeh1,*, seyed mahmood ghaffari2 caryologia. international journal of cytology, cytosystematics and cytogenetics 72(1): 15-21, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-247 citation: ramanpreet, r. chand gupta (2019) meiotic studies in genus withania pauquy, from indian thar desert. caryologia 72(1): 15-21. doi: 10.13128/cayologia-247 received: 19th july 2018 accepted: 20th december 2018 published: 10th may 2019 copyright: © 2019 ramanpreet, r. chand gupta. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. meiotic studies in genus withania pauquy, from indian thar desert ramanpreet*, raghbir chand gupta department of botany punjabi university, patiala, 147002, punjab, india * corresponding author: ramanbrar247@gmail.com abstract. detailed male meiosis has been made in two species of genus withania collected from desert regions of rajasthan, india. the study revealed 2n=48 for both the species. the meiotic analysis of w. somnifera is reported to be normal whereas, w. coagulans showed abnormal meiosis with the presence of abnormalities like spindle irregularities, chromatin transfer, laggards and irregular microsporogensis with the presence of monads, dyads, triads, polyads and tetrads with micronuclei which further lowered the pollen fertility and giving rise to varying size pollen grains. both the species are widely used for various medicinal purposes by local/tribal people of the state. keywords. withania, medicinal plant, abnormal meiotic behavior, indian thar desert. introduction the genus withania belongs to the family solanaceae. the family comprises of 100 genera and about 2500 species (hunziker 2001, olmstead et al. 2008). the genus is distributed throughout the tropical and sub-tropical regions of the world with 26 species (ahmad 2014). w. somnifera is well distributed in india with and is growing well in dry parts of tropical and subtropical regions extending to the elevation of 1500 m. but w. coagulans is rare and endangered plant species found only in few localities in rajasthan. both the species are well distributed in east of mediterranean regions extending to south asia. regarding the medicinal properties of the species, a composite ayurvedic medicine “liv 52” commonly used as intestinal infections is a hepatoprotective herbal preparation and contains extracts from w. coagulans and w. somnifera (mishra et al. 2013). w. coagulans is commonly known as “indian cheese maker” since fruits and leaves of the plants are used as coagulant, as it contain an enzyme called withanin, having milk coagulating activity (naz et. al. 2010). in some parts of india and pakistan, both the species are used as blood purifier, for cleaning teeth and plant smoke is inhaled for relief in toothache (dymock et al. 1972, krishnamurthi 1969). the plant possesses 16 ramanpreet, raghbir chand gupta antitumor, antimicrobial and anti-inflammatory properties. since the plant is toxic in nature so it should be used with caution (purohit and yyas 2004). flowers of its species are used to treat nervous exhaustion, insomnia and impotence. the meiotic course of the species reveals the presence of 2n=48 from west pakistan (baquar 1967) as well as outside india. w. somnifera commonly known as “ashwangandha” or “indian ginseng” is extensively studied from india by bir et al. (1978) bir and sidhu (1979 and 1980) from punjab plains, koul et al. (1976) from jammu and kashmir, madhavadian (1968) from tamil nadu, bhaduri (1933), datta et al. (2005), iqbal and datta (2007) from west bengal. it is also extensively worked out from pakistan by baquar (1967) and khatton and ali (1982) and from saudi arabia by al-turki et al. (2000). earlier meiotic studies reveals the presence of intraspecific diploid cytotype (2n=24), tetraploid cytotype (2n=48) and hexaploid cytotypes (2n=72). the karyotype analysis of the species shows seven groups of the chromosomes with occurrence of metacentric and sub-metacentric types (samaddar et al. 2012). the species also shows polysomatomy (2n=12, 18, 24, 36, 48, 72) with predominance of 2n=48.whereas cytologically, w. coagulans is not as much explored. there is no cytological report of it from the studied area. present research work is undertaken by keeping in view the existence of cytological diversity. materials and methods floral buds were fixed in freshly prepared carnoy’s fixative (6 parts of absolute alcohol: 3 parts of chloroform: 1 part of glacial acetic acid) for 24-48 hours. afterwards, these were transferred to 70% ethyl alcohol and stored in refrigerator at 4˚c until use. for chromosomal preparations, anthers were crushed and tapped to prepare a smear of pollen mother cells (pmcs) in 1% acetocarmine (belling 1921). a number of pmcs were observed and chromosome counts were confirmed. in case of species with meiotic abnormalities, large numbers of pmcs are observed to confirm frequency of various abnormalities. pollen fertility was observed by mounting the pollen grains in 50% glycerol-aceto carmine (1:1) solution (marks 1954). pollen grains with stained nuclei were taken as fertile and viable, whereas, unstained pollen grains marked as sterile ones. pollen grain size was measured using an occulometer. the photomicrographs of the pmcs and pollen grains were taken from the temporary slides by using, nikon 80i digital imaging system. the species are collected from different localities of district churu and jodhpur. w. somnifera is more frequent in its appearance as compared to w. coagulans, which is a rare medicinal plant and is collected only from single locality of the studied area. the plant specimens were identified with the help of various floras such as flora of north east rajasthan (sharma and tiagi 1979), flora of the indian desert (bhandari 1978), flora of rajasthan (shetty and singh 1993) and by comparing the plant specimens with the samples lying in of herbaria of department of botany, punjabi university,  patiala (pup) and botanical survey of india, jodhpur (bsi). the identified voucher specimens of plants have been deposited in the herbarium, department of botany, punjabi university patiala (pun), india. results detailed cytological investigation is carried out in two species of genus withania i.e., w. somnifera and w. coagulans. meiotic studies of w. somnifera reveals the presence of 24 bivalents at metaphase-i (fig. 1). pmcs also show the presence of equal segregation of 24:24 chromosomes at anaphase-i (fig. 2). the meiotic course is normal with high pollen fertility (97.66%). the meiosis of w. coagulans depicts the presence 24 bivalents at metaphase-i (fig. 3). these chromosomes are unable to segregate on the spindle and are scattered all over the cytoplasm (fig. 4). a very few the cells also shows equal segregation of 24:24 chromosomes at anaphase-i (fig. 5). the meiotic course reveals spindle irregularities in the pmcs as the chromosomes are unable to move towards the poles (figs. 6, 7) and several chromosomes remain in the cytoplasm to the form of laggards (fig. 8) at anaphase-i. the spindle abnormalities (figs. 9, 10) and laggard formation is also observed at anaphase-ii stages of meiosis (fig. 10). further the formation of bridges (fig. 11) is also observed at telophase-ii. all these abnormalities lead to the formation of multipolarity (fig. 12) as chromosomes are not able to move towards their respective poles and remain in the cytoplasm. microsporogenesis is highly abnormal with the presence of monad (fig. 13), dyad (fig. 14) and triad (fig. 15). chromatin transfer within tetrads (fig. 16), polyad (fig. 17) and polyads with micronuclei (fig. 18) is also observed. pollen grains with unequal size (fig. 19) and fertile and sterile pollen are also observed (fig. 20) which leads to low pollen fertility. the majority of the pmcs depicted abnormal spindle formation which resulted in irregular arrangement of bivalents at the spindle plate during metaphase-i and segregation of chromosomes during anaphase-i/teolophase-i and anaphase-ii/telophase-ii. chromosomes also lack the ability of congregation at a single pole and 17meiotic behavior of a tetraploid cytotype of brazilian nightshade figs. 1-20. withania somnifera 1) pmc at metaphase-i showing 24 bivalents; 2) pmc at anaphase-i showing 24:24 chromosomes; w. coagulans 3-4) pmc at metaphase-i showing 24 bivalents; 5) pmc at anaphase-i showing 24:24 chromosomes; 6-7) pmc showing spindle irregularities with scattered chromosomes (scattered chromosomes arrowed); 8) pmc at anaphase-i showing laggards (laggards arrowed); 9, 10) pmcs at anaphase-ii showing spindle irregularities in form of laggards (laggards arrowed); 11) pmc at telophase-ii showing laggards and bridges (bridge arrowed); 12) pmc showing multipolarity; 13) monad; 14) dyad; 15) triad; 16) chromatin transfer within tetrad (chromatin transfer arrowed); 17) chromatin transfer within polyad (chromatin transfer arrowed); 18) polyad with micronuclei (micronuclei arrowed); 19) unequal sized pollen grains; 20) sterile and fertile pollen grains. scale bar=10µm. 18 ramanpreet, raghbir chand gupta remained scattered in the cytoplasm or in small groups. chromosomes in these pmcs failed to reach their respective poles and constitute micronuclei during late telophase stages and sporad formation. irregular spindles in these plants are also depicted in the meiocytes, which showed multipolar presence of chromosomes. discussion and conclusions meiosis is most sensitive stage in the life cycle for all sexual species and has direct relevance to natural selection; it leads to the formation of gametes, contributes to genome stability and generates genetic diversity. the process of meiosis depends upon interrelated events of homologous chromosome recognition, intimate association, synapsis and recombination (hamant et al. 2006, de muyt et al. 2009). in plants, it is affected by various genetic and environmental factors (ahmad et al. 1984, viccini and carvalho, 2002, sun et al. 2004, bajpai and singh 2006, rezaei et al. 2010). there are various meiotic abnormalities which hinder the path of normal meiosis and are the cause of changes in the morphology and genetic constitutions of the plant. the evolution of vascular plants is dependent upon the variation in chromosome numbers which may be caused due to genomic mutations especially polyploidy (auto or allopolyploidy) (soltis et al. 2009, bedini et al. 2012). there are number of research papers on the phenomena of polyploidy, emphasizing its origin, impact and role in speciation (stebbins 1985, ramsey and schemske 1998, otto and whitton 2000, cifuentes et al. 2010, jiao et al. 2011). the autotetraploids are generally characterized by the presence of quadrivalents due to homology of 4 sets of chromosomes, whereas, in allopolyploids there is normal pairing because of existence of two separate sets of chromosomes. on the other hand in segmental allotetraploids due to the partial homology of two genomes there is low frequency of quadrivalent formation. in the present study w. somnifera shows normal bivalent formation in all the pmcs, without any quadrivalent formation which indicates its allotetraploid behavior. however, the absence of quadrivalents does not confirm that it is an allotetraploid because there are many artificially produced autotetraploids where there is only bivalent formation because the formation of quadrivalents depends upon many other factors such as localization of chiasmata, small size of chromosomes, and presence of some suppressor genes etc., which does not allow the pairing between the homeologous chromosomes (morrison and rajhathy 1960, gottschlk 1978). on the other hand in w. coagulans the meiosis is highly abnor19meiotic behavior of a tetraploid cytotype of brazilian nightshade mal with the presence of spindle abnormalities which indicates the absence of multivalents and also indicates that it might be hybrid or more probably due the presence of specific genes which interfere in the pairing and functioning of spindle (baum et al.1992, risso-pascotto et al. 2003; kumar and singhal 2008; singhal and kaur 2009). the basic function of the spindle is to attach at kinetochore and separate the chromosome or chromatids at anaphases (wadsworth et al. 2011), these attach to the centromeres (qu and vorsa 1999) and rearrange the chromosomes on the equatorial plate and bring them together at metaphase-i (qu and vorsa 1999). but, if due to some factors (genetic or enviornmental) the spindle activity fails then chromosomes are unable to line up in the equator and then separate at anaphases of the meiosis, which leads to abnormal meiotic course. earlier, a number of plants have been reported with abnormalities like irregular spindle activity, cytomixis and chromatin stickiness leading to abnormal microsporogenesis (baum et al. 1992, caetano-pereira and pagliarinini 2001, kumar and singhal 2008, rai and kumar 2010, singhal and kaur 2009). spindle irregularities are generally divided into 4 categories-multipolar, monopolar, radial and apolar. multipolar spindles are those in which 3 or more poles are formed, in monopolar only one spindle formation takes place, in radial spindles ends are located at the periphery and near the equator (shamina et al. 2000a) and apolar spindles have randomly oriented set of fibers (shamina et al. 2000b, seriukova et al. 2003). abnormalities like cytomixis, chromosome stickiness, unoriented bivalents, laggards, bridges which ultimately lead to abnormal microsporogenisis with the production of dyads, triads, polyads, tetrads with micronuclei, and sterile and fertile pollen grains. acknowledgments this study was funded by department of biotechnology (dbt), new delhi, dbt-ipls project with reference number bt/pr 4548/nf/22/146/2012. the authors are also thankful to head, department of botany, punjabi university, patiala, for all the necessary laboratory facilities. references ahmad, q. n., britten, e. j. and byth, d. e. 1984. effect of interacting genetic factors and temperature on meiosis and fertility in soybean×glycine soja hybrids. can. j. genet. cytol. 26: 50-56. al-turki, t. a., filfilan, s. a. and mehmood, s. f. 2000. a cytological study of flowering plants from saudi arabia. willdenowia 30: 339-358. anesini, c. and perez, c. 1993. screening of plants used in argentine folk medicine for antimicrobial activity. j. ethnopharmacol. 39: 119-128. bajpai, a. and singh, a. k. 2006. meiotic behavior of carica papaya l. spontaneous chromosome instability and elimination in important cvs in north indian conditions. cytologia 71: 131-136. baquar, s. r. 1967. cytomorphological studies in the family solanaceae from west pakistan. genetica 38: 388-397. baum, m. e., lagudah, e. s. and appels, r. 1992. wide crosses in cereals. annu. rev. plant physiol. plant mol. biol. 43:117-143. bedini, g., garbari, f. and peruzzi, l. 2012. does chromosome number count? mapping karyological knowledge on italian flora in a phylogenetic framework. j. syst. evol. 298: 739–750. belling, j. 1921. on counting chromosomes in pollen mother cells. american naturalist. 55: 573-574. bhaduri, p. n. 1933. chromosome numbers of some solanaceous plants of bengal. j. ind. bot. sci. 12: 56-64. bhandari, m. m. 1978. flora of indian desert. pp. 471. scientific publishers. jodhpur. bir, s. s. and sidhu, m. 1979. cytological observations in weed flora of orchards of patiala district, punjab. recent res. plant sci. 7: 261-271. bir, s. s. and sidhu, m. 1980. cyto-palynological studies on weed flora of cultivable lands of patiala district (punjab). j. palynol. 16: 85-105. bir, s. s., kumari, s., shoree, s. p. and saggoo, m. i. s. 1978. cytological studies in certain bicarpellatae from north and central india. j. cytol. genet. 13: 99-106. caetano-pereira, c. m. and pagliarini, m. s. 2001. a new meiotic abnormality in zea mays multiple spindles associated with abnormal cytokinesis in both divisions. genome 44: 865-871. cifuentes, m., grandont, l., moore, g., chèvre, a. m. and jenczewski, e. 2010. genetic regulation of meiosis in polyploidy species: new insights into an old question. new phytol. 186: 29-36. datta, a. k., mukherjee, m. and iqbal, m. 2005. persistent cytomixis in ocimum basilicum l. 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m a d pour, ghasem karimzadeh, seyed mahmood ghaffari (2022). karyomorphology, genome size, and variation of antioxidant in twelve berry species from iran. caryologia 75(4): 133-148. doi: 10.36253/caryologia-1633 received: april 18, 2022 accepted: december 04, 2022 published: april 28, 2023 copyright: © 2022 saeed mohammadpour, ghasem karimzadeh, seyed mahmood ghaffari. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyomorphology, genome size, and variation of antioxidant in twelve berry species from iran saeed mohammadpour1, ghasem karimzadeh1,*, seyed mahmood ghaffari2 1 department of plant genetics and breeding, col lege of agriculture, tarbiat modares university, p.o. box 14115-336, tehran, iran 2 institute of biochemistry and biophysics, university of tehran, p.o. box 13145-1384, tehran, iran *corresponding author. e-mail: karimzadeh_g@modares.ac.ir abstract. twelve berry species, including rubus fruticosus cv. qaemshahr, rubus occidentalis cv. qaemshahr, and morus alba cv. mashhad, morus rubra cv. karaj, fragaria vesca subsp. vesca, ribes nigrum, ribes rubrum, ribes uva-crispa; lycium barbarum, lycium infaustum, lycium ruthenicum, and vaccinium corymbosum were surveyed for karyomorphology analysis, monoploid genome size, and antioxidant activity. results indicated that all species were diploid (2n = 2x = 14, 16, 24, and 28). among these species, chromosome counts and karyomorphology parameters of three cultivars including: r. fruticosus cv. qaemshahr (2n = 2x = 14), r. occidentalis cv. qaemshahr (2n = 2x = 14), and m. alba cv. mashhad (2n = 2x = 28) are reported here for the first time. the flow cytometric mean monoploid 2cx dna of berry species was 2.35 pg, varied from 0.68 (rubus occidentalis cv. qaemshahr) to 5.15 pg (lycium ruthenicum). the average antioxidant capacity of berry species was obtained 32.8 μmol g-1. their average total phenol and flavonoid contents were 4.98 mg g-1 (3.08-8.61 mg g-1), and 5.18 (2.4710.63 mg g-1), respectively. keywords: blueberry, raspberry, cytogenetic, antioxidant, karyomorphology, flow cytometry. introduction berries are economically important crop in many countries (umdale et al. 2020). interest in berries has recently increased because they are excellent sources of health-promoting vitamins, anti-oxidants, polyphenols, especially anthocyanins, micronutrients, fiber, and other valuable nutrients (song and sing, 2004). berries are low in calories and are high in moisture and fiber (basu et al. 2010). they contain natural antioxidants such as vitamins c and e, and micronutrients such as folic acid, calcium, selenium, alpha and beta carotene, and lutein (basu 2019). however, berry production is deficient, even though soil and climate conditions in iran are excellent for the intensive cultivation of berry fruits (nestby et al. 2019). some of the berry cultivars that 134 saeed mohammadpour, ghasem karimzadeh, seyed mahmood ghaffari have recently been introduced to iran are potentially profitable alternative and non-conventional fruit crops. among those are cultivars of the blueberry (vaccinium corymbosum l.), the goji berry (lycium barbarum, l. infaustum, l. ruthenicum), and the raspberry (rubus occidentalis) (ahmadu and ahmad 2021). in iran, the production of blackberry (rubus fruticosus) relies mainly on the collection of the berries from stands of native varieties that grow wild in the mountains. habitat destruction has led to a reduction in the supply of native blackberries. although these native wild varieties are not as suitable for intensive culture as the new cultivars, they are still very valuable high dietary value and breeding potential. they can be a versatile raw material for the food processing and pharmaceutical industries. recently, interest in growing various berry fruits has been increasing among either home gardeners, or small farmers. berry culture is increasing in the country’s mountainous, where soil and climate conditions are more favorable for intensive berry culture than in the lowland areas (nestby et al. 2019). the consumption of berry fruits and their contribution to improving cardiovascular health is a significant issue (blumberg et al. 2013). the commonly consumed berries in the united states, including blackberry, black raspberry, blueberry, goji berry, cranberry, red raspberry, and strawberries (yang et al. 2019). display quotations of over 40 words or as needed. berry fruits are deciduous shrubbery that grows in different parts of the world (donno et al. 2015). for instance, goji berry grows in china, tibet, argentina, chile, southern africa, and other parts of asia, and its fruits are 1-2 cm long, bright orange-red ellipsoid berries. goji berry is widely distributed in warm regions of the world, in particular, in the mediterranean area and southwest and central asia. it is also cultivated in north america and australia as a hedge plant (potterat 2010). mulberry (morus alba) is native to china, but is also found worldwide on not native continents, such as europe, africa, north america, and south america. currant (ribes spp.), is widely cultivated across temperate europe, russia, new zealand, parts of asia, and to a lesser extent north america (steffen et al. 2015). the main centers of diversity for blueberry are distributed in europe (poland, germany, france, the netherlands, spain, and sweden), new zealand, mexico, and north america. karyotype analysis and chromosome observation are necessary to elucidate phylogenetic relationships, structure, function, organization, and evolution of plant genes and genomes (amosova et al. 2019). for those reasons, many studies must be performed to observe plant chromosomes. higher similarity in chromosome shapes suggests a closer phylogenetic relatedness. the base chromosome number in rubus spp. (r. fruticosus, and r. occidentalis), ribes spp. (r. nigrum, r. rubrum, and r. uva-crispa), lycieae (l. barbarum, l. infaustum, and l. ruthenicum), moraceae (m. alba, and m. rubra) is x = 7, 8, 12, and 14, respectively. the base chromosome number in strawberry (fragaria vesca) and blueberry (vaccinum corymbosum) is x = 7, and 14, respectively (zong et al. 2021). most species are diploid with 2n = 24. cytological information, including the number of chromosome and karyotypes, is available for many american and asian lycium and several species in southern african (bernardello et al. 2008). monoploid genome size in the form of base-pair was calculated based on the formula proposed by doležel et al. (2003), when 1 pg of dna represents 978 mega base pairs (mbp). monoploid genome size was considered as the amount of dna of one chromosome set, 1cxvalue, with chromosome base number x, and holoploid genome size as the amount of dna of the whole chromosome complement, 1c-value, with chromosome number n, irrespective of the degree of generative polyploidy (greilhuber et al. 2005; mahdavi and karimzadeh 2010; karimzadeh et al. 2011; abedi et al. 2015; tavan et al. 2015). in recent decades, significant progress has been made in the use of flow cytometry in various fields of botany. this growth is due to the advantages of flow cytometry such as high speed, ease of sample preparation, and analysis of inactive mitotic tissues. despite these advantages, the need for fresh plant materials may often prevent the further development of flow cytometry in field research (suda and trávníček 2006). flow cytometry is known as the most powerful, reliable, and quick method for estimating the genome size (2c dna) for a wide range of plant communities (e.g. garcia et al. 2004; doležel and bartoš 2005; doležel et al. 2007; loureiro et al. 2007; mahdavi and karimzadeh 2010; karimzadeh et al. 2010, 2011; abedi et al. 2015; tavan et al. 2015; tarkesh esfahani et al. 2016, 2020; javadian et al. 2017; hamidi et al. 2018; zarabizadeh et al. 2022). the term “c-value” refers to the constant amount of dna of an unreplicated haploid chromosome complement (swift 1950). monoploid genome size was considered as the amount of dna of one chromosome set, 1cx-value, with chromosome base number x (greilhuber et al. 2005). the mean monoploid 2cx dna value was 2.35 pg. on the other hand, the monoploid genome size (1cx dna) varied from 420.54 mbp (s3; rubus occidentalis cv. qaemshahr) to 2518.35 mbp (s9; lycium ruthenicum). reports of 2cx dna in some berry species have been reported, including s3; rubus occidentalis = 0.60 pg, s5; ribes rubrum = 1.94 pg, and s6; ribes 135karyomorphology, genome size, and variation of antioxidant in twelve berry species from iran uva-crispa = 1.88 pg (meng and finn 2002; chiche et al. 2003). antioxidants are compounds that effectively prevent oxidation in a variety of ways and, by slowing down the oxidation rate, significantly increase the oxidation period (dorman et al. 2003). from one perspective, antioxidants fall into two main categories: synthetic and natural. phenolic compounds are one of the best sources of natural antioxidants (dorman et al. 2003). because the use of such plant resources is effective in delaying oxidation and reducing chronic diseases, mutations, and cancer (briskin 2000). in new research works in the field of natural antioxidants, much attention has been paid to essential oils and extracts of medicinal plants because a wide range of medicinal plants and their aromatic compounds as natural sources with antioxidant properties, (moure et al. 2001; tepe et al. 2006). it is generally recognized that oxygen free radicals (ros) are associated with many potential risks, including parkinson’s disease, cancer, alzheimer’s, as well as aging (liochev 2013; kim et al. 2016).berry species are popular around the world as a ‘super fruit’ due to their nutritional value, elevated levels of bioactive phenolic molecules, and excellent sensory evaluation (kalt et al. 2020). in addition to these essential nutrients, berries contain a wide range of antioxidant phenolic molecules such as phenolic acids, flavonoids, flavonols, flavanols, anthocyanins, proanthocyanidins, and ellagitannins (prior et al. 1998; piljacžegarac et al. 2009; nile and park 2014; skrovankova et al. 2015). cranberries ranked first in polyphenol content among the commonly consumed fruits in north america which relate to high antioxidant activity (vinson et al. 2001). the consumption of lingonberries and bilberries has been proved in preventing human cancer which is ascribed to the high levels of phenolic and anthocyanin compounds (faria et al. 2010; yang and kortesniemi 2015). the average total antioxidant capacity, total phenol content, total flavonoid content of berries were 32.79, 4.98, and 5.18 μmol g-1, respectively. in the study of islam et al. (2017) on genus lycium, the mean of the antioxidant activity for red goji berry (s7; lycium barbarum) and black goji berry (s9; lycium ruthenicum) were measured as 16.65 and 34.28 µmol g-1, respectively. in the report of mustafa ahmed et al. (2022) on genus vaccinium, the mean of total phenol content, total flavonoid content, and antioxidant capacity were reported as 3.73 mg g-1, 2.57 mg g-1, and 17.67 µmol g-1, respectively. in the present study, we provide a detailed karyotype analysis for 12 berries species. our goals were to confirm the number of chromosomes, karyotype determination, estimate the nuclear genome size, assess the phenolic profile, antioxidant properties, and flavonoid content of these twelve species, and provide scientific insight into the phenolic and antioxidant functions of these species to consumers and nutraceutical industry. materials and methods plant materials seeds were kindly provided by the cultivation development company of berries (mazandaran, iran). the culture medium used was ms (murashige and skoog 1962) with macroelements at 1/3 (rache and pacheco 2010), 1/8, and 1/16 of its original concentration (nh4no3: 1650 mg l-1, kno3: 1900 mg l-1, kh2po4: 170 mg l-1, cacl2h2o: 440 mg l-1 and mgso4.7h2o: 370 table 1. specifications of berry species collection sites (berry spp.) used in this research. species codes english name scientific name localities longitude (e); latitude (n) altitude (m) s1 strawberry fragaria vesca subsp. vesca iran, mazandaran, sari 52°59’42.83”; 36°32’25.67” 83 s2 blackberry rubus fruticosus cv. qaemshahr iran, qaemshahr 52°51’28.29”; 36°27’19.11” 282 s3 black raspberry rubus occidentalis cv. qaemshahr iran, qaemshahr 52°51’28.29”; 36°27’19.11” 282 s4 black currant ribes nigrum usa, arizona, pinal 111°17’04.21”; 32°48’58.34” 660 s5 red currant ribes rubrum usa, arizona, pinal 111°17’04.21”; 32°48’58.34” 660 s6 goose berry ribes uva-crispa usa, arizona, pinal 111°17’04.21”; 32°48’58.34” 660 s7 red goji berry lycium barbarum usa, arizona, pinal 111°17’04.21”; 32°48’58.34” 660 s8 goji berry lycium infaustum usa, arizona, pinal 111°17’04.21”; 32°48’58.34” 660 s9 black goji berry lycium ruthenicum usa, arizona, pinal 111°17’04.21”; 32°48’58.34” 660 s10 blueberry vaccinium corymbosum usa, maine 68°59’05.21”; 45°11’18.34” 426 s11 white mulberry morus alba cv. mashhad iran, karaj, mohammadshahr 51°00’13.27”; 35°44’23.12” 1228 s12 red mulberry morus rubra cv. karaj iran, karaj, mohammadshahr 51°00’13.27”; 35°44’23.12” 1228 136 saeed mohammadpour, ghasem karimzadeh, seyed mahmood ghaffari mg l-1). the medium was adjusted to ph = 5.8 and autoclaved for 20 min at 121ºc. these plants were grown in a greenhouse at the college of agriculture, tarbiat modares university (tmu). voucher specimens of the examined species are preserved in the herbarium of the research institute of forests and rangelands of iran (tari) and the tarbiat modares university of iran (tmu). also, the images of species are shown in figure figure 1. the picture of berry species that studied in this research. 137karyomorphology, genome size, and variation of antioxidant in twelve berry species from iran 1. all cultures were maintained in a growth chamber at 25 ºc, using a 16 h light/8 h dark photoperiod. the light was supplied, using white fluorescent lamps at an intensity of 50 µmol m-2 s-1. karyomorphology the best method to study mitosis for preparing a karyotype is root tip meristem tissue, which was used for cytogenetic studies. for the cytological preparations, growing roots about 1 cm long were removed at the appropriate time (when the largest number of cells are in metaphase) and pretreated in 0.002 mol 1−1 8-hydroxyquinoline for 4h at room temperature (rt) in the dark to induce metaphase arrest, followed by washing twice (each for 5 min) in distilled h2o and fixing in fresh 3:1 (v/v) absolute ethanol: glacial acetic acid for 24 h at 4 °c. the fixed roots were hydrolyzed in 1 m hcl for 7-10 min at 60°c washed two times (each for 5 min) in distilled h2o and stained in 1% (w/v) aceto-orcein for 3 h or in 4% (w/v) hematoxylin for 4 h at rt. five well-spread monolayer metaphase plates from different individuals were analyzed per species. highresolution microscopic digital photographs were taken, using an olympus bx50 microscope (olympus optical co., tokyo, japan), equipped with an olympus dp12 digital camera (olympus optical co.). eight chromosomal parameters were either measured as long (l) and short (s) arm lengths or calculated as chromosome length (cl), arm ratio (ar; l/s), r-value (s/l), total chromosome volume (tcv = πr2 cl), where r = average chromosome radius, percentage of total chromosome form (f%), and centromeric index (ci = s/cl). idiograms were drawn from the mean values, and chromosome types were determined, using the formula of levan et al. (1964). for karyotypic analysis, five parameters, including karyotype total form percentage (tf% = ʃs/ ʃcl × 100), coefficient of variation of total chromosome length (cvcl% = (total cl standard deviation/total cl mean) × 100), mean centromeric asymmetry (mca = a × 100), where the degree of karyotype asymmetry (a = [ʃ(l-s/l+s(/n] × 100(, stebbins (1971) asymmetry categories (st), and romero-zarco (1986) indices: intrachromosomal asymmetry index (a1) and interchromosomal asymmetry index (a2), were measured. flow cytometric genome size estimation relative dna content was determined, using pistained flow cytometry. young leaves of the analyzed individuals and a reference standard (either solanum lycopersicum; 2c = 1.96 pg., petroselinum crispum; 2c = 4.45 pg., or zea mays; 2c = 5.43 pg) were co-chopped with a razor blade in a glass petri dish, containing 1 ml of ice-cold wpb buffer (woody plant buffer; 0.02 m tris-hcl, 4 mm mgcl2.6h2o, 2 mm edta na2.2h2o, 86 mm nacl, 10 mm sodium metabisulfite, 1% pvp10, and 1% (v/v) triton x-100, ph 7.5) (loureiro et al. 2007). the crude nuclei suspension was filtered through a 50-µm nylon mesh. rnase and propidium iodide (each 50 µg ml−1) was then added. after incubation for two min at rt, the relative fluorescence intensity of nuclei was analyzed. at least 5000 nuclei were analyzed in each measurement with cv% (coefficient of variation; %) values below 5.0%. subjects were randomly selected for the flow cytometric analysis. the dna amount of a sample was calculated based on the values of the g1 peak means (doležel et al. 2003, 2007; doležel and bartoš 2005) as follows: sample 2cx dna (pg) = (sample g1 peak mean/standard g1 peak mean) × standard 2c dna (pg). the obtained data were initially checked for the normality test and analyzed, using spss (version 22) and minitab 17. the karyotypic, flow cytometric, and phytochemical content data were first tested for the normality and then analyzed based on a completely randomized design (crd) with 5, 3, and 3 replications, respectively. the results were statistically evaluated by analysis of variance (anova) and expressed as mean. means comparisons were performed, using the lsd test with spss; differences were considered statistically significant at p ≤ 0.01 and p ≤ 0.05. linear regression analysis was carried out to find out the relationship between monoploid 2cx dna values and some traits, using minitab 17. chromatographic analysis the method used to determine total polyphenol content (tpc) was based on folin-ciocalteu phenol reagent and spectrophotometric determination at 765 nm. the method used to determine the total flavonoid content (tfc) was based on aluminum chloride and spectrophotometric determination at 420 nm. antioxidant activity in the berries fruit pulp was evaluated by a 2,2-diphenyl1-picrylhydrazyl (dpph) assay (laczkó-zöld et al. 2018). total antioxidant capacity (tac) the scavenging activity for dpph radicals was determined using spectrophotometric analysis accord138 saeed mohammadpour, ghasem karimzadeh, seyed mahmood ghaffari ing to a modified method of lalegani et al. (2018). briefly 100 µl sample solution was added to 900 µl of 0.1 m dpph solution and mixed thoroughly at rt. absorbance at 517 nm was determined after 30 min. total phenolic content (tpc) the tpc was estimated, using the folinciocalteu colorimetric method described by barreca et al. (2016). a 20 µl of methanolic extract was added to 2 ml of deionized water, and 100 µl of dilutions folinciocalteu was then added. after 1-8 min (average 5 min), 300 µl of sodium carbonate 7% (w/v) was added to it, and the absorbance was measured in the dark at 765 nm after incubation for 2 h at rt. quantification was done based on the standard curve of gallic acid. results were expressed as equivalent of the gallic acid (gae), i.e., mg gallic acid g-1 dw. total flavonoid content (tfc) the tfc was estimated, using the aluminum chloride method described by barreca et al. (2016). in this method, a 600 µl of methanolic extract was added to 600 µl of 2% (w/v) aluminum chloride, and after 10 min, the absorbance was measured at 420 nm. quantification was done based on the standard curve of quercetin. the tfc was calculated and expressed as quercetin equivalents, i.e., mg quercetin g-1 dw. results karyomorphologic analysis in the current study, one chromosome type “m” formed karyotype formulas of “14m” for fragaria vesca subsp. vesca (s1), rubus fruticosus cv. qaemshahr (s2), and rubus occidentalis cv. qaemshahr (s3), “16m” for ribes nigrum (s4), ribes rubrum (s5), and ribes uvacrispa (s6), “24m” for lycium barbarum (s7), lycium infaustum (s8), lycium ruthenicum (s9), and vaccinium corymbosum (s10), and “28m” for s11 and s12 (morus alba cv. mashhad (s11), morus rubra cv. karaj (s12). the base chromosome number in fragaria vesca subsp. vesca, rubus fruticosus cv. qaemshahr, rubus occidentalis cv. qaemshahr; is x = 7; ribes nigrum, ribes rubrum, ribes uva-crispa x = 8; lycium barbarum, lycium infaustum, lycium ruthenicum, vaccinium corymbosum x = 12; morus alba cv. mashhad, morus rubra cv. karaj x = 14, and all of these species are diploid with 2n = 14, 16, 24, and 28. anova shows significant (p < 0.01) differences between the berries species for the most studied chromosomal parameters (table 2). karyotypes of somatic complement and the idiograms of the haploid completable 2. anova for chromosomal parameters of berry species. s.o.v. df ms s l cl ar r-value f% tcv ci species 11 7.413** 8.825** 20.685** 0.128** 0.128** 23.661** 49.778** 0.199** error 593 0.039 0.046 0.11 0.017 0.017 0.121 0.422 0.057 total 604 **significant difference at p < 0.01 figure 2. somatic chromosomes of 12 berry species. scale bar = 5 μm. 139karyomorphology, genome size, and variation of antioxidant in twelve berry species from iran ment of studied berries are demonstrated in figures 2 and 3, respectively. the mean value of chromosome length (cl) was 2.83 μm, varying from 1.06 μm (morus rubra cv. karaj; s12) to 3.75 μm (ribes nigrum; s4). the mean tcv was 1.6 μm, ranging from 0.10 μm (s10) to 6.19 μm (s1). the mean ci of the complement varied from 46% (s12) to 51% (s5). the studied berry species showed different symmetrical groups according to various karyotypic symmetrical indices. for instance, the highest value of total form percentage of karyotype (tf%) was detected in s5 (50.51%; table 3; the most symmetric), and the lowest value was identified in s12 (46.28%; the most asymmetric). the highest and the lowest values of coefficient of variation (cvcl%) were identified on s11 (18.03%; the most asymmetric) and s3 (11.98%; the most symmetric), respectively. on the other hand, karyotypes of all these species were classified in 1a class of stebbins classification (stebbins 1971; table 3). the basic chromosome number for these berries is consistent with previously published data. the ploidy level agreed with prior and this study’s f low cytometric data. despite rapid and convenient estimation of the ploidy level, using monoploid genome size measurements, chromosome counting is necessary to reliably infer the ploidy level when the dna content of polyploids fails to match the ratio expected from closely related diploids. for more detailed studies of asymmetry, romero-zarco’s (1986) indices of a1 and a2 were also calculated (table 3; figure 4). table 3 also shows two more asymmetry indices of mca and cvcl. the a1 and a2 indices illustrate four groups. the scatter plot of mca vs cvcl illustrates three groups. to determine total variation in berries and parameters quota in total variation, the principal component analysis (pca) of the karyotypic parameters was performed. the single dendrogram constructed based on karyotype similarities (figure 3) shows four major clusfigure 3. idiograms of haploid chromosomes of 12 berry species. table 3. karyotypic symmetry formula and flow cytometric genome size of 12 berry species. total form percentage of karyotype (tf%), mean centromeric asymmetry (mca%), coefficient of variation of chromosome length (cvcl%), karyotype formula (kf), stebbins asymmetry categories (st), intrachromosome asymmetry index (a1), interchromosome asymmetry index (a2), m: metacentric, m: metacentric. means with the same symbol letter in the “2cx dna (pg)” column are not significantly different (p > 0.01), using lsd test. species asymmetry indices (romero-zarco, 1986) st kf cvcl% mca% tf% 2cx dna (pg) a1 a2 s1 0.46 0.13 1a 14m 13.38 4.37 47.79 2.10d s2 0.47 0.18 1a 14m 17.71 4.91 47.59 1.55g s3 0.47 0.12 1a 14m 11.98 4.73 47.66 0.86i s4 0.39 0.14 1a 16m 14.21 4.31 47.86 1.86e s5 0.33 0.16 1a 16m 16.17 4.54 50.51 1.94e s6 0.39 0.15 1a 16m 14.61 4.30 47.98 1.94e s7 0.10 0.16 1a 24m 15.82 5.35 47.38 3.93b s8 0.09 0.17 1a 24m 16.65 4.73 47.68 3.82b s9 0.09 0.16 1a 24m 16.51 4.75 47.60 5.15a s10 0.11 0.16 1a 24m 15.63 5.91 47.07 2.53c s11 0.00 0.18 1a 28m 18.03 7.31 46.37 1.65f s12 0.00 0.16 1a 28m 16.05 7.58 46.28 1.18h figure 4. dendrogram shows relationships of similarity among studied 12 berry species related to all chromosomal characteristics, constructed using euclidean distance and single method; cophenetic correlation r = 0.75. 140 saeed mohammadpour, ghasem karimzadeh, seyed mahmood ghaff ari ters. th e fi rst cluster consists of s11 and s12, the second cluster consists of s7-s10, and the third cluster consists of s2-s6, while s1 is separated within the fourth cluster. nuclear genome size estimation th e resultant fl ow cytometric data were fi rst tested for normality test and then analyzed according to a completely randomized design (crd) with 3 replicate cells. th e monoploid genome size (2cx dna) of these twelve species (fragaria vesca subsp. vesca, rubes fruticosus cv. qaemshahr, rubus occidentalis cv. qaemshahr, ribes nigrum, ribes rubrum, ribes uva-crispa, lycium barbarum, lycium infaustum, lycium ruthenicum, vaccinium corymbosum, morus alba cv. mashhad, morus rubra cv. karaj) were 2.1, 1.55, 0.68, 1.86, 1.94, 1.94, 3.93, 3.82, 5.15, 2.53, 1.65, and 1.18 pg, respectively (table 3). th e diff erences in nuclear dna contents among these analyzed species were statistically signifi cant (p < 0.01). th e highest value was detected in lycium ruthenicum (s9, 5.15 pg), while rubus occidentalis cv. qaemshahr (s3) was the lowest (0.68 pg), and the mean total value was 2.36 pg (table 3). th e histograms obtained for analyzing nuclear dna contained two peaks (figure 6). th e left peaks in s7 to s10 refer to the known samples reference standard, and the right peaks to the unknown samples. in other species, the peaks on the left refer to unknown specimens, and the peaks on the right refer to the reference standard of known specimens. young leaves of berries spefigure 5. scatter diagram of 12 berry species: romero-zarco asymmetry indices (a), cvcl and mca (b). figure 6. diagram resulted from principal components analysis 1 (highly related to the position of centromere) and 2 (strongly related to the length of the complements) of studied berry species. figure 7. histograms of relative fluorescence intensity were obtained, using wpb isolation buff er aft er simultaneous analysis of nuclei isolated from standard plant and berry species plants. th e mean channel number and coeffi cient of variation (cv%) value of each peak are also given. 141karyomorphology, genome size, and variation of antioxidant in twelve berry species from iran cies contain many cytosolic compounds such as phenolic acids and flavonoids, which can interfere with the fluorescent staining of nuclear dna. to compensate this problem, a new isolation buffer, wpb, was developed to analyze problematic or woody species because the sodium metabisulfite and pvp-10 in wpb act as reducing and binding agents to prevent the action of interfering phenols and secondary metabolites. this buffer was found to be suitable for the analysis of lycium species, as evidenced by the cv of dna peak and flow cytometric histograms of relative fluorescence intensity. phylogenetic analysis revealed that lycium species are included in a monophyletic group, indicating a very close evolutionary relationship. this study discovered significant differences in the genome size among these three species (l. barbarum, l. ruthenicum, and l. infaustum). it was also shown in ribes, rubus, and morus species. there is excellent genome size variation between species. it has been proposed that having a large genome has direct physiological consequences for plants, such as earlier flowering time, larger stomata size, and longer life cycles than small genomes. the flowering time may be partly affected by differences in dna content. phytochemical studies anova shows significant (p < 0.01) differences between the berries species for the three studied phytochemical traits, including total antioxidant capacity (tac), total phenol content (tpc), and total flavonoid content (tfc). the mean comparisons are shown in table 4, using lsd test at 0.01 probabilty level. the average total antioxidant capacity (tac; table 4) of berries was 32.79 μmol g-1, ranging from 6.92 μmol g-1 (s1; fragaria vesca subsp. vesca) to 63.84 μmol g-1 (s3; rubus occidentalis cv. qaemshahr). the average total phenol content (tpc; table 4) of berries was 4.98 μmol g-1, ranging from 3.08 μmol g-1 (s12; morus rubra cv. karaj) to 8.61 μmol g-1 (s9; lycium ruthenicum). the average total flavonoid content (tfc; table 4) of berries was 5.18 μmol g-1, ranging from 2.43 μmol g-1 (s12; morus rubra cv. karaj) to 10.63 μmol g-1 (s9; lycium ruthenicum). black raspberry (s3; rubus occidentalis cv. qaemshahr) in terms of all three phytochemical traits, had the highest score among all berry species. interestingly, the level of antioxidant activity of black raspberry (s3; rubus occidentalis cv. qaemshahr) had statistically significant difference among all studied berry species in the present study at a probability level of 1%. for example, it had 6.6-fold increase over strawberry (s1; fragaria vesca subsp. vesca) and a 24% increase over black currant (s4; ribes nigrum). in the next positions, goose berry (s6; ribes uva-crispa) and black currant (s4; ribes nigrum) had the highest values, respectively. moreover, strawberry (s1; fragaria vesca subsp. vesca) and goji berry (s8: lycium infaustum) had the lowest one. discussion our results provided the basic genetic, genomic and phytochemical information for these species, which are helpful for the construction of genetic and physical maps and whole-genome sequencing in the future and provide scientific insight into the phenolic and antioxidant functions to consumers and the nutraceutical industry. for the first time, some of these species’ chromosome number and karyotype were determined in iran. chromosome numbers 2n = 2x = 14, 16, 24, and 28 in these berry species are consistent with other reports. differences in the karyotype formula of these species, which are geographically, climatically, and habitually different from each other, indicating the existence of chromosomal structural changes in the process of karyotype evolution and species formation in these species. it varies according to geographical and climatic conditions. structural changes in the speciation process of these species can be changed such as displacements, inversions, and other structural changes. according to stebbins table 4. information obtained from phytochemical evaluation of studied 12 berry species. total antioxidant capacity (tac), total flavonoid content (tfc), total phenol content (tpc). in each column, means with the same symbol letter are not significantly different (p > 0.01), using lsd test. species tac (µmol g-1) tfc (mg g-1) tpc (mg g-1) s1 9.62h 2.47f 3.08f s2 38.40c 5.40c 5.75b s3 63.84a 9.60a 7.92a s4 51.57b 6.50b 5.82b s5 24.56e 3.43d 3.73d s6 52.19b 9.23a 7.68a s7 25.06e 3.45d 4.07c s8 16.38g 2.57ef 3.17ef s9 34.95c 10.63a 8.61a s10 27.50de 2.77e 3.28e s11 29.11d 3.70d 3.59d s12 20.32f 2.43f 3.08f average (range) 32.79 (9.62-63.84) 5.18 (2.43-10.63) 4.98 (3.08-8.61) 142 saeed mohammadpour, ghasem karimzadeh, seyed mahmood ghaffari (1971), cytogenetic studies can be used to understand better the relationships between species and different populations of a species and to orient the evolutionary tendencies of plants. each of these plants shows a unique adaptation to the environment in which they grow. as this compromise increases, new varieties or species may appear in plant communities. this study developed an important tool to assess berries’ chemical composition and bioactivity, using different chromatographic methods for its fruits’ comprehensive authentication and quality control. in the present study, all chromosomal parameters were significantly different. differences between species for all karyological parameters confirm the intraspecific chromosomal variations. these results are in agreement with those reported by chen et al. (2013). in the current study, one chromosome type (“m”) formed karyotype formulas of “14m” for s1-s3 (fragaria vesca subsp. vesca, rubus fruticosus cv. qaemshahr, rubus occidentalis cv. qaemshahr), “16m” for s4-s6 (ribes nigrum, ribes rubrum, ribes uva-crispa), “24m” for s7-s10 (lycium barbarum, lycium infaustum, lycium ruthenicum, vaccinium corymbosum), and “28m” for s11 and s12 (morus alba cv. mashhad, morus rubra cv. karaj). similar to our findings, chen et al. (2013) reported the same one chromosome type in lycium species. in the present study, the average total length of chromosomes (cl) in the studied species is 2.83 μm. karyotype symmetry was more indicative of evolutionary traits in the studied berries species, regardless of ploidy levels. to determine the karyotypic symmetry, several properties were examined in 12 species. based on tf%, s6 with the highest value (47.98%) has the most symmetric, and s12 with the lowest value (46.28%) has the most asymmetric karyotype. gradual changes and changes in tf% values may be due to chromosomal abnormalities. structural changes in chromosome morphology probably due to chromosome duplication or translocation (exchange and displacement) between chromosomes (das and teng 1998). according to the cvcl% index, s3 (11.98%) has the most symmetrical karyotype and s11 (18.03%) has the most asymmetric karyotype among other species. overall, the coefficient of variation in the sample was low due to the metacentric nature of most chromosomes, indicating the symmetry and the uniformity of these karyotypes. in general, it can be concluded, species that are more evolutionarily advanced have variations in chromosome type and size and are therefore asymmetrically karyotypically. this asymmetry is exacerbated by the displacement of chromosomal fragments. karyotypic dissimilarity in terms of karyotype symmetry measurement parameters can lead to failure in reproduction and inadequate seed production in the offspring while preventing successful intraspecific crosses. in other words, the results of such crosses may be somewhat sterile. in the study of chen et al. (2013) on the genus lycium, the chromosomal base number for red goji berry (s7; l. barbarum) and black goji berry (s9; l. ruthenicum) was reported to be 12, which is in complete agreement with the findings of the present study (table 3). costich et al. (1993) worked on the blueberry plant (s10; vaccinium corymbosum) showed that the chromosomal base number was 12, which is consistent with the findings of the current study. for the genus ribes, the base chromosome number 8 was reported by chiche et al. (2003), which is matched for the three different species of this genus (s4; r. nigrum, s5; ribes rubrum, s6; r. uva-crispa) in the present study. on the other hand, karyotypes of all species were classified in 1a (table 3) . for more detailed studies of asymmetry, romero-zarco (1986) indices of a1 and a2 were also calculated. the scatter diagram of these indices (figure 4) presents four species groups. according to the analyzed asymmetry indices, s3 was demonstrated as the most symmetric species, while s2 and s11 were among the most asymmetric species. it has been suggested that karyotypes with more asymmetry have a derived status in comparison to those with more symmetrical morphology (lakshmi et al. 1984). for example, differences in chromosome length (cl) may indicate the occurrence of cyclic changes in genome size during the diversification of the genus. thus, the study of asymmetry indices and variation in genome size is a valuable means for the establishment of the evolutionary relationship between the species and the origin of diversification of the populations (karimzadeh et al. 2011). to determine the total diversity in the population and the quota of the parameters in the total diversity, principal component analysis (pca) of the karyotypic parameters was performed. it shows that the first two main components make up 82% of the cumulative changes and they are predicted in a two-dimensional graph (figure 5). the single dendrogram constructed on the basis of karyotype similarities (figure 3) shows four major clusters. the first cluster consists of s11 and s12, the second cluster consists of s7-s10, and the third cluster consists of s2-s6, while s1 is separated within the fourth cluster. the arrangement of pca species from this experiment is fully consistent with the analysis obtained by single grouping analysis. therefore, the results of this study proposed that species within a cluster have the most homology in chromosomal variations. for this purpose, a cross between s7 and s8 or s9 is suggested because they have the most similarity in their chromosomal characteristics. 143karyomorphology, genome size, and variation of antioxidant in twelve berry species from iran the mean monoploid 2cx dna value was 2.35 pg in the studied species, verifying intraspecific genome size variation. on the other hand, the monoploid genome size (1cx dna) varied from 420.54 mbp (s3; rubus occidentalis cv. qaemshahr) to 2518.35 mbp (s9; lycium ruthenicum). according to the results (chen et al. 2013), red goji berry (s7; lycium barbarum) and black goji berry (s9; lycium ruthenicum) had 2cx dna values of 4.35 pg and 5.45 pg, respectively. in the present study, the genome size for the those was 3.93 pg and 5.15 pg, correspondingly, which is almost in agreement with the findings of the previous report. the existence of some deviation from this species may be due to the inter-species and intersex diversity. knowing genome size may be useful in genome research and studies of relationships between dna content, physiology, and plant ecology (thiem and sliwinska 2003). reports of 2cx dna in some berry species have been published, including s3; rubus occidentalis = 0.60 pg, s5; ribes rubrum = 1.94 pg, and s6; ribes uva-crispa = 1.88 pg (meng and finn 2002; chiche et al. 2003). in the present study (table 3), the monoploid genome size (2cx dna) for the latter three species is 0.68 pg, 1.86 pg, and 1.94 pg, respectively, which is in complete agreement with the findings of the two previous reports. in the present study, the existence of a statistically significant difference in the monoploid genome size indicates the interspecific and intersex diversity among berry species. on the other hand, the correlation between 2cx dna with environmental conditions (longitude, latitude, and altitude), chromosome length, and the chromosome number showed that 2cx dna in the studied berry species had a significant correlation with the chromosome length and chromosome number, and there were no significant correlations between 2cx dna with environmental conditions. hence, it is concluded that the genome size of berries species is independent of changes in environmental conditions. antioxidant properties estimated by antioxidant assays (dpph) showed significant differences among different concentrations. evaluation of total polyphenol content in samples is a widely used method to determine the number of antioxidants in the samples. a rapid, simple, and inexpensive method to measure the antioxidant capacity of food involves the use of the free radical 2,2-diphenyl-1-picrylhydrazyl (dpph). dpph radicals are frequently utilized in antioxidant studies. dpph is widely used to test the ability of compounds to act as free radical scavengers or hydrogen donors and evaluate the antioxidant activity of foods. antioxidants in a sample can scavenge the dpph radicals. a gradual reduction in absorbance is observed, indicating that dpph radicals are being scavenged. through adding samples, which are rich in antioxidants, to a dpph solution. therefore, the percentages we have presented pertain to dpph radical scavenging capacity, which is directly proportional to antioxidant capacity. the total phenolic content of dry fruits showed a wide range, with values ranging from 3.08 mg gae/g (s1; fragaria vesca subsp. vesca) to 8.61 mg gae/g (s9; lycium ruthenicum) shown in table 4. by comparing these data, it can be concluded that the s3 (rubus occidentalis cv. qaemshahr) has a high amount of antioxidants. the total flavonoid content (tfc) of dried fruits showed a wide range, with values ranging from 2.43 g qe/g (s12; morus rubra cv. karaj) to 10.63 g qe/g (s9; lycium ruthenicum) as shown in table 4. based on the above studies and considering the diversity of these 12 species in terms of cytology and phytochemistry, it is concluded that some of these species are prone to enter the daily food basket. black raspberry (s3; rubus occidentalis cv. qaemshahr) had the highest value in terms of all three phytochemical traits, followed by goose berry (s6; ribes uva-crispa) and black currant (s4; ribes nigrum), respectively. strawberry (s1; fragaria vesca subsp. vesca) and red mulberry (s12; morus rubra cv. karaj) had the lowest value. we report that black raspberry (s3; rubus occidentalis cv. qaemshahr) is a remarkable source of antioxidant compounds compared to other fruits. hence, research supports deep-colored fruits as potent antioxidant sources. berries and dried fruit compose a relatively small part of the average diet, but they are important antioxidant sources. highly pigmented berries have the highest antioxidant activity. such fruits are rich in antioxidant compounds that are known for their enhanced stability and bioaccessibility. based on our findings and the cited literatures, it can be suggested that black raspberries are among the fruits that provide antioxidants. in the study of islam et al. (2017) on genus lycium, the mean of total phenol content, total flavonoids content, and antioxidant activity for red goji berries (s7; lycium barbarum) were measured as 3.16 mg g-1, 2.83 mg g-1, and 16.65 µmol g-1 respectively. these values were 8.33 mg g-1, 11.03 mg g-1, and 34.28 µmol g-1 respectively for black goji berry (s9; lycium ruthenicum). in the present study (table 4), these values were 4.07 mg g-1, 3.45 mg g-1, and 25.06 µmol g-1 respectively, for s7 with a significant increase of 29%, 22%, and 50% in all three cases, respectively, compared to the previous report of red goji berry (s7; lycium barbarum). also for s9, these values are reported 8.61 mg g-1, 10.63 mg g-1, and 34.95 µmol g-1, respectively, which is almost equal to the previous report for (s9; lycium ruthenicum). in the study of mustafa ahmed et al. (2022) on genus vaccinium, the mean of total phenol 144 saeed mohammadpour, ghasem karimzadeh, seyed mahmood ghaffari content, total flavonoid content, and antioxidant capacity were reported as 3.73 mg g-1, 2.57 mg g-1, and 17.67 µmol g-1, respectively. in the present study (table 4), these values were 3.28 mg g-1, 2.77 mg g-1, and 27.50 µmol g-1, respectively which are almost in agreement with those in the previous report. in the current study, the correlation was carried out between phytochemical traits with chromosome number, chromosome length, the monoploid genome size (2cx dna), and environmental conditions (latitude and altitude). the results showed no significant correlation between phytochemical traits of species with environmental conditions, but there was a significant correlation between phytochemical traits with chromosome number, antioxidant activity with genome size, total phenol with chromosome length, and total flavonoid with chromosome length (table 5). from these results, it is inferred that phytochemical traits in the studied species are independent of changes in environmental conditions. for traits that had a significant correlation between them in the table above, linear regression analysis was performed (figure 7). figure 7 shows that the monoploid genome size (2cx dna) has a direct linear relationship with chromosome length and chromosome number of berry plants (figures a, and b). antioxidant has an inverse linear relationship with the genome size and chromosome number (figures c, and d). it means that berries with fewer chromosomes and smaller genome sizes produce more antioxidants. phenol and flavonoid have an inverse linear relationship with the chromosome number (figures e, and f ), but a direct relationship with the chromosome length (figures g, and h). for further study, the correlation between genome size and chromosome length for four species (s7, s8, s9, s10) with the same chromosome number (2n = 2x = 24) was also calculated and was significant at the 1% probability level (r = 0.89**). therefore, figure 8 shows the relationship of the direct linear regression between the monoploid genome size and chromosome length for the four species mentioned above. in another study, the effect of different ploidy levels on the quantity and quality of essential oils of different species of berry was investigated and their genetic modification was provided. it is suggested that these berry species should be compared with other species in iran in terms of cytogenetics and phytochemistry. it is suggested that due to significant differences in genome size and morphology, further research should pin-point rdna (5s and 45s) sites and additional repetitive dna elements, using fluorescence in situ hybridization (fish) to better uncover the processes involved in the chromosome evolution of these twelve berry species. acknowledgments thanks to tarbiat modares university for financial support, and to the cultivation development company 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international journal of cytology, cytosystematics and cytogenetics volume 75, issue 3 2022 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 72(2): 91-95, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-239 citation: n.m. travenzoli, i.c. de oliveira barbosa, g.a. carvalho-zilse, t.m.f. salomão, d.m. lopes (2019) karyotypic description and repetitive dna chromosome mapping of melipona interrupta latreille, 1811 (hymenoptera: meliponini). caryologia 72(2): 91-95. doi: 10.13128/cayologia-239 published: december 5, 2019 copyright: © 2019 n.m. travenzoli, i.c. de oliveira barbosa, g.a. carvalho-zilse, t.m.f. salomão, d.m. lopes. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyotypic description and repetitive dna chromosome mapping of melipona interrupta latreille, 1811 (hymenoptera: meliponini) natália martins travenzoli1, ingrid cândido de oliveira barbosa2, gislene almeida carvalho-zilse2, tânia maria fernandes salomão3, denilce meneses lopes1,* 1 laboratório de citogenética de insetos, departamento de biologia geral, universidade federal de viçosa, cep 36570-900, viçosa, minas gerais, brazil 2 grupo de pesquisas em abelhas, coordenação de biodiversidade, instituto nacional de pesquisas da amazônia (inpa), cep 69067-375, manaus, amazonas, brazil 3 laboratório de biologia molecular de insetos, departamento de biologia geral, universidade federal de viçosa, cep 36570-900, viçosa, minas gerais, brazil *corresponding author: denilce.lopes@ufv.br abstract. heterochromatic patterns in the genus melipona vary among subgenera species. melikerria is the only subgenus that presents species with different content of heterochromatin. thus, the cytogenetic knowledge of other species of this subgenus is important for the understanding of karyotype evolution in melipona. here, we describe the karyotype of melipona (melikerria) interrupta based on the chromosomal heterochromatic patterns, at and gc richness, mapping sequences of rdna, microsatellites and telomeric regions. we observed 2n=18 chromosomes, with a high heterochromatin content rich in at and euchromatic regions rich in gc base pairs. the high gc content was observed at interstitial region near the junction of the stained euchromatin and heterochromatin of the first chromosomal pair, the same region marked for the rdna 18s locus. microsatellites hybridized only on euchromatin regions and the telomeric probe on terminal regions of all chromosomes. melipona (melikerria) interrupta belongs to previous described heterochromatic group ii, suggesting there has been an increase in heterochromatin content in melikerria. the m. quinquefasciata, belonging to the same subgenus as melipona (melikerria) interrupta, has low content of heterochromatin and appears to be evolving independently. so, the differences in the content heterochromatin, in the marker regions of cma3 and the rdna 18s locus in species of melikerria is an important feature to be investigated further. keywords. cytogenetics, fluorescence in situ hybridization (fish), heterochromatin, melikerria, stingless bee. 1. introduction bees of the genus melipona illiger, 1806, are eusocial insects belonging to the meliponini tribe and occur throughout the neotropical region (michener 2007; camargo and pedro 2013). this genus is represented by 73 species, 43 92 natália martins travenzoli et al. of which can be found in brazil (camargo and pedro 2013; pedro 2014). morphologically, the genus melipona is grouped into four subgenera: eomelipona, melikerria, melipona stricto sensu, and michmelia (camargo and pedro 2013). the melikerria, melipona stricto sensu, and michmelia subgenera are considered monophyletic, whereas eomelipona is polyphyletic according to molecular phylogenies (rasmussen and cameron 2010; ramírez et al. 2010). cytogenetically, only 28 of all the melipona species have had their karyotypes described. these species are characterized by a conserved diploid number of 2n = 18 chromosomes in females and n = 9 in males, except for m. seminigra merrilae and m. seminigra pernigra, with 2n = 22 (francini et al. 2011). in addition, the pattern of heterochromatin distribution in some melipona species differs from that observed in the majority of species in the meliponini tribe (reviewed in tavares et al. 2017; cunha et al. 2018; silva et al. 2018). the genus can be divided into two groups based on the pattern of distribution and content of heterochromatin: group i is composed of species with low heterochromatin content present only in the pericentromeric regions, similar to that in the other meliponini; and group ii comprised by species with a high heterochromatin content that covers large extensions of their chromosomes (rocha and pompolo 1998; rocha et al. 2002). from all the analyzed species, those belonging to the eomelipona and melipona stricto sensu subgenera present low levels of heterochromatin, whereas all those of the subgenus michmelia have high levels of heterochromatin (rocha and pompolo 1998; rocha et al. 2002; rocha et al. 2003; lopes et al. 2011; cunha et al. 2018). however, melikerria has only three species described cytogenetically that present the two patterns: melipona fasciculata smith, 1854 and melipona grandis guérin, 1844, with high content of heterochromatin and melipona quinquefasciata lepeletier, 1836 with a low content (rocha et al. 2002; rocha 2002; lopes et al. 2011), making heterochromatic evolution in this group difficult to elucidate. thus, cytogenetic studies with other species of the subgenus melikerria are needed since they may help to elucidate the processes leading to the chromosomal alterations in the genus. the aim of this study was to characterize the karyotype of melipona (melikerria) interrupta based on the heterochromatin distribution pattern and chromosomal regions rich in guanine-cytosine (gc) and adeninethymine (at) base pairs, as well as mapping the ribosomal 18s dna sites, the microsatellites ga(15), gag(10), caa(10), and cgg(10), and the regions containing telomeric ttagg(6) sequences. 2. material and methods larvae of melipona (melikerria) interrupta were collected from three colonies in itacoatiara, amazonas, brazil and kept in the meliponary of the instituto nacional de pesquisas da amazônia (inpa), manaus, amazônia, brazil. mitotic chromosomes were obtained from the larval brain ganglia at the last larval instar as described by imai et al. (1988), and stained with giemsa. the heterochromatin regions were visualized by the c-band technique (sumner 1972) and the dapi and cma3 fluorochromes were used according to schweizer (1980). fifteen individuals were used, with 10 metaphases being analyzed on average for each slide. the images were obtained with an olympus bx60 epifluorescence microscope, using olympus q-color3™ software olympus® images. fluorescent in situ hybridization (fish) was performed according to pinkel et al. (1986), with modifications: (metaphase chromosomes were denatured in 70%/2xssc formamide at 75 °c for 5 min; the probes were hybridized with chromosomes in 20 µl of hybridization mix and heated for 10 min at 85 o c). the 18s ribosomal dna probe was labeled with digoxigenin11-dutp (roche applied science) and the signal was detected wit h anti-digoxigenin-rhodamine (roche applied science). this probe was obtained by polymerase chain reaction (pcr) amplification, using the primers f1(5′-gtcatatgttgtctcaaaga-3′) and 18sr1.1 (3′-tcta attttttca a agta a acgc-5′) designed for the species melipona quinquefasciata (pereira 2006). the microsatellites ga(15), gag(10), caa(10), cgg(10), and ttagg(6) were labeled directly with cy3 in the 5′ regions (sigma, st. louis, mo, usa). the metaphase images were obtained with an olympus bx53 microscope fitted with an olympus dp73f camera, using the cellsens imaging software. 3. results and discussion the chromosome number observed in m. (melikerria) interrupta was 2n = 18 (fig. 1a) similar the number finding by barbosa (2018), which does not differ from that reported by kerr (1969, 1972). the c-band revealed a karyotype with high heterochromatin content (fig. 1b), making visualization of the centromere difficult. thus, we could not identify chromosome morphology to determine the karyotype of this species. based on the heterochromatic patterns, m. (melikerria) interrupta can be classified as belonging to the group composed of species with more than 50% of heterochromatin in their chro93karyotypic description and repetitive dna chromosome mapping of melipona interrupta mosomes, designated as group ii by rocha and pompolo et al. (1998). the species m. (melikerria) fasciculata and m. (melikerria) grandis were classified as belonging to this group (lopes et al. 2011; andrade-souza et al. 2018) as well as m. (melikerria) interrupta. unlike, m. (melikerria) quinquefasciata has a low heterochromatin content (rocha et al. 2007), indicating that the karyotype of m. quinquefasciata may be evolving independently or have been the karyotype plesiomorphic within the melikerria. so, in this subgenus may have been an increase in the levels of heterochromatin in the karyotype of the species. in fact, it has been, suggested that independent amplification of heterochromatin or differentiation in distinct melipona subgenus (piccoli et al. 2018). staining with the base-specific fluorophores, dapi and cma3, indicated that the heterochromatic regions were dapi+ (fig. 1c) and the euchromatic regions were cma3+. stronger labeling with cma3 was seen in the interstitial region near the euchromatin and heterochromatin junction of the first chromosomal pair (fig. 1d), coincident with the hybridization site of the 18s rdna probe (fig. 2a). staining with dapi has shown that heterochromatin in eusocial bees is generally at-rich (brito et al. 2003; rocha et al. 2003; lopes et al. 2011; godoy et al. 2013). this characteristic seems to be shared by melipona species that contain either high or low levels of heterochromatin. although the numbers of cma3+ and rdna markers on a single pair of chromosomes are conserved traits in the genus (cunha et al. 2018; andrade-souza et al. 2018), the chromosomal positions differ among species, even in those with a high heterochromatin content. according to the results of cma3+ and rdna probe, species with a low heterochromatin content exhibited pericentromeric markings in the first chromosome pair, while group ii species showed terminal markings (reviewed in cunha et al. 2018). although m. (melikerria) interrupta is classified as belonging to group ii, cma3+ and rdna markings were observed in the interstitial region of the first chromosomal pair. melipona (melikerria) fasciculata and m. (melikerria) grandis, which belongs to the same subgenus, also showed cma3+ markings in this region (lopes et al. 2011; andrade-souza et al. 2018), different to that observed in the other species with high heterochromatin content (reviewed in cunha et al. 2018). these results suggest that variation in the positions of gc rich regions and 18s rdna sites may be results of divergent heterochromatin evolutionary pathways in the melipona as suggested by cunha et al. (2018) and picolli et al (2018). as the markings in other species of group ii is at the chromosome end, the occurrence of rearrangements, such as inversion, would result in a portion of heterochromatin at the ends of the chromosome. however, we did not find heterochromatin in these regions suggesting that the position of cma3 and rdna no result of chromosome inversion events. the microsatellite probes ga(15), gag(10), caa(10), and cgg(10) labeled only euchromatin regions (fig. 2b-e), while the telomeric probe ttagg(6) showed staining in the terminal regions of the chromosomes of m. (melikerria) interrupta (fig. 2f ). this staining pattern in regions of euchromatin was also observed in the chromosomal mapping of melipona scutellaris latreille, 1811 using different repetitive dna sequences (ca(15), gac(10), and taa(10)) (piccoli et al. 2018). the ttagg(6) labeling on the terminal regions of the chromosomes indicated the presence of the ttagg sequence in the fig. 1. mitotic metaphase chromosomes of melipona (melikerria) interrupta stained with giemsa (a), cbanding (b), dapi (c), and cma3 (d). scale bar = 5 μm. fig. 2. patterns obtained in metaphase chromosomes of melipona (melikerria) interrupta with fish using the following repetitive dna probes: 18s (a), ga(15) (b), gag(15) (c), caa(10) (d), cgg(10) (e), and ttagg(6) (f ). in blue: chromosomes stained with dapi. in red: regions hybridized with probes. scale bar = 5 μm. 94 natália martins travenzoli et al. telomeric sites in the karyotype of m. (melikerria) interrupta. studies have shown that ttagg (sahara et al. 1999) and tcagg (mravinac et al. 2011) were observed in telomeres of several hymenoptera species, including apidae (apis mellifera) (meyne et al. 1995; sahara et al. 1999) and formicidae (tapinoma nigerrimum, myrmecia spp. and acromyrmex striatus) (meyne et al. 1995; lorite et al. 2002; frydrychová et al. 2004; pereira et al. 2018). however, in many other hymenoptera the ttagg sequence was not observed in the telomeres of the chromosomes, suggesting that it has been lost and recovered in apidae and formicidae or that multiple losses of this region have occurred throughout the evolutionary history of the groups (menezes et al. 2017). in this study, we report for the first time the telomeric sequence based on the fish technique on a bee species of meliponini tribe. this information added to the cytogenetic characteristics of melipona already described in the literature may contribute to the understanding of karyotype evolution in these bees. we conclude that m. (melikerria) interrupta is classified as belonging to group ii based on heterochromatic patterns, which suggests an increase in the amount of heterochromatin in the subgenus melikerria. it further suggests that karyotype the m. quinquefasciata be plesiomorphic or this may be evolving independently in the group, and that the differences in the cma3 and 18s marker regions interstitial, reported in the two species of melikerria subgenus with high heterochromatin content, are an important feature to be further investigated. 5. acknowledgment the “coordenação de aperfeiçoamento de pessoal de nível superior (capes)”, “fundação de amparo à pesquisa do estado de minas gerais (fapemig)” and the “fundação de amparo à pesquisa do estado do amazonas (fapeam)”. 6. bibliographic references andrade-souza v, duarte omp, martins ccc, santos is, costa mgc, costa ma. 2018. comparative molecular cytogenetics in melipona illiger species (hymenoptera, apidae). sociobiology. 65 (4): 696-705. barbosa ico. 2018. caracterização citogenética e análise do perfil de metilação em melipona seminigra e melipona interrupta (hymenoptera, apidae). dissertation, instituto nacional de pesquisas da amazônia. 42pp. brito rm, caixeiro apa, pompolo sg, azevedo gg. 2003. cytogenetic data of partamona peckolti (hymenoptera, apidae, meliponini) by c banding and fluorochrome staining with da/ cma3 and da/dapi. genet. mol. biol. 26 (1): 53-57. camargo jmf, pedro srm. 2013. meliponini lepeletier, 1836. in moure, js, urban, d. and melo, ga r (orgs). catalogue of bees (hymenoptera, apoidea) in the neotropical region online version. available at http://www.moure.cria.org.br/catalogue. accessed 18 january 2019. cunha ms, travenzoli nm, ferreira rp, cassinela ek, silva h, salomão tmf, lopes dm. 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res. 75 (1): 304-306. tavares mg, lopes dm, campos lao. 2017. an overview of cytogenetics of the tribe meliponine (hymenoptera: apidae). genetica 145 (6): 1-18. caryologia international journal of cytology, cytosystematics and cytogenetics volume 72, issue 2 2019 firenze university press karyotype analysis of a natural lycoris double-flowered hybrid jin-xia wang1, yuan-jin cao1, yu-chun han1, shou-biao zhou1,2, kun liu1,* insights on cytogenetic of the only strict african representative of genus prunus (p. africana): first genome size assessment, heterochromatin and rdna chromosome pattern justine germo nzweundji1, marie florence sandrine ngo ngwe2, sonja siljak-yakovlev3,* assessment of cytotoxicity and mutagenicity of insecticide demond ec25 in allium cepa and ames test arzu özkara cytogenetic effects of fulvic acid on allium cepa l. root tip meristem cells özlem sultan aslantürk evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of allium test ioan sarac1, elena bonciu2,*, monica butnariu1, irina petrescu1, emilian madosa1 fluorescence in situ hybridisation study of micronuclei in c3a cells following exposure to elf-magnetic fields luc verschaeve1,2,*, roel antonissen1, ans baeyens3, anne vral3, annemarie maes1 phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities nicoleta anca şuţan1,*, irina fierăscu2, radu fierăscu2, deliu ionica1, liliana cristina soare1 telomeric heterochromatin and meiotic recombination in three species of coleoptera (dorcadion olympicum ganglebauer, stephanorrhina princeps oberthür and macraspis tristis laporte) anne-marie dutrillaux, bernard dutrillaux* a whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of populus trichocarpa l. subjected to different stresses alberto vangelisti#, gabriele usai#, flavia mascagni#, lucia natali, tommaso giordani*, andrea cavallini differences in c-band patterns between the japanese house mice (mus musculus) in hokkaido and eastern honshu hikari myoshu, masahiro a. iwasa* karyotypic description and repetitive dna chromosome mapping of melipona interrupta latreille, 1811 (hymenoptera: meliponini) natália martins travenzoli1, ingrid cândido de oliveira barbosa2, gislene almeida carvalho-zilse2, tânia maria fernandes salomão3, denilce meneses lopes1,* caryologia. international journal of cytology, cytosystematics and cytogenetics 75(3): 123-134, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1728 caryologia international journal of cytology, cytosystematics and cytogenetics citation: marlon garcia paitan, maricielo postillos-flores, luis rojas vasquez, maria siles vallejos, alberto lópez sotomayor (2022). nuclear dna content and comparative fish mapping of the 5s and 45s rdna in wild and cultivated populations of physalis peruviana l. caryologia 75(3): 123-134. doi: 10.36253/caryologia-1728 received: july 03, 2022 accepted: november 29, 2022 published: april 5, 2023 copyright: © 2022 marlon garcia paitan, maricielo postillos-flores, luis rojas vasquez, maria siles vallejos, alberto lópez sotomayor. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. nuclear dna content and comparative fish mapping of the 5s and 45s rdna in wild and cultivated populations of physalis peruviana l. marlon garcia paitan*, maricielo postillos-flores, luis rojas vasquez, maria siles vallejos, alberto lópez sotomayor research group on genetic resources (recgen), faculty of biological sciences, universidad nacional mayor de san marcos, lima, peru *corresponding author. e-mail: marlon.garcia@unmsm.edu.pe abstract. physalis peruviana l. often known as goldenberry, has increased its commercial growth in the international market in recent years due to its nutritional value and antioxidant potential. this situation has enabled countries such as peru to increase their production in order to meet the global demand. however, investigations about the genetic diversity of cultivated and wild populations of goldenberry are still in their early stages. fish mapping of 5s and 45s rdna loci and flow cytometry estimation of nuclear dna content were used to assess genetic differences between wild and cultivated goldenberry populations from ayacucho and cajamarca. the majority of metaphases had six 5s rdna sites for all populations and two and four 45s rdna sites for the cultivated and wild populations, respectively. we were able to characterize nine different types of chromosomes based on their morphology, fluorescence, rdna location, and conservation across populations by analyzing the chromosomes that contained rdna. furthermore, cultivated populations had more nuclear dna (13.262±0.087 pg) than wild populations (12.955±0.086 pg). the results show genetic differences between wild and cultivated populations of goldenberry at molecular cytogenetic level as well as in genome size. these findings establish a precedent for future cytogenetic and genomic studies in goldenberry populations, enabling future breeding programs. keywords: goldenberry, fish, rdna, chromosomes, flow cytometry. 1. introduction physalis peruviana l., also known in latin america as “aguaymanto,” “uchuva,” or “uvilla,” is a plant in the solanaceae family that originated as well as diversified in the andes mountains. it produces orange-yellow berryshaped fruits covered by a calyx. these fruits are high in vitamin c, carotenoids, phenolic compounds with antioxidant precursors, and withanolides, which have anticancer and antitumor properties (singh et al. 2019). because of these characteristics and the increased international demand for organic food, the production of physalis peruviana l. in peru has quickly grown in recent years, establishing it as the second-largest producer of goldenberry 124 marlon garcia paitan et al. in latin america. however, the amount of goldenberry production compared to colombia, the leading producer in latin america, and other countries outside the american continent remains limited (sierra y selva exportadora 2021). to increase commercial competitiveness, genetic improvement programs that increase fruit quality and production must be implemented, and our understanding of the genetic diversity of physalis peruviana l. in cultivated and wild populations will determine the success of these programs. cytogenetic studies provide a foundation for the characterization of germplasm resources and offer guidance in selecting the parental cultivars for plant breeding programs (herrera 2007). furthermore, for future complex genomic projects such as cloning of genes of interest or genome sequencing, genome size data is essential to design effective projects (doležel et al. 2007). most cytogenetic studies in physalis peruviana l. have used classical staining techniques to determine the chromosome number, where 2n=48 predominates (rodríguez and bueno 2006; sánchez 2014; liberato et al. 2014; trevisani et al. 2018), while other studies report mixoploid plants (sánchez 2014), and somatic aneuploidy (carbajal et al. 2021). karyotypic formulas of some populations have been previously reported (azeez and faluyi 2019; carbajal et al. 2021). however, since goldenberry chromosomes are small, numerous, and have similar morphology, the karyotype determination and its analysis are considered limited when classical staining techniques are used. fish is a molecular cytogenetic technique for mapping specific sequences on chromosomes by hybridizing probes with their complementary sequences. 5s and 45s ribosomal dna (rdna) probes have been widely used to perform cytogenetic characterization and to understand intraspecific and interspecific evolutionary relationships at chromosomal level by determining the position and number of signals on metaphase chromosomes, even in species with numerous and small chromosomes (herrera 2007). this is due to the nature of rdna sequences, which are tandemly repeated dna regions in the genome with high transcription rates and a predisposition for unequal recombination, which makes them inherently unstable and prone to variation in copy number and location in the genome (salim and gerton 2019). previously, the number of 5s rdna signals in two cultivated populations from the same region has been studied at the molecular cytogenetic level in physalis peruviana l. (siles et al. 2021). however, no studies have been conducted to analyze the number and position of the 5s and 45s rdna sites to determine if there are any chromosomal differences between wild and cultivated populations as a consequence of the domestication of the species. plant genome size varies enormously and is considered a biodiversity trait that provides valuable information for systematic and evolutionary studies (pellicer et al. 2018). it is also useful for determining the genetic characterization of germplasm and plant identification because of these characteristics (jedrzejczyk and rewers 2020). flow cytometry is a quick and easy way to estimate nuclear dna content, making it a powerful tool for accelerating selection processes in plant breeding and providing critical data for sequencing projects (cipriansalcedo et al. 2020). given the foregoing, the goals of this study were to characterize and analyze the genetic differences between wild and cultivated populations of physalis peruviana l. from the regions of ayacucho and cajamarca through the mapping of 5s and 45s rdna by fish, and to estimate the nuclear dna content using flow cytometry. these two approaches will set a precedent as potential tools for programs to improve this genetic resource in peru and, as a result, improve the international competitiveness of peru against other producing countries. 2. methods and materials 2.1 plant material and preparation of metaphase chromosomes seeds were collected from wild and cultivated populations of ripe goldenberry fruits in the cajamarca and ayacucho regions (figure 1). plant material was transported to the genetics laboratory of the universidad nacional mayor de san marcos (unmsm) and correctly identified as physalis peruviana l. by the unmsm natural history museum. (certificates 224-usm-2015 and 001-usm-nhn -2022). we modified the protocols described by aguilera et al. (2016), aliyeva-schnorr et al. (2015), and carbajal et al. (2021) in order to obtain mitotic metaphase chromosomes. goldenberry seeds were germinated, and roots with an approximate size of 4 mm were treated with 0.03 percent colchicine for 80 minutes at room temperature before being submerged in distilled water for 60 minutes at 37 °c and fixed in an ethanol solution: acetic acid (3:1) at -20 °c for at least one day. subsequently, the roots were washed in cold distilled water twice for 5 minutes, followed by two washes in cold citrate buffer (0.01 m at ph 4.6) for 5 minutes. they were then macerated in a 2% cellulase solution (from aspergillus niger, sigma) and a 10% liquid pectinase solution (from aspergillus niger, sigma) dissolved in 40% glycerol in 0.01m citrate buffer at ph 4.6, at 37 °c for 1 hour. then, they were washed four times with cold citrate buffer for 5 minutes (0.01 m at ph 125nuclear dna content and comparative fish mapping of the 5s and 45s rdna in physalis peruviana l. 4.6) and twice in ethanol 90° for 5 seconds. the apex of each root was then transferred to a slide, a drop of 45 percent acetic acid was placed on it, and the “squash” technique was performed. after that, the slide was frozen with dry ice to remove the coverslip slide, then air-dried and examined under a phase-contrast microscope to confirm the presence of cells with properly separated metaphase chromosomes. finally, the selected slides were immersed in absolute ethanol at -20 °c until they were used. 2.2 amplification of rdna genomic dna was extracted from seedlings grown from goldenberry seeds using the gf-1 plant dna extraction kit (vivantis, malaysia). to obtain the 5s rdna probe, 5s rdna amplification was performed using the primers pr5s14 (5’-ggcgagtagtactaggatccgtgac-3’) and pr5s15 (5’-gcttaacttcggagttctgatggga-3’) reported by volkov et al. (2001). because the 45s rdna locus is too large to be completely amplified, it was decided to use the 18s rdna gene as a probe. since 18s rdna is present within the gene structure of the 45s rdna, mapping it would also show the location of the 45s rdna locus. the following primers were used to achieve 18s rdna amplification: primers f-566 (5’-cagcagccgcggtaattcc-3’) and r-1200 (5’-cccgtgttgagtcaaattaagc-3’) which were previously reported by hadziavdic et al. (2014). the pcr reaction mixture for both cases had a final volume of 20 µl containing 1x pcr buffer, 1.5 mm mgcl2, 0.2 µm dntps, 0.2 µm of each primer, 1 u of taq polymerase (abm, canada), and 30 ng of genomic dna. the pcr program for 5s rdna began with an initial denaturation at 95 °c for 9 minutes, followed by 45 cycles of 95 °c for 30 seconds, 58 °c for 30 seconds and 72 °c for 45 seconds, and a final extension at 72 °c for 5 minutes. on the other hand, 18s rdna involved an initial denaturation at 95 °c for 9 minutes, followed by 42 cycles of 95 °c for 30 seconds, 62 °c for 30 seconds and 72 °c for 60 seconds, and a final extension at 72 °c for 5 minutes. the amplicons were purified using the genejet pcr purification kit (thermo scientific™) and quantified by spectrophotometry. 2.3 labelling of dna probes the 5s rdna amplicons were labeled with biotin14-datp for 90 minutes using the bionick labeling system commercial kit (invitrogen, usa), and the 18s rdna was labeled for 180 minutes with digoxigenin11-dutp using the dig-nick translation mix commercial kit (roche, switzerland). 2.4. fish and signal detection the fish methodology used was slightly modified from that provided by poggio et al. (2000). the slides with the chromosome preparation were treated with rnases and 4% (w/v) paraformaldehyde. the hybridization solution contained 15 μl of formamide, 6 μl of 50% dextran sulfate, 3 μl of 20x ssc, 1 μl of salmon dna (10 mg/ml), 1μl of 10% sds, and 50 ng of each 5s and 45s rdna probe, with a final volume of 30 μl. this solution was applied to the chromosome preparations, and the hybridization program was as follows: 75 °c for 7 minutes, 55 °c for 6 minutes, 45 °c for 5 minutes, and 37 °c for 12 hours inside a hybridizer (biobase, hs-500). after the hybridization, the following washes were performed to remove nonspecific binding: 2x ssc at 42 °c for 5 minutes, 20% formamide in 0.1x ssc at 42 °c for 10 minutes, 0.1x ssc at 42 °c for 5 minutes, 2x ssc for 5 minutes at 42 °c, and finally three washes with 0.2% (v/v) tween 20 in 4x ssc for 5 minutes at room temperature. the 5s probe was detected with a neutravidinoregon-green 488 conjugate (thermo scientific™) while the 18s probe was detected with anti-digoxigenin-rhodamine (roche). the slides were mounted with slowfigure 1. map of peru showing sampling locations for wild and cultivated populations of physalis peruviana l. 126 marlon garcia paitan et al. fade™ gold antifade mountant (invitrogen™), which contained dapi and an antifade solution. the slides were observed and photographed under an epifluorescence microscope (zeiss axio scope.a1). for dapi f luorophore, bp 340/30 excitation and bp 510/90 emission filters were used; for oregon green 488, bp 470/40 excitation and bp 540/50 emission filters were used; and for rhodamine, bp 560/40 excitation and bp 630/75 emission filters were used. we specifically worked with cells in metaphase that contained 48 recognizable chromosomes to avoid variation in the results attributable to any somatic aneuploidy present in the meristematic tissue of the root of the species, a phenomenon already observed in prior studies (carbajal et al. 2021; franco et al. 2021). the following programs were utilized: gimp 2.10.14 was used for picture overlaying and chromosomal individualization; ideokar1.2 (mirzaghaderi and marzangi 2015) for morphological measurements of chromosomes and the production of ideograms; and imagej for fluorescence quantification of probe signals. the latter was achieved in accordance with fitzpatrick (2021). 2.5 determination of nuclear dna content each wild and cultivated population had eight plants evaluated. these were obtained after a three-month seed culture in pots. flow cytometry was used to determine the nuclear genome size of each plant in duplicate using pisum sativum cv. ‘ctirad’ (2c = 9.09 pg dna) as an internal reference standard. the nuclear suspension preparation protocol and nuclear dna content estimation were performed as described by doležel et al. (2007). young leaves of the standard and goldenberry were utilized as plant material in the streamlined two-step process for nuclei suspension preparation. furthermore, propidium iodide was employed as a fluorochrome. the nuclear dna content was estimated using an attune nxt flow cytometer (thermo fisher scientific) and the following formula: 2c value of the sample (pg de adn) = !"#$ &'()*)'$ '+ *," -. &"#/ '+ *," (#0&1" !"#$ &'()*)'$ '+ *," -. &"#/ '+ *," 2"+"2"$3" (*#$4#24 x 9.09pg 3. results 3.1 number of 5s and 45s rdna signals in wild and cultivated populations fish was used to identify the 5s and 45s rdna probe signals in the metaphases of physalis peruviana l. populations (figure 2). for each population, 25 metaphases with 48 chromosomes were analyzed. after studying the metaphases of all the populations, a total of five combinations of the number of 5s and 45s rdna sites were identified; three of these combinations were detected in wild populations (figure 2a, b, e, f, i, j) and four combinations in cultivated populations (figure 2c, d, e, g, h, k, l). there were no significant variations in the number of 5s and 45s rdna sites across wild populations (p=0.422), nor between cultivated populations (p=0.085), however, there were significant differences across populations from the same region (p<0.01). furthermore, it was discovered that the predominant rdna combination in wild populations was six 5s rdna sites and four 45s rdna sites, whereas in cultivated populations it was six 5s rdna sites and two 45s rdna sites (figure 3). in general, a higher frequency of six 5s rdna sites is observed in all goldenberry populations, while for 45s rdna a number of two sites predominates for cultivated populations and 4 sites for wild populations. 3.2 characterization of chromosomes with rdna loci the 12 best metaphases with clear and distinct rdna signals in relation to their chromosomal arms and fluorescent signals were selected from the 25 metaphases per population, considering at least one metaphase that showed each combination of the number of 5s and 45s rdna sites from each wild and cultivated population. the morphological characterization, individualization, and pairing with their homologous chromosomes that displayed rdna signals were performed in each of the selected metaphases (figure 4, figure s1). based on the study of the chromosomes that showed rdna signals in all populations, nine types of chromosomes were identified according to their morphological features, the position of the 5s or 45s rdna locus, the fluorescence intensity of the rdna probe, and their presence in the populations (figure 5 and table 1). the n chromosomes are those that are found in all populations: n1 has a metacentric morphology and the 5s rdna in the p arm; n2 is submetacentric and has the 45s rdna in the p arm, and n3 is acrocentric and has the 5s rdna in the q arm. the s chromosomes, which stand for wild, are found specifically in wild populations: s1 has a submetacentric morphology and 45s rdna in the q arm; s2 is metacentric and has 45s rdna in the q arm; s3 is metacentric and has 5s rdna in the p arm. it should be noted that, while n1 and s3 are described in the same way, the difference between them is determined by the length of the chromosome (table 1). c chromosomes are those found solely in cultivated populations: c1 has a submetacentric 127nuclear dna content and comparative fish mapping of the 5s and 45s rdna in physalis peruviana l. morphology and has the 5s rdna in the q arm, whereas c2 has a metacentric/submetacentric morphology and has the 5s rdna in the q arm, and a lower fl uorescence intensity than c1. th e morphology of the c2 chromosome is attributable to the fact that only one c2 chromofigure 2 fluorescent in situ hybridization signals of the 5s (green) and 45s (red) rdna probes in physalis peruviana l. metaphase chromosomes. th e numbers at the bottom left of each image represent the number of 5s (green) and 45s (red) sites found in the metaphases. combinations of the number of 5s and 45s rdna sites identifi ed in the population of wild ayacucho are shown in a, e, and i; wild cajamarca is shown in b, f, and j; cultivated ayacucho is shown in c, g, and k; and cultivated cajamarca is shown in d, e, h, and l. th e scale bar measures 10 μm. figure 3 th e frequency of 5s and 45s rdna sites observed in the 25 metaphases of each goldenberry population. diff erent letters at the top of each frequency circle show signifi cant diff erences (fisher’s exact test, p<0.05). figure 4 chromosomes that showed hybridization of the 5s and 45s rdna probes between the best 12 metaphases for each population, encompassing at least one metaphase for each of the combinations of the number of 5s and 45s rdna sites reported for each population of goldenberry. m=metacentric, sm=submetacentric, acro=acrocentric. 128 marlon garcia paitan et al. some was observed in each of the cultivated populations, resulting in a metacentric morphology in ayacucho and a submetacentric morphology in cajamarca (figure 6). the cc chromosome, found only in the cultivated cajamarca population, has a submetacentric morphology and the 45s rdna in the p arm, similar to n2, although it differs in terms of chromosome length and rdna probe fluorescence intensity (table 1). the types of chromosomes that contain rdna are conserved among wild populations. however, there is a difference in the observed frequency of chromosomes s1, which is higher in cajamarca, and s2, which is higher in ayacucho (figure 6). additionally, in ayacucho, the 45s rdna locus of s1 chromosome is found in the centromeric position, whereas in cajamarca, it is found in the interstitial position. the cc chromosome was found only in cajamarca among the cultivated populations, while the other chromosomes were found in both groups with no variations in frequency or position of the rdna (figure 6). 3.3 size of the nuclear genome all f low cy tometric analyses showed high-resolution histograms with a coefficient of variation (cv) of the g0/g1 peaks between 1.63% and 2.85% (mean 2.04%). representative histograms are shown with the peaks corresponding to the g0/g1 nuclei of physalis peruviana l. and the internal standard pisum satifigure 5 characterization of the nine types of chromosomes that showed rdna probe hybridization based on their existence in populations, morphology, size, presence of the 5s or 45s locus, position of the rdna locus, and rdna probe fluorescence intensity, m=metacentric, sm=submetacentric, and acro=acrocentric. figure 6 the average ideogram of the types of chromosomes that included rdna. the proportion observed in the total of the metaphases analyzed by each population is indicated beneath each chromosome. equal symbols above the percentages indicate a significant difference between the observed frequencies of both populations (fisher’s exact test, p<0.05). m=metacentric, sm=submetacentric, and acro=acrocentric. 129nuclear dna content and comparative fish mapping of the 5s and 45s rdna in physalis peruviana l. vum cv. ctirad of the wild and cultivated populations in figure 7. the nuclear dna estimation content based on the amount of the 2c internal standard (9.09pg of dna) displayed a substantial variation between both groups (table 2). 4. discussion the determination of the number of rdna sites and their distribution on chromosomes by fish has proven to be a reliable molecular cytogenetic marker when establishing genetic relationships of plant species that contain a significant number of small and homogenous chromosomes (su et al. 2020). the current study table 1. morphometric and fluorescence data of the nine types of chromosomes found in physalis peruviana l. populations. rdna-bearing chromosome n chromosome length arm ratio log (dna probe fluorescence intensity) cm position of rdna locus n1 48 3.120 ± 0.416 1.363 ± 0.161 3.943 ± 0.265 m 5s -> p n2 48 2.436 ± 0.332 2.191 ± 0.265 3.909 ± 0.404 sm 45s -> p n3 45 1.499 ± 0.230 3.919 ± 0.641 3.078 ± 0.307 acro 5s -> q s1 10 2.516 ± 0.341 2.189 ± 0.240 3.383 ± 0.318 sm 45s -> q s2 12 2.417 ± 0.216 1.471 ± 0.112 3.371 ± 0.164 m 45s -> q s3 24 2.241 ± 0.287 1.284 ± 0.167 3.995 ± 0.319 m 5s -> p c1 24 3.002 ± 0.461 2.220 ± 0.292 3.930 ± 0.240 sm 5s -> q c2 1 2.908 ± 0.106 1.951 ± 1.238 3.180 ± 0.001 m/sm 5s -> q cc 2 1.922 ± 0.047 2.027 ± 0.366 3.212 ± 0.062 sm 45s -> p the values are expressed as mean ± standard deviation. the “n” sample values are per chromosomal pair. cm = chromosome morphology; m = metacentric; sm = submetacentric; acro= acrocentric. figure 7 fluorescence histograms of the nuclei of goldenberry and pisum sativum cv. ctirad populations (2c = 9.06 pg as internal reference standard) were analyzed simultaneously. each peak denoted by the numbers 1 or 2 corresponds to the populations of nuclei in the g0/g1 phase of each species. each histogram includes the percentage of the coefficient of variation (cv) and average absolute fluorescence for each of these peaks. 130 marlon garcia paitan et al. found a substantial difference in the number of 5s and 45s rdna sites between wild and cultivated populations of goldenberry (figure 3). this is the first mapping of the 45s rdna in physalis peruviana l., where a number of two, four, and six sites have been reported for all the populations, prevailing a number of four sites in wild populations and two sites in cultivated populations. this intraspecific variation at the species level, as well as its variability among the populations analyzed, in terms of the number of 45s rdna sites, can be explained by its nature as a fragile location within the plant genome (huang et al. 2012). this trait increases the likelihood of chromosomal breakage in the rdna region, resulting in a chromosomal rearrangement. furthermore, because of its repetitive nature, it is an unstable genomic region that promotes homologous recombination (rosato et al. 2016), resulting in increased variability in the amount of 45s rdna sites within the genome. this variability has also been observed in other solanaceae species, such as solanum spp., which presents between 2 and 4 sites (pendinen et al. 2008; rego et al. 2009; moyetta et al. 2017); capsicum spp., which presents between 2, 4, 6, 8 up to 16, 24, 28, 36 sites (youn-kyu et al. 1999; kwon and kim 2009; romero-da et al. 2017); nicotiana spp. likewise shows considerable variety, reporting between 2, 4, 6, and, to a lesser extent, 8 and 10 sites (lim et al. 2000; nakamura et al. 2001; kitamura et al. 2001; matyasek et al. 2003; kovarik et al. 2004). all of this suggests that the number of 45s rdna sites of 2, 4, and 6 reported in the populations studied in this research is within the predicted range for the solanaceae family. the variation between wild and cultivated populations was influenced by the unstable character of the 45s rdna region and was favored by the selective breeding process throughout the domestication of the species (doebley et al. 2006). in comparison to the number of 45s rdna sites, the number of 5s rdna sites has been found to remain reasonably consistent throughout most angiosperm and gymnosperm taxa (garcia et al. 2017). this is consistent with the findings of the current study, which found a predominance of six 5s rdna sites in all goldenberry populations, with a small fraction of four and seven sites in the cultivated populations (figure 3). similarly, siles et al. (2021) discovered a prevalence of six 5s rdna sites in goldenberry cultivated populations. however, other solanaceae species have a lower number of 5s rdna sites, such as solanum spp., which has a prevalence of two 5s rdna sites and, to a lesser extent, four sites (pendinen et al. 2008; rego et al. 2009; aguilera et al. 2016; romero-da et al. 2017), capsicum spp., which typically presents two sites (youn-kyu et al. 1999; park et al. 2000; kwon and kim 2009; aguilera et al. 2016; romero-da et al. 2017); nicotiana spp., which generally alternates between two and four sites (nakamura et al. 2001; kitamura et al. 2001; fulneček et al. 2002; matyasek et al. 2003; kovarik et al. 2004), and cestrum spp., which has two 5s rdna sites (fregonezi et al. 2006; fernandes et al. 2009; urdampilleta et al. 2014). these data indicate that goldenberry populations have more 5s rdna sites than other species in the family. however, not enough studies have been conducted on this clade or the physalis genus to be conclusive. furthermore, the presence of a considerable number of 5s rdna sites may be due to ribosomal gene copy number amplification during crossing over or transposition events (kapitonov and jurka 2003; su et al. 2020). in accordance to prior research (garcia et al. 2017), the majority of angiosperm species have more 45s rdna sites than 5s rdna sites; this is also shown with molecular cytogenetic results in the solanaceae family to a higher extent (vitales et al. 2017). this contrasts with the findings in the goldenberry populations analyzed in this study, which revealed six 5s rdna sites and two/four 45s rdna sites (figure 3). therefore, these results will be helpful as precedents for the molecular cytogenetic characterization of the genus physalis and the cytotaxonomic understanding of the solanaceae family. the nine types of chromosomes that contain rdna (figure 5) allowed cytogenetic distinction of wild and cultivated populations of goldenberries (figure 6). these discrepancies are indistinguishable when only the chromosomal number (2n=48) is taken in consideration. therefore, fish significantly reduces the number of chromosomes that must be studied to determine cytogetable 2. flow cytometry was used to quantify the nuclear dna content and genome size of wild and cultivated populations of physalis peruviana l. using pisum sativum cv. ctirad as the internal reference standard. population n(r) average values cv (%)nuclear dna content 2c ± de (pg) genome size 1c ± de (x 109 pb)** wild 8(2) 12.955 ± 0.086* 6.335 ± 0.041* 2.124±0.360 cultivated 13.262 ± 0.087* 6.485 ± 0.042* 1.960±0.216 the n represents the sample number, while the r represents the number of repetitions per sample, cv: the coefficient of variation of g0/g1 peaks in flow cytometry histograms. * indicates a significant difference between values of the same column p < 0.05. ** 1pg dna equal to 0.978×109 bp, according to doležel et al. (2007). 131nuclear dna content and comparative fish mapping of the 5s and 45s rdna in physalis peruviana l. netic differences, emphasizing its relevance in the study of plant species with multiple chromosomes (su et al. 2020). the existence of different chromosomes in wild (s1, s2, and s3) and cultivated (c1, c2, and cc) populations could be attributed to multiple breeding events that happened throughout the domestication of the species, leading to chromosomal rearrangements (bashir et al. 2018; su et al. 2020). there is no variation in the presence of rdna-containing chromosomes between the two wild populations, implying that there is no structure between these populations. however, discrepancies in the frequency of chromosomes s1 and s2 may imply a slight genetic distinction between the two wild populations as a result of their geographical separation (figure 1). the presence of the cc chromosome in cajamarca could be explained by the same trait found in cultivated populations (figure 6). the 5s rdna locus is present in three different chromosomal sites: telomeric (n1 and s3), interstitial (c1 and c2), and centromeric (n3) (figure 5). the dispersed distribution of 5s rdna has already been observed in numerous mappings of plant chromosomes (vitales et al. 2017), which could be linked to the activity of mobile genetic elements such as transposons, which have been detected in association with ribosomal gene sequences (kapitonov and jurka 2003; raskina et al. 2004). the 45s rdna, on the other hand, has a more restricted position, with telomeric (n2 and cc) and interstitial (s1 and s2) sites reported (figure 6). this more restricted variability is typical of angiosperms, and it has also been revealed that the 45s rdna ancestral location is terminal (garcia et al. 2017). the 45s rdna locus is known to be involved in the creation of nucleoli during the cell cycle, especially if it is positioned in the satellite sections of the chromosomes since it correlates with the existence of nucleolar organizing areas (long and dawid 1980; lopez et al. 2021). if the species has a large number of 45s sites, however, not all of them will be functional (grabiele et al. 2018; báez et al. 2020). in the current investigation, no satellites correlated with an active 45s rdna locus were found in the analyzed chromosomes, however, the n2 chromosome is likely to contain a functional locus because it is conserved in all goldenberry populations and is the only type of chromosome that has a 45s rdna locus in the ayacucho cultivated population. garcia et al. (2017) propose that if a karyotype has a single 5s or 45s rdna locus, that locus must be functional. the fluorescence intensity of the rdna probes that are highlighted in the differentiation of chromosome types c1 and c2, as well as between cc and n2, could be related to the number of rdna tandem repeats that they have between the loci, implying that low fluorescence would indicate a comparatively low number of repeats (prado et al. 1996). furthermore, events such as amplification, deletion, and unequal crossing over are known to change the number of repeats and result in variations in fluorescence signals (su et al. 2020). differences in the amount of nuclear dna between wild and cultivated populations of goldenberries may be due to the domestication process of the species, since artificial selection during domestication affects the evolution of its genome (díez et al. 2013). the results reported in this study (table 2) for the wild (12,955 pg) and cultivated (13,262 pg) populations are higher than those reported by liberato et al. (2014) in colombian accessions (between 5.77 pg and 8.12 pg). the values of the cultivated populations in this study are remarkably close to those published by trevisani et al. (2018) in colombian (13.29 pg), brazilian (13.32 pg), and peruvian populations (13 pg). it is important to note that all of these studies report a 2n=48 chromosome number. this could imply that they are related to the same population of goldenberries, however, more research into the molecular cytogenetics of these groups is needed to prove it. according to the plant genome size database (pellicer and leitch 2020), estimations of the amount of 2c dna for the solanaceae family are 5.68 ± 5.27 pg. this would place the physalis peruviana l. populations studied here in the same family as the solanaceae species with large genome sizes. expansion of transposons, particularly retrotransposons with lengthy terminal repeats, has been identified as one of the main causes of genome size disparities in plants (michael 2014), in addition to genome doubling events during speciation (wendel et al. 2016). based on the amount of nuclear dna content in the goldenberry, all of our findings would be indicative of potential biodiversity and should be considered for future genomic investigations. this is the first study in peru to use molecular cytogenetics and genome size to compare wild and cultivated goldenberry populations. in conclusion, the molecular cytogenetic technique revealed a genetic variation in the number of 45s rdna sites between wild and cultivated populations, as well as different types of chromosomes containing rdna. this genetic difference was also detected when the amount of nuclear dna in the populations was compared. finally, the findings show no difference between populations from different places, both for cultivated and wild, suggesting that they probably originated from the same population. this study also underlines the utility of these methodologies for analyzing and characterizing the genetic variability of physalis peruviana l. populations, and it will be valuable for future genetic, genom132 marlon garcia paitan et al. ic, and phylogenetic research on this species, as well as the design of genetic improvement programs. geolocation information the cultivated populations of goldenberry were collected from the province of huanta (department of ayacucho) (gps: 13°01’50.9”s, 74°10’ 14.3”w) and the province of san pablo (department of cajamarca) (gps: 7°05’43.9”s, 78°49’13.9”w), the wild populations from the province of huamanga (department of ayacucho) (gps: 13°03’53.5”s, 74°09’13.1”w) and san marcos (department of cajamarca) (gps: 7°20’47.3”s, 78°06’0.3”w). the research procedure was developed in the faculty of biological sciences of the unmsm, lima (gps: 12°03’35.1”s, 77°04’55.6”w). acknowledgments we gratefully acknowledge the assistance of ing. gean carlo ciprian salcedo of the program for research and social projections in cereals and native grains of the universidad nacional agraria la molina in measuring the genome size using flow cytometry. the authors would like to express their gratitude to the agroandino company in cajamarca and the andrea family in ayacucho for providing us with goldenberry samples. finally, we would like to express our gratitude to the unmsm vice rectorate for research and postgraduate studies for providing study financing. funding the vice rectorate for research and postgraduate studies at the national university of san marcos supported this research under the research projects program for research groups (n°. rr-03202-r-18, project code b18100191) and the program for the promotion of undergraduate thesis. 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capsicum. genes & genetic systems. 74(4): 149–157. online supplementary material figure s1. chromosomes from the 12 best metaphases among the populations of goldenberries studied. https://drive.google.com/file/d/1fzgeergtuxs2gkd588n8m3cswzf 8ss1_/view?usp=sharing caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 3 2022 firenze university press chromosome mapping of repetitive dnas in the picasso triggerfish (rhinecanthus aculeatus (linnaeus, 1758)) in family balistidae by classical and molecular cytogenetic techniques kamika sribenja1, alongklod tanomtong1, nuntaporn getlekha2,* chromosome number of some satureja species from turkey esra kavcı1, esra martin1, halil erhan eroğlu2,*, fatih serdar yıldırım3 l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations selma tabur1,*, nai̇me büyükkaya bayraktar2, serkan özmen1 characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques kristel ramírez-matadamas1, elva irene cortés-gutiérrez2, sergio moreno-limón2, catalina garcía-vielma1,* contributions of species rineloricaria pentamaculata (loricariidae:loricariinae) in a karyoevolutionary context a cius¹, ca lorscheider2, lm barbosa¹, ac prizon¹, ch zawadzki3, la borin-carvalho¹, fe porto4, alb portela-castro1,4 cadmium induced genotoxicity and antioxidative defense system in lentil (lens culinaris medik.) genotype durre shahwar1,2,*, zeba khan3, mohammad yunus khalil ansari1 biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts and evaluation of their biosafety potential denisa manolescu1,2, georgiana uță1,2,*, anca șuțan3, cătălin ducu1, alin din1, sorin moga1, denis negrea1, andrei biță4, ludovic bejenaru4, cornelia bejenaru5, speranța avram2 polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel (foeniculum vulgare mill.) egizia falistocco methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. sazada siddiqui*, sulaiman a. alrumman comparative study and genetic diversity in malva using srap molecular markers syamand ahmed qadir1, chnar hama noori meerza2, aryan mahmood faraj3, kawa khwarahm hamafaraj4, sherzad rasul abdalla tobakari5, sahar hussein hamarashid6,* nuclear dna 2c-values for 16 species from timor-leste increases taxonomical representation in tropical ferns and lycophytes inês da fonseca simão1, hermenegildo ribeiro da costa1,2,3, helena cristina correia de oliveira1,2, maria helena abreu silva1,2, paulo cardoso da silveira1,2,* nuclear dna content and comparative fish mapping of the 5s and 45s rdna in wild and cultivated populations of physalis peruviana l. marlon garcia paitan*, maricielo postillos-flores, luis rojas vasquez, maria siles vallejos, alberto lópez sotomayor identification of genetic regions associated with sex determination in date palm: a computational approach zahra noormohammadi1,*, masoud sheidai2, seyyed-samih marashi3, somayeh saboori1, neda moradi1, samaneh naftchi1, faezeh rostami1 comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 133-144, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-169 citation: s. tarkesh esfahani, g. karimzadeh, m. reza naghavi (2020) in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.). caryologia 73(1): 133-144. doi: 10.13128/caryologia-169 received: february 15, 2019 accepted: february 23, 2020 published: may 8, 2020 copyright: © 2020 s. tarkesh esfahani, g. karimzadeh, m. reza naghavi. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 1 department of plant breeding and biotechnology, faculty of agriculture, tarbiat modares university (tmu), p. o. box. 14115-336, tehran, iran 2 department of agronomy and plant breeding, college of agricultural and natural resources, university of tehran, karaj, iran *corresponding author. e-mail: karimzadeh_g@modares.ac.ir abstract. papaver bracteatum lindl. grows as a wild perennial medicinal plant in northern iran and is known mainly for its high amounts of the pharmaceutically valuable alkaloid of thebaine. in vitro production of tetraploid p. bracteatum through colchicine treatment of imbibed seeds is reported. resulted tetraploid and mixoploid plants were effectively identified by chromosome counting and flow cytometry technique. the chromosome number in diploid and successfully induced tetraploids were confirmed to be 2n=2x=14 and 2n=4x=28, where their calculated 2c dna values were 6.15±0.03 and 11.95±0.07 pg, respectively. the highest induction efficiency was obtained by colchicine concentration of 0.05% and the treatment duration of 24 h. the effects of colchicine toxicity on plant survival and growth were proportional mainly to its concentration rather than duration of exposure to colchicine. tetraploid plants possessed significantly larger and less frequent leaf stomata as well as a larger cell size. these attributes may serve as criteria for preliminary screening of p. bracteatum populations for ploidy level. keywords. persian poppy, seed, tetraploid, colchicine, flow cytometry, 2c dna value. introduction persian poppy (papaver bracteatum lindl.; 2n=2x=14) is a wild perennial medicinal plant belonging to the papaveraceae family, section oxytona that grows natively in the alborz mountains in the north of iran in altitudes higher than 1800 m on the slopes facing the caspian sea (sharghi and lalezari 1967). it is mainly known for the high amounts of the valuable isoquinoline alkaloid thebaine as the main secondary metabolite in different organs particularly in roots and capsules (nyman and bruhn 1979; madam 2011) while some 20 other alkaloids are reported to be present in this species only in trace amounts (wu and dobberstein 1977). persian poppy seeds contain 45-48% oil rich in nutritionally valuable unsaturated fatty acids, so the other important usage of the plant is in the food industry (madam 2011). 134 saeed tarkesh esfahani, ghasem karimzadeh, mohammad reza naghavi seddigh et al. (1982) reported mean seed yield and seedoil yield of p. bracteatum to be 90 and 40 kg ha-1, respectively. successful polyploidy induction has been reported in various medicinal and ornamental plants with the aim of producing plants with improved agronomical, phytochemical or economically important characteristics. application of anti-mitotic agents such as colchicine (chen and gao 2007; sakhanokho et al. 2009; majdi et al. 2010; omidbaigi et al. 2010a, b; kaensaksiri et al. 2011; wu et al. 2011; marzougui et al. 2011; tavan et al. 2015; javadian et al. 2017; sadat noori et al. 2017), oryzalin (bouvier et al. 1994; thao et al. 2003; kermani et al. 2003; lehrer et al. 2008; sakhanokho et al. 2009), amiprophosmethyl (rodrigues et al. 2011) and trifluralin (eeckhaut et al. 2002) has been reported as the most common procedure for in vitro polyploidy induction in plants with colchicine being the most commonly used anti-mitotic agent. polyploid plants have been found to be valuable genetic resources due to possession of superior agronomic and phytochemical traits over their diploid progenitors. they have therefore attracted increasing attention in breeding programs, agriculture and medicinal plants industries. some of the more frequently reported advantages of induced polyploid plants include larger vegetative and reproductive organs such as leaves and flowers (chen and gao 2006; majdi et al. 2010; tang et al. 2010; gantait et al. 2011; miller et al. 2012), darker green leaves with a higher chlorophyll content and photosynthesis capacity (kulkarni and borse 2010; gantait et al. 2011), increased tolerance to environmental stresses (natuli and zobolo 2008), increased production of secondary metabolites (dhawan and lavania 1996; kaensaksiri et al. 2011; xu et al. 2013; tavan et al. 2015; javadian et al. 2017), increased expression of important genes and enzymes (adams et al. 2003; mishra et al. 2010; miller et al. 2012; xu et al. 2013) and delayed florescence time (gu et al. 2005). on the other hand, decreased fertility, increased level of mitotic disruptions and pollen sterility (liu et al. 2012) and facilitated biological invasion (beest et al. 2012) are reported as the most important unfavorable consequences of induced polyploidy. in vitro polyploidy induction in several papaveraceae plants has been previously studied mainly with the aim of obtaining an increased content of medicinal alkaloids. mishra et al. (2010) successfully induced tetraploidy in papaver somniferum l. and reported a significant enhancement in the morphine content and increased expression level of important genes involved in alkaloid biosynthesis pathway in tetraploid plants. milo et al. (1987) reported the induction of tetraploidy in p. bracteatum through colchicine treatment of apical meristems and subsequent production of triploid plants by crossing induced tetraploids to diploid plants. they suggested the ploidy breeding and tetraploidy induction as the most promising approach for development of thebaine-rich poppy lines. in their studies, they selected the plants with different ploidy levels through chromosome counting and cytological techniques. so an effective, easy and clearly described method for in vitro polyploidy induction in p. bracteatum and effective discrimination of tetraploid and mixoploid results based on flow cytometric (fcm) technique and 2c dna value is not reported yet. consequently, we report for the first time the in vitro production of autotetraploid p. bracteatum by colchicine treatment of imbibed seeds followed by fcm identification of polyploidy. the differences in the dna c-value, anatomical and morphological traits between diploid and induced tetraploid plants were also measured and their capability for being employed as reliable indicators of ploidy level in the plant populations was described. material and methods plant material seeds of mature persian endemic papaver bracteatum plants were collected in polour region (latitude 35° 52’ 16.99” n, longitude 52° 04’ 38.62” e, altitude 2489 ± 50 m) from hillsides of alborz mountains in northern iran. the seeds from each individual plant were collected separately and kept in small plastic bags. since p. bracteatum is a self-incompatible totally cross-pollinating plant (nyman and bruhn 1979), the seeds which originated from each individual plant were considered as progenitors for future colchicine-treated plants. the seeds of each maternal plant were collected separately so that the eventual comparisons between different ploidy levels could be conducted between two half-sib plants rather than two plants with completely different genetic backgrounds. for this purpose, all treated seeds in polyploidy induction assay were selected from the seeds of an individual maternal plant. polyploidy induction seeds obtained from one capsule from an individual p. bracteatum plant were sterilized by immersing in ethanol 70% (v/v) for three 30 s times, followed by sodium hypochlorite 5% (v/v) for 7 min. the seeds were then rinsed with distilled water for 5 min and transferred on 135in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) two layers of moistened filter papers in glass petri dishes and irrigated regularly with distilled water to allow water imbibition. germination process progressed up to the radicle emergence. after 8-10 days, the imbibed seeds (swollen seeds without a well-defined radicle apex) were transferred to tubular penicillin vials containing 1000 µl of colchicine solutions (sigma-aldrich corporation, mo, usa) with different concentrations comprising 0.00, 0.025, 0.050, 0.075, 0.10 and 0.20% (w/v). the vials were placed on a shaker with a rotation speed of 95 rpm and shaken for predetermined durations, including 4, 8, 12, 24, 36, 48, 72, and 168 h. at the end of treatment duration, the treated seeds were washed thoroughly with distilled water for 3 × 3-min and transferred to a 250-ml glass baby food jars containing 40 ml of ½ murashige and skoog (1962) medium with 1 g l-1 charcoal. the latter was used in order to prevent the seed phenolic compounds from interfering with the emergence and growth of new seedlings. seven treated seeds were cultured in each jar (representing one replication for a given treatment in data analysis). the seedlings were re-cultured once a month on a new medium with the above-mentioned composition and conditions. after three months, the plants with 6-7 true developed leaves were transferred to 500 g pots containing sand, commercial potting soil and vermiculite as the main components mixed with a ratio of 2:2:1 orderly. flow cytometry analysis one cm2 of young, healthy and fully green developed leaf material from each examined persian poppy plant together with about 1/3-1/2 in area of leaf material from pisum sativum cv. ‘citrad’ (2c dna = 9.09 pg; doležel et al. 1998) as an internal reference standard, were chopped into small pieces by a sharp razor blade in a 100 mm glass petri dish, containing one ml of woody plant buffer (wpb; loureiro et al. 2007). the resultant nuclear suspension was filtered through a partec (partec, münster, germany) 30 µm-nylon mesh, followed by treating with 50 µg ml-1 rnase (sigma-aldrich corporation, mo, usa) and 50 µg ml-1 propidium iodide (pi, fluka) as dna staining agent, and then incubated for two min at room temperature. to determine nuclear 2c dna amount, the nuclei suspension was analysed by a bd facscanto ii flow cytometer (bd biosciences, bedford, ma, usa), using bd facsdivatm software. output data were then transferred to a flowing software version 2.5.0 to be editable in partec flomax ver. 2.4e (partec, münster, germany). the measurements of relative fluorescence intensity of stained nuclei were performed on a linear scale, analysing at least 5,000 nuclei for each sample. the absolute dna amount of a sample was calculated based on the values of the g1 peak means (doležel et al. 2003, 2007; doležel and bartoš 2005; mahdavi and karimzadeh 2010; karimzadeh et al. 2010, 2011; abedi et al. 2015) as follows: sample 2c dna (pg) = (sample g1 peak mean/standard g1 peak mean) × standard 2c dna (pg) the analysed samples were classified based on the fcm results into diploid (2x), tetraploid (4x) and mixoploid (2x and 4x) samples. chromosome counting the ploidy status of the induction results were additionally confirmed by microscopic chromosome, counting in 10 randomly sampled plants from each class of ploidy level. root tips from confirmed diploid and tetraploid plants were pretreated with α-bromonaphthalene for 1 h at 24 °c, followed by rinsing with distilled water for 3 × 3 min. the pretreated roots were then fixed in carnoy solution (3:1 ethanol:glacial acetic acid) and stored at 4 °c followed by washing in distilled water, hydrolyzing with 1 n hcl for 8 min at 65 °c and staining with 1% (w/v) aceto-orcein for 1 h. treated 1-2 mm long root tips were excised and squashed on slide glass, with a drop of 45% (v/v) acetic acid, and protected with a cover slip. chromosome counts were analysed by observation under a bx51 olympus light microscope (olympus optical co., tokyo, japan), equipped with an olympus dp12 digital camera (olympus optical co., tokyo, japan) using wh10x (fn22) eyepiece and 100x objective lens. anatomical and morphological analysis to assess any possible relations between anatomical traits and ploidy level in p. bracteatum, leaf cell size as well as the dimensions and the frequency of leaf stomata were measured on the lower epidermis of 20 fully developed leaves each taken from an individual plant in each confirmed class of ploidy. data on stomata dimensions were measured on 60 stomata from leaves of the plants in each class of ploidy level. the stomata were visualized by the impression method (majdi et al. 2010; tavan et al. 2015). the density of the stomata was counted at 200x and the area of stomata were measured at 1000x magnification, using high resolution microscopic digital photographs taken with an olympus dp12 digital camera (olympus optical co., tokyo, japan) interfaced to 136 saeed tarkesh esfahani, ghasem karimzadeh, mohammad reza naghavi an olympus bx50 (olympus optical co., tokyo, japan) microscope. the stomata and the cells in three random microscopic fields per each leaf were counted and measured. the measurements of stomata dimensions, including area and large diameter (length) as well as the cell area were determined, using the captured images and the uthscsa imagetool program (university of texas health science center at san antonio, texas, usa). data analysis the data regarding the effect of time and concentration of the colchicine treatment on survival rate and polyploidy induction were analysed through a completely randomized design (crd) with three replications. mean comparison was carried out by using least significant difference (lsd) test at p<0.05. seeds germination percentage was calculated as gp = (number of germinated seeds / total planted seeds) * 100. tetraploidy induction efficiency was assessed using the method reported by bouvier et al. (1994) and majdi et al. (2010) as follows: induction efficiency = % seedling survival × % tetraploidy induction for induction efficiency calculation, a seedling was considered as survived if it persisted for three months after being treated with colchicine and had enough green leaf area to be analysed by fcm. the resultant data were first tested for normality with the kolmogorov-smirnov test. the logarithmic transformation was then used for both stomata area and stomata frequency data. mean comparisons between two different ploidy levels for anatomical traits were conducted, using student’s t test. all statistical analysis were conducted, using spss 18 (chicago: spss inc., 2009). results survival and the growth of colchicine-treated seeds the toxic effects of colchicine on the treated persian poppy (papaver bracteatum lindl.) plants were different in terms of their survival and consecutive growth. some treated seeds could no longer survive colchicine treatment, as they remained as dark necrotic non-emerging seeds without any root appearance. whereas some others could survive in the colchicine and remained alive initially at the time of transferring to the medium, but their seedlings could not keep normal growing and turned to dark brown-colored necrotic tissues during later three weeks after being transferred. the degree of mortality caused by different colchicine either concentrations or durations were variable. the toxic effects of colchicine treatment on the survival and on the growth of the seeds were therefore assessed 30 days after treatment induction. the general colchicine-induced mortality was divided and expressed as two different criteria including seed mortality and seedling mortality (table 1). seed mortality was calculated based on non-emerging seeds which could not survive in the colchicine treatment, while seedling mortality was reflected by the emerged seedlings with inhibited successive growth. the results showed that increased colchicine concentration significantly increased the level of lethal effects (table 1, fig. 1), so that only 4.76±2.38% seed survival but no subsequent growth was observed in the seeds treated with the 0.2% (w/v) colchicine solution. while no seedling growth was identified in the 0.2% colchicine treatment, the survival and the growth in the seeds treated with other concentable 1. results of the analysis of variance (anova) for the effect of colchicine concentration and treatment duration on the mortality of papaver bracteatum seeds. source of variation mean squares seed mortality seedling mortality colchicine concentration (c) 13097.26** 13173.03** treatment duration (d) 126.22n.s. 162.97* c*d interaction 116.51* 70.15n.s. **, * and n.s. indicate significant at 1%, 5% level and and not significant, respectively. figure 1. effect of colchicine concentration on seed survival and seedling growth of treated papaver bracteatum explants. all values are in percentage. mean values specified by the same latter are not significantly different at p<0.05 by lsd test. letters with a prime symbol designate mean differences in seedling growth. 137in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) table 2. effect of colchicine concentration and treatment duration on the percentage (mean ± standard error) of seed mortality, seedling mortality, diploid (2x), mixoploid (2x+4x), and tetraploid (4x) explants in papaver bracteatum. colchicine concentration (%) exposure duration (h) seed mortality (%) seedling mortality (%) 2x (%) 2x + 4x (%) 4x (%) 0.000 4 0.00 ± 0.00 4.76 ± 4.76 95.24 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 8 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 12 0.00 ± 0.00 4.76 ± 4.76 95.24 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 24 0.00 ± 0.00 4.76 ± 4.76 95.24 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 36 0.00 ± 0.00 9.52 ± 9.52 90.48 ± 9.52 0.00 ± 0.00 0.00 ± 0.00 48 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 72 0.00 ± 0.00 4.76 ± 4.76 95.24 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 168 0.00 ± 0.00 14.29 ± 8.25 85.71 ± 8.25 0.00 ± 0.00 0.00 ± 0.00 0.025 4 4.76 ± 4.76 4.76 ± 4.76 90.48 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 8 14.29 ± 8.25 0.00 ± 0.00 85.71 ± 8.25 0.00 ± 0.00 0.00 ± 0.00 12 0.00 ± 0.00 9.52 ± 9.52 90.48 ± 9.52 0.00 ± 0.00 0.00 ± 0.00 24 14.29 ± 8.25 4.76 ± 4.76 80.95 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 36 0.00 ± 0.00 4.76 ± 4.76 95.24 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 48 0.00 ± 0.00 14.29 ± 8.25 85.71 ± 8.25 0.00 ± 0.00 0.00 ± 0.00 72 0.00 ± 0.00 4.76 ± 4.76 95.24 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 168 4.76 ± 4.76 9.52 ± 4.76 85.71 ± 8.25 0.00 ± 0.00 0.00 ± 0.00 0.050 4 9.52 ± 4.76 14.29 ± 8.25 33.33 ± 17.17 19.05 ± 4.76 23.81 ± 12.60 8 28.57 ± 8.25 4.76 ± 4.76 19.05 ± 4.76 23.81 ± 12.60 23.81 ± 12.60 12 28.57 ± 14.29 9.52 ± 4.76 0.00 ± 0.00 23.81 ± 4.76 38.10 ± 9.52 24 4.76 ± 4.76 4.76 ± 4.76 23.81 ± 17.17 33.33 ± 4.76 33.33 ± 17.17 36 23.81 ± 9.52 9.52 ± 9.52 19.05 ± 12.60 19.05 ± 4.76 28.57 ± 16.50 48 19.05 ± 9.52 19.05 ± 12.60 38.10 ± 12.60 14.29 ± 0.00 9.52 ± 9.52 72 0.00 ± 0.00 19.05 ± 4.76 57.14 ± 0.00 23.81 ± 4.76 0.00 ± 0.00 168 28.57 ± 8.25 19.05 ± 4.76 28.57 ± 16.50 14.29 ± 0.00 9.52 ± 9.52 0.075 4 28.57 ± 14.29 14.29 ± 0.00 9.52 ± 9.52 33.33 ± 4.76 14.29 ± 8.25 8 9.52 ± 4.76 19.05 ± 4.76 14.29 ± 8.25 23.81 ± 4.76 33.33 ± 4.76 12 42.86 ± 8.25 4.76 ± 4.76 14.29 ± 0.00 28.57 ± 8.25 9.52 ± 9.52 24 9.52 ± 4.76 9.52 ± 4.76 23.81 ± 4.76 23.81 ± 4.76 33.33 ± 9.52 36 38.10 ± 4.76 9.52 ± 4.76 4.76 ± 4.76 23.81 ± 4.76 23.81 ± 12.60 48 33.33 ± 4.76 14.29 ± 8.25 9.52 ± 4.76 19.05 ± 4.76 23.81 ± 4.76 72 38.10 ± 4.76 14.29 ± 8.25 19.05 ± 9.52 19.05 ± 4.76 9.52 ± 4.76 168 33.33 ± 9.52 14.29 ± 0.00 19.05 ± 12.60 9.52 ± 4.76 23.81 ± 4.76 0.100 4 33.33 ± 9.52 19.05 ± 4.76 0.00 ± 0.00 23.81 ± 9.52 23.81 ± 4.76 8 33.33 ± 4.76 19.05 ± 12.60 9.52 ± 4.76 14.29 ± 0.00 23.81 ± 9.52 12 47.62 ± 4.73 14.29 ± 4.25 0.00 ± 0.00 14.29 ± 0.00 23.81 ± 4.76 24 23.81 ± 9.52 28.57 ± 8.25 4.76 ± 4.76 19.05 ± 4.76 23.81 ± 4.76 36 28.57 ± 8.25 33.33 ± 12.60 9.52 ± 9.52 14.29 ± 0.00 14.29 ± 8.25 48 28.57 ± 14.29 28.57 ± 8.25 14.29 ± 8.25 14.29 ±0.00 14.29 ± 8.25 72 38.10 ± 9.52 28.57 ± 8.25 4.76 ± 4.76 9.52 ± 4.76 19.05 ± 4.76 168 47.62 ± 9.52 28.57 ± 0.00 9.52 ± 4.76 4.76 ± 4.76 9.52 ± 4.76 0.200 4 76.19 ± 12.60 23.81 ± 12.60 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 8 90.48 ± 9.52 9.52 ± 9.52 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 12 95.24 ± 4.76 4.76 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 24 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 36 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 48 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 72 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 168 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 138 saeed tarkesh esfahani, ghasem karimzadeh, mohammad reza naghavi trations varied widely, where the mean germination percentage and the mean percentage of developed seedlings in the 0.1% treatment were 64.88±3.22 and 39.88±2.72, respectively (table 2). these values were 95.24 ± 1.86 and 88.69 ± 2.27 in the 0.025% treatment, respectively (fig. 1). flow cytometry analysis of ploidy level the ploidy level of colchicine-treated plants was determined by fcm analysis. all treated plants with apparently normal growth and development were classified into three main classes including diploids (2x), mixoploids (2x+4x) and tetraploids (4x). as shown in related histograms (fig. 2), diploid and tetraploid plants revealed a peak at the position of channels 50 and 95 of the relative fluorescent intensity respectively, while mixoploids revealed two peaks with variable heights at both channels 50 and 95. the 2c dna contents of the diploid and induced tetraploid plants were estimated as 6.15±0.03 (fig. 2a) and 11.95±0.07 pg (fig. 2c), respectively. the ploidy status of the resultant events was additionally confirmed by microscopic chromosome counting in plants with different ploidy levels. it was indicated that all diploid plants had a chromosome number of 2n=2x=14 (fig. 3a), whereas all tetraploids had 2n=4x=28 (fig. 3b). in the present study, significant differences between induction treatments were seen based on the induction efficiency data. the results showed that both higher concentrations of colchicine and longer durations of exposure to colchicine resulted in a significantly larger percentage of tetraploid plants (table 3). the highest induction efficiency (31.29) was yielded by 0.05% (w/v) colchicine concentration at exposure duration of 24 h. the next two most efficient treatments were 0.75% (w/v)-24 h and 0.05% (w/v)-12 h with induction efficiency values of 27.89 and 25.85 respectively (figs. 4, 5). anatomical and morphological characteristics stomata data measured on the leaves of plants in each class of ploidy showed that the stomata size in figure 2. flow cytometric histogram of the relative fluorescence intensity of nuclei isolated from papaver bracteatum plants. histograms show the nuclei isolated form diploid (a), mixoploid (b), and induced tetraploid (c) plants. the s in each histogram indicates the peak resulted by the cells of the pisum sativum cv. ‘citrad’ (2c dna=9.09 pg) used as the internal standard. figure 3. chromosome numbers of surveyed papaver bracteatum plants. number of chromosomes in diploid (2n=2x=14) (a) and artificially induced tetraploid (2n=4x=28) (b) plants are compared. table 3. results of the analysis of variance (anova) for the effect of colchicine concentration and treatment duration on the polyploidy induction rate in papaver bracteatum. source of variation mean squares colchicine concentration (c) 16.98** treatment duration (d) 2.57* c*d interaction 1.11n.s. **, * and n.s. indicate significant at 1%, 5% level and and not significant, respectively. (a) (a) (b) (b) (c) 139in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) tetraploid plants was larger than that in diploids (p<0.01; figs. 6a, 6b). the average stomata area for the diploid and tetraploid (figs. 7a, b, respectively) plants was 531.44±24.02 and 868.98±55.66 µm2 respectively indicating a 63.51% increase in stomata area of tetraploid plants. it was also included that the stomata length in tetraploid plants (40.46±1.33 µm) was 30.06% larger than that in diploids (31.10±0.89 µm). in addition, a significant difference was identified between diploid and polyploid plants in the stomatal density (p<0.01), where the average number of stomata per square millimetre in the leaves of diploid and tetraploid (figs. 7c, d, respectively) plants was 236.18±10.54 and 157.14±3.78, respectively (fig. 6c). in other words, tetraploidy induction caused a 50.3% decrease in stofigure 6. comparison of anatomical traits between diploid and tetraploid plants of papaver bracteatum at the cellular level. the stomata length (a), stomata area (b), stomata frequency (c), and stomata cell area (d) are significantly changed in induced tetraploid plants. mean values specified by different letters are significantly different at p<0.05 by students’s t test. bars show standard errors. figure 4. effect of colchicine concentration and treatment duration on tetraploidy induction efficiency in papaver bracteatum. bars show standard errors. figure 5. effect of colchicine concentration and treatment duration on the contribution (%) of seed mortality, seedling mortality, diploid, mixoploid, and tetraploid explants produced during polyploidy induction in papaver bracteatum. figure 7. impressions illustrating the size and density of the stomata in the leaf lower epidermis of papaver bracteatum plants. the smaller stomata size in diploid (a) than in induced tetraploid (b) plants and the lower stomata density in diploid (c) than in tetraploid (d) plants are illustrated. (a) (a) (b) (b) (c) (c) (d) (d) 140 saeed tarkesh esfahani, ghasem karimzadeh, mohammad reza naghavi mata density in studied p. bracteatum plants. data regarding the cell size showed that the average leaf cell area in diploid and tetraploid p. bracteatum plants was 1187.20±73.93 and 1515.12±118.31, respectively indicating a 27.62% increase in epidermal cell area of tetraploid plants (p < 0.05; fig. 6d). unlike in the stomatal morphology, tetraploid plants exhibited no remarkable differences in visible morphological traits such as plant height, shoot or leaf thickness and growth rate compared to their diploid counterparts (fig. 8). discussion the results regarding the colchicine effects on survival of seeds and seedlings are in agreement with those reported in various plant species e.g. berberis thunbergii (lehrer et al. 2008), tanacetum parthenium (majdi et al. 2010) and thymus persicus (tavan et al. 2015), indicating that the toxic effects of colchicine as an anti-mitotic chemical is largely influenced by the colchicine concentration (table 1). the toxic effects of colchicine on the treated persian poppy were expressed as two different criteria including seed mortality and seedling mortality. the results showed that increased colchicine concentration significantly increased the level of both lethal effects and decreases the ability of treated plants to survive and grow (table 2). the c-value index defined as the dna content of an unreplicated haploid chromosome complement (i.e. a gamete) is highly useful in systematics, genome size estimation and many other biological fields related to eukaryotic organisms (doležel and bartoš 2005). during normal mitosis two daughter cells each with 2c dna content is formed (doležel et al. 2003). therefore, the 2c dna content of a tetraploid cell which is resulted by mitotic arrest is expected to be about two times that of the progenitor diploid cells. in the present study, the calculated 2c dna contents of the diploid and tetraploid plants were estimated as 6.15±0.03 (fig. 3a) and 11.95±0.07 pg (fig. 3b), respectively, indicating the successful induction of tetraploidy. furthermore, these results suggest the effectiveness of fcm-based analysis as a rapid and reliable strategy for discriminating p. bracteatum from other identified or unidentified papaver species with similar morphological traits and different 2c dna values. tetraploid induction efficiency has frequently been used in previous studies (bouvier et al. 1994; lehrer et al. 2008; majdi et al. 2010) as a measure for identifying the most effective treatments capable of inducing complete and stable polyploidy. it is known as reliable index because it takes into account not only the rate of conversion of diploidy tetraploidy, but the survival rate of successful tetraploid events (lehrer et al. 2008). our results showed that both higher concentrations of colchicine and longer durations of exposure to colchicine resulted in a significantly larger percentage of tetraploid seedlings, but their interaction effect was non-significant (table 3). there are several reports about the evaluation of the effects by the concentration of the anti-miotic agent and treatment duration as the two main determining factors in polyploidy induction efficiency in different plant types. for instance, stanys et al. (2006) working on chaenomeles japonica reported that with both colchicine and oryzalin as the anti-mitotic agents, the efficiency of ploidy induction was mainly dependent on the concentration of anti-mitotic agent rather than its exposure duration. on the other hand, several authors suggested that the polyploidy induction efficiency is associated with both optimum concentration and the duration of anti-miotic (gu et al. 2005; tang et al. 2010; tavan et al. 2015). the results of fcm analysis showed that mixoploid plants could be expected to form a large contribution of polyploids sometimes as high as 33% of inducfigure 8. comparison of visible morphological traits at the whole plant level. the illustrated diploid (a) and tetraploid (b) papaver bracteatum plants are all at the same age (195 d) and acclimated under similar environmental condition. (a) (b) 141in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) tion results obtained by in vitro polyploidy induction in p. bracteatum (table 2; fig. 5). in polyploidy induction works, high percentage of mixoploid results are generally known as a drawback of the procedure because their unstable polyploidy state often reverts partially or totally to the diploid condition after successive cell division cycles. it occurs mainly because the remaining diploid cells proliferate at higher rates than the tetraploid ones (mergen and lester 1971). during artificial polyploidy induction, colchicine influences actively dividing cells in the treated tissues and polyploidization therefore occurs unequally among explant cells, leading to the occurrence of mixoploids and chimeras (wan et al. 1989). accordingly, a low growth rate and intrinsically stunted development in treated plants are expected to aggregate these effects particularly in short exposure durations leading to a higher proportion of mixoploid events among polyploidy induction results. chakraborti et al. (1998) suggested that in in vitro induction methods, the occurrence of mixoploids may have been minimized by growing the treated plants under more favorable conditions. they stated that the uniformity of environmental factors like temperature and photoperiod may favor the synchronous division of meristematic cells and help minimize the mixoploid events leading to a high tetraploidy rate (chakraborti et al. 1998). the ratio of tetraploid to diploid cells based on the analysed fcm data can be calculated as a measure of the contribution of tetraploid cells within a mixoploid event. the values higher than one indicate a higher percentage of tetraploid cells than the diploids. in the present study, the obtained values indicated a high degree of variability in polyploidy induction capability of colchicine in p. bracteatum (detailed data not shown because of the large number of hits and lack of a significant interaction). in addition, it was interestingly observed that varying values of the ratio of tetraploid to diploid cells might be obtained by the same concentration-duration combination. for example, both values of 0.19 and 1.75 which indicate a low and a high contribution of tetraploid cells respectively, were seen in the mixoploid events resulted by the 0.05%24h treatment combination. so despite of favorable and controlled environmental factors within in vitro induction and growth environments, large differences between induction capabilities of certain concentration-duration combinations were revealed by the variable degrees of mixoploidy obtained by the same treatment combination. these results indicate that the effectiveness of anti-mitotic agent in polyploidy induction in p. bracteatum can be determined mainly by the explant and cell-related factors rather than those related to the induction environment. however, the rather high percentage of mixoploid events yielded by in vitro colchicine treating of p. bracteatum explants as well as the wide range of the ratio of tetraploid to diploid cells within mixoploid events can be explained by the incomplete effects of colchicine on meristematic cells in the treated explants. these incomplete effects could be aggravated by the intrinsically stunted growth of p. bracteatum. indeed, short exposure times and lower concentrations of the anti-mitotic agent during polyploidy induction may in turn decrease the chance for complementation of polyploidization process leading to a higher degree of mixoploidy (wan et al. 1989). a recent study by tavan et al. (2015) on thymus persicus has showed that the mixoploid events which were produced during polyploidy induction in thymus persicus had a considerable capability for producing pharmaceutically important compounds. they reported significantly increased production of medicinal triterpenoids in both tetraploid and mixoploid results as compared to their diploid progenitors, where mixoploid plants interestingly yielded significantly higher contents of betulinic acid, oleanolic acid and ursolic acid even than successfully induced tetraploids (tavan et al. 2015). additionally, successful generation of tetraploid plants from mixoploid progenitors using tetraploid cells of leaf callus through callus-based techniques and tissue culture strategies has been previously reported in various plant species such as humulus lupulus l. (roy et al. 2001), astragalus membranaceus (chen and gao 2007) and echinacea purpurea l. (dahanayake et al. 2010). likewise shao et al. (2003) reported that further subculture of mixoploid events resulted by in vitro colchicine treatment of shoots in punica granatum l. resulted in their separation to tetraploid and diploid progenies. this strategy has been recommended to be employed when an anti-mitotic agent generates a high degree of mixoploid events during tetraploidy induction. in general, the mixoploids can therefore be considered as valuable sources of genetic material in ploidy breeding programs. they particularly can be employed for certain plant species which are expected to produce high numbers of mixoploid events during polyploidy induction works. application of tissue culture techniques in p. bracteatum has been well established, where successful in vitro regeneration pf this species using callus derived from seedlings (ilahi and ghauri 1994), roots, seeds and cotyledons (rostampour et al. 2010) as well as through cell suspension culture (farjaminezhad et al. 2013) and hairy roots (sharafi et al. 2013) have previously been reported. therefore, it allows combination polyploidy induction strategies and tissue culture techniques to achieve higher goals in p. bracteatum breeding programs. 142 saeed tarkesh esfahani, ghasem karimzadeh, mohammad reza naghavi these obtained results indicating increased stomata size (fig. 6a) and decreased stomata frequency (fig. 6c) in tetraploid plants as compared to their diploid progenitors are in agreement with those of several previous reports in different plant types. furthermore, differences in stomata size and density are frequently reported to successfully discriminate plants with different ploidy levels, where polyploid plants are often known to have, on average, a lower stomata number per leaf area unit and increased size of the stomata and guard cells (de carvalho 2005; tang et al. 2010; gantait et al. 2011; aina et al. 2012) as well as an increased number of chloroplasts in stomatal guard cells (ewald et al. 2009). gantait et al. (2011) suggested that the lower density of stomata in polyploid plants was due to the larger stomata and epidermal cell size, as well as reduced stomata differentiation. the cell size is also reported to be related to polyploidy level and to be significantly different between induced tetraploid plants and their diploid progenitors (melaragno et al. 1993). it is suggested as a potential anatomical indicator of ploidy level being capable of discriminating plants in a mixed population of tetraploid and diploids (zeng et al. 2006). the results regarding the cell size in the plants with different ploidy levels showed that the tetraploid p. bracteatum plants had significantly larger leaf epidermal cells than diploid plants (figs. 6b, 6c). these results confirm previous reports where larger cell dimensions were reported for tetraploid plants as compared to their diploid progenitors in various plant species such as fortunella crassifolia, citrus sinensis (zeng et al. 2006), tanacetum parthenium (majdi et al. 2010) and thymus persicus (tavan et al. 2015). the results obtained in this study indicated that certain anatomical traits such as leaf epidermal cell and stomata size and frequency may serve as reliable criteria for screening of p. bracteatum plants for ploidy level. however, other routinely suggested anatomical, morphological or physiological charachteristics such as chlorophyll florescence, flower size and morphology, pollen grain size, chloroplast density, enzymatic activity, etc. have to be evaluated precisely before being employed as potential indicators of ploidy level in quick evaluation and successful screening of large numbers of p. bracteatum plants. because in the present study, unlike in the stomatal morphology, the colchicine-treated plants did not exhibit any remarkable differences in visible morphological traits such as plant height, shoot or leaf thickness and growth rate (fig. 8). these observations were in agreement with those reported by milo et al. (1987) who stated that there were no significant differences between p. bracteatum plants with different ploidy levels in the morphological traits such as plant height, flower size or in the height of the flowering stem. however, the artificially induced tetraploid plants of p. bracteatum were reported to flower later than diploid plants. their capsules also matured significantly later than their diploid plants (milo et al. 1987). the lack of visible morphological distinctions between diploid and tetraploid p. bracteatum plants might be attributed to the high morphological variation observed in this species particularly in its natural habitat. wild p. bracteatum plants naturally exhibit high variation in plant size, growth rate and other visible characteristics. so, the new polyploid variants are likely to still fall within the wide variation range that already exists in wild p. bracteatum populations. consequently, like their diploid progenitors, tetraploid plants are expected to reveal a wide range of morphological variation. hence, ploidy statuses of the induction results need to be confirmed by quick, easy and reliable criteria such as flow cytometry techniques rather than morphological measures. in conclusion, polyploidy was successfully induced in diploid persian poppy (papaver bracteatum lindl.) through colchicine treatment of newly germinated seeds. tetraploid and mixoploid progenies were quickly and effectively recognized by fcm technique and the 2c dna content. both colchicine concentration and exposure duration were known as determining factors in success of in vitro tetraploidy induction. morphological traits like stomata size and frequency and cell size were significantly associated with ploidy level in persian poppy and were known as reliable criteria for preliminary screening of mixed populations based on ploidy level. further research works are in progress to study the ploidy level dependent secondary metabolites and potentially major candidate 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pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 73(3): 13-19, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/10.13128/caryologia-197 caryologia international journal of cytology, cytosystematics and cytogenetics citation: i̇. genç, ş. kültür (2020) karyological analysis of twelve euphorbia species from turkey. caryologia 73(3): 13-19. doi: 10.13128/10.13128/caryologia-197 received: march 26, 2019 accepted: april 13, 2020 published: december 31, 2020 copyright: © 2020 i̇. genç, ş. kültür. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyological analysis of twelve euphorbia species from turkey i̇lker genç*, şükran kültür department of pharmaceutical botany, faculty of pharmacy, i̇stanbul university, i̇stanbul, turkey *corresponding author. e-mail: gencilker1@gmail.com abstract. karyotypes of 12 euphorbia species were studied and described for the first time; euphorbia cheiradenia, e. pannonica, e. pestalozzae, e. petrophila, e. pisidica, e. thessala and e. yildirimli. karyological analyses indicate relationships among the species with respect to their asymmetry indices. most of the investigated taxa are diploids with 2n = 2x = 18. e. macroclada and e. smirnovii showed tetraploid cytotypes 2n = 4x = 36. all karyotypes are symmetrical, consisting of metacentric and submetacentric chromosomes. the chromosomes range in size from 0.79 µm to 2.20 µm. the total haploid chromosome length (thl) ranges from 8.75 μm (e. terracina) to 16.78 μm (e. petrophila). principal coordinate analysis with five uncorrelated parameters was performed to determine the karyological relationships among the taxa. keywords: chromosome number, karyotype asymmetry, pithyusa, esula, tetraploid, karyosystematics. introduction genus euphorbia (euphorbiaceae), with over 2000 species, is the secondlargest genus of flowering plants in the world (bruyns et al. 2006; horn et al. 2012). the use of euphorbia species as medicine and poison are of greatest biocultural importance and the most-valued medicinal use of euphorbia species is in the treatment of digestive and respiratory complaints, inflammation and injuries (ernst et al. 2015). additionally, euphorbia taxa have been used in turkish folk medicine to treat rheumatism, swelling and especially as a wart remover (baytop 1984). there are also various biological effects as antioxidant, antimicrobial and wound healing activities (barla et al. 2006, 2007; özbilgin et al. 2018, 2019). the genus is represented in turkey by two subgenera, e. subg. chamaesyce raf. and e. subg. esula pers., with a total of 120 taxa (öztekin 2012; genç and kültür 2016). euphorbia subg. esula comprises about 480 species, most of which are annual or perennial herbs (riina et al. 2013). according to the recent classification of riina et al. (2013), the subgenus is represented by 14 sections in the turkey (genç and kültür 2018). the chromosome numbers in these two subgenera are very variable. various chromosome numbers have been reported for the subg. esula (2n = 10, 14 i̇lker genç, şükran kültür 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 36, 40, 42, 44, 56, 60, 64, 72) and sect. pithyusa (2n = 16, 18, 28, 36, 40, 72) worldwide (riina et al. 2013). the chromosome numbers of 64 taxa, which are distributed in turkey naturally have not been determined yet. up to now, the chromosome numbers of 12 euphorbia taxa were reported from turkey (radcliffesmith 1976; strid 1987; kesercioglu et al. 1990; iyer 1991; vicens et al. 1991; rice et al. 2015; genç and kültür 2017). considering that the number of chromosomes of the vast majority of these species is not yet clear, there is a remarkable gap in this area. this study aimed to determine the chromosome numbers and karyomorphology of 11 perennial euphorbia sect. pithyusa species distributed in turkey. in addition, e. terracina (sect. pachycladae) was also included in the present study as an external group. materials and methods twelve euphorbia species were analysed in this study. seeds of 12 species were collected from mature capsules in their natural habitats as given in table 1. voucher specimens are deposited in i̇stanbul university, faculty of pharmacy herbarium (iste). for each population, 9-27 mature seeds were selected from at least 5 individuals (inflorescence). the seeds germinated on wet filter paper in petri dishes at room temperature. the root tips meristems were excised at 9-9.30 am. mitotic chromosomes were prepared from root tips and pre-treated with 8-hydroxyquinoline at +4 °c for 24 h. roots were fixed for a minimum of 2 h in absolute ethanol:glacial acetic acid, (3:1,v/v), hydrolysed at 60 °c in 1 n hcl for 15 min and stained with feulgen reagent. finally, root tips were squashed in a drop of 1% aceto-orcein. permanent microscopic slides were prepared with entellan (rapid mounting medium / merck darmstadt germany). microphotographs of good quality metaphase plates were taken using an olympus bx53 (tokyo, japan) microscope equipped with a high-resolution digital camera. metaphase observations and chromosome measures were made using the image analysis systems kameram (argenit microsystems, i̇stanbul, turkey). the chromosome number and karyotype details were studied in five to fourteen well-spread metaphase plates from different individuals; mean values were used for the analysis. chromosome pairs were identified and arranged on the basis of chromosome/chromatid length and any other evident karyomorphological features. the nomenclature used for describing karyotype followed levan et table 1. localities and voucher data of euphorbia taxa examined in the present study. species voucher specimens previous counts (rice et al. 2014) e. cheiradenia boiss. & hohen. şanlıurfa, tektek mountain national park, 460m, 18.6.2015, i̇.genç 2398, ş.kültür. -e. macroclada boiss. eskişehir-antalya road, roadsides, 10.8.2015, i̇.genç 2472. 18 e. niciciana borbás ex novák karabük, bolkuş village, 220 m, 03.vi.2015, i̇.genç 2290, g. ecevit genç. 18, 18+1 e. pannonica host i̇stanbul, dağyenice-kalfaköy, roadsides, 110m, 18.vii.2016, i̇.genç 2477, g. ecevit genç. -*e. pestalozzae boiss. antalya, saklıkent, 1780m, 30.vii.2016, i̇.genç 2499. -e. petrophila c.a.meyer kastamonu, kastamonu-sinop roadsides, 720m, 14.vii.2015, i̇.genç 2419, g. ecevit genç. -*e. pisidica hub.-mor. & m.s.khan burdur, altınyayla-gölhisar road, dirmil pass, 1585m, 23.vi.2014, i̇.genç 2114, g. ecevit genç. -e. seguieriana necker ağrı, between doğubeyazıt-çaldıran, roadsides, 2600m, 24.vii.2015, i̇.genç 2453. 16, 18, 40 e. smirnovii geltman erzincan, ergan mountain, 1510m, 25.vii.2015, i̇.genç 2468, a. kandemir. 18 (genç & kültür 2017) e. thessala (form.) degen & dörf. kırklareli, kırklareli-pınarhisar roadside, 190m, 5.vi.2015, i̇.genç 2304, g. ecevit genç. -*e. yildirimlii dinç eskişehir, sivrihisar, aşağıkepen village, gypsum slopes, 900m, 26.viii.2014, i̇.genç 2267, g. ecevit genç. -e. terracina l. bursa, mudanya, söğütlüpınar village, s.l., 7 vıı 2014, i̇.genç 2192, s. yüzbaşıoğlu 18, 20, 36 * the species endemic to turkey. 15karyological analysis of twelve euphorbia species from turkey al. (1964). to determine the karyological relationships among taxa, we performed principal coordinate analysis (pcoa) with fi ve uncorrelated parameters as suggested by peruzzi and altınordu (2014). th ese parameters are basic chromosome number (x), total haploid length (thl), mean centromeric asymmetry (mca) it is calculated as the mean (l-s)/(l+s)×100 where, for each chromosome, l is the length of long arm and s is the length of short arm; coeffi cient of variation of chromosome length (cvcl=(l+s)×100) and coeffi cient of variation of centromeric index (cvci =s/(l+s) ×100) (paszko 2006; peruzzi et al. 2009; zuo and yuan 2011; peruzzi and eroğlu 2013). th e soft ware past 3.03 (hammer et al. 2001; hammer 2018) was used to perform this analysis. seed and gland morphology in the classifi cation of the genus euphorbia is one of the most important taxonomic characters. th erefore, in order to evaluate the resulting pcoa graphic in terms of the seed and gland morphological characters, some symbols are given to the studied species (table 2). table 2. pcoa graphic symbols of some important taxonomic characteristics for euphorbia. seed surface smooth seed surface not smooth cyathial glands horned cyathial glands hornless figure 1. somatic chromosomes of the studied taxa. (a) e. cheiradenia; (b) e. macroclada; (c) e. niciciana; (d) e. pannonica; (e) e. pestalozzae; (f ) e. petrophila; (g) e. pisidica; (h) e. seguieriana; (i) e. smirnovii; (j) e. thessala; (k) e. yildirimli; (l) e. terracina. (scale bars 2 μm). 16 i̇lker genç, şükran kültür mitotic metaphase chromosomes are given in figure 1. ideograms of these taxa are arranged in order of centromere position and then decreasing the length of homologue chromosome pairs (figure 2). results karyomorphological details (shortest chromosome length; longest chromosome length; mean long arm length; mean short arm length; karyotype formula) of 12 species of euphorbia are listed in table 3. the chromosome numbers, total haploid length, mean centromeric asymmetry, coefficient of variation of chromosome length and the coefficient of variation of centromeric index of the species are also summarized in table 4. metaphase plates and their related ideograms of the studied species are presented in figures 1 and 2. karyotype analysis revealed the basic chromosome number x=9 for all species. most of the species showed diploid cytotypes 2n = 18 but e. macroclada and e. smirnovii showed tetraploid cytotypes 2n = 4x = 36 (figure 1). the chromosomes were mainly metacentric with centromeres localized in the median position. the karyotypes of four species (e. cheiradenia, e. macroclada, e. smirnovii, e. yildirimli) included one submetacentric chromosome. only karyotype of e. petrophila included two submetacentric chromosomes. the ratio of the shortest and the longest chromosome lengths ranged from 0.79 μm (e. smirnovii) to 2.20 μm (e. petrophila). the ratio of the shortest and the longest total haploid chromosome length (thl) ranged from 8.75 μm to 16.78 μm in e. terracina and e. petrophila, respectively. the smallest mean short arm length (q) was observed in e. terracina (0.44 μm) and e. seguieriana (0.51 μm), the largest mean long arm length (p) was observed in e. petrophila (1.07 μm) (table 3). figure 2. haploid ideograms of the studied taxa. (a) e. cheiradenia; (b) e. macroclada; (c) e. niciciana; (d) e. pannonica; (e) e. pestalozzae; (f ) e. petrophila; (g) e. pisidica; (h) e. seguieriana; (i) e. smirnovii; (j) e. thessala; (k) e. yildirimli; (l) e. terracina. table 3. karyotype analysis of the investigated euphorbia taxa. sc–lc q (μm) mean (±sd) p (μm) mean (±sd) p+q mean (±sd) karyotype formula e. cheiradenia 1.35–2.12 0.76(±0.10) 0.98(±0.14) 1.75(±0.22) 16 m + 2 sm e. macroclada 1.21–1.89 0.67(±0.09) 0.85(±0.13) 1.53(±0.20) 32 m + 4 sm e. niciciana 0.94–1.53 0.55(±0.08) 0.65(±0.10) 1.20(±0.17) 18 m e. pannonica 1.05–1.60 0.63(±0.08) 0.68(±0.08) 1.31(±0.16) 18 m e. pestalozzae 0.98–1.44 0.54(±0.04) 0.67(±0.09) 1.21(±0.13) 18 m e. petrophila 1.59–2.20 0.79(±0.12) 1.07(±0.12) 1.86(±0.18) 14 m + 4 sm e. pisidica 1.10–1.64 0.63(±0.06) 0.75(±0.10) 1.38(±0.16) 18 m e. seguieriana 0.88–1.42 0.51(±0.06) 0.61(±0.11) 1.11(±0.16) 18 m e. smirnovii 0.79–1.52 0.52(±0.10) 0.62(±0.14) 1.14(±0.22) 32m + 4 sm e. thessala 1.15–1.78 0.65(±0.06) 0.79(±0.13) 1.44(±0.19) 18 m e. yildirimli 1.13–1.74 0.66(±0.08) 0.80(±0.13) 1.47(±0.18) 16 m + 2 sm e. terracina 0.80–1.14 0.44(±0.03) 0.54(±0.07) 0.97(±0.09) 18 m abbreviations:sc: the shortest chromosome length; lc: the longest chromosome length; p: mean long arm length; q: mean short arm length; sd: standard deviation; m: metacentric; sm: submetacentric. 17karyological analysis of twelve euphorbia species from turkey euphorbia petrophila, with relatively high intrachromosomal (mca= 14.90) and e. pannonica (4.17) with low intrachromosomal asymmetry, also e. smirnovii with high interchromosomal (cvcl=19.65) and e. petrophila (9.49) low interchromosomal asymmetry were observed. intrachromosomally, seed surface smooth and cyathial glands horned species are more asymmetrical than seed surface smooth and cyathial glands hornless species (table 4). twelve euphorbia species were analysed by pcoa (cumulative variance explained by the first two axes: 88.21%). and, the species with the same seed and cyathial gland morphology tend to cluster together except e. smirnovii (figure 3). the most important characters in recognizing these groups as distinct resulted thl, mca and cvcl. discussion the number, size, and asymmetry of chromosomes are important characteristics that help explain the phylogenetic relationships of species (eroğlu et al., 2013). karyotype data for seven taxa (3 endemic) are reported for the first time in the present study. various chromosome numbers have been reported for the sect. pithyusa (2n=16, 18, 28, 36, 40, 72) (riina et al 2013). however, most of the investigated taxa in this study have the same chromosome number (2n=18), only e. macroclada and e. smirnovii showed tetraploid cytotypes 2n = 4x = 36. according to riina et al. (2013) chromosome number of e. terracina is 18 and our results supported this report. however, even though e. terracina has 18 chromosomes, it differs from the other investigated taxa in smaller chromosomes (thl=8.75). table 4. chromosome numbers and karyo-morphometric parameters, symmetry indices for investigated taxa. 2n x thl mca cvcl cvci e. cheiradenia 18 9 15.71 12.56 12.51 6.82 e. macroclada 36 9 13.73 11.37 12.89 7.73 e. niciciana 18 9 10.78 8.43 14.61 3.34 e. pannonica 18 9 11.77 4.17 12.40 1.53 e. pestalozzae 18 9 10.86 10.52 10.87 5.16 e. petrophila 18 9 16.78 14.90 9.49 10.44 e. pisidica 18 9 12.41 8.06 11.25 2.99 e. seguieriana 18 9 10.01 8.49 14.33 4.73 e. smirnovii 36 9 10.30 7.71 19.65 7.96 e. thessala 18 9 12.93 9.56 13.16 4.64 e. yildirimli 18 9 13.21 9.37 12.35 7.54 e. terracina 18 9 8.75 9.85 9.76 3.93 abbreviations: thl: total haploid length; mca: mean centromeric asymmetry; cvcl: coefficient of variation of chromosome length; cvci: coefficient of variation of centromeric index. figure 3. pcoa analysis based on five quantitative karyological parameters of investigated taxa. 18 i̇lker genç, şükran kültür the chromosome number of e. macroclada was reported 2n = 2x = 18 by lessani and chariat-panahi (1979) and chariat-panahi et al. (1982). tetraploid cytotype is reported for the first time for this species. different chromosome numbers were reported for e. seguieriana subsp. seguieriana (2n= 16, 18, 40) (rice et al. 2015). in this study, the chromosome number of this species is reported to be 2n = 2x = 18. euphorbia terracina is a bit separated from other taxa according to the pcoa analysis. so, our results support the position of e. terracina in a distinct section (e. sect. pachycladae), as suggested by riina et al. (2013). three groups are examined in pcoa graph as group i (taxa with seed surface not smooth and cyathial glands horned), group ii (taxa with seed surface smooth and cyathial glands horned) and group iii (taxa with seed surface smooth and cyathial glands hornless). but e. smirnovii is also partially out of these groups (figure 3). as a conclusion, further studies on the chromosome morpholog y of euphorbia taxa should be performed. thus, it can be seen more clearly how species are grouped according to the chromosome morphology. in our opinion, studies on the chromosome morphology of euphorbia will contribute to the taxonomy of the genus. acknowledgements this study was supported by the scientific and technological research council of turkey (tubitak) project number 114z125. references barla a, birman h, kültür ş, öksüz s. 2006. secondary metabolites from euphorbia helioscopia and their vasodeppressor activity. turk. j. chem. 30: 325–332. barla a, öztürk m, kültür ş, öksüz s. 2007. screening of antioxidant activity of three euphorbia species from turkey. fitoterapia 78: 423–425. baytop t. 1984. türkiyede bitkilerle tedavi [therapy with medicinal plants in turkey]. i̇stanbul: i̇stanbul university press. turkish. bruyns pv, mapaya rj, hedderson t. 2006. a new subgeneric classification for euphorbia (euphorbiaceae) in southern africa based on its and psba-trnh sequence data. taxon 55: 397–420. chariat-panahi ms, lessani h, cartier d. 1982. etude caryologique de quelques espèces de la flore de l’iran. rev. cytol. biol .veg. bot. 5: 189–197. ernst m, grace om, saslis-lagoudakis ch, nilsson n, simonsen ht, rønsted n. 2015. global medicinal uses of euphorbia l.(euphorbiaceae). j. ethnopharmacol. 176: 90–101. genç i̇, kültür ş. 2016. euphorbia akmanii (euphorbiaceae), a new species from turkey. phytotaxa 265(2): 112–120. genç i̇, kültür ş. 2017. morphological and caryological features of euphorbia smirnovii geltman. trak univ journal of nat sci. 18(2): 133–136. genç i̇, kültür ş. 2018. seed morphology of perennial taxa of euphorbia section pithyusa (euphorbiaceae) in turkey. phytotaxa 336(3): 263–271. hammer ø, harper dat, ryan pd. 2001. past: paleontological statistics software package for education and data analysis. palaeontol electron. 4(1): 9. hammer ø. 2018. past 3.19. http://folk.uio.no/ohammer/past [accessed march 2018] horn jw, van ebw, morawetz jj, riina r, steinmann vw, berry pe, wurdack kj. 2012. phylogenetics and the evolution of major structural characters in the giant genus euphorbia l. 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plant syst evol. 297: 141–145. caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 27-35, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-279 citation: c. alaca, a. özdemir, b. bozdağ, c. özdemir (2020) cytogenetic effects of c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. and mathematical analysis. caryologia 73(1): 27-35. doi: 10.13128/ caryologia-279 received: may 31, 2019 accepted: february 26, 2020 published: may 8, 2020 copyright: © 2020 c. alaca, a. özdemir, b. bozdağ, c. özdemir. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. cytogenetic effects of c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. and mathematical analysis cihangir alaca1, ali özdemir1, bahattın bozdağ2, canan özdemir2,* 1 celal bayar university, science and art faculty, mathematics department, manisa, turkey 2 celal bayar university, science and art faculty, biology department, manisa, turkey *corresponding author. e-mail: cozdemir13@gmail.com abstract. xylene is a readily flammable and poisonous liquid with a chemical formula of c6h4 (ch3)2. it is used as raw material or auxiliary raw material in many industrial products such as dye, pencil, agricultural chemicals, rubber, fiber, glue and diaper. in this study, cytogenetic effects of xylene, on the meristematic cells of root tips of v. faba l. used as food have been investigated. for this purpose, the seeds of the plant have been treated with xylene solutions prepared in different concentrations for different time periods. chromosomes at the root tips have been looked and the effect of xylene has been determined. the abnormalities as chromosome breaking, chromosome dispersion, bridge chromosome, chromosome adherence, ring chromosome have been observed. abnormalities have been seen at each treatment depended on the time periods. in addition to these visible damages of xylene in the study, possible damages on chromosomes carrying genetic codes of living beings to future generations have been investigated and mathematical analyzes has been made. the results obtained have been evaluated statistically. keywords. abnormalities, chromosome, mathematical analysis, xylene. 1. introduction xylene is a colorless, characteristic solvent odorous and liquid form raw material with a chemical formula of c6h4 (ch3)2 and molecular weight of 106.17 gr / mol. it is formed by bonding two methyl groups to benzene and it is a readily flammable and poisonous liquid. xylene may leak to surface, surface water or groundwater, where it may remain for months or more. xylene is widely used industry and medical technology as a solvent, but concerns about its safety have been raised from time to time (jenifer 1994). health and safety authorities in most countries recommend a threshold limit value (tlv) of 100 ppm in the working environment. xylene vapour is absorbed rapidly from the lungs, and xylen1. e liquid and vapour are absorbed slowly through the skin. of the xylene absorbed, about 95% 28 cihangir alaca et al. is metabolised in the liver to mha and 70 to 80% of metabolites are excreted in the urine within 24 hours. differences are suspected between animal species, and between animals and humans, in the metabolism of, and sensitivity to, xylene (langman 2009). there have been different studies about that the cytogenetic effects of some metals and chemical substances except to xylene on plant in literature. various chemical substances which may be used in medicine, biology and agricultural fields can affect negatively growth of both plants beside their positive effects (i̇nceer et al. 2003; kıran and şahin 2005). i̇nceer and beyazoğlu (2000) have investigated cytogenetic effects of copper chloride on root cells of vicia hirsuta (l.) s.f. gray and they detected that this compund affects cell division negatively and also leads to chromosomel abnormalities. the researchers reported that compounds with mercury affect spindle threads during cell division in v. faba and allium cepa l. (leonard et al. 1983). some researchers have made some investigations about the effects of heavy metal pollution on plants, resulted from different factors at environment and enterence of these elements into soil and plant (çelik et al. 2004; özdemir 2008; özdemir et al. 2015; şutan 2018). in this study, we investigated the effects of xylene used as raw materials or auxiliary raw materials in many industrial products such as dye, pencil, agrochemicals, rubber, fibers, glue and diaper on chromosomes of v. faba used as food. faba bean is one of the most important grain legumes in the world because of its multiple uses and its ability to grow over a wide range of climatic conditions (kursheed et al.2018) the results of the research have been determined mathematically and the as statistical have been evaluated. for this purpose, xylene solutions have been prepared in different concentrations, and the seeds of the v. faba have been germinated with treatment with these solutions. chromosomes at at the root tips have been looked and the effect of xylene has been determined. the environment where xylene, a carcinogenic substance, is present at 100 ppm or more than 435 mg/m3 in air is harmful to human health (haglund et al. 1980). in addition to these visible damages of xylem in the study, possible damages on chromosomes carrying genetic codes of living beings to future generations have been investigated and mathematical analyzes have been made. the results obtained have been evaluated statistically. 2. material and methods in our study, we determined the concentration of xylene by taking into consideration the application period and the level of harm to human health in the literature. the amounts given in the literature belong to the direct xylene effect of the human. we tried to determine this effect by applying on seed of v. faba, which commonly used by people as food. thus, we used 10 ml / l, 10 ml / l, 12h and 24h values for the application. the seeds of v. faba have been treated with these concentrations of xylene during 12 and 24 hours. then, the seeds have been has beenhed by distiled water and germinated in petri dish at 20-25 ºc. the root tips obtained, have been put in 70 % ethyl alcohol after the fixation of they. stock root tips have been stained by feulgen method (darlington and la cour 1976) and have been got ready for microscopic examination. homoligous areas have been chosen on these preparations for cytogenetic examination; the cells in these areas have been counted and the number of mitotic cells have been also detected. chromosomel abnormalities have been tried to detected in the cells counted. preparates has been photographed with motorized leica dm 3000 microscope. chromosome abnormalities detected in the study have been coded as a, b, c, d, e, f, g, h and i (table 3). the concentrations and times of treatment have been coded as 1-4 (10 ml/l -12h): 1, (10 ml/l -24h): 2, (100 ml/l -12h): 3, (100 ml/l -24h): 4 we used following formulas for calculating the mitotic index and the percentage of total abnormalities. in this the study the cell numbers in per unit area (24x24mm) have been considered. number of dividing cells mitotic index = x 100 total number of cells number of cells with abnormal chromosome % of total abnormalities = x 100 total number of cells statistical analyses have been performed using minitab software package. 3. results at the end of the study, it has been observed that different concentrations of xylene treatment on the seeds have been increased mitotic cell division at the different periods of time, compared with the control group (figure 1). this situation has been reached on the top point at 12 hour of 100ml/l treatment. at the 12 hour 29cytogenetic effects of c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. for 100ml/l of treatment, mitotic cell division has been decreased. mitotic division increased again at the 12 and 24 hour of 100ml/l of treatment (table 1,2). in the cells of the root tips of treated with xylene investigated seeds various chromosomel abnormalities as sticky chromosome, ring chromosome, chromosome breaking, bridge chromosome, vagrant chromosome, polar deviation, binucleated cell and scattered anaphase at different stages of mitotic division have been detected (figure 2-8). total abnormalities (%) has been observed high level all treatment for 24 hours of time according to 12 hours of time (table 1-3). the number of different chromosome abnormalities have been observed the highest level at 10ml/l and 24 hour treatment time. whereas the at least number of different chromosome abnormalities have been observed at 10ml/l and 12 hour treatment time. the chromosomel abnormalities with the highest percentage have been seen in sticky chromosome and ring chromosome. bridge chromosomes have been seen as single, binary, triple and multiple bridge shaped in the all treatment except to control and 12h100ml/l treatment. chromosome shrinking, ring chromosome and chromosome breaking have been seen all treatment times except to 10ml/l and 12 hour and control. binucleated cell and scattered anaphase have been seen in the all treatment except to control and 12h10ml/l treatment (table 1-3; figure 3,7) also, according to the statistical results derived, there is a considerable positive relation between the increase concentration of treatment and the mitotic index (%). on the other hand, there are positive relation between the time of treatment and the chromosome abnormality (%) (table1-3). the statistical analysis of the results are shown in tables 1-8. according to table 4 and 8 based on the pearson’s correlation and analysis of variance method figure 1. photomicrographs of v. faba root tip meristem cells. normal mitotic phases: (a) prophase, (b) metaphase, (c) anaphase, and (d) telophase. table 1. the mitotic index and total chromosome abnormalities in the root tip cells of v. faba. control 10 ml/l 100 ml/l 12h 24h 12h 24h mitotic index (%) ± sd 12.02 ± 7.84 25.23 ± 11.12 20.02 ± 10.12 32.02 ± 17.14 27.03 ± 19.64 total abnormalities (%) 0.00 03.13 10.21 07.05 18.03 the number of different chromosome abnormalities 0.00 2 9 6 4 s.d.standart deviation, time (h): hour. table 2. number (%) of cells in each mitotic stage of v. faba roots treated with xylene. stages (%) control 10 ml/l 100 ml/l 12h 24h 12h 24h prophase 10.00 22.12 18.00 27.06 20.02 metaphase 1.30 2.03 1.20 1.60 1.70 anaphase 0.82 0.72 0.87 0.13 1.20 telophase 0.42 0.02 0.60 1.12 2.02 time (h): hour. 30 cihangir alaca et al. figure 2. the xylene induced abnormalities: anaphase bridges figure 3. the xylene induced abnormalities: binucleated cells table 3. the xylene induced chromosome abnormalities in the root tip cells of v. faba. chromosome abnormalities (%) 10 ml/l 100 ml/l 12h (1) 24h (2) 12h (3) 24h (4) sticky chromosome a 1.30 2.30 1.50 3.20 ring chromosome b 1.30 3.20 1.20 2.10 chromosome breaking c 0.53 2.80 0.60 1.50 bridge chromosome d 0.00 1.21 1.10 4.30 vagrant chromosome e 0.00 0.00 1.40 3.20 polar deviation f 0.00 0.00 0.30 2.20 binucleated cell g 0.00 0.20 1.30 0.70 scattered anaphase h 0.00 0.10 0.10 0.80 treatment time (h): hour. abbreviations: a-h: codes of chromosome abnormalities. abbreviations: 1-4 : codes of treatment (10 ml/l -12h): 1, (10 ml/l -24h): 2, (100 ml/l -12h): 3, (100 ml/l -24h): 4. 31cytogenetic effects of c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. (correlation), there are important correlations among (a-b, d; c-f; d-f, d-h; e-f; f-h) the investigated chromosome abnormalities at levels of 0.01 and 0.05 (table 4, 8). according to table 5, based on the pearson’s corfigure 4. the xylene induced abnormalities: chromosome breaking. figure 5. the xylene induced abnormalities: vagrant chromosome. table 4. pearson’s correlation based on chromosome abnormalities. a b c d e f g b 0,966 0,034* c 0,938 0,062 0,894 0,106 d 0,012* 0,988 0,195 0,805 0,264 0,736 e 0,342 0,658 0,539 0,461 0,117 0,883 0,916 0,084 f 0,266 0,734 0,419 0,581 0,031* 0,969 0,964 0,036 0,950 0,050* g 0,220 0,780 0,441 0,559 0,313 0,687 0,326 0,674 0,579 0,421 0,301 0,699 h 0,142 0,858 0,298 0,702 0,161 0,839 0,985 0,015 0,921 0,079 0,991 0,009** 0,249 0,751 * significant at the level of p< 0.05. ** significant at the level of p< 0.01. abbreviations: a-i : codes of chromosome abnormalities. table 5. pearson’s correlation based on chromosome abnormalities. 1 2 3 2 0,851 0,007** 3 0,412 0,272 0,310 0,514 4 0,028* 0,135 0,470 0,762 0,750 0,239 * significant at the level of p< 0.05. ** significant at the level of p< 0.01. abbreviations: 1-4 : codes of treatment. 32 cihangir alaca et al. relation method there are important correlations among (1-2; 1-4) the treatment time and treatment concentrations at levels of 0.01 and 0.05 according to table 9, based on the analysis of variance method, there are important correlations among only 1-2 the treatment time and treatment concentrations at levels of 0.01. 4. discussion in this study, we studied the cytogenetic effects of xylene on which human beings are exposed in different way on chromosome of plant. for this aim we used meristematic cells of root tips belonging to the v. faba figure 6. the xylene induced abnormalities: ring chromosome. figure 7. the xylene induced abnormalities: polar deviation and scattered anaphase. 33cytogenetic effects of c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. which widely used as food by humans. the researchers have pointed out that allim cepa has as an advantage due to its large chromosomes, easily observed with a light microscope (bonciua 2019). for the same reason, figure 8. the xylene induced abnormalities: sticky chromosome. table 6. regression analysis: a versus b. the regression equation is c5 = 0,899 + 0,435 c6 predictor coef se coef t p constant 0,8995 0,1588 5,66 0,030 c6 0,43508 0,08255 5,27 0,034 s = 0,140751; r-sq = 93,3%; r-sq(adj) = 89,9% analysis of variance source df ss ms f p regression 1 0,55038 0,55038 27,78 0,034 residual error 2 0,03962 0,01981 total 3 0,59000 abbreviations: a-b : codes of chromosome abnormalities table 7. regression analysis: 1 versus 2. the regression equation is c1 = 0,065 + 0,370 c2 predictor coef se coef t p constant -0,0654 0,1645 -0,40 0,705 c2 0,36970 0,09332 3,96 0,007 s = 0,334601; r-sq = 72,3%; r-sq(adj) = 67,7% analysis of variance source df ss ms f p regression 1 1,7570 1,7570 15,69 0,007 residual error 6 0,6717 0,1120 total 7 2,4288 abbreviations: 1-2: codes of treatment. 34 cihangir alaca et al. we have used v. faba which has this feature in this study. we think that the results of the study are quite important. because chromosomes are the passwords that keep the viability. we have been determined that xylene has been caused to some chromosamal abnormality on root tip cells of the plant as bridge chromosome, chromosome breaking, sticky chromosome, ring chromosome, vagrant chromosome, scattered anaphase. similarly, the researchers investigated the cytotoxic effect of benzene and thinner, the toxic chemical used in the painting of steel furniture such as xylene. the effect were evaluated using root tip cells of allium cepa. they observed chromosomal abnormalities induced were early separation, exclusion, laggard, sticky bridge and persistent bridge (barbhuiya et al. 2018). some other the researchers reported that copper chloride has caused to some chromosamal abnormality on root tip cells of vicia hirsuta (l.) gray. according to the same researchers the most observed abnormalityes have been chromosome adherence and bridge chromosome (i̇nceer and beyazoğlu 2000). in other study, it has been determined that increase of the lead (pbcl2) concentrations cell division has been decreased, several mitotic anomalies such as c mitosis, lagging chromosomes, multipolar anaphases and chromosome bridges on root tip cells of lens culinaris medik (kıran and şahin 2005). in another study, it has been determined that the frequency of mitotic cell division have been affected by uranium depending on the different treating time and uranium led to chromosomel abnormalities in the v. faba cells (özdemir et al. 2008). similarly, in our study, the mitotic division of the seed treated with xylene gradually increased in comparision to the control group. on the other hand, the chromosome abnormalities vary in parallel with the concentration while the chromosome abnormalities changes irregularly with the time periods of the treatment in our study. as a result of the study, xylene has been shown to induce cleavage in plant meristematic cells and cause abnormal cell division and chromosomel abnormalities. statistical analysis have been performed using analysis of variance, regression and pearson correlation tests. the differences have been evaluated with the same tests. the reason for the application of this statistical method is to see if there is a difference between the groups on the variables studied. these statistical methods have been used to test the differences between two or more groups for our investigated. the results have been taken into account in the significance evaluations at p <0.05 and p <0.01 levels. thus, we have tried to prove and evaluate the results obtained from laboratory studies numerically. also, according to the statistical results derived, different concentrations of xylene treatment on the seeds have been increased mitotic cell division at the different periods of time, compared with the control group. however, mitotic division of these treated seeds decreased with increasing application time. this result shows that the application time in mitotic division is important. on the other hand there is a considerable positive relation between the treatment time (hour) and the chromosome abnormality (%). as shown in the tables according to analysis of variance, regression analysis and pearson correlation tests it has been found that there have been statistically important differences at levels of 0.01p between scattered anaphase and polar deviation (table 4,6,8). we can say that these chromosomal abnormalities are interrelated and trigger each other. we did not find any detalied study on the effect of xylene on plant chromosomes except for a few studies on the effect of xylene on animal cells in our literature review (mohtashamipur et al. 1985; dean 1985; nise 1991). in literatüre some researchers have done studies table 8. correlation between 8 investigated chromosome abnormalities (analysis of variance). ms f-value probability significance a-b 0.5503 27.78 0.034 * a-c 0.5187 14.56 0.062 ns a-d 45.134 482.2 0.050 * a-f 0.0418 01.15 0.734 ns b-f 0.5120 0.430 0.581 ns b-h 0.2580 0.190 0.702 ns c-d 0.2370 0.150 0.736 ns d-f 9.5300 26.47 0.036 * d-h 9.9522 66.84 0.015 ** e-f 6.2427 18.71 0.050 * f-h 3.3103 115.7 0.009 ** g-h 0.0624 0.130 0.751 ns ms: mean square; *p<.05; **p<.01; a-h: codes abnormalities; ns: not significant. table 9. correlation between treatment time and concentrations (analysis of variance). ms f-value probability significance 1-2 1,7570 15,69 0,007 ** 1-3 0,4128 1.230 0,310 ns 1-4 0,0398 0.100 0,762 ns 2-3 0,9520 0.480 0,514 ns 2-4 0.2340 0.110 0,750 ns 3-4 0.4380 0.710 0,239 ns ms: mean square; **p<.01; abbreviations: 1-4: codes of treatment; ns: not significant. 35cytogenetic effects of c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. on the health damages of xylene through various routes of exposure. the researchers indicated that xylene, an aromatic hydrocarbon, is widely used in industry and medical laboratory as a solvent and it is a flammable liquid that requires utmost care during its usage. also researchers pointed that prolonged exposure to xylene leads to a significant amount of solvent accumulation in the adipose and muscle tissue ( rajan and malathi 2014). thus, we think that we are trying to close this deficiency in the literature with this study. the researchers observed the damage of chromosome in bone marrow cells of rats after dosing with xylene (lebowitz et al. (1979). similarly donner et al. (1980) exposed rats to technical-grade xylene by inhalation at 300 ppm, 6 h/day, 5 days/week for up to 18 weeks. the chromosome damage has been detected in animals examined after 9, 14 or 18 weeks exposure. the occurrence of these disorders in the chromosomes shows that this negativity will be transmitted from generation to generation. people who have been reluctantly exposed to xylene could have similar effects. therefore, the use of this chemical in our lives should make us think. references barbhuiya s. n, barhoi d, datta s.k, and giri1 s. 2018. two major components of steel fabrication industry, benzene and thinner induce cytotoxicity in allium cepa l. root cells. cytologia 83(2): 155–158. https://doi.org/10.1508/cytologia.83.155 bonciu e, firbas p, fontanetti c. s., jiang w, karaismailoğlu m c, liu d, menicucci f, pesnya d.s., popescu a, romanovsky a.v., schiff s, ślusarczyk j, de souza c. p., srivastava a., sutan a., papini a. 2018. an evaluation for the standardization of the 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malathi n. 2014. health hazards of xylene: a literature review. j clin diagn res. 8(2): 271-274. şutan n. a., uţă, g., bărbuceanu, d. 2018. oxidative stress and cytogenetic effects in root tip cells of allium cepa l. induced by alcoholic extracts of leptinotarsa decemlineata (say). caryologia 1-9. caryologia international journal of cytology, cytosystematics and cytogenetics volume 73, issue 1 2020 firenze university press karyotypic investigation concerning five bromus species from several populations in iran sara sadeghian, ahmad hatami, mehrnaz riasat high genetic diversity and presence of genetic structure characterise the endemics ruta corsica and ruta lamarmorae (rutaceae) marilena meloni1, caterina angela dettori2, andrea reid3, gianluigi bacchetta2,4,*, laetitia hugot5, elena conti1 cytogenetic effects of c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. and mathematical analysis cihangir alaca1, ali özdemir1, bahattın bozdağ2, canan özdemir2,* clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 73(3): 103-110, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-252 caryologia international journal of cytology, cytosystematics and cytogenetics citation: m. kaur aulakh, m. inder singh saggoo (2020) an adverse effect of meiotic abnormalities on spore fitness in medicinal fern glaphyropteridopsis erubescens (wall. ex hook.) ching. caryologia 73(3): 103-110. doi: 10.13128/ caryologia-252 received: may 10, 2019 accepted: june 23, 2020 published: december 31, 2020 copyright: © 2020 m. kaur aulakh, m. inder singh saggoo. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. an adverse effect of meiotic abnormalities on spore fitness in medicinal fern glaphyropteridopsis erubescens (wall. ex hook.) ching mandeep kaur aulakh*, manjit inder singh saggoo department of botany, punjabi university, patiala-147002, punjab, india *corresponding author. e-mail: mandeepbot@gmail.com abstract. glaphyropteridopsis erubescens plants were collected from parvati valley, himachal pradesh, india. meiotic investigations in the accessions collected from different areas of himachal pradesh revealed normal meiosis and good spore fertility. however, the accessions collected from parvati valley has depicted meiotic anomalies and high spore abortion index. all populations shared the same cytological status of 2n = 72, which is in line with previous records. individuals depicted normal behavior at diakinesis and metaphase-i but later stages were abnormal. it includes chromatin stickiness, interbivalent connections, early and late disjunction. the formation of laggards at anaphase-i lead to series of abnormalities such as chromatin bridges, random sub-grouping of chromosomes at anaphase-ii, chromatin fragmentation and irregular sporogenesis. large numbers of micronuclei were present in triads and tetrads along with pycnotic nuclei. empty and some abnormal sporangia with heterogenous spores were also observed. keywords: glaphyropteridopsis, abnormal, meiosis, sporogenesis, pteridophyte, parvati valley. introduction pteridophy tes are treasures of valuable secondary metabolites that helped them to withstand harsh environmental conditions. medicinally, they are reported to have antioxidant, antimicrobial, antiviral, anti-inflammatory, antitumor and anti-hiv properties (baskaran et al. 2018). fern and fern-allies are the major medicinal resource of phenols, flavonoids, alkaloids and steroids along with some unique phytochemicals having numerous industrial applications (shinozaki et al. 2008). the tribes of north west india are using crosiers, rhizomes and fronds of adiantum capillus-veneris, actiniopteris radiata, adiantum caudatum, adiantum philippense, adiantum venustum, diplazium esculentum, pteris vittata, pteris wallichiana, asplenium trichomanes, asplenium nidus and angiopteris evecta etc. in different formulations of drugs and ointments. thelypteris species have been used to 104 mandeep kaur aulakh, manjit inder singh saggoo cure cold, itching, nerve pain, rheumatism, swellings, spermatorrhea, body pain, backache, cuts and wounds, bone fracture, sprain, gastric trouble, malaria fever, body pain, sprain, boils, cancer and asthma (sureshkumar et al. 2018). north west himalayas are facing major threat to biodiversity due to habitat destruction and pollution. they are the reservoirs of medicinal ferns as they are habitat specific and needs special care if grown in nurseries and botanical gardens. some of high altitudinal ferns are nearly impossible to grow outside their natural habitat like presently studied fern. so, evaluation of wild accessions from different parts will be helpful in finding new and stable morphotypes and chemotypes among them. the reproductive success of medicinal ferns largely depends upon spore viability. this can be influenced by meiotic behavior and sexual status of the species. meiotic abnormalities such as chromosomal fragmentation, bridges, laggards, micronuclei and cytomixis are known to cause sterility in number of plant species. plant chromosomes exhibit different types of aberrations caused by mutagens, radiations or temperature etc. and they acts as sensitive indicators to environmental pollutants. so, higher plant systems acts as an indicator of possible genetic damage caused by environmental mutagens (grant 1978). this paper deals with meiotic investigation of glaphyropteridopsis erubescens (wall. ex hook.) ching (=thelypteris erubescens (wall.) ching (thelypteridaceae), an important medicinal fern collected from disturbed areas of parvati valley. this family comprises 20 genera and 1000 species growing in forests with ravines of low mountains, between altitudes of 800-2000m, distributed mainly in china, taiwan, japan, myanmar, pakistan, philippines, vietnam, nepal and bhutan. in india, g. erubescens grows well in shady habitats of himachal pradesh, uttarakhand, sikkim, darjeeling, arunachal pradesh, meghalaya and jammu and kashmir. plants of g. erubescens are 2−3m tall, the rhizomes are stout and they are decumbent, woody and glabrous. fronds are clustered, stipes are 1−2 m, thicker than 1 cm, ribbed, glabrous, throughout stramineous and often reddish. the pinnae are in 40−50 pairs per frond, opposite, sessile and proximal several pairs strongly oblique distally. this fern has noteworthy medicinal potential as leaf decoction is used to treat indigestion, dough of fronds is applied externally for rheumatism and root powder is used as an antidote in scorpion bite in nigeria (nwosu 2002). powder of dried rhizome mixed with rice water can be internally administered for gonorrhea, especially for leucorrhea by the people of deoprayag area in garhwal himalayas, india (gaur and bhatt 1994). g. erubescens is a natural source of kaempferol derivative kaempferol-3-o-α-l-rhamnoside involved in a variety of signaling pathways. kaempferol has potential to significantly modulate a variety of signaling pathways that are induced in many adverse clinico pathological conditions (kashyap et al. 2017). in the north east of india, fresh fronds of fern locally called “pire uneu” is used in the making of fermented cakes called marcha, amylolytic mixed starters similar to other regions of north east such as hamei of manipur, pham, ipoh and phab of arunachal pradesh, humao of assam and thiat of meghalaya (tamang et al. 2012). during exploratory surveys to analyze diversity in medicinal plants of north west india, we had collected accessions from parvati valley (figure 1). its vernacular name is “vaarne” and rhizomes were being used to prepare decoction for reproductive disorders by local people. parvati valley is situated between 31°21’21’’−32°25’0’’n and 76°56’30’’−77°52’20’’e in south east of kullu, altitude ranging between 1100−5550m above mean sea level. the valley is under extreme biotic and abiotic pressure due to rampant tourism, proliferation of concrete structures, construction of roads, dams, piles of garbage, hippie culture, drug trafficking (marijuana or hash) which had affected the biodiversity in recent years. all these activities result in degradation of forests and pose serious threat to fern inhabitants along with other plant communities (sharma 2005). meiotic abnormalities were found to be consequence of extreme environmental factors in some species. abortion in microspores of isoetes sinensis was linked to anthropogenic activities causing habitat fragmentation figure 1. (a) map showing the geographical location of himachal pradesh in india; (b) map depicting various localities of himachal pradesh (arrowed kullu district of parvati valley); (c) localities visited for the collection of accessions from parvati valley (highlighted with star). 105an adverse effect of abnormalities on spore fitness in glaphyropteridopsis and decrease in numbers drastically (heng-chang et al. 2007). the present study aims to analyze the abnormal meiotic course and spore abortion index in this fern from disturbed locations within parvati valley, himachal pradesh, india. the present work is the first attempt to study the cytology of this fern species from parvati valley. materials and methods the material for meiotic studies was collected from wild plants growing at various localities of parvati valley, himachal pradesh, india in monsoon season (july to september). pinnae from fronds with young sporangia were fixed in carnoy’s fixative ethanol: chloroform: glacial acetic acid (6:3:1, v/v) for 24 h at room temperature and then transferred to 70% ethanol and stored under refrigeration until use. to study meiotic course, young sporangia were squashed in 1% acetocarmine. a number of freshly prepared slides per plant were carefully examined and several spore mother cells (smcs) were observed to determine chromosome number and frequency of meiotic abnormalities at different stages. specimens were dried by keeping them in the folds of blotting sheets to remove excess moisture and they were supervised timely to keep them away from fungal growth or any other type of contamination. mature spores taken onto the microscopic slide with the help of needle were treated with 1:1 glycerol and acetocarmine mixture (marks 1954). the procedure followed for observations on number of spores per sporangium was as given by huang et al. (2011). minimum number of sporangia (8-10) was dealtwith at a time. well stained, normal looking spores were observed for calculating average spore size and spore fertility. distorted, deformed and poorly stained spores were taken as sterile. the spore abortion index (sai) was calculated (hornych and ekrt 2017). photomicrographs of smcs and spores were taken from the freshly prepared slides using magnus mlx microscope. identification was done from fern floras (beddome 1870; dhir 1980 and khullar 1994), ef loras and by comparing specimens with already deposited specimens of prof. s. s. bir at herbarium, department of botany, punjabi university, patiala. finally, pressed and dried voucher specimens were deposited in the herbarium, department of botany, punjabi university, patiala and their accession numbers (pun) were obtained (table 1). results and discussion the cytological information for thelypteridaceae is available for 51 species and in genus thelypteris, there are seven basic chromosome numbers, viz. 36, 35, 34, 32, 31, 30 and 27 (loyal 1963). meiotic behavior was studied in six populations of g. erubescens from parvati valley, himachal pradesh, india. the chromosome count of the presently investigated fern is in line with previous reports of n = 36 from the himalayas (verma and loyal 1960; loyal 1961; loyal 1963; mehra and khullar 1980; khullar et al. 1983, 1988), nepal (roy et al. 1971) and south india (irudayaraj and manickam 1987). meiosis is dynamic cellular process which maintains genome stability and integrity. any error in meiotic process can lead to variations in chromosomal structure and ploidy level. plant species are most acceptable material for meiotic studies because of availability of more genetic resources (cai and xu 2007). the accessions collected from parvati valley depicted meiotic anomalies and high spore abortion index (figure 2) while large number of populations from other parts of north west india were normal. smcs showed a high degree of abnormalities at all stages of meiosis such as stickiness, unoriented bivalents, laggards, early and late disjunction table 1. populations with altitude, voucher specimen, percentage of abnormal smcs in g. erubescens. populations with altitude pun* meiotic abnormalities (%) stickiness unoriented bivalents laggards bridges early disjunction late disjunction interbivalent connections chromatin fragmentation hp: jia (1,125m) 4867 36.31 7.89 13.15 11.84 3.94 5.26 7.89 19.73 hp: manikaran road (1,423m) 4868 32.30 7.69 6.15 10.76 9.23 4.61 6.15 9.23 hp: bharain (1,889m) 4869 25.53 12.76 10.63 6.38 hp: shamshi (1,111m) 4870 35.71 14.28 9.52 4.76 7.14 9.52 hp: kasol (2,619m) 4871 42.37 6.77 5.08 8.47 3.38 10.16 hp: manikaran sahib (1,760m) 4872 46.77 3.22 6.45 4.83 1.61 8.06 pun*= accession number herbarium code of department of botany, punjabi university, patiala, hp=himachal pradesh, india. 106 mandeep kaur aulakh, manjit inder singh saggoo figure 2. (a) g. erubescens growing wild; (b) smc showing 36ii bivalents at diakinesis; (c) smc at metaphase-i showing 36ii ; (d) chromatin stickiness at metaphase-i; (e) stickiness prevailing at anaphase-i; (f ) smc with unoriented bivalent at metaphase-i (arrow indicating one unoriented bivalent); (g) bivalents encircled showing early disjunction at metaphase-i; (h) arrow pointing to single chromatin bridge at anaphase-i; (i) arrows showing multiple bridges at anaphase-i; (j) extreme clumping of bivalents into amorphous mass with random sub grouping of chromatin material in marked area; (k) arrow indicating laggards at anaphase-i along with agglomerisation of chromatin; (l) smc at telophase-i with several fragments resulting from breakage; (m) three vagrants indicated by arrows at anaphase-i; (n) arrow showing one vagrant at telophase-i; (o) numerous (9) micronuclei at tetrad stage; (p) polyad with marked pycnotic nuclei; (q) empty sterile sporangium; (r) abnormal sporangium with varied number of spores (instead of expected 64 spores); (s) heterogenous spores; (t) group of sterile spores. scale bar = 10µm. 107an adverse effect of abnormalities on spore fitness in glaphyropteridopsis of chromosomes, interbivalent connections, chromatin bridges and chromatin fragmentation which leads to abnormal sporogenesis (table 1, 2). smc with n = 36 bivalents were recorded at diakinesis (figure 2b) and metaphase-i (figure 2c). chromatin stickiness prevailed at almost every stage, more prevalent at metaphase-i (figure 2d) and anaphase-i (figure 2e) in every population ranging from mild to severe (chromosomes appeared to be an amorphous mass with no identity) affecting 25−47% of smcs. chromatin stickiness can lead to serious consequences of spore sterility as these abnormalities were found to be interlinked to each other. this abnormality becomes evident at metaphase-i stage when chromosomes form intense chromatin mass but if it continues to anaphase-i obviously it will pose difficulty in bivalent separation, forming chromatin bridges. the maximum percentage of stickiness was present in population collected from manikaran sahib (hp) (46.77%). it has certainly affected pollen viability in many other species as they are unbalanced through irregular chromosome segregation and fragmentation (golubovskaya 1989 and rao et al. 1990). the bivalents which failed to orient themselves on the equatorial plane due to spindle abnormalities were present as unoriented bivalents (figure 2f ). the maximum percentage of unoriented bivalents was found to be present in population collected from shamshi (hp) i.e. 14.28% and minimum in case of manikaran sahib population (3.22%). this random orientation of chromosomes and their consequent sub-grouping (figure 2j) results due to irregular spindle activity while a single and transient spindle during mitosis and meiosis ensures the proper chromosome segregation (caetano-pereira and pagliarini 2001). interbivalents connections involving 3−4 bivalents was another phenomenon observed in four populations in which maximum percentage was in kasol (hp) population (10.16%). these interbivalent connections were meant to keep the bivalents together before spindle formation in order to guarantee orientation of chromosomes in division plane but fusion of heterochromatic regions will result in formation of chromatic knots (viinikka and nokkala 1981). these diffused connections between the bivalents are associated with origin of polyploid species (malgwi et al. 1997). early disjunction at metaphase-i (figure 2g) and late disjunction at anaphase-i was found to be the possible reason for the presence of laggards and chromatin bridges at later stages. it has been found that late disjunction was present in three populations only ranging from 1−5%, and early disjunction i.e. within range of 3−9%. we came across two types of non-synchronization disjunction of bivalents early and late which arises due to different rates of terminalisation of various chromosomes of complement (darlington 1937), changed homolog y of chromosomes (koul 1971) or absence of coordination between chromosome and spindle (sharma 1976). early disjunction of bivalents does not affect the normal distribution of chromosomes at anaphase i but late disjunction which is more pronounced in hybrids and diploids with meiotic abnormalities will lead to bridges and laggard formation and consequently pollen malformation (wang et al. 2004). this abnormal phenomenon is quite significant as it can lead to formation of gametes with numerical variations in chromosome number (singhal et al. 2010). laggards at anaphase-i (figure 2k) are noticeable phenomenon as compared to other abnormalities involving 5−13% of smcs, with maximum percentage in case of jia (hp) population (13.15%). sometimes these laggards were found to be stretched in between two poles forming single (figure 2h) or multiple chromatin bridges (figure 2i). bhattacharjee (1953) observed bridge like contable 2. sporogenesis of populations with spore abortion index and spore size. populations* sporogenesis jia manikaran road bharain shamshi kasol manikaran sahib number of sporads observed 98 103 84 92 106 107 normal tetrad number (%) 45(45.91) 61(59.22) 52(61.90) 51(55.43) 60(56.60) 65(60.75) tetrad with micronuclei (%) 40(40.81) 31(30.09) 15(17.86) 21(22.83) 29(27.36) 20(18.69) tetrad with micronuclei+pycnotic nuclei (%) 8(8.16) 6(5.82) 10(11.90) 16(17.39) 12(11.32) 10(9.35) triad with micronuclei (%) 5(5.10) 5(4.85) 7(8.33) 4(4.25) 5(4.71) 12(11.21) spore abortion index 55.47±2.64 45.17±1.93 38.30±2.39 49.80±3.06 43.63±1.31 37.55±0.52 spore size (µm) 42.42×33.42 39.51×29.57 41.85×32.38 39.74×29.01 42.72×33.83 38.76×29.02 42.56×32.26 38.71×28.97 42.35×31.64 39.86×29.65 42.23×32.09 38.67×29.82 populations* = all the populations were collected from different localities of parvati valley. 108 mandeep kaur aulakh, manjit inder singh saggoo figuration which might be originated due to interlocking of chromosomes starting from prophase, persists till metaphase and during its separation at anaphase stringing out of cy toplasmic strands between them, resulting in one or two false bridge like configurations. these bridge fragment configuration is considered to be the result of crossing over in heterozygous paracentric inversions (saylor and smith 1966). this may lead to unequal distribution of chromosomes resulting in abortion and heterogenous pollen grains. chromatin fragmentation resulting from breakage of chromosomes was observed at anaphase-i and telophase-i (figure 2l) and some vagrants were observed at anaphase-i (figure 2m) and telophase-i (figure 2n). the possible causes of presence of laggards at anaphase were asynapsis, desynapsis, failure of chiasma formation and premature disjunction of bivalents (gupta and priyadarshan 1982) and abnormal spindle formation (tarar and dhyansagar 1980). these laggards when permanently failed to reach poles often constituted micronuclei during microsporogenesis (jiang et al. 2011). one such example is the case of diploid species of clematis flammula l. where meiotic abnormalities and cytomixis had resulted in pollen malformation (kumar et al. 2008). extra chromosomes were reported in case of ophioglossum but in present case chromatin fragmentation were found to be present at anaphase-i and telophase-i stages, so we cannot confirm them as extra chromosomes (goswami and khandelwal 1980). the incidence of meiotic aberrations (e.g. irregular disjunction resulting from univalent and multivalent formation) and other processes may lead to the production of non-viable spores (ramsey and schemske 2002). the effect of abnormal meiotic behavior on spore fertility seems to be independent of ploidy status of plant and it varies with each species. the observations regarding the effect of meiotic abnormalities on reduced pollen fertility in flowering plants have been made by daniela et al. 2005; guan et al. 2012; jaryal et al. 2015; kumari and saggoo 2017; mandal and nandi 2017; ramanpreet and gupta, 2019 and andrada 2019. sexual species of ferns usually produced normal fertile spores whereas hybrids, apomicts, triploids etc. produce unbalanced predominantly aborted spores (hornych and ekrt 2017). it has been seen that if the population is seriously affected by the abnormalities, its spore abortion index will be also high. the population collected from jia (hp) is highly abnormal with more than 75% of smcs affected resulting in high spore abortion index (55%). due to presence of large number of micronuclei at tetrad stage (figure 2o), triad stage and polyad with pycnotic nuclei (figure 2p) sporogenesis was severely affected. empty (figure 2q), abnormal sporangia (figure 2r), group of sterile spores (figure 2t) were present along with heterogenous spores (figure 2s) leading to low spore fertility. more investigations in row on meiotic abnormalities in different fern species will be helpful in drawing conclusions about their origin whether these are genetic, physiological or environmental. the general paradigm that can be drawn from the present observations is the fact that large percentage of meiotic abnormalities will lead to abnormal sporogenesis in this species. so, we can conclude that spore sterility may not pose problem in diploid populations unless they are seriously affected by meiotic abnormalities. these studies will be significant in studying the effect of meiotic abnormalities on reproductive behavior and calculating spore abortion index in fern species. further, cytotaxonomic studies can solve the problems regarding scientifically correct identification of medicinal plants which is crucial for their appropriate use in pharmaceutical industry. nowadays, conservation of medicinal plants is top most priority by conservation biologists and government agencies. thus, evaluation of meiotic course of these plants from unexplored areas is also significant in conservation 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5, 2019 copyright: © 2019 l. verschaeve, r.antonissen, a. baeyens, a. vral, a. maes. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. fluorescence in situ hybridisation study of micronuclei in c3a cells following exposure to elf-magnetic fields luc verschaeve1,2,*, roel antonissen1, ans baeyens3, anne vral3, annemarie maes1 1 sciensano, risk and health impact assessment service, brussels, belgium 2 department of biomedical sciences, university of antwerp, antwerp, belgium 3 department of basic medical sciences, ghent university, b‑9000 ghent, belgium *corresponding author: luc.verschaeve@sciensano.be abstract. human c3a cells were exposed to extremely low frequency (50 hz) magnetic fields (elf-mf’s) up to 500 µt. they were subjected to the micronucleus assay using a fluorescence in situ hybridization (fish) technique with an in-house pancentromere probe. we found no increased frequency in micronucleated cells and no change in the proportion of centromere positive over centromere negative micronuclei compared to the unexposed control cells. these results are in accordance with some, but in contradiction with other previously published investigations underlining that effects of environmental elf-emf’s on cellular dna may be very subtle and that small changes or environmental influences may determine the outcome of a (geno)toxicity study. interestingly, a low-level (5µt) exposure resulted in less than the background micronucleus frequency. keywords. 50 hz magnetic fields, fish staining, micronuclei, centromere staining, genotoxicity. introduction overall, there is little experimental or theoretical evidence that extremely low frequency magnetic fields (elf-mf’s) from power lines or other man made sources in the environment can be genotoxic. given the level of energy involved, it is difficult to accept that they are able to directly interact with genomic structures. the results of most in vitro and in vivo genetic toxicology studies involving elf-mf’s have been negative and therefore there is a general consensus that they, especially at normal (moderate) exposure levels, are not directly mutagenic (bergqvist et al. 2003; vijayalaxmi and prihoda 2009). yet, some papers did report effects suggesting that elf magnetic fields may interact with dna or, most often, with dna-damaging agents, hence being co-genotoxic (e.g., tofani et. al. 1995; lai and singh 1997; singh and lai 1998; bergqvist et al. 2003; cho and chung 2003; ding et al. 2003; 46 luc verschaeve et al. moretti et al. 2005; vijayalaxmi and obe 2005; juutilainen et al. 2006; ehc 2007; ruiz-gómez and martinez-morillo 2009; markkanen 2009; udroiu et al. 2006). according to some of these studies elf-magnetic fields are able to enhance, but not to start a mutagenic (dna damaging) effect. some of the above mentioned papers also indicate that emf-mf’s exposure may, alone or in conjunction with another agent, be able to promote the occurrence of aneuploidy caused by an aneugen via a mechanism involving the neuroendocrine system (maes et al. 2016a). jin et al. (2015) however, provided evidence that elf-mf’s alone do not induce either g2/m arrest or aneuploidy, even when administered in combination with different stressors. only a few papers reported so far on possible aneugenic or co-aneugenic effects of electromagnetic fields (udroiu et al. 2006; maes and verschaeve 2012; maes et al. 2016a). on previous investigations (maes et al. 2016a,b) we reported increased levels of especially nuclear buds and large micronuclei in cells that were exposed to 50 hz elf-mf’s. this indicated that the magnetic fields may, at least in particular cells, situations and exposure levels induce gene amplification (buds) and aneuploidy. in the present paper we further explore this possibility by using fluorescence in situ hybridisation (fish) with a pan-centromeric probe. the main objective was to verify our previous results and to investigate whether potential elf-mf’s induced micronuclei (mn) were predominantly centromere-positive or centromere-negative, respectively suggesting an aneugenic or clastogenic effect. materials and methods elf‑mf exposure unit the exposure unit was a cylindrical coil (380 turn coil, 42 cm long, 20 cm inner diameter) which allowed the exposure of cell cultures to a nearly constant magnetic field (with a tolerance of a few percent). with this device, cell cultures could be exposed to different 50 hz magnetic field amplitudes ranging from 0 up to about 2500 µt. more details about the exposure unit can be found elsewhere (maes et al. 2000; verheyen et al. 2003; mineur 2009). we exposed cell cultures to magnetic fields of 5, 10, 50, 100 and 500 µt. the ambient magnetic field was 0.02±0.01 mt. cell cultures and elf‑mf exposure human hepatic c3a cells (brunschwig chemie b.v, amsterdam, the netherlands) were grown in 24 well plates in dulbecco’s modified eagle’s culture medium supplemented with 10% foetal calf serum. the cell density was 200.000 cells/well. plates were incubated at 37°c and 5% co2. humidity was maintained using a water bath containing milli-q water inside the incubator. after 24 h of incubation, a magnetic field producing a determined magnetic flux density (5, 10, 50, 100, or 500 µt) was applied for another 24 h. following exposure to the magnetic field, cells were blocked in their binucleated (bn) telophase stage with cytochalasin b (4.5 µg/ml, merck). another 24h later cells were fixed with methanol/acetic acid (3/1) and spread onto well-cleaned microscope slides. magnetic flux densities were chosen based on our previous experiments (maes et al. 2000, 2016a,b; verheyen et al. 2003). each exposure was accompanied by its own unexposed (negative) control culture (0 µt). methyl methane sulfonate (mms, 15µg/ml) was used as a positive control. it was found to induce micronuclei as expected (results not shown). both control cultures were incubated away from the coil at a distance where no elfmf, other than the ambient field could be measured. two independent investigations were conducted. in the first experiment magnetic flux densities of 5, 10, 50, 100 and 500 µt were investigated. the second study was conducted on new exposed cell cultures and fresh slides using magnetic flux densities of 5, 50 and 500 µt. fluorescence in situ hybridization (fish) in the first set of experiments, fish was performed on the slides using an in-house pan-centromeric probe, labelled with spectrum orange. for more details about this probe and the fish protocol we refer to baeyens et al. (2011) and vral et al. (2016). in the repeat study, fish was performed using a fitc-labeled pna (peptide nucleic acid) probe, specific for centromeric sequences, from panagene (centfam 5nmol, pn-cn001-005 eurogentec, belgium). the protocol, described in detail by m’kacher et al. (2014), for centromere staining of dicentric chromosomes was followed. at the end of both fish procedures, the slides were counterstained and mounted with dapi-vectashield (h-1200, labconsult, belgium). fish-dapi stained slides were analysed with the metafer 4 platform (metasystems gmbh, altlussheim, germany) connected to a motorized zeiss axioimager m1 microscope (zeiss, oberkochen, germany). detailed information regarding the msearch slide scanning procedure, stage movement, focusing and image acquisition are detailed in willems et al. (2010). for analyses of micronuclei the mnscore module for metafer msearch was used. this software allows automated mn scoring in binucleated (bn) cells using a 10x objective. the auto47fluorescence in situ hybridisation study of micronuclei in c3a cells following exposure to elf-magnetic fields matically selected bn cells were then checked manually (false bn cells and false positive or negative micronuclei were removed) and only confirmed bn cells with micronuclei were scanned via the autocapt image acquisition software using a 40x objective. the autocapt images were then manually viewed for the presence of centromeres. bad/non-interpretable fish images were rejected. for more details about the mn-centromere analysis we refer to vral et al. (2016 ). the number of investigated cells was set to 2.500 but the actual number of analysed cells was sometimes less (see table 1). two slides (approximately 1.250 cells) were analysed per exposure. results and discussion the results of the two independent investigations are summarized in table 1. the table data indicate that micronucleus frequencies were not increased following elf-mf’s exposures up to 500 µt. according to this analysis we observe no clear differences in number of mn between the tested situations. no clear difference or trend in the percentage of centromere-negative or centromere-positive mn can be observed as well. according to these data 50 hz elf-mf’s do not change the proportion of centromere-positive over centromere-negative mn, neither do they induce elevated micronucleus frequencies. this does not coincide with our previous results on the same cells that were exposed in the same way to magnetic fields of the same magnetic flux densities. in our previous investigation (maes et al. 2016b) we also performed two independent experiments but slides were stained with giemsa. currently we used a fluorescence in situ hybridisation procedure but there is no reason why the different staining methods should influence the results. both investigations were also performed by the same persons ruling out the possible variation in micronucleus frequencies obtained by individual laboratories and scorers (fenech et al. 2003). as mentioned before, the literature reveals the presence of positive as well as negative results following exposure of cells or organisms to (weak) elf-mf’s. we, for example, did not find increased micronucleus frequencies in peripheral human white blood cells after exposure to elf-mf’s up to 800 µt using the same experimental set up (verheyen et al. 2003). moreover, loberg et al. (2000) also did not find evidence for the hypothesis that magnetic fields interact with genotoxic agents to induce adverse biological effects in either normal or genetically susceptible human cells. the same holds true for the investigation of ding et al. (2003) where chinese hamster ovary cells were exposed to a 60 hz elf-mf at 5 mt field strength. in this investigation, mn were evaluated by immunofluorescence staining using anti-kinetochore antibodies from the serum of scleroderma (crest syndrome) patients. no statistically significant difference in the frequency of mn was observed between sham exposed and 24 h elf-mf’s exposed cells. the number of spontaneous kinetochorepositive and kinetochore-negative mn was, as in our present investigation, not affected by exposure to an elf magnetic field alone. however, kesari et al. (2016) reported increased micronucleus frequencies at 10 and 30 µt in sh-sy5y neuroblastoma cells using a f low cytometry method. other examples of positive and negative results on elf-mf’s exposed cells and organisms were presented by heredia-rojas et al. (2017). actually, the absence of independent replication has been a consistent feature of experimental studies searching for biological effects of weak elf-(electro) mf’s (elf-emf’s). as pointed out by foster and skufca (2016) many scientific results can’t be replicated, leading to serious questions about what’s true and false in the world of research. many reasons, especially involving statistical inadequacies or different experimental factors table 1. summary of two independent experiments on elf-mf’s exposed c3a cells following fish-staining of micronuclei. number of binucleated cells mn/1000 cells mn+ mn% cm+ % cmmn+/ mnexperiment 1 5 µt 2500 10 5.2 4.8 52 48 1.1 control 2500 19.2 14 5.2 72.17 27.08 2.7 10 µt 2500 12 7.2 4.8 60 40 1.5 control 2500 14 8.8 5.2 62.86 37.14 1.7 50 µt 2500 18 14.4 3.6 80 20 4 control 2500 13.6 11.2 2.4 82.35 17.65 4.7 100 µt 2500 16.8 12.8 4 76.19 23.81 3.2 control 2500 18.8 13.6 5.2 72.34 27.66 2.6 500 µt 2500 12 8 4 74.29 25.71 2 control 2500 14 10.4 3.6 66.67 33.33 2.9 experiment 2 5 µt 3432 20.69 15.15 5.54 73.24 26.76 2.7 control 3423 21.62 16.36 5.26 75.68 24.32 3.1 50 µt 1629 22.71 14.12 2.46 89.19 10.81 5.7 control 1093 20.13 16.47 3.66 81.82 18.18 4.5 500 µt 2193 22.8 15.04 7.75 66 34 1.9 control 3021 28.8 16.88 5.3 81.61 18.39 3.2 48 luc verschaeve et al. or errors of unknown nature, can be evoked. effects of environmental elf-emf’s on cellular dna are believe to be very subtle (heredia-rojas et al. 2018) and therefore small experimental changes or environmental influences may determine the outcome of a (geno)toxicity study. also, different cell types (e.g., healthy lymphocytes versus a cancer cell line), but especially the different physiological state of the cells may account for different susceptibilities and consequently different results (fenech 1998). it was for example shown that cells from aged donors and leukemic patients respond to elfemf’s exposure differently than ‘other’ (normal) cells (cadossi et al. 1992), and that dna damage was found in cells from turner syndrome patients but not in cells from healthy individuals (scarfi et al. 1997a). the same authors found that turner syndrome subjects showed a lower spontaneous and mitomycin c-induced micronucleus frequency, in comparison with healthy subjects (scarfi et al. 1996). on the other hand, in another publication they did not report a different response between normal cells and cells from turner syndrome patients (scarfi et al. 1997b). the viability of goldfish that were infected by a parasite increased substantially when the fish were exposed to very low levels of elf-mf’s (cuppen et al. 2007) giving another example of possible different effects according to the health status of a cell or organism. some more cases are also reported in the literature. we used hepg2/c3a cells mainly because they have nitrogen metabolizing activity comparable to perfused rat livers, which was an important asset in some of our other studies. they are a clonal derivative of hep g2 hepatocellular carcinoma cells, with an unstable chromosome number of 45-60. perhaps another batch and cell passage may also be responsible for small physiological changes that ultimately may influence the mn frequency. although cellular passaging was not found to influence significantly hascs’s secretome properties (serra et al., 2018), increased cell passage number was found to alter p-glycoprotein expression in caco2 cell (senarathna and crowe, 2015). previously, gloy et al. (1994) already reported that the response of membrane voltage to atp and angiotensin ii in rat mesangial cells was influenced by cell culture conditions and passage number. furthermore, peiser et al. (1993) reported that more micronuclei were always detected in cells of higher passages than of lower passages showing that metabolic and genetic characteristics of permanently growing cells differ remarkably depending on the culture passage. unfortunately, we do not recall whether the cells used in our independent investigations were from a different batch and/or different cell passages. the different background levels of mn found in our different investigations may yet show that there are some differences in cell behaviour from one experiment to the other. in our previous investigation (maes et al., 2016b) we obtained background mn yields of 5-9mn/1000 bn cells (giemsa stain; experiment done in 2014-2015), whereas we now had background micronucleus frequencies of approximately 13-19mn/1000 bn cells in the first experiment (2016) and 20-29mn/1000 bn cells (2018) in the second experiment (fish staining). examples of micronucleated c3a cells are given in figure 1. an important finding of our previous investigation (maes et al. 2016b) was also that low-level elf-mf exposures resulted in micronucleus frequencies that were lower than in the unexposed control cells. actually, this was also observed here. however, this was only substantial (and statistically significant according to the binomial test described by kastenbaum and bowman 1970) in our first experiment. here the micronucleus frequency in cells exposed to 5 µt was 10mn/1000 bn cells compared to almost the double (19.2mn/1000 bn cells) in the controls. this may possibly indicate that low-level exposures to elf-mf’s, as environmental stimuli, can activate dna repair mechanisms which then result in the repair of ‘spontaneous’ dna damage which is not repaired in unexposed cells. this may be more or less comparable to the adaptive response which was already described in earlier investigations. adaptive response is a phenomenon in which cells that were pre-exposed to extremely low and non-toxic doses of a toxic agent build-up a resistance to the damage induced by subsequent exposure to a fig. 1. examples of binucleated c3a cells with micronuclei following fish staining. centromere positive cells are at the left. 49fluorescence in situ hybridisation study of micronuclei in c3a cells following exposure to elf-magnetic fields higher and toxic dose of the same, similar (in action) or another toxic agent (vijayalaxmi et al. 2014). there are indications that elf-emf’s may also elicit such response and therefore may even have beneficial instead of detrimental properties, at least after short exposure times. some previous research also proposed that low-frequency magnetic fields might play a positive role in cardiac tissue against ischemia reperfusion injury via regulating ros production and no/onoo− balance (ma et al. 2013). although the present study was not able to associate elf-mf’s with genotoxicity it is evident that there is no consensus reached yet on the alleged association between elf-electromagnetic fields (and elf-mf’s in particular) and adverse health effects in humans. yet, this remains an important issue, especially in view of the transition from nuclear and fossil to renewable energy sources for power production which leads to the necessity to optimize and expand the existing power grid and to construct several high voltage ac and dc power lines across the concerned countries (for the time being this is for example the case in germany where nuclear power plants are all about to be dismantled). acknowledgements the authors wish to thank ms. johanna aernoudt and ms. toke thiron of the department of basic medical sciences of ghent university for their excellent practical assistance. this paper was part of the research performed within the bbemg (belgian bioelectromagnetics group); 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representative of genus prunus (p. africana): first genome size assessment, heterochromatin and rdna chromosome pattern justine germo nzweundji1, marie florence sandrine ngo ngwe2, sonja siljak-yakovlev3,* assessment of cytotoxicity and mutagenicity of insecticide demond ec25 in allium cepa and ames test arzu özkara cytogenetic effects of fulvic acid on allium cepa l. root tip meristem cells özlem sultan aslantürk evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of allium test ioan sarac1, elena bonciu2,*, monica butnariu1, irina petrescu1, emilian madosa1 fluorescence in situ hybridisation study of micronuclei in c3a cells following exposure to elf-magnetic fields luc verschaeve1,2,*, roel antonissen1, ans baeyens3, anne vral3, annemarie maes1 phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities nicoleta anca şuţan1,*, irina fierăscu2, radu fierăscu2, deliu ionica1, liliana cristina soare1 telomeric heterochromatin and meiotic recombination in three species of coleoptera (dorcadion olympicum ganglebauer, stephanorrhina princeps oberthür and macraspis tristis laporte) anne-marie dutrillaux, bernard dutrillaux* a whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of populus trichocarpa l. subjected to different stresses alberto vangelisti#, gabriele usai#, flavia mascagni#, lucia natali, tommaso giordani*, andrea cavallini differences in c-band patterns between the japanese house mice (mus musculus) in hokkaido and eastern honshu hikari myoshu, masahiro a. iwasa* karyotypic description and repetitive dna chromosome mapping of melipona interrupta latreille, 1811 (hymenoptera: meliponini) natália martins travenzoli1, ingrid cândido de oliveira barbosa2, gislene almeida carvalho-zilse2, tânia maria fernandes salomão3, denilce meneses lopes1,* caryologia. international journal of cytology, cytosystematics and cytogenetics 72(2): 37-43, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-256 citation: i. sarac, e. bonciu, m. butnariu, i. petrescu, e. madosa (2019) evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of allium test. caryologia 72(2): 37-43. doi: 10.13128/cayologia-256 published: december 5, 2019 copyright: © 2019 i. sarac, e. bonciu, m. butnariu, i. petrescu, e. madosa. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of allium test ioan sarac1, elena bonciu2,*, monica butnariu1, irina petrescu1, emilian madosa1 1 banat’s university of agricultural science and veterinary medicine “king michael i of romania” timisoara, romania 2 department of agricultural and forestry technology, university of craiova, craiova, romania *corresponding author: elena.agro@gmail.com abstract. the present study aimed to evaluate the cytogenetic effects induced by heavy metals nickel (ni) and lead (pb) to crop plants, using the allium sativum (garlic) as a test plant. for this purpose, were used solutions of nickel nitrate ni(no3)2 and lead nitrate pb(no3)2 at concentrations of 50, 150 and 450 ppm for 72 hours, along with an untreated control variant immersed in plain water. the biological material was immersed from the beginning in the tested solutions. the results obtained showed a strong inhibitory effect of these heavy metals on the process of rhizogenesis, as well as a significant mitodepresive effect in the meristematic cells, both phenomena being correlated with increasing concentration of the tested solutions. at the same time, several types of chromosomal aberrations (c-mitosis, vagrants, star-anaphase, star-telophase, fragments, clumping, stickiness, bridges) have been recorded in all treatment variants. the presence of these chromosomal aberrations in all treatment variants indicates the aneugenic effects of nickel nitrate and lead nitrate in the meristematic cells of a. sativum. the results suggest the ecotoxicity potential of nickel and lead on plants even at low concentrations and confirm the suitability of a. sativum as a test plant for assessing the cytotoxicity and genotoxicity of heavy metals to plants. keywords. genotoxicity, chromosomal aberrations, lead, mitotic index, nickel. introduction the concept of heavy metals has refers to any metallic chemical element that has a relatively high density and is toxic or poisonous in low concentrations. as natural water pollutants, heavy metals are among the most toxic pollutants due to their prolonged persistence in solutions and the difficulty of being converted into insoluble compounds in surface waters (bilal et al. 2014). the contamination of soil by heavy metals is also a major environmental problem (lassoued et al. 2014) and have a toxic action on the aquatic organisms, meanwhile inhibiting the self-purification processes (nemcsók 38 ioan sarac et al. et al. 2010). heavy metals are dangerous because they tend to bioaccumulate (coroian et al. 2017; hariri et al. 2018; marinova et al. 2018). bioaccumulation means the increase in time in biological organisms of the concentration of the substance in an amount compared to the concentration of this substance in the environment. the presence of heavy metals in soil, is one important factor that can cause altered physiological and metabolic processes to plants or disturbing the metabolism of essential elements (dong et al. 2006; mohanpuria et al. 2007; wójcik and tukiendorf 2014; petrescu et al. 2015; sarac et al. 2015; georgieva et al. 2018; nikolova and georgieva 2018). symptoms of heav y metal toxicity are the result of harmful effects of metals on physiological processes including: inhibiting respiration and photosynthesis, altering the plant-water relationship that causes stress, decreased plasma membrane permeability in the root cells, adverse effects on the metabolic activities of enzymes (arduini 1994). lead (pb) is one of the ubiquitously distributed most abundant toxic elements in the soil. pb inhibits the activity of enzymes at cellular level by reacting with their sulfhydril groups (yadav 2010). high lead exposure is harmful, particularly for children; its effects include damage to the nervous system, liver and kidney damage and developmental delays. also, the lead exposure is associated with an increased risk of several cancers, in particular, meningioma, brain cancer, and kidney cancer (liao et al. 2016). nickel (ni) is considered to be an essential micronutrient for plants (eskew et al. 1983) but at excess concentrations this metal becomes toxic for majority of plant species and triggers oxidative damage (zornova et al. 1999; nakazawa et al. 2004; gajewska et al. 2006; sachan and lal 2017). on the other hand, some authors reported cytotoxic effects even at low doses (20 to 100µm) of nickel ions as well as antioxidative enzyme changes in allium cepa roots (gantayat et al. 2017). concentrations of ni could increase by human activities such as application of phosphate fertilizers and pesticides (gimeno-garcía et al. 1996) or industrial and agricultural wastewater discharges, domestic sewage discharge and atmospheric deposition (yan et al. 2018). the vegetal meristematic tissues that are used for testing the effects of chemicals on chromosomes should be easy to obtain and less expensive. from this point of view, the species a. sativum and a. cepa are well suited to cytogenetic studies because the meristematic roots appear lightly, have relatively large chromosomes in small numbers and can be easily observed by optical microscope (doroftei et al. 2010; bonciu 2012; bonciu et al. 2018). materials and methods plant material the biological material consisted of garlic bulbs, clean and without traces of pests or diseases that had been spread in several bulbils. they were cleaned from the dried leaves and formed by removing any roots after which they were transferred to small glass bottles containing the heavy metal solutions: nickel nitrate ni(no3)2 and lead nitrate pb(no3)2 in concentrations of 50, 150 and 450 ppm for each of them. three treatment variants with 4 repetitions were performed for each of the heavy metals experienced, along with an untreated control immersed in plain water. in each variant, four garlic bulbils were immersed directly into the treatment solutions for 72 hours, time required for the meristematic roots to be emitted. microscopic preparations after sampling, the meristematic roots were fixed with a mixture of absolute ethyl alcohol and glacial acetic acid in a volume ratio of 3: 1 for 16 hours in the refrigerator, followed by acid hydrolysis with 1 n hcl for 5 minutes and hcl 50% consisting of equal parts of hcl and distilled water for 16 min at room temperature. roots’ staining was performed by the feulgen technique with schiff’s reagent; the staining time was 90 minutes, followed by the intensification of the coloration in plain water for 20 minutes. statistical analyses after 72 hours, the meristematic roots were counted and measured at each variant. the cytogenetic effects of heavy metals were assessed by calculating the mitotic index (mi) and analysing the chromosomal aberrations observed in the various stages of mitosis. the microscopic preparations have been studied using a microscope with digital camera kruss (kruss manufacturer hamburg, germany). five preparations for each variant and 500 cells were analysed for calculating the mitotic index and the chromosome aberration frequency. statistical analysis was done using ms excel 2007. the obtained data were analysed statistically with oneway analysis of variance (anova). the differences between treatment means were compared using the lsd-test at a probability level of 0.05% subsequent to the anova analysis. the mitotic index and chromosomal aberrations were calculated using the following formulas: 39evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of allium test mitotic index (mi%) = total number of cells in division / total number of analysed cells x 100; chromosomal aberrations (ca%) = total number of aberrant cells / total number of cells in division × 100. results the treatment of a. sativum bulbils with nickel and lead depending on the concentration negatively influenced the process of issuing meristematic roots. the treated roots was smaller in size and they had a smaller number than the control. thus, their number decreased as the concentration of the heavy metal solutions tested in all variants increased: from 46 registered roots to the control variant, to 14-28 roots to the variant treated with ni(no3)2 respectively 5-18 roots to the variant treated with pb(no3)2 (figure 1). the results showed that both heavy metals treatments caused a decrease in mi at all the treatment groups (table 1). thus, the value of the mi decreased with the increase concentration of heavy metal solutions. the intensity of mitotic activity was decreasing in order of treatment with lead nitrate to nickel nitrate treatment. however, the strongest mitodepresive effect was seen in the treatment of pb(no3)2 at the concentration of 450 ppm, when mi was 5.32%, ie with 46.4% lower mitotic activity compared to the control variant. heavy metals tested induced a high number of ca when compared with control. the increase of ca was dependent on the increasing treatment concentrations (table 1). the types of ca identified in meristematic cells of a. sativum were the following: c-mitosis (figure 2a); fragments and vagrants (figure 2b); star-anaphase; star-telophase (figure 2c); clumping; stickiness (figure 2d); bridges (figure 2e). as can be seen from the data of table 1, the most common types of ca were stickiness, c-mitosis and bridges, and the least frequent were vagrants chromosomes. compared with the control variant, the total ca rate recorded insignificant values for the variant treated with 50 ppm ni(no3)2, significant for the variant treated with 150 ppm ni(no3)2 and distinctly significantly positive for the variant treated with 450 ppm ni(no3)2. on the other hand, in case of the pb(no3)2 treated variants compared to the control variant, the total ca recorded significantly positive values for the variant treated with 50 ppm pb(no3)2 and strongly positive for the variants treated with 150 and 450 ppm pb(no3)2 respectively. discussion we chose to study the cytotoxic effects of ni and pb heavy metals on a. sativum because, according to many authors (saxena et al. 2004; gul et al. 2006; unyayar et al. 2006; liu et al. 2009), the evaluation of ca in a. sativum meristematic roots is a reliable biotest example that can be applied to detect a wide range of genetic damage. at a macroscopic level, heavy metals induced inhibition of the growth of meristematic garlic roots. generally, heavy metals are known to decreasing the plant growth and ground cover (mcgrath et al. 2001). some effects of lead on growth, physiology, metabolism and yield attributes of plants are the following: inhibition in seed germination, fresh and dry biomass, leaf area, chlorophyll and growth to helianthus annuus (mahmood et al. 2013); decline in growth, chlorophyll, carotenoids and prolinecontent to brassica juncea (john et al. 2009); decrease in plant growth, root hair to vigna unguiculata (kopittke et al. 2007), etc. in our experiment, reducing the number of meristematic root has been accentuated with increasing con 0 50 150 450 50 150 450 46 28 21 14 18 9 5 0 50 100 150 200 250 300 350 400 450 500 concentration (ppm) number of meristematic roots after 72 h fig. 1. the inhibitory effect of different concentrations of ni(no3)2 and pb(no3)2 on the rhizogenesis to a. sativum. a b c d e fig. 2. some chromosomal aberrations identified in meristematic cells of a. sativum exposed to ni(no3)2 and pb(no3)2: c-mitosis (a), fragments and vagrants (b), star-telophase (c), stickiness in telophase (d), anaphase bridge (e). 40 ioan sarac et al. centrations of test solutions (especially to treatments with lead nitrate). proportionately with increasing concentrations, intensity of the mitotic division has been decreased to all variants, as an active protection reaction of the plants exposed to heavy metals action. these findings are in agreement with doroftei et al. (2010) who have tested the cytogenetic effects of lead nitrate to a. cepa. the lead and nickel inhibited cell division to other plants too, like zea mays (kozhevnikova et al. 2007). the root growth is an integrative process depending on whole-organism signalling and individual growth trajectories of cells (beemster et al. 2003). the intensity of the mitotic division is directly related to the growth of plant roots; conceptually, mitotic division in the apical root meristem provides cells during longitudinal growth (sanz et al. 2012). the number of dividing cells in the root apical meristem generates a cell flux with importance in modulating root growth (baskin, 2013). studies of cell length profiles have shown that the proliferative fraction of dividing cells in the apical root meristem proliferation domain is indistinguishable from one, even in response to moderate levels of stress (ivanov and dubrovsky 2013). the results of this study highlight the increasing of ca frequencies to a. sativum dependent on different concentrations of nickel nitrate and lead nitrate. stickiness, c-mitosis and bridges were most often identified in all treatment variants but the highest frequency was recorded to variants treated with lead nitrate. according to some authors, sticky chromosomes might have resulted from increased chromosome contraction and condensation or possibly from depolymerisation of dna and partial dissolution of nucleoproteins (kuras et al. 2005; turkoglu 2013b). asita and mokhobo (2013) suggested that the induction of allium’s sticky chromosomes under the influence of pesticides indicates abnormal dna condensation, abnormal chromosomal wrapping, and inactivation of the axes, and all these anomalies cell division can have adverse effects on the environment. c-mitosis indicates a chemical-inhibited spindle formation similar to the effect of colchicine and induction of these aberrations suggests a turbogenic effect (shahin and el-amoodi 1991; turkoglu 2013b). regarding the anaphase bridges, these chromosomal aberration cause structural chromosome mutations and may lead to loss of genetic material (george 2000; pampalona et al. 2016). according to turkoglu (2013b), bridges could form due to dicentric chromosome presence or due to the breakage and fusion of chromosomes and chromatids. in our experiment, the stickiness had a frequency of 3.62-5.42% at the nickel nitrate treatment, while for treatment with lead nitrate the frequency of these ca was at the level of 6.28-7.40% at all concentrations (50, 150, 450 ppm). sticky chromosomes can probably lead to cell death (singh 2015). these results suggest the strong genotoxic effect of lead nitrate even at low concentrations of 50 and 150 ppm. the current findings agreed well with other reports which showed cytotoxicity and genotoxicity of lead in plant cells (choudhury and panda 2004; arya et al. 2013). some nickel compounds have been established as human carcinogens (coogan et al. 1989), but at the same time, some plant seeds, such as soybeans, can act as protectors when introduced into diet of treated mice, by reducing the percentage of ca (fahmy et al. 2014). certain hormones can act as agents for inducing resistance table 1. mitotic index, type and percentage of mitotic aberrations induced by some heavy metals on the meristematic roots to a. sativum. treatment / exposure time (hours) conc. (ppm) mi ± sd (%) ca (%) total aberrations (%)c-m v s-a s-t f cl s b ct / 72 0 11.45±0.5 2.35 0 0 0 0 0 0 0 2.35 ni(no3)2 / 72 50 10.66±0.4 1.61 0.65 0.42 0 0 0 3.62 2.31 8.61 150 8.13±0.3 1.90 0.83 1.81 0.83 0 1.23 5.11 3.22 14.93* 450 6.84±0.8 3.15 1.40 2.73 1.43 2.11 3.41 5.42 3.91 23.56** pb(no3)2 / 72 50 8.73±0.4 4.65 1.62 3.90 1.70 2.80 3.96 6.28 4.82 29.73** 150 6.51±0.8 6.23 2.11 4.31 2.82 3.70 4.20 6.50 5.31 35.18*** 450 5.32±0.5 7.51 2.64 4.85 4.20 4.21 6.52 7.40 6.10 43.43*** ct = control; conc. = concentration; mi = mitotic index; sd = standard deviation; ca = chromosomal aberrations; c-m = c-mitosis; v = vagrants; s-a = star-anaphase; s-t = star-telophase; f = fragments; cl = clumping; s = stickiness; b = bridges; the differences between treatment means were compared using the lsd-test at a probability level of 0.05%: *significant at p<0.05, **significant at p<0.01, ***significant at p<0.001 as compared to the control variant. 41evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of allium test of plants to heavy metal toxicity. thus, some authors have reported that brassica juncea plants sprayed with 28-homobrassinolide hormone, showed improved resistance against the some heavy metal toxicity (hayat et al. 2007). also, the organic acids (citrate and malate) have been reported to have a role in the plant protection against heavy metal stress (haydon et al. 2007). ca called c-mitosis had a frequency of 1.61-3.15% at the nickel nitrate treatment, while for treatment with lead nitrate the frequency of c-mitosis was at the level of 4.65-7.51%, these results demonstrating the high genotoxic potential of lead to plants. c-mitosis is the result of damaged mitotic apparatus due to genotoxic substances in the cells and is stimulated by many chemicals (fiskesjö 1993; firbas and amon 2014). cytogenetic tests on a. sativum reveal a decrease in the mitotic index following heavy metal treatments. mitosis analysis indicates the occurrence 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sandrine ngo ngwe2, sonja siljak-yakovlev3,* assessment of cytotoxicity and mutagenicity of insecticide demond ec25 in allium cepa and ames test arzu özkara cytogenetic effects of fulvic acid on allium cepa l. root tip meristem cells özlem sultan aslantürk evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of allium test ioan sarac1, elena bonciu2,*, monica butnariu1, irina petrescu1, emilian madosa1 fluorescence in situ hybridisation study of micronuclei in c3a cells following exposure to elf-magnetic fields luc verschaeve1,2,*, roel antonissen1, ans baeyens3, anne vral3, annemarie maes1 phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities nicoleta anca şuţan1,*, irina fierăscu2, radu fierăscu2, deliu ionica1, liliana cristina soare1 telomeric heterochromatin and meiotic recombination in three species of coleoptera (dorcadion olympicum ganglebauer, stephanorrhina princeps oberthür and macraspis tristis laporte) anne-marie dutrillaux, bernard dutrillaux* a whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of populus trichocarpa l. subjected to different stresses alberto vangelisti#, gabriele usai#, flavia mascagni#, lucia natali, tommaso giordani*, andrea cavallini differences in c-band patterns between the japanese house mice (mus musculus) in hokkaido and eastern honshu hikari myoshu, masahiro a. iwasa* karyotypic description and repetitive dna chromosome mapping of melipona interrupta latreille, 1811 (hymenoptera: meliponini) natália martins travenzoli1, ingrid cândido de oliveira barbosa2, gislene almeida carvalho-zilse2, tânia maria fernandes salomão3, denilce meneses lopes1,* caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 163-178, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-186 citation: k. saha, r.k. sinha, s. sinha (2020) karyological studies in thirteen species of zingiberacaeae from tripura, north east india. caryologia 73(1): 163-178. doi: 10.13128/caryologia-186 received: march 11, 2019 accepted: february 23, 2020 published: may 8, 2020 copyright: © 2020 k. saha, r. kumar sinha, s. sinha. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha cytogenetics and plant biotechnology laboratory, department of botany, tripura university, suryamaninagar-799022, tripura, india *corresponding author. e-mail: saha.kshn@gmail.com abstract. tripura being a state in north east india belongs to indo-burma bio-diversity hotspot and is considered as a centre of origin of many species of zingiberaceae. alpinia calcarata, alpinia malaccensis, alpinia nigra, amomum aromaticum, amomum koenigii, amomum maximum, curcuma amada, curcuma caesia, curcuma longa, curcuma picta, hedychium coccineum, hedychium coronarium and hedychium thyrsiforme are found in wild state in different geographical locations of tripura. their karyotypes were analyzed both at interspecific and intraspecific levels. the somatic chromosome number of alpinia spp. and amomum spp. was found to be 2n = 4x = 48. the curcuma spp. represented by c. amada had 2n = 42 chromosomes and three other species viz., c. caesia, c. longa and c. picta had 2n = 63 chromosomes having x=21, indicating that polyploidy is a common feature in this genus. the somatic chromosome number of hedychium spp. was found to be 2n = 34 chromosomes having a basic no. x = 17. chromosomal data based clustering pattern and their sub-grouping at intra-specific level validates the taxonomic status of these species. gower’s similarity matrix is an indicator of cryptic changes leading genus specific karyotype conservatism. keywords. indo-burma bio-diversity hotspot, zingiberaceae, karyotype, polyploidy, cryptic changes. introduction the state of tripura (20°56’-24°34’ north latitude and 91°10’-92°21’ east longitude) is situated in the sub-himalayan region of north east india and belongs to indo-burma biodiversity hotspot of the world (myers et al. 2000; mao et al. 2009). the diverse vegetation of this region includes a good number of plants, which are endemic either to the state or to the north-eastern region of india (deb 1981). in india, the zingiberaceae family includes 22 genera and about 200 species which are distributed in andaman and nicobar island, western ghats and eastern himalaya region including north-eastern india (jain and prakash 1995; sabu 2006). many natural useful products such as food, dyes, medicines, spices and condiments are obtained from the members of this family (nayak 2002; arambewela et al. 2004; sahoo et al. 2014; rahman and islam 2015). the local people of south asian countries also use 164 kishan saha, rabindra kumar sinha, sangram sinha several plant products of zingiberaceae in traditional medicines (jayaweera 1982; prakash and mehrotra 1996; gupta et al. 1999; kala 2005; bhuiyan et al. 2010; roy et al. 2012; sarangthem et al. 2013; rajkumari and sanatombi 2018). some species of zingiberaceae are also highly valued as ornamental plants because of their showy scented flowers (jatoi et al. 2007; gao et al. 2008). deb (1983) recorded nine genera having 24 different species of this family from this phyto-geographical region. however, alpinia galanga, hedychium ellipticum and hedychium marginatum could not be found in specific localities as was earlier reported by deb (1983). probably, these species have eventually perished due to anthropological disturbances. in-spite of such habitat destruction, five more species viz., curcuma caesia, curcuma picta, hedychium coccineum, amomum aromaticum (syn. a. jainii s. tripati & vedprakash), amomum koenigii (syn. amomum corynostachyum. wall.) and amomum maximum are found in different locations which were not reported by deb (1983). thus, 13 different species belonging to four genera viz., alpinia, amomum, curcuma and hedychium of zingiberaceae are now available in tripura. these are alpinia calcarata (haw.) roscoe, alpinia malaccensis (burm.f.) roscoe, alpinia nigra (gaertn.) burtt, amomum aromaticum roxb. (syn. amomum jainii s. tripati and vedprakash), amomum koenigii j.f.gmel. (syn. amomum corynostachyum wall.), amomum maximum roxb., curcuma amada roxb., curcuma caesia roxb., curcuma longa l., curcuma picta roxb. ex skornick, hedychium coccineum buch.-ham. ex sm., hedychium coronarium j. koenig. and hedychium thyrsiforme sm. morphologically these species are quite distinct in their rhizome, floral and fruit characters (deb 1983; vanchhawng and lalramnghinglova 2016). delineation of plant species based on morphological characters alone is not adequate (larsen and smith 1978) and so, for characterization of species at interand intra-specific levels cytological tools are now effectively used to understand the taxonomic relationship and evolutionary patterns between and within species (yoshikane and naohiro 1991; joseph et al. 1999). previously, cytological works on zingiberaceae were mainly focussed on curcuma spp. and various researchers from time to time (raghavan and venkatasubban 1943; chakraborti 1948; sato 1948; sharma and bhattacharya 1959; sato 1960; ramachandran 1961,1969; fedorov 1969; prana 1977; prana et al. 1978; nambiar 1979; mandi 1990; ardiyani 2002; skornickova et al. 2007; bhadra and bandhyopadhyay 2015; lamo and rao 2017; bhadra et al. 2018) reported the existence of different ploidy levels, ranging from diploid (2n = 42) to tetraploid (2n=84), having a basic number x=21. in addition, aneuploid cytotypes in curcuma spp. had also been reported (das et al. 1999, sugiura (1931, 1936), sato 1960). cytological studies previously carried out in alpinia spp. and amomum spp. were mainly concentrated on the determination of somatic chromosome numbers having 2n = 4x= 48 chromosomes (raghavan and venkatasubban 1943; chakravorti 1948; sharma and bhattacharya 1959; ramachandran 1969). but, from the literature it is evident that the detailed karyotype analysis in different species of alpinia and amomum was very meagre (chen et al. 1988; joseph 1998). in hedychium spp. the occurrence of different ploidy levels (2n=34, 51 and 68) was reported in various cytological studies (sharma and bhattacharyya 1959; bhattacharyya 1968; ramachandran 1969; mukherjee 1970; mahanty 1970; khoshoo 1979; gao et al. 2008). tripura being a part of indoburma hotspot is considered as a centre of origin of many indian species of zingiberaceae but till date, cytological investigation of these species have not been carried out from this region. the present study is the first attempt to assess the karyotypic relationship in different species of zingiberaceae at interand intraspecific level from the sub – himalayan region of tripura and for providing additional information to the chromosomal database of zingiberean plants of indian origin. materials and methods plant materials we examined two populations (described as pop-i and pop-ii) of 13 different wild species of zingiberaceae viz., alpinia calcarata (haw.) roscoe, alpinia malaccensis (burm.f.) roscoe, alpinia nigra (gaertn.) burtt, amomum aromaticum roxb. (syn. amomum jainii s. tripati and vedprakash), amomum koenigii j. f. gmel. (syn. amomum corynostachyum wall.), amomum maximum roxb., curcuma amada roxb., curcuma caesia roxb., curcuma longa l., curcuma picta roxb. ex skornick, hedychium coronarium j. koenig., hedychium coccineum buch.-ham. ex sm. and hedychium thyrsiforme sm. growing in different locations in tripura. they were grown in the experimental garden, department of botany, tripura university for future research. all the plant species were independently authenticated by the taxonomy experts. herbarium of each species has been submitted in tripura university herbarium with respective voucher numbers (table s1). preparation of somatic chromosomes the somatic chromosome preparation was carried out with modified aceto-orcein staining technique 165karyological studies in thirteen species of zingiberacaeae from tripura, north east india (sharma and sharma 1980). young healthy root tips of each species were pre-treated in a mixture of saturated solution of para dichloro-benzene (p-db) and 0.002m 8hydroxyquinoline (1:1) at 12-150c for 6 hrs. the root tips were then washed with distilled water and kept in acidulated alcohol (mixture of 1nhcl and absolute ethyl alcohol in 1:1 ratio) for 1 hour. thereafter, root tips were kept in 45% acetic acid for 20 minutes. after a thorough wash with distilled water, root tips were treated with 5% cellulase (sigma cat. no. 22178) and 5% pectinase (sigma cat. no. 17389) mixture (1:1) in citrate buffer (ph 4.8) for 3 hours at 370c. enzyme treated root tips were then washed with double distilled water and stained with 2% aceto-orcein : 1nhcl (9:1) mixture for overnight and finally squashed in 45% acetic acid. the well spread metaphase plate was captured using zeiss make axio (lab.a1) microscope and zen software was used for determining the length of short and long arm of individual chromosome of the species studied. study of nucleoli in somatic cells nucleolar staining was carried out following the technique of fernandez-gomez et al. (1969). initially root tips were fixed in 10% formol :1% hydroquinone (1:1) solution for 2 hrs. these were thoroughly washed in distilled water and immersed in 2% silver nitrate solution at 60ºc in dark for overnight. agno3 treated root tips were again kept in formol-hydroquinone (1:1) solution for 1 hour and finally squashed in 45% acetic acid. data analysis in preparing the numerical data of karyotype, three well spread metaphase plates of each species were compared. in cases, where the length and the arm ratio varied the mean was taken to calculate the value of centromeric index (f%). the centromeric index, tf%, inter-chromosomal asymmetry index (a2), coefficient of variation of chromosome length (cvcl), and mean centromeric asymmetry (mca) were calculated by the following formulae:centromeric index (f%) = s/(l+s)×100 (levan et al. 1964);tf% = (∑s/∑cl) × 100 (huziwara 1962);inter-chromosomal asymmetry index (a2) = scl/ xcl (zarco 1986); degree of karyotype asymmetry (a) = [∑ (l-s)/(l+s)]/n (watanabe et al. 1999); coefficient of variation of chromosome length (cvcl) = a2×100 (paszko 2006); mean centromeric asymmetry (mca) = a×100 (peruzzi and eroglu 2013); (s = length of short arm, l = length of long arm, cl = chromosome length, sclstandard deviation of chromosome length, xcl mean of chromosome length, n – haploid number of chromosome complement). along with stebbins asymmetry indices, the inter and intra-chromosomal asymmetry indices were measured statistically (zarco 1986; watanabe et al. 1999) and the relationship among the species was explained by means of bi-dimensional scattered plot (peruzzi and eroglu 2013).to determine the karyological relationship among taxa upgma mediated dendrogram was constructed using the software past 3.03 (hammer 2013). in addition, multivariate principal co-ordinate analysis (pca) was also performed using different parameters of numerical data of karyotypes (table 1) of the respective species and their populations (peruzzi and altinordu 2014). results due to difficulty in obtaining suitable metaphase chromosome spreads with the conventional aceto-orcein staining technique, a new protocol has been developed to determine the somatic chromosome number and to analyze the detailed karyotype of all the taxa studied. the somatic chromosomes of alpinia spp., amomum spp., curcuma spp. and hedychium spp. could be classified into three distinct morphological types. type a: short chromosomes (1.33 µm – 2.66 µm) bear two constrictions, primary and secondary, one is nearly median (m) to sub-median (sm) and the other is terminal (t) in position. type b: chromosomes are short in size and their length range from 0.96 µm – 2.79 µm. the position of centromere is sub-median (sm) to median (m) (f% >33.33%). type c: chromosomes are short in size and their length range from 1.86 µm – 2.13µm. the position of centromere is sub-median (sm) (f% <33.33%). analysis of karyotypes except in alpinia nigra, which had two pairs of c type of submetacentric chromosomes, karyograms (figure s1) of rest of the 12 species showed the presence of different combinations of a and b types chromosomes as classified in the present study. the detailed analyses of the karyotypes of pop-i and pop-ii of alpinia spp., amomum spp., curcuma spp. and hedychium spp. reveal the following data: alpinia calcarata-pop-i: somatic chromosome number 2n=48 (figure 1a); number of chromosomes bearing secondary constric166 kishan saha, rabindra kumar sinha, sangram sinha tion 2; range of chromosome length – (1.06 µm – 2.79 µm); total chromosome length – 90.43 µm; ratio of largest and smallest chromosome – 2.63:1; mean arm ratio (l/s) –1.38; karyotype formula a2 (2m) b46 (6m +30m+10sm); stebbins category – 1b; tf% 42.61; coefficient of variation of chromosome length (cvcl) – 28.44; mean centromeric asymmetry (mca) – 30.00. alpinia calcarata-pop-ii: somatic chromosome count 2n=48 (figure 1b); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.06 µm – 2.79 µm); total chromosome length – 91.20 µm; ratio of largest and smallest chromosome – 2.63:1; mean arm ratio (l/s) –1.39; karyotype formula a2 (2m) b46 (6m+34m+6sm) stebbins category – 1b; tf% – 42.44; cvcl – 27.71; mca – 30.23. alpinia malaccensis-pop-i: somatic chromosome count 2n=48 (figure 2a); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.16 µm – 2.14 µm); total chromosome length – 82.10 µm; ratio of largest and smallest chromosome – 1.84:1; mean arm ratio (l/s) – 1.33; karyotype formula a2 (2m) b46 (36m+10sm); stebbins category – 1a; tf% – 43.57; cvcl – 18.17; mca – 26.00. alpinia malaccensis-pop-ii: somatic chromosome number 2n=48 (figure 2b); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.16 µm 2.26 µm); total chromosome length – 85.74µm; ratio of largest and smallest chromosome – 1.95:1; mean arm ratio (l/s) –1.33; karyotype formula a2 (2m) b46 (4m+36m+6sm); stebbins category – 1a; tf% – 43.65; cvcl – 17.79; mca – 25.41. table 1. karyological parameters for cluster analysis and gower’s similarity coefficient matrix. name of the species and population scn hcl (µm) bcn tf% mca cvcl alpinia calcarata, pop-i 48 45.22 12 42.61 30.00 28.44 alpinia calcarata, pop-ii 48 45.60 12 42.44 30.23 27.71 alpinia malaccensis, pop-i 48 41.05 12 43.57 26.00 18.17 alpinia malaccensis, pop-ii 48 42.87 12 43.65 25.41 17.79 alpinia nigra, pop-i 48 44.06 12 40.29 38.83 23.00 alpinia nigra, pop-ii 48 44.14 12 40.27 38.91 22.45 amomum maximum, pop-i 48 33.06 12 44.75 20.76 18.61 amomum maximum, pop-ii 48 32.93 12 44.79 20.84 17.82 amomum aromaticum, pop-i 48 30.61 12 45.39 18.45 21.75 amomum aromaticum, pop-ii 48 30.57 12 45.52 17.94 20.93 amomum koenigii, pop-i 48 39.50 12 44.56 23.67 26.46 amomum koenigii, pop-ii 48 39.32 12 44.28 22.87 26.48 hedychium coronarium, pop-i 34 20.72 17 47.32 10.72 15.91 hedychium coronarium, pop-ii 34 20.78 17 47.24 11.06 15.11 hedychium coccineum, pop-i 34 26.40 17 46.68 13.27 15.51 hedychium coccineum, pop-ii 34 26.46 17 46.47 14.11 15.40 hedychium thyrsiforme, pop-i 34 19.13 17 46.60 13.56 15.24 hedychium thyrsiforme, pop-ii 34 19.70 17 46.61 13.69 14.88 curcuma amada, pop-i 42 31.05 21 47.02 11.91 12.52 curcuma amada, pop-ii 42 32.35 21 46.96 12.17 12.77 curcuma caesia, pop-i 63 34.29 21 47.98 12.05 12.08 curcuma caesia, pop-ii 63 34.37 21 48.11 11.35 12.08 curcuma picta, pop-i 63 44.67 21 47.96 12.23 13.16 curcuma picta, pop-ii 63 44.43 21 47.94 12.34 12.66 curcuma longa, pop-i 63 44.16 21 47.01 16.78 11.18 curcuma longa, pop-ii 63 44.04 21 47.13 17.23 11.01 scn – somatic chromosome number; hcl-total chromosome length of the haploid complement; bcnbasic chromosome number; tf% total form percentage; mca mean centromeric asymmetry; cvcl coefficient of variation of chromosome length. 167karyological studies in thirteen species of zingiberacaeae from tripura, north east india figures. 1a-13b. mitotic metaphase chromosomes of thirteen zingiberaceae species from tripura. 1alpinia calcarata; 2a. malaccensis; 3a. nigra; 4amomum maximum; 5a. aromaticum; 6a. koenigii; 7curcuma amada; 8c. caesia; 9c. picta; 10c. longa; 11h. coronarium; 12h. coccineum; 13h. thyrsiforme (a – pop i; b – pop ii); scale bars = 5µm. 168 kishan saha, rabindra kumar sinha, sangram sinha alpinia nigra-pop-i: somatic chromosome number 2n=48 (figure 3a); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.09 µm – 2.80 µm); total chromosome length – 88.12 µm; ratio of largest and smallest chromosomes – 2.57:1; mean arm ratio (l/s) – 1.57; karyotype formula – a2 (2sm) b42 (4m+26m+12sm) c4 (4sm); stebbins category – 2b; tf% – 40.29; cvcl – 23.00; mca – 38.83. alpinia nigra-pop-ii: somatic chromosome number 2n=48 (figure 3b); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.09 µm – 2.72 µm); total chromosome length – 88.28 µm; ratio of largest and smallest chromosome – 2.50:1; mean arm ratio (l/s) – 1.57; karyotype formula a2 (2sm) b42 (4 m+26 m+ 12 sm) c4 (4 sm); stebbins category – 2b; tf% – 40.27; cvcl – 22.45; mca – 38.91. amomum maximum-pop-i: somatic chromosome number 2n=48 (figure 4a); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.06 µm – 1.94 µm); total chromosome length – 66.12 µm; ratio of largest and smallest chromosome – 1.83:1; mean arm ratio (l/s) –1.27; karyotype formula a2 (2m) b 46(14m+26m+6sm); stebbins category – 1a; tf% – 44.75; cvcl – 18.61; mca – 20.76. amomum maximum-pop-ii: somatic chromosome number 2n=48 (figure 4b); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.06 µm – 1.94 µm); total chromosome length – 65.86 µm; ratio of largest and smallest chromosome – 1.83:1; mean arm ratio (l/s) –1.26; karyotype formula a2 (2m) b 46(16m+24m+6sm); stebbins category – 1a; tf% – 44.79; cvcl – 17.82; mca – 20.84.̀ amomum aromaticum-pop-i: somatic chromosome number 2n=48 (figure 5a); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.06 µm – 2.10 µm); total chromosome length – 61.22 µm; ratio of largest and smallest chromosome – 1.98:1; mean arm ratio (l/s) – 1.22; karyotype formula a2 (2m) b 46 (16m+30m); stebbins category – 1a; tf% – 45.39; cvcl – 21.75; mca – 18.45. amomum aromaticum-pop-ii: somatic chromosome number 2n=48 (figure 5b); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.06 µm – 2.10 µm); total chromosome length – 61.14 µm; ratio of largest and smallest chromosome – 1.98:1; mean arm ratio (l/s) –1.21; karyotype formula a2 (2m) b 46(16m+30m); stebbins category – 1a; tf% – 45.52; cvcl – 20.93; mca – 17.94. amomum koenigii-pop-i: somatic chromosome number 2n=48 (figure 6a); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.06 µm – 2.39 µm); total chromosome length – 79.00 µm; ratio of largest and smallest chromosome – 2.25:1; mean arm ratio (l/s) – 1.27; karyotype formula a2 (2m) b 46(12m+28m+6sm); stebbins categorization – 1b; tf% – 44.56; cvcl – 26.46; mca – 23.67. amomum koenigii-pop-ii: somatic chromosome number 2n=48 (figure 6b); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.06 µm – 2.39 µm); total chromosome length – 78.64 µm; ratio of largest and smallest chromosome – 2.25:1; mean arm ratio (l/s) –1.28; karyotype formula a2 (2m) b 46(8m+32m+6sm); stebbins category – 1b; tf% – 44.28; cvcl – 26.48; mca – 22.87. curcuma amada-pop-i: somatic chromosome number 2n=42 (figure 7a); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.35 µm – 1.91 µm); total chromosome length – 62.10 µm; ratio of largest and smallest chromosome – 1.41:1; mean arm ratio (l/s) –1.13; karyotype formula a2 (2m) b 40 (10m+30m); stebbins category – 1a; tf% – 47.02; cvcl – 12.52; mca – 11.91. curcuma amada-pop-ii: somatic chromosome number 2n=42 (figure 7b); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.37 µm – 1.91 µm); total chromosome length – 64.70 µm; ratio of largest and smallest chromosome – 1.39:1; mean arm ratio (l/s) – 1.14; karyotype formula a2 (2m) b 40(10m+30m); stebbins category – 1a; tf% – 46.96; cvcl – 12.77; mca – 12.17. curcuma caesia-pop-i: somatic chromosome number 2n=63 (figure 8a); number of chromosomes bearing secondary constriction 3; range of chromosome length – (0.96 µm – 169karyological studies in thirteen species of zingiberacaeae from tripura, north east india 1.33 µm); total chromosome length – 68.58 µm; ratio of largest and smallest chromosome – 1.39:1; mean arm ratio (l/s) –1.09; karyotype formula – a3 (3m) b 60(30m+30m); stebbins category – 1a; tf% – 47.98; cvcl – 12.08; mca – 12.05. curcuma caesia-pop-ii: somatic chromosome number 2n=63 (figure 8b); number of chromosomes bearing secondary constriction 3; range of chromosome length – (0.96 µm – 1.33 µm); total chromosome length – 68.73 µm; ratio of largest and smallest chromosome – 1.39:1; mean arm ratio (l/s) – 1.08; karyotype formula – a3 (3m) b 60(33m+27m); stebbins category – 1a; tf% – 48.11; cvcl – 12.08; mca – 11.35. curcuma picta-pop-i: somatic chromosome number 2n=63 (figure 9a); number of chromosomes bearing secondary constriction 3; range of chromosome length – (1.10µm – 1.67 µm); total chromosome length – 89.34 µm; ratio of largest and smallest chromosome – 1.52:1; mean arm ratio (l/s) – 1.10; karyotype formula – a3 (3m) b60 (42m+18m); stebbins category – 1a; tf% – 47.96; cvcl – 13.16; mca – 12.23. curcuma picta-pop-ii: somatic chromosome number 2n=63 (figure 9b); number of chromosomes bearing secondary constriction 3; range of chromosome length – (1.10 µm – 1.63 µm); total chromosome length – 88.86 µm; ratio of largest and smallest chromosome – 1.48:1; mean arm ratio (l/s) – 1.10; karyotype formula – a3 (3m) b60 (42m+18m); stebbins category – 1a; tf% – 47.94; cvcl – 12.66; mca – 12.34. curcuma longa-pop-i: somatic chromosome number 2n=63 (figure 10a); number of chromosomes bearing secondary constriction 3; range of chromosome length – (1.17 µm – 1.70 µm); total chromosome length – 88.32 µm; ratio of largest and smallest chromosome – 1.45:1; mean arm ratio (l/s) –1.13; karyotype formula – a3 (3m) b60 (15m+45m); stebbins category – 1a; tf% – 47.01; cvcl – 11.18; mca – 16.78. curcuma longa-pop-ii: somatic chromosome number 2n=63 (figure 10b); number of chromosomes bearing secondary constriction 3; range of chromosome length – (1.18 µm – 1.70 µm); total chromosome length – 88.08 µm; ratio of largest and smallest chromosome – 1.44:1; mean arm ratio (l/s) – 1.13; karyotype formula – a3 (3m) b60 (15m+45m); stebbins category – 1a; tf% – 47.13; cvcl – 11.01; mca – 17.23. hedychium coronarium-pop-i: somatic chromosome number 2n=34 (figure 11a); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.06 µm – 1.65 µm); total chromosome length – 41.14 µm; ratio of largest and smallest chromosome – 1.56:1; mean arm ratio (l/s) –1.18; karyotype formula a2 (2m) b 32(18m+14m); stebbins category – 1a; tf% – 47.32; cvcl – 15.91; mca – 10.72. hedychium coronarium-pop-ii: somatic chromosome number 2n=34 (figure 11b); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.06 µm – 1.60 µm); total chromosome length – 41.56 µm; ratio of largest and smallest chromosome – 1.51:1; mean arm ratio (l/s) –1.18; karyotype formula a2 (2m) b32 (16m+16m); stebbins category – 1a; tf% – 47.24; cvcl – 15.11; mca – 11.06. hedychium coccineum-pop-i: somatic chromosome number 2n=34 (figure 12a); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.32 µm – 2.08 µm); total chromosome length – 52.80 µm; ratio of largest and smallest chromosome – 1.58:1; mean arm ratio (l/s) –1.22; karyotype formula a2 (2m) b32 (18m+14m); stebbins category – 1a; tf% – 46.68; cvcl – 15.51; mca – 13.27. hedychium coccineum-pop-ii: somatic chromosome number 2n=34 (figure 12b); number of chromosomes bearing secondary constriction 2; range of chromosome length – (1.32 µm – 2.08 µm); total chromosome length – 52.92 µm; ratio of largest and smallest chromosome – 1.58:1; mean arm ratio (l/s) – 1.22; karyotype formula a2 (2m) b32 (18m+14m); stebbins category – 1a; tf% – 46.47; cvcl – 15.40; mca – 14.11. hedychium thyrsiforme-pop-i: somatic chromosome number 2n=34 (figure 13a); number of chromosomes bearing secondary constriction 2; range of chromosome length – (0.96 µm – 1.44 µm); total chromosome length – 38.26 µm; ratio of largest and smallest chromosome – 1.50:1; mean arm ratio (l/s) –1.21; karyotype formula a2 (2m) b32 (12m+20m); stebbins category – 1a; tf% – 46.60; cvcl – 15.24; mca – 13.56. 170 kishan saha, rabindra kumar sinha, sangram sinha hedychium thyrsiforme-pop-ii: somatic chromosome number 2n=34 (figure 13b); number of chromosomes bearing secondary constriction 2; range of chromosome length – (0.96 µm – 1.44 µm); total chromosome length – 39.40 µm; ratio of largest and smallest chromosome – 1.50:1; mean arm ratio (l/s) –1.21; karyotype formula a2 (2m) b32 (12m+20m); stebbins category – 1a; tf% – 46.61; cvcl – 14.88; mca – 13.69. discussion somatic chromosome number 2n=48 having a basic number x=12 was found to be constant in alpinia calcarata, alpinia malaccensis and alpinia nigra as was reported by earlier researchers (raghavan and venkatasubban 1943; chakravorti 1948, 1952; ramachandran 1969; chen 1988; joseph 1998). the different proportions of metacentric and submetacentric chromosomes present in the somatic chromosome complements of these three species are in partial agreement with the reports of joseph (1998). at intra-specific (pop-i and pop-ii) level, the karyotype of each species is more or less homogeneous having one pair of chromosomes bearing secondary constriction, a characteristic of the karyotype itself. on the contrary, at inter-specific level, their karyotypes differ though the chromosomes are mostly metacentric and submetacentric in nature. according to stebbins categorization, the karyotype of a. malaccensis, a. calcarata and a. nigra falls under category 1a, 1b and 2b, respectively. therefore, the reduction in size of some of the chromosomes in relation to other has occurred in chromosome complements of a. calcarata and a. nigra. moreover, the presence of two pairs of submetacentric chromosomes having f% < 33.33 in pop-i and pop-ii of a. nigra indicates that their karyotypes are slightly asymmetric. thus, in alpinia spp. a tendency from symmetric to asymmetric karyotype is observed. the somatic chromosome count 2n=48 is the distinctive character of amomum species which corroborates previous findings (sharma and bhattacharya 1959; chen et al. 1988). due to limited cytological studies in these three species, little information was available regarding the detailed chromosomal architecture. in general, chromosomes are short in size where one pair of metacentric chromosomes possessed secondary constriction. the karyotype of a. aromaticum, a. koenigii and a. maximum at inter-specific and intraspecific levels exhibit gross similarity in the types of chromosomes present, number of chromosomes bearing secondary constriction, tf%, total chromosome length and l/s arm ratio. the arm ratio l/s and tf% are the indicators of symmetric type of karyotype. according to stebbins (1971) categorization, the karyotype of a. aromaticum and a. maximum falls under 1a while that of a. koenigii belonging to category 1b indicates minor deviation in the size of largest to smallest chromosome ratio. the identical karyotype formula a2b46 without any pair of acrocentric or telocentric chromosomes having arm ratio l/s ˃2:1 suggests accentuated chromosomal homology. in curcuma complex, the somatic chromosome number of c. amada was found to be 2n=42 with a basic number x=21 which corroborates previous reports (sharma and bhattacharya 1959; ramachandran 1961, 1969; islam 2004; joseph 1998; bhadra and bandhyopadhyay 2015; lamo and rao 2017). at intra-specific level, the karyotype of pop-i and pop-ii of c. amada is almost identical and symmetrical (1a) in nature indicating the absence of acrocentric or telocentric chromosomes. based on asymmetry index value, bhadra and bandyopadhyay (2015, 2018) reported that the karyotype of c. amada falls under stebbins category 1b which may be due to its occurrence in different eco-climatic zone. the somatic chromosome number 2n=63 of c. caesia and c. longa is in agreement with previous reports (ramachandran 1961, 1969; joseph et al. 1999; nair and sasikumar 2009; nair et al. 2010; lamo and rao 2014; bhadra and bandyopadhyay 2015; lamo and rao 2016, 2017). the karyotype of these two species are identical both at interand intraspecific levels. according to stebbins formula, their karyotypes belong to category 1a which is not in agreement with the report of bhadra and bandyopadhyay (2015, 2018). the somatic chromosome count of 2n=63 chromosomes of c. picta is a first report and the karyotypes of pop-i and pop-ii of c. picta exhibit gross similarity in the types of chromosomes present, number of chromosomes with secondary constriction, tf%, total chromosome length and mean arm ratio (l/s). according to the degree of asymmetry, the karyotype of c. picta belongs to the category 1a. there are different views regarding the basic number of curcuma spp. according to one school, the different species of curcuma have a basic number x=21 (sharma and bhattacharya 1959; ramachandran 1961, 1969; islam 2004; joseph 1998; chen et al. 2013; bhadra and bandhyopadhyay 2015; lamo et al. 2016) which has been derived from a combination of x=12 and x=9 (ramachandran 1961; lamo and rao 2017). the other group (sato 1960; skornickova et al. 2007) proposed that the basic number of curcuma is x=7 suggesting, thereby, all the species of curcuma studied, having somatic chromosome number 2n=63 are nonaploid and c. amada with 2n=42 chromosomes is a hexaploid species. apparently, there is no conflict between x=7 and x=21 in curcuma complex. but the absence of cytotypes 171karyological studies in thirteen species of zingiberacaeae from tripura, north east india in multiple of x such as 2x, 3x, 4x and 5x in natural populations and/or cultivars of curcuma species suggests that the basic number x=21 is deep seated in curcuma spp. from which the diploid and triploid species have eventually evolved in the due course of evolution. the occurrence of 2x, 3x, 4x, 5x and 6x cytotypes is not uncommon in flowering plants and in the monocot genus dioscorea, valid cytotypes having 2x, 3x, 4x, 5x etc. are found in the natural population (muthamia et al. 2014). in the light of this knowledge, the acceptance of x=7 in curcuma complex is questionable. moreover, if x= 7 is accepted as basic number in curcuma spp., then their natural cytotypes having 9x, 12x and 15x chromosomes would have more copy number of genes. in such a situation, the regulation of dosage compensation having excess copy of genes in their cytotypes is beyond any elucidation. the somatic chromosome count 2n=34 chromosomes found in pop-i and pop-ii of hedychium coccineum and h. coronarium having a basic number x=17 supports previous findings (mukherjee 1970; mahanty 1970; gao et al. 2008). sharma and bhattacharya (1959) reported a cytotype of h. thyrsiforme having 2n = 24 chromosomes with basic number x=12. in the present investigation, the presence of 34 chromosomes recorded in most of the somatic cells (modal number) of h. thyrsiforme indicates that the diploid chromosome number is, indeed 2n=34 as was reported by mahanty (1970). this also suggests that all the three hedychium species growing in this region are stabilized with a basic chromosome number x=17. it is imperative that the karyotypes of the six individuals studied are almost identical and have the same karyotype formula a2b32, but the absence of acrocentric and telocentric chromosomes infers the symmetric nature of karyotypes justifying their inclusion under stebbins category 1a. karyotype conservatism without any structural alteration leading to the formation of acrocentric or telocentric chromosomes in these species having a basic number x=17 reveals a karyotype stasis. information regarding the number of chromosomes having secondary constriction in all the taxa of zingiberaceae studied by previous researchers is minimum (bhattacharyya 1957; mandi 1990; joseph et al. 1999; islam 2004) and in some of the previous investigations there was no reference with regard to the number of chromosomes possessing secondary constriction. for resolving this problem, ag-nor study (figure s2) has been carried out for the first time. there are different views regarding the maximum number of nucleoli per cell and the number of chromosomes bearing secondary constriction present in somatic chromosome complements of flowering plants (sharma and ghosh 1954; sato 1980). however, the coincidence between the number of chromosomes possessing secondary constriction and the maximum number of nucleoli per cell indicates the relationship between the presence of secondary constriction and the ability of those chromosomes to form the maximum number of nucleoli in all the taxa studied. the presence of three nucleoli in triploid species of curcuma is also an indicator of such a relationship. the interand intra chromosomal asymmetry based on stebbins quali-quantitative method reveals that the karyotypes of most of the species are symmetrical in nature. a progressive asymmetry is, however, observed in amomum koenigii (1b), alpinia calcarata (1b) and alpinia nigra (2b). undoubtedly, stebbins (1971) quali-quantitative method is the determinant of evaluation of the karyotype asymmetry index but based on this data alone, it would not be possible to ascertain the genetic relationship between different taxa of the same family. the bi-dimensional (figure 14) scatter plot (peruzzi and eroglu 2013) drawn against mean centromeric asymmetry (mca) and coefficient of variation of chromosome length (cvcl) clearly indicates that the two tribes alpinieae (represented by alpinia spp. and amomum spp.) and zingibereae (represented by curcuma spp. and hedychium spp.) of zingiberaceae (angiosperm phylogeny group – 2009) have distinct karyotypes in which alpinia spp. and amomum spp. show comparatively high inter-chromosomal and intra-chromosomal asymmetry indices (zarco 1986; watanabe et al.1999; paszko 2006) than those of curcuma spp. and hedychium spp. the dendrogram reveals that alpinia spp., amomum spp., curcuma spp. and hedychium spp. with their respective populations formed four separate clusters (figure 15). figure 14. bi-dimensional scattered plot against mca (x axis) and cvcl (y axis). 172 kishan saha, rabindra kumar sinha, sangram sinha the pca illustrates no overlap and thus, the distinctive position of each genus with respect to other is evident (figure 16). the karyotype data, therefore, validates the taxonomic position of alpinia spp., amomum spp., curcuma spp. and hedychium spp. the chromosomal data of alpinia spp., amomum spp., curcuma spp., and hedychium spp. suggest that they might have originated from a common ancestry but eventually through gradual changes, evolved as separate species in the due course of time. gower’s (1971) similarity matrix points towards a possible rearrangement of the chromosomal architectures in 13 different taxa studied. such chromosomal rearrangements are possibly associated with cryptic changes maintaining a high syntenic value. conclusion the present study has been focussed on t he detailed kar yot y pe ana lysis of two populations of alpinia calcarata, a. malaccensis, a. nigra, amomum aromaticum, a. koenigii, a. maximum, curcuma amada, c. caesia, c. longa, c. picta, hedychium coccineum, h. coronarium, and h. thyrsiforme of zingiberaceae, collected from different geographical locations of tripura. the somatic chromosome number of alpinia and amomum is found to be 2n = 48 having a basic number of x=12 chromosomes. in hedychium spp. the diploid chromosome number is 2n=34 with basic number x=17 chromosomes. the present findings reveal that in majority of the somatic cells of curcuma caesia, curcuma longa and curcuma picta, 63 chromosomes are present indicating their triploid nature. in contrast, diploid somatic chromosome number (2n=42) is recorded in curcuma amada. stebbins quali-quantitative analysis indicates that except in alpinia nigra, the karyotype of other 12 species is symmetric in nature. the accentuated chromosome homology at intra-species level is the intrinsic characteristic of their karyotypes. the inter-chromosomal and intra-chromosomal asymmetry indices, based on statistically based method, suggest that alpinia spp. and amomum spp. have higher interand intra-chromosomal asymmetry in comparison to curcuma spp. and hedychium spp. the dendrogram and pca derived data clearly validate the taxonomic position of these four genera investigated. gower’s similarity matrix endorses the cryptic changes leading to karyotype conservatism at species level of each genus. acknowledgements ks is grateful to ugc, new delhi for providing bsr fellowship. disclosure of statement the authors declare that they have no conflict of interest. figure 15. upgma dendrogram based on gower’s similarity matrix of six karyological parameters. figure 16: principle coordinate analysis of selected species in 2d plot. 173karyological studies in thirteen species of zingiberacaeae from tripura, north east india references angiosperm phylogeny group. 2009.  an update of the angiosperm phylogeny group classification for the orders and families of flowering plants: apg iii.  bot j linean soc. 161 (2): 105–121.  arambewela lsr, arawwawala ldam, ratnasooriya wd. 2004. antinociceptive activities of aqueous and ethanolic extracts of alpinia calcarata rhizomes in rats. j ethnopharmacol. 95 (2-3):311–316. ardiyani m. 2002. systematic study of curcuma l.: turmeric and its allies. 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(zingiberaceae) in mizoram, north east india. international journal of waste resources 6 (3): 234. watanabe k, yahara t, denda t, kosuge k. 1999. chromosomal evolution in the genus brachyscome (asteraceae, astereae): statistical tests regarding correlation between changes in karyotype and habit using phylogenetic information. j plant res. 112:145-161. yoshikane i, naohiro n. 1991. karyotypes of fragaria nubicola and f. daltoniana. cytologia 56:453-457. zarco cr. 1986. a new method for estimating karyotype asymmetry. taxon 35: 526–530. 176 kishan saha, rabindra kumar sinha, sangram sinha supplementary file (appendices) figure s1. karyogram of thirteen zingiberaceae species from tripura. 1alpinia calcarata; 2a. malaccensis; 3a. nigra; 4amomum maximum; 5a. aromaticum; 6a. koenigii; 7curcuma amada; 8c. caesia; 9c. picta; 10c. longa; 11h. coronarium; 12h. coccineum; 13h. thyrsiforme (a – pop i; b – pop ii); scale bars = 5µm. 177karyological studies in thirteen species of zingiberacaeae from tripura, north east india figure s2. agimpregnated somatic cells of thirteen zingiberaceae species. 1alpinia calcarata; 2a. malaccensis; 3a. nigra; 4amomum maximum; 5a. aromaticum; 6a. koenigii; 7curcuma amada; 8c. caesia; 9c. picta; 10c. longa; 11h. coronarium; 12h. coccineum; 13h. thyrsiforme (a – pop i; b – pop ii). 178 kishan saha, rabindra kumar sinha, sangram sinha table s1. details of plant samples collected from different geographical locations of tripura. name of the species herbarium voucher no. locations and altitudes alpinia calcarata tuh – 460 (pop-i) suryamaninagar, (34 mt.) 23045.0’44.74’’ n, 91015.0’54.29’’e tuh – 2001 (pop-ii) baramura, (110 mt.) 23049.0’0.70’’ n, 91031.0’2.33’’e alpinia malaccensis tuh – 466 (pop-i) debtamura, (48.0 mt.) 23033.0’2.50’’n, 91038.0’5.40’’e tuh – 2002 (pop-ii) atharamura, (134 mt.) 23029.0’54.10’’n, 91037.0’34.30’’e alpinia nigra tuh – 2049 (pop-i) mohanpur, (30.0 mt.) 23049.0’28.50’’n, 91026.0’45.0’’e tuh – 2003 (pop-ii) chabimura, (110 mt.) 23033.0’18.20’’n, 91037.0’0.98’’e amomum aromaticum tuh – 2055 (pop-i) tulamura,(46 mt.) 23023.0’11.3’’n, 91026.0’10.7’’e tuh – 2004 (pop-ii) unakoti, (200 mt.) 24023.0’11.3’’ n, 9204.0’13.0’’e amomum maximum tuh – 1407 (pop-i) atharamura foot hills, (45 mt.) 23053.0’2.10’’n, 91042.0’12.5’’e tuh – 2005 (pop-ii) jampui hills, (191 mt.) 23059.0’4.63’’ n, 92017.0’50.6’’e amomum koenigii tuh470 (pop-i) dhuptali, (40 mt.) 23023.0’11.3’’ n, 91026.0’15.1’’e tuh2006 (pop-ii) baramura,(120 mt.) 23050.0’4.0’’ n, 91036.0’38.96’’e curcuma amada tuh – 465 (pop-i) suryamaninagar, (21 mt.) 23045.0’44.27’’ n, 91015.0’53.38’’e tuh – 2007 (pop-ii) howaibari,(105 mt.) 23049.0’0.60’’ n, 91034.0’04.61’’e curcuma longa tuh – 1407 (pop-i) belkum para, (48 mt.) 23053.0’2.1’’ n, 91043.0’13’’e tuh – 2008 (pop-ii) jampui hills, (163 mt.) 2402.0’30.21’’ n, 92016.0’42.34’’e curcuma caesia tuh466 (pop-i) suryamaninagar, (30 mt.) 23045.0’43.13’’ n, 91015.0’52.17’’e tuh2009 (pop-ii) sepahijala, (30 mt.) 23039.0’35.74’’ n, 91018.0’8.54’’e curcuma picta tuh – 459 (pop-i) kamarikhala, (47 mt.) 23033.0’3.0’’ n, 91038.0’5.30’’e tuh – 2010 (pop-ii) baramura foot hills (102 mt.) 23048.0’41.24’’ n, 91030.0’54.24’’e hedychium coronarium tuh – 458 (pop-i) college tilla, (25 mt.) 23049.0’46.72’’ n, 91017.0’36.28’’e tuh – 2011 (pop-ii) baramura, (105 mt.) 23049.0’0.10’’ n, 91033.0’3.54’’e hedychium coccineum tuh – 461 (pop-i) paikhola, (45 mt.) 23022.0’27.8’’ n, 91030.0’48.30’’e tuh – 2012 (pop-ii) karbook, (106 mt.) 23021.0’38.90’’ n, 91042.0’12.15’’e hedychium thyrsiforme tuh507 (pop-i) jampui hills, (120 mt.) 23059.0’4.7’’ n, 92017.0’56.0’’e tuh2013 (pop-ii) manpui, (290 mt.) 2408.0’5.9’’ n, 92016.0’39.0’’e caryologia international journal of cytology, cytosystematics and cytogenetics volume 73, issue 1 2020 firenze university press karyotypic investigation concerning five bromus species from several populations in iran sara sadeghian, ahmad hatami, mehrnaz riasat high genetic diversity and presence of genetic structure characterise the endemics ruta corsica and ruta lamarmorae (rutaceae) marilena meloni1, caterina angela dettori2, andrea reid3, gianluigi bacchetta2,4,*, laetitia hugot5, elena conti1 cytogenetic effects of c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. and mathematical analysis cihangir alaca1, ali özdemir1, bahattın bozdağ2, canan özdemir2,* clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 37-44, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-269 citation: s. siddiqui, s. al-rumman (2020) clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l.. caryologia 73(1): 37-44. doi: 10.13128/caryologia-269 received: may 25, 2019 accepted: february 23, 2020 published: may 8, 2020 copyright: © 2020 s. siddiqui, s. al-rumman. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman department of biology, college of science, king khalid university, abha 61413, saudi arabia *corresponding author. e-mail: sasdeky@kku.edu.sa abstract. pesticides are highly noxious materials.their poisonousness might not be unequivocally precise to the target entities but can unfavorably disturb various procedures in the non-target host plants. in the present study, the effect of application of clethodim on pollen sterility and meiotic anomalies of pisum sativum l are studied. pisum sativum l seeds are treated with different concentrations of clethodim varying from 0.01%, 0.02%, 0.03%, and 0.04% for exposure time of 1 hour and their effect on pollen sterility and chromosomal anomalies were investigated. the outcomes reveal that treatment of clethodim on pisum sativum l seeds induces pollen sterility (ps) and chromosomal anomalies (ca) in a dose-dependent manner. also in clethodim treated seeds, an elevation in the proportion of abnormal meiotic phases were observed which was time and concentration dependent. secondary association (sea), precociuos separation (ps), clumped nuclei (cnu) were reported in metaphase i & ii, stickiness (stc), bridges (br) and laggards (lg) in anaphase i & ii. the results of the present study reveal that frequently used herbicide clethodim has a substantial cytotoxic effect on meiotic cells of pisum sativum l. keywords. herbicide, clethodim, pollen sterility, meiotic abnormality, pisum sativum l. introduction the application of herbicides has led to an alteration in the phytosociological properties of weeds and to a choice of biotypes resilient to herbicides. in addition, it also causes effects in the health of humans and environment although they are categorized as an extremely efficient means in the regulation of weeds. according to he et al. (2012), all over the world, the most applied chemical materials are the herbicides. in the 90’s, the worldwide pesticide sales continued to stay moderately persistent, amongst 270 to 300 billion american dollars, 79% of this amount is related to herbicides. amongst the three foremost groups of pesticides i.e. herbicides, fungicides and insecticides; herbicides have taken the top slot since 2007 (zhang et al. 2011). in order to upsurge agricultural output, the application of herbicides for regulating weeds is the most widely used practice in worldwide agriculture. nevertheless, when these chemical substances are applied in an unrestrained 38 sazada siddiqui, sulaiman al-rumman way, they can influence and disturb other organisms, particularly aquatic creatures (nwani et al. 2011; van bruggen et al. 2018). earlier reports have shown that much of the noxious effects of herbicides on plants and animals were inadequately explored (chevreuil et al. 1996; kim and feagley 1998; abdel-rahman et al. 1999). as a result of the absence of evidences about the effect of herbicides in the biotic environments, they can also epitomize a setback to health of human beings (munger et al. 1997; gorell et al. 1998). the influence of pesticide in the environments is determined by its noxiousness, concentration and dispersion manner (van der werf 1996). the mutagenic effects of the herbicides can result from several reactions within the organism, as a direct action of the compound on the nuclear dna; incorporation in the dna during cell replication; interference in the activity of the mitotic or meiotic division, resulting in incorrect division of the cell (timbrell 1999). a few herbicides affect directly the elongation, cell differentiation and cell division of plants. herbicide clethodim falls into the cyclohexanedione oxime class. from post-emergence, clethodim is active as an herbicide against various species of grass weeds mostly in sunflower, soybean, cotton and other broad-leaved plants (edwards 2005). earlier studies have emphasized the adverse effects of the herbicides on plant physiology and cytogenetics. maleic hydrazide (mh) is one of the few herbicides which prevents the synthesis of proteins and nucleic acids (siddiqui et al. 2008). mutagenic action of glyphosate, alachor and maleic hydrazide has been reported by siddiqui et al. (2012), diclofopmethyl and lindane has been stated by anila and ditika (2013) and anilofos has been described by arzu et al. (2014) on root tip cells of plant. pisum sativum l, (fabaceae) is a plant useful in the manure production, ayurvedic medications and food (davies et al. 1985). it is one of the most used leguminous plant and is a source of protein. there are no studies available in the literature which have assessed the adverse effects of clethodim on pisum sativum l, a potential multipurpose crop. in the current investigation, we have assessed the noxious consequences of clethodim on meiotic cells of pisum sativum l. materials and methods seeds and chemical pisum sativum l seeds, variety arkil were obtained from the indian council of agricultural research bbhoj krishi vigyan kendra, near village naktara, p. o. bankhedi, nh-86 ext., raisen sagar road, bhopal, india. clethodim herbicide was obtained from the delta chemical company based in riyadh, saudi arabia. treatment of pisum sativum l with clethodim healthy and even sized seeds of pisum sativum l were taken and submerged for 6 hours in double distilled water of ph 6.7. in a glass beaker of 250 ml, seeds of pisum sativum l were immersed for 1 hour in 150 ml clethodim solutions of various concentrations (0.01, 0.02, 0.03 and 0.04%). the seeds were shaken frequently for providing ample air to the seeds. for eliminating any remaining amount of clethodim, the treated seeds were properly cleaned with double distilled water. for control, a set of seeds were submerged in distilled water. in order to eradicate any remaining chemical sticking to the seed coat, the seeds were cleaned with tap water. each set of seeds comprising of 10 seeds per pot were seeded in pots having a height of 24 cm and width of 17 cm. the entire experiment was repeated thrice under similar conditions. pollen grain analysis for experiments related to pollen grains, f lower buds of similar age were collected from treated plants and control group. they were fixed in 70% alcohol. for identifying pollen sterility as well as fertility, pollens were stained with 1% propionocarmine. pollens having uniform size and shape which were stained dark purple in colour and filled with nuclei and cytoplasm were considered as fertile whereas pollen grains which were stained pale yellow colour or colourless, having irregular size and shape and without nuclei and cytoplasm were defined as sterile. pollen sterility was calculated as the ratio of non viable pollen grains to the total number of pollens and expressed in percentage. no. of non viable pollen grains no. of sterile pollen grains = x 100 total no. of pollen grains meiotic analysis to assess the effect on meiotic cells, flower buds of similar size were fixed in 1:3 acetic acid saturated with iron and absolute alcohol for 24 hours and than passed to 70% alcohol. the meiotic cell preparations were prepared by traditional acetocarmine squash technique (man39clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. ton 1950). by squashing the anther in an acetocarmine stain, slides were prepared. in normal butnyl alcohol (nba) series, permanent slides were made and they were mounted in canada balsam and than dried up at 45°c. statistical analysis statistical analysis was performed employing one way anova test using gpis software 1.13 (graphpad, california, usa) to detect the significance of differences of variables. all values are expressed as mean ± se. results the occurrence of pollen sterility and meiotic chromosomal abnormalities were listed in (figs 1, 2 and table 1). it is clear from the results that clethodim was proficient in inducing pollen sterility and different types of meiotic chromosomal abnormalities such as secondary association (sea) (fig. 2, a and b), precociuos separation (ps) (fig. 2, c and d), and clumped nuclei (cnu) (fig. 2, e and f) in metaphase i & ii and stickiness (stc) (fig. 2, g and h), bridges (br) (fig. 2, i and j) and laggards (lg) (fig. 2, k and l) in anaphase i & ii. effect of clethodim on pollen sterility of pisum sativum l. the observation reveals that the percentage of pollen mother cells (pmcs) showing pollen sterility increased with increase in concentrations of clethodim at different exposure times and was null in control (fig. 1). a lower percentage of pollen sterility were observed at 0.01% concentration which was 60% at one hour and it was highly significant (p>0.001) when compared to control. in addition, at highest concentration high increase in pollen sterility were reported which reached 83% at one hour in clethodim treated pmcs. pollen sterility is a sign of disturbance in reproductive process which was observed in clethodim treated pollen mother cells of pisum sativum l at 1 hour (fig. 1). the increase in the ratio of sterile pollen grains in the clethodim treated groups as the dose increases might be due to their toxic effects on pollen grains. effect of clethodim treatment on chromosomal aberration (ca) of pisum sativum l cytological analysis also documented numerous anomalies in the pmcs of clethodim treated plants at one hour, as shown in table 1. different types of ca such as sea, ps, and cnu were noticed in metaphase i & ii and stc, br and lg were observed in anaphase i & figure 1. pollen sterilty of pisum sativum l pmcs exposed to different concentrations of clethodim for 1 hour. ¥ p≤0.001 compared to control. data are mean of three replicates ± se; 0.0 = control group. table 1. percentage of meiotic abnormality in metaphase i & ii and anaphase i & ii plate of pisum sativum l in pmcs exposed to different concentrations of clethodim for 1 hour. (%) concentration time treatment pmc cells total metaphase i/ii (mean ±s.e) anaphase i/ii (mean ±s.e) sec. asso. pre.sep. clumped nuclei stickiness bridges laggards total anomalies 1 h 0.00 110 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 00.0 ± 0.0 0.01 125 1.5 ± 0.34 2.6 ± 0.29 3.5 ± 0.2 1.8 ± 0.3 2.4 ± 0.4 1.4 ± 0.2 13.2 ± 1.73 0.02 115 2.8 ± 1.20 3.8 ± 0.93 4.6 ± 0.9 2.2 ± 0.7 3.1 ± 0.7 2.8 ± 0.6 19.3 ± 5.03 0.03 128 3.2 ± 1.70 4.2 ± 1.04 6.4$ ± 1.3 3.5 ± 1.2 3.8 ± 0.3 3.4 ± 1.2 24.5 ± 7.34 0.04 130 6.5# ± 2.80 6.1$ ± 2.1 7.4$ ± 2.4 6.7¥# ± 2.0 6.5# ± 2.7 6.2$# ± 2.1 39.4$ ± 14.1 p≤ 0.05; $ p≤0.01; ¥ p≤0.001 compared to control. data are mean of three replicates ± se; 0.0 = control group. 40 sazada siddiqui, sulaiman al-rumman ii (table 1 and fig. 2). cytological investigations clearly revealed that the level of cas gradually increased along with increasing concentrations of clethodim at one hour. studies of different stages of meiotic division shows that almost all the stages of division were affected to a certain extent. the percentage of formation of sea, ps, and cnu in metaphase i & ii were reported maximum in 0.04% (6.5% significant p≤0.05, 6.1% very significant p≤0.01 and 7.4% very significant p≤0.01) as compared to control, at one hour and reported minimum in 0.01% (1.5%, 2.6% and 3.5%) at one hour of clethodim treated plants. the percentage of formation of stc, br, and lg in anaphase i & ii were reported maximum in 0.04% (6.7% very significant p≤0.01 and significant p≤0.05, 6.5% significant p≤0.05 and 6.2% very significant p≤0.01) as compared to control at one hour and reported minimum in 0.01% (1.8%, 2.4% and 1.4%) at one hour of clethodim treated plants. increased incidence of cas were in the following order for 1 hour clethodim treatment: cnu > stc > br = sea > lg > ps. discussion in the present study, the occurrence of laggards and bridges in anaphase i & ii were strictly correlated with sterility. similar finding were reported by liang et al. (1967) on sorghum by atrazine treatment which affected meiotic stability. çalli (2008) reported that the equation pro (22.5% famoxadone+30% cymoxanil) fungicide instigates abnormalities in the meiosis of pollen grains and thus resulting in pollen sterility. dubey et al. (1977) reported a decrease in viability of about 60% in the pollen of eggplants, indicated by the joint effect of a dinitro herbicide and two organophosphate insecticides. fungicide propiconazole produced damaging effects on pollen tube development and pollen germination of tradescantia virginiana (he et al. 1995). rana and swaminathan (1964) and ramanna (1974) found that any deviance in cytokinesis or karyokineses could generate non-viable microspores. sinha and godward (1969;1972) found that translocations were liable for reduced pollen fertility. in h.orientalis, tube growth and in vitro pollen germination was disturbed and affected by the herbicide a e dcb ji hgf k l figure 2. (a-l) meiotic aberrations induced by clethodim in pisum sativum l pmcs. (a-b) secondary association; (c-d) precocious separation; (e-f) clumped nuclei at metaphase i & ii, (g-h) sticky chromosome; (i-j) bridges; (k-l) laggards at anaphase i & ii in pmcs in pisum sativum l. 41clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. quizalofop-p-ethyl (qpe) treatments. especially, the maximum qpe concentration instigated alterations in the morphological attributes of h. orientalis. pollen germination is decreased by three times by utmost vigorous qpe application. within the pollen tube morphological anomalies were also detected (deveci et al. 2017). pisum sativum l and allium cepa l are generally used in order to check the genotoxicity and cytotoxicity of several chemicals (bonciu et al. 2018 a and b; siddiqui 2018). previous works have revealed that herbicides have mutagenic effect on pisum sativum l and allium cepa l (siddiqui et al. 2008; rosculet et al. 2019). cytological abnormalities in plants can be a useful monitoring method for the recognition of chemicals present in the environment that might cause a genetic threat. in the current study, we found numerous kinds of chromosomal irregularities such as sea, ps, cnu in metaphase i & ii, lg, br, and stc in anaphase i & ii in pisum sativum l after treatments with clethodim of one hour. the prevalence of secondary association has been found in treated plants. incidence of secondary association is a regular attribute occurring primarily because of the existence of more than two homologous chromosomes. in the course of secondary pairing, there is a debate on the participation of homologous chromosomes amongst the researchers. hirayoshi (1957) differed with the postulation that contemplates the participation of homologous chromosome in secondary association and after investigating thorough outcomes in zizanieae and oryzeae offered the inference that secondary association might be an incidence effective under physico and bio-chemical effects and has got no relation to the exact homology of chromosomes. secondary pairing within bivalents is well thought to be a sign of polyploid attribute of a species as found in kidney bean (girjesh and nitu, 2014), ocimum (mukherjee and datta 2005), uraria picta (bhattacharya and datta 2010). as per stebbins (1950), secondary association may be contemplated to be a phenomenon that illustrates the polyploid attribute of a species but detailed phylogenetic estimations may not be framed from this since secondary pairing within bivalents is substantially altered by further chromosomal alterations. in the present study, the occurrence of precocious movement also appeared enhanced with rising clethodim concentations. precocious separations were formed due to unstable spindle process or inactivation of spindle formation and interrupted homology for pairing of chromosomes that might lead to the precocious motion of chromosomes (umar and singh 2003). it might also be attributed due to the occurrence of breakage of chromosomes or because of the heavy metals breaching the protein moiety of the nucleoprotein backbone. in the current study, the prevalence of clumped nuclei also get boosted with rising clethodim concentations. because of the turbulences at the cytochemical level, clumped nuclei were created. clumping of the chromosomes can occur with regular arrangement at metaphase and separation may result in the creation of bridges. some types of gene mutations that cause incorrect coding of some non-histone proteins involved in chromosomal organization can lead to chromosomal clumping. there is a possibility that the mutagen reacts with the available histone proteins and results in an alteration in the surface property of the chromosomes because of incorrect folding of dna and hence causing the chromosomes to stick or clump (grant 1978). stickiness was also reported with increase in the clet hodim concentrations. stick iness could occur because of incomplete detachment of the nuclear proteins and changes in their design of association or because of incomplete detachment of the nucleoproteins and amendment in their design of association or because of depolymerization of nucleic acids instigated by clethodim treatment. stickiness might result from disorders in cytochemical balance reaction (dewitte et al. 2010; rosculet et al. 2019). depolymerisation of nucleic acids due to herbicidal treatment or by incomplete detachment of nucleoproteins (kaufman et al. 1955) or by the limited separation of nucleoprotein variation in their design of organization (evans 1962) may cause stickness. numerous bridges were formed in anaphase i & ii in the treated plants. bridges were probably created by breaking and combination of chromosomes bridges which augmented with the treatment of clethodim. formation of chromosomal bridges could occur because of the chromosomal stickiness and consequent failure of free anaphase division or because of inversion of chromosome fragments or irregular translocation (gomurgen 2000). rosculet et al. (2019) reported that the origin of bridges were mainly because of the merger amongst broken chromosomes. the laggards noticed within the current investigation could be due to the failure of the movement of chromosomes or because of the deferred termination of stickiness of the ends of chromosomes. lagging of chromosomes might occur because of the interruption of the motion of bivalents towards equatorial plate at metaphase i. the utmost regular occurence was the lagging of single univalent (zeyad et al. 2019). barthelmess (1957) reported that more than one lagging chromosomes in meiosis might occur because of the interruption of the prometaphase motion of chromosomes supplemented by 42 sazada siddiqui, sulaiman al-rumman union of the centromeric to the adjoining inner surface of plasma. bridges and laggards might have been formed because of deferred terminalisation of stickiness of the ends of chromosomes (kaur and grover 1985). creation of micronuclei at telophase i is caused by laggards. generation of micronuclei at telophase ii is caused by laggards or acentric fragments and thus it results in the alteration in numeral and size of pollen grains ensuing from mother cells. as reported in previous findings, our outcomes suggest the possibility of these chemicals to instigate meiotic anomalies (namrata and alka 2014; das et al. 2018). the chromosomal abnormalities caused by these herbicides might be because of their impeding effect on spindle proteins and their capability to instigate swap over of sister chromatids (tartar et al. 2006; siddiqui et al. 2009). previous investigations have revealed that free radicals might be the reason for genomic uncertainty in cells. damages in dna, disarray in the cytoskeleton and disproportion in energy metabolism might result in chromosomal anomalies as reactive oxygen species are extremely unstable (siddiqui et al. 2012; siddiqui 2015). due to genetic and physiological disarrays, numerous types of meiotic abnormalities might have been created. it has become clear from the above findings that clethodim used in the current work is probably capable of instigating several types of chromosomal anomalies. conclusion within the experimental settings applied in the current work, clethodim displayed a sturdy genotoxic effect on pisum sativum l. it is obligatory to do additional research work on the attribute of the harvests resulting from seeds and plants subjected to these chemicals in relation to their nutritive worth and their vulnerability to acclimatize with diseases. additionally, it is significant to note that the pesticide preparations encompassing these active constituents may or may not have a parallel effect on the plant cells. regarding this, supplementary researches are essential to evaluate their outcome on biological procedures. acknowledgments we are grateful to deanship of scientific research at king khalid university, abha, kingdom of saudi arabia for providing technical and administrative support. declaration the authors declare no conflict of interest. funding the authors (sazada siddiqui and suleiman alrumman) extend their appreciation to the deanship of scientific research at king khalid university for funding this work through research groups program under grant number r.g.p.1/49/39. refrences abdel-rahmam ar, wauchope rd, truman cc, dowler cc. 1999. runoff and leaching of atrazine and alachlor on a sandy soil as affected by application in sprinkler irrigation. j of environ sci and health-b: pesti and food cont. 34:381–396. anila md, ditika k. 2013. cytotoxic and genotoxic potency screening of two pesticides on allium cepa l. proc tech. 8:9–26. arzu o, dilek a, yasin e, sevim fe. 2014. potential cytotoxic effect of anilofos by using allium cepa assay. cytotechnology. 67:783–791. barthelmess a. 1957. chemish induzierte multipalare mitosen. protoplasma. 48:546–561. bhattacharya a, datta ak. 2010. secondary chromosome associations in uraria picta (jacq.) dc. 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ruta lamarmorae (rutaceae) marilena meloni1, caterina angela dettori2, andrea reid3, gianluigi bacchetta2,4,*, laetitia hugot5, elena conti1 cytogenetic effects of c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. and mathematical analysis cihangir alaca1, ali özdemir1, bahattın bozdağ2, canan özdemir2,* clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 72(3): 3-10, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-317 citation: s. tabur, m.d. yurtlu, s. özmen (2019) role of humic acid against salt-induced cytotoxicity in hordeum vulgare l.. caryologia 72(3): 3-10. doi: 10.13128/cayologia-317 published: december 13, 2019 copyright: © 2019 s. tabur, m.d. yurtlu, s. özmen. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. role of humic acid against salt-induced cytotoxicity in hordeum vulgare l. selma tabur*, merve dündar yurtlu, serkan özmen süleyman demirel university, arts and sciences faculty, department of biology, 32260 isparta, turkey *corresponding authors: taburs@gmail.com abstract. the effect of humic acid, which is an replace by a biostimulant on mitotic activity and chromosome behaviors in meristem cells of hordeum vulgare l. germinated under different salt concentrations were investigated. in the parallel to increasing salt concentrations, mitotic index partly decreased and observed the higher number of chromosomal abnormalities as compared to control. also, it was determined that the mitotic index of seeds pretreated with only humic acid increased by 30% according to control and by 42% of mitotic aberrations. whereas, humic acid along with salt significantly inhibited to mitotic index with parallel to increasing salt concentrations. moreover, the frequency of chromosomal aberrations in seeds germinated in humic acid and salty medium significantly decreased according to its own control. humic acid revealed to a successful performance in ameliorating of the detrimental effect of salinity in the all concentrations studied. humic acid application at 0.35 m salinity displayed perfectly successful by reaching to the same abnormality percentage of control. keywords. barley, chromosomal aberrations, cytotoxicity, humic acid, mitotic activity, salinity. introduction it is a known fact to affect of abiotic stresses on plant growth and development. one of the most common environmental stress factors is salinity, which an increasing problem of many arid and semiarid areas of the world. approximately 20% of the world’s cultivated land and accounts for over 6% of the world total area is threatened by salinity (fao 2015). abiotic stresses, such as drought, salinity, extreme temperatures, chemical toxicity and oxidative stress are serious threats to agriculture and the natural status of the environment. increased salinization of all arable land is expected to have devastating global effects, resulting in 30% land loss within the next 25 years, and up to 50% by the year 2050 (wang et al. 2003). it has been known for a long time that salt adversely affects plant growth and development, hindering seed germination, seedling growth (çavuşoğlu and ergin 2015), enzyme activity (dash and panda 2001), dna, rna and protein synthesis (anuradha and rao 2001) and mitosis (tabur and demir 2010 a, b; çavuşoğlu et al. 2016). however, recent investigations are focus4 selma tabur, merve dündar yurtlu, serkan özmen ing more on the mechanisms of salt tolerance in plants (munns and tester 2008).the most efficient way to minimize the detrimental effects of salinity on plant breeding is the development of varieties with high salinity tolerance. hence, researchers have used various plant growth regulators and leaf extracts to reduce or eradicate negative effects of salinity on seed germination, seedling growth (çavuşoğlu and ergin 2015), and mitotic activity (tabur and demir 2010 a, b; çavuşoğlu et al. 2016). however, in spite of substantial efforts, the outcome is still disappointingly poor due to the physiological and genetic complexity of this trait, the lack of reliable screening tools, and most importantly, the lack of a comprehensive understanding of the mechanisms behind salinity tolerance (zhu et al. 2016). the data relating to mitotic activity are also mostly paradox. the main ingredient of organic substances in the soil is humus. the most active biochemical substance of humus is humic acid. humic substances have been known that the germination and growth of plants has stimulated. humic substances can pass through micropores of biological or artificial membrane systems, facilitate the transport of trace elements in plant roots and behave like growth hormones in plants (chen et al. 2004). therefore, humic substances are evaluated as a biostimulant by du jardin (2012) who conducted a bibliographic analysis of plant biostimulants. biostimulants are derived from natural or biological sources and can i) enhance plant growth and development when applied in small quantities; ii) help improve the efficiency of plant nutrients, as measured by either improved nutrient uptake or reduced nutrient losses to the environment, or both; or act as soil amendments to help improve soil structure, function, or performance and thus enhance plant response (du jardin 2012). humic acid has positive effects on plant growth and nutrition (calvo et al. 2014). in cytophotometric studies of the dna, it was seen that humic substances increased the amount of dna synthesis in the interphase nucleus of the meristematic cells in plants (gorova et al. 2005). furthermore, humic substances are accepted to be a plant growth promoter, particularly by changing the root structure and growth dynamics (jindo et al. 2012; canellas and olivares 2014). under both normal and salt conditions, there have been many investigations related to seed germination, seedling growth (çavuşoğlu and ergin 2015), root development (sivananthi and paul 2014), plant growth and mineral nutrient uptake (khaled and fawy 2011), and also some metabolic changes (el-bassiouny et al. 2014). however, there is only limited research on the effect of humic acid on cell divison (feretti et al. 2012) and the protective role of against effects of mutagenic and genotoxic of various environmental conditions and chemicals (gichner et al. 1990; ferrara et al. 2000). in particular, no data have been recorded about effects of humic acid on mitotic activity and chromosomal aberrations in salinity conditions. in the study reported here, the influence of humic acid pretreatment on mitotic activity and chromosome behaviors in root meristems of barley seeds exposed to salinity stress were investigated. so, we have aimed to clarify to some extent to what extent humic acid can alleviate salt stress, whether it stimulates cells to enter the mitosis division or not and also whether it causes any changes in the structure and behavior of chromosomes or not. material and methods in the present study, barley seeds (hordeum vulgare l. cv. bülbül 89) were used. the barley seeds were subjected to surface sterilization before used. for this, seeds were kept in 1% sodium hypochlorite for ten minutes, then washed with distilled water five times and dried on filter paper at room temperature. preparation of solutions and germination of seeds nacl and humic acid used in the experiments were obtained from merck and sigma-aldrich firm respectively. as test solution, 28 mg/l humic acid were used due to promote germination in the best way against the inhibitory effect of salt. concentrations of nacl were 0.25, 0.30 and 0.35 m (molar). these salt levels and the concentration of humic acid used were determined in the result of a preliminary study. primarily, plump-looking, robust and approximately equal-sized 20-25 barley seeds were selected. then, sterilized seeds were soaked in test tubes filled with 28 mg/l humic acid and distilled water (control) at constant volume (50 ml) for 24 h at room temperature. at the end of this pretreatment session, the seeds from every application were arranged in 10 cm petri dishes covered with two sheets of filter paper moistened with 7 ml of distilled water and different salt concentrations. petri dishes were transferred in an incubator to germinate at 20 ± 1°c in continuous dark for several days. cytogenetical analyses when the root tips were 0.5–1 cm long, they were cut off, pretreated with a saturated solution of paradi5role of humic acid against salt-induced cytotoxicity in hordeum vulgare l. chlorobenzene for 4 h at 20°c, fixed with carnoy’s fluid i (absolute ethanol: glacial acetic acid, 3:1, v/v) for 24 h, and stored in 70% alcohol at 4°c until used. then, the root tips were hydrolyzed in 1 n hcl at 60°c for 18 min, stained with feulgen for 1 h, and squashed in 45% acetic acid (sharma and gupta 1982). after 24 h, microscopic slides were made permanent by mounting in balsam. the mitotic phases and mitotic aberrations were photographed (100×) with a digital camera (olympus c-5060) mounted on an olympus cx41 microscope. data analyses and statistical evaluations to determine the effect of humic acid and salt on the mitotic index, at least 3000 cells (approx. 1000 per slide) were scored in control and in treated groups. chromosomal aberrations were calculated for each concentration as the percentage of 300 dividing cells counted. statistical analysis related to all parameters was performed by using spss program according to duncan’s multiple range test at the level of significance p ≤ 0.05 (duncan 1955). results effects of humic acid on mitotic index and chromosome aberrations in normal conditions barley seeds pretreated with 28 mg/l humic acid were germinated at 20°c in distilled water and slides were prepared with the root tips obtained. the mitotic index values calculated as a result of the cell counting procedures performed in these slides were presented in table 1. humic acid pretreatment caused a 30% increase in the mitotic index of barley seeds germinated in nonstress conditions as compared to those of the control group. in other words, the mitotic index of seeds treated with humic acid was even higher than seeds germinated in distilled water. as a result of cytological analyzes, chromosomal aberrations in meristem cells of barley seeds germinated at 20°c in distilled water (control) were statistically insignificant. that is, all mitotic phases were normal (fig. 1). however, the frequency of chromosomal aberrations in seeds germinated in distilled water after humic acid pretreatment was remarkably higher than that in the control (table 2). for example, while the rate of chromosomal aberrations in control seeds was 0.04%, it was 0.42% in seeds pretreatmented with humic acid. a resulting of the application of humic acid alone, chromosomal aberrations such as fragment formation, lagging chromosome, anaphase and telophase bridges, fault polarization in telophase and anaphase were frequently observed (fig. 2). effects of humic acid on mitotic index and chromosome aberrations in salinity conditions mitotic index scores obtained from this study made to determine the activity degree of humic acid on the table 1. mitotic index of barley seeds germinated in distilled water and different nacl concentrations after humic acid pretreatment. mitotic index (%) nacl (m, molar) control humic acid (28 mg/l) 0.0 (distilled water) *0.19 ± 0.13bcd 0.27 ± 0.04e 0.25 0.19 ± 0.03bcd 0.17 ± 0.03abcd 0.30 0.18 ± 0.01abc 0.18 ± 0.02bcd 0.35 0.18 ± 0.03abc 0.12 ± 0.02ab the pretreatment process of seeds was performed by soaking 24 h in constant volumes of distilled water (control) or humic acid. as test solution, 28 mg/l humic acid was used. different salt concentrations (0.25, 0.30, 0.35 m nacl) were exogenously applied to germination medium. data are the means of three replications ± standard deviation. * shows values with insignificant difference (p ≤ 0.05) for each column shown with same letters fig. 1. normal mitosis phases in root tips meristems of barley germinated in distilled water (control). (a) prophase; (b) metaphase (2n = 14); (c) anaphase; (d) telophase. scale bar = 10μm. 6 selma tabur, merve dündar yurtlu, serkan özmen mitotic index of barley seeds germinated under salt stress was summarized in table 1. the mitotic index value of barley seeds was statistically decreased at especially high concentrations as parallel to the increase of salt concentration as compared with control. it was found that the seeds applied alone humic acid shows a considerable increase on the mitotic index as compared with seeds germinated in distilled water. that is, the addition of 28 mg/l humic acid increased the mitotic index by 30%. however, humic acid pretreatment caused a significant decrease in the mitotic index with increasing salt concentrations. the mitotic index value (0.12) in root meristems of the seeds germinated at the highest salt concentration (0.35 m) after treated with humic acid reduced to a large extent (table 1). considering all of the application groups, it was determined that humic acid pretreatment together with salinity decreased the mitotic index at 0.25 m and 0.35 m salinity, while it was at the same level with its own control at 0.30 m salinity (table 1). in addition to, chromosomal aberration scores were presented in table 2. while there is a chromosomal aberration that can be ignored statistically in control seeds germinated in distilled water, screening mitotic divisions revealed the many numbers of chromosomal aberrations as parallel to increasing salt concentrations. it was determined that chromosomal aberrations at the highest salt concentration (0.35 m) were higher approximately 60% according to those in distilled water. at the same time, alone humic acid pretreatment caused a 42% increase in chromosomal aberrations as compared to control. however, the frequency of chromosomal aberrations of seeds germinated at different salt concentrations after treatment with humic acid showed a decrease which can be considered statistically significant from those of the seeds germinated in distilled water after treatment with humic acid. namely, humic acid has been quite successful in mitigating the detrimental effect of salt stress on chromosomal aberrations at all the salt concentrations studied here. moreover, humic acid application at the highest salt concentration (0.35 m) significantly reduced the detrimental effect of salt stress and reached the percentage of the same abnormality (0.04) as the seeds germinated in distilled water (table 2). the most prominent chromosomal aberrations in seeds belonging to all application groups were the disorganizations in anaphase and telophase such as bridges, table 2. frequency of chromosomal aberrations of barley seeds germinated in distilled water and different nacl concentrations after humic acid pretreatment chromosomal aberrations (%) nacl (m, molar) control humic acid (28 mg/l) 0.0 (distilled water) 0.04 ± 0.18a 0.42 ± 0.05cd 0.25 0.25 ± 0.27bc 0.22 ± 0.01bc 0.30 0.35 ± 0.07c 0.30 ± 0.15bc 0.35 0.58 ± 0.05d 0.04 ± 0.04a the pretreatment process of seeds was performed by soaking 24 h in constant volumes of distilled water (control) or humic acid. as test solution, 28 mg/l humic acid was used. different salt concentrations (0.25, 0.30, 0.35 m nacl) were exogenously applied to germination medium. data are the means of three replications ± standard deviation. * shows values with insignificant difference (p ≤ 0.05) for each column shown with same letters fig. 2. chromosomal aberrations in root meristems of barley seeds germinated in distilled water and different nacl concentrations after treatment with 28 mg/l humic acid: a) micronucleus; b) granulation c) disorderly prophase; d) disrupted equatorial plate; e) uncoiling chromosomes f ) fragments g) sticky chromosomes; h) bridges in anaphase and lagging chromosome; ı) fault polarization in anaphase; j) vagrant chromosome in anaphase (arrow); k) alignment anaphase; l) bridges in telophase; m) fault polarization in telophase n) distant poles in telophase; o) vagrant chromosome in telophase. scale bar = 10 μm. 7role of humic acid against salt-induced cytotoxicity in hordeum vulgare l. lagging chromosome, fault polarization, distant poles, vagrant chromosomes and alignment anaphase (fig. 2h–o). in addition, other chromosomal aberrations observed were the presence of micronucleus, granulation, disorderly prophase, disrupted equatorial plate, uncoiling chromosomes, fragments, sticky chromosomes (fig. 2a–g). the minimal common aberrations were micronucleus, granulation and disrupted equatorial plate. discussion it is well known that humic substances stimulate germination in seeds of various species by increasing enzymatic activities in seed tissues during germination and decreasing mitotic activity under normal conditions (feretti et al. 2012). however, no information has been found on effects of humic acid on mitotic activity and chromosomal aberrations especially under saline conditions. all the data on the effect of humic acid on these parameters in saline conditions are presented for the first time in this study. in the present work, it was determined that humic acid pretreatment alone in non-stress conditions significantly increase the mitotic index of barley seeds as compared to distilled water (table 1). whereas, feretti et al. (2012) have suggested in their study using various plant tests (allium cepa test, tradescantia and vicia faba micronucleus test) that alone humic acid pretreatment decreases the mitotic index in studied two solutions. because of the differences in the findings of these researchers may be due to the species of plant studied or the concentration of the humic acid used. however, our findings have been endorsed by the data expressed that the humic substances increase the amount of dna synthesis in meristematic cells in plants (gorova et al. 2005). in addition, we found that the application of alone humic acid increased the chromosome abnormality rate by 42% under normal conditions (table 2). our this finding is consistent with ferretti et al. (2012)’s findings. for this reason, we can reach the result that alone humic acid pretreatment increases the mitotic index value in barley seeds germinated in distilled water but may have a genotoxic effect because of the negativity that it has shown on chromosome behavior. this reveals the fact that alone humic acid application can create mutations in various types over time. as is known, plant growth and development are adversely affected by salinity. high salinity is an important factor negatively inf luencing plant growth and development even in most halophy tes. at present, approximately 20% of cultivated lands in the world are affected by salinity (fao 2015). generally, it is suggested that salinity impairs seed germination, retards plant development and reduces productivity. in some cases, the plant dies before completing the life cycle. there have been numerous investigations conducted to explore the effects of salt on plant growth, but mechanisms of salt stress have not yet been explained precisely (munns and tester 2008; zhu et al. 2016). we determined that mitotic index in root meristems of barley seeds significantly decreased with increasing salt concentrations (table 1). the inhibitory effects of salt stress on mitotic activity are known for a long time (lutsenko et al. 2005). salt induced-inhibition of cell division may relate to osmotic effect and ion uptake (munns and tester 2008 ), inhibition of dna, rna and protein synthesis (anuradha and rao 2001), distruption the activity of enzymes required for cells (miller et al. 2010) and hinderance of mitosis division (tabur and demir 2010 a, b; çavuşoğlu et al. 2016). it is worth mentioning again that the relation between salinity and mitotic activity was confirmed by the present work. in our study, it was also detected that there was a remarkable increase in all kinds of chromosomal aberrations at the root meristems of barley parallel to the rise of salt concentrations (table 2). the detrimental effects of salt stress on chromosomal aberrations in plants have been studied for over the past decade. these recent studies have shown that the higher concentrations of nacl has chromotoxic effects and increases the percentage of total aberrations (tabur and demir 2010 a, b). furthermore, it was reported that these high salt concentrations delayed mitosis and caused various anaphase aberrations in barley (tajbakhsh et al. 2006) and in onion (çavuşoğlu et al. 2016). there is no relevant literature data relating to effects of humic acid on either mitotic activity or/and chromosomal abnormalities in saline conditions. the present study is the first one revealing the cytogenetic responses to the salt stress of humic acid. however, there are a few studies about the effect of humic acid application against the genotoxic effects of various chemicals such as n-nitrous compounds, maleic hydrolase and some disinfectants (gichner et al. 1990; ferrara et al. 2000). these studies have argumented that humic acid exhibits an anticlastogenic or antimutagenic activity in different plants. ferretti et al. (2012) determined that humic acid alone reduces the mitotic index and has genotoxic effects. however, there could not be made explanation for the effect of humic acid on these disinfectants since these investigators have not determined any evidence of the genotoxic effect of disinfectants alone. 8 selma tabur, merve dündar yurtlu, serkan özmen in the present work, we analyzed that humic acid pretreatment in salt stress conditions was not sufficiently successful on the mitotic index of barley seeds, but exhibit a performance very successfully statistically on chromosome behaviors. although humic acid application alone was caused a significant increase (42%) of chromosomal aberrations in root meristems of barley seeds germinated in distilled water, it has shown remarkable success in alleviating a large majority of these abnormalities caused by salinity in salt stress conditions. in parallel to salt concentrations rise, humic acid was reduced the detrimental effects of salinity and caused complete elimination of chromosome abnormalities at the highest salt concentration studied. that is, the application of 28 mg/l humic acid at 0.35 m salinity achieved an excellent success on the negative effect of salt stress, reaching to the same percentage (0.04) as the seeds germinated in distilled water. the important point here is that humic acid should be used at the appropriate concentrations, considering the negative effects on chromosome behaviors when it was used in non-stressed conditions. humic acid application alone under nonstressed conditions may have functioned as a stimulator by triggering the synthesis of proteins necessary for normal cell division and by accelerating the mitotic cycle. the acceleration of the mitotic cycle may have led to a number of disruptions during the cell division and a significant increase of chromosomal abnormalities. as is known, external stimulator growth regulator applications are useless under normal conditions where there is no stress (tabur and demir 2010 a). therefore, it is not surprising that the application of humic acid under stress conditions slows down mitotic activity in parallel with salt concentrations rise and eventually alleviating the negative effects of stress by regulating chromosome behaviors, and even removing (at 0.35 m salinity). in addition, we can explain as follows the reason why various chromosomal aberrations observed during the microscopic examination of the root meristem cells of seeds belonging to all the applications: in general, accurate chromosome segregation in mitosis requires that sister kinetochores attach to microtubules emanating from opposite spindle poles. because kinetochore attachment is a stochastic process, it is error prone and can result in chromosome malorientation (rieder and salmon 1998). mitotic irregularities such as disorderly prophase, fault or distant polarization, alignment anaphase, vagrant chromosome and bridges may be mainly the result of the above mentioned reasons or spindle dysfunction. generally, such abnormalities constitute a significant portion of chromosomal aberrations. the formations of micronuclei are likely the consequence of vagrant chromosomes and fragments. also, some researchers reported that mns, indicators of chromosomal genotoxicity and instability, are formed from one or more chromosomes (bonciu et al. 2018). it is known that fragments are considered as structural changes in chromosomes and that chromosomes are affected by physical or chemical agents outside normal conditions (el-ghamery et al. 2000). it has been reported that certain regions of chromosomes are broken by reacting with chemical substances and these regions are particularly heterochromatic regions (rieger et al. 1973). abnormal chromatin condensation expressed as chromatin granulation is concerned with the inhibition of enzymes and histone proteins. while laggard chromosomes could be the result of the failure of spindle apparatus to organize in normal way, sticky chromosomes may result from the improper folding of the chromatin fibres (klášterská et al. 1976). according to some researchers, chromosomal stickiness is a marker of the toxic effect on chromatin (fiskesjö and levan 1993). the prophase and metaphase cells with uncoiled chromosomes may be due to disorderly chromosome contractions. the disrupted equatorial plate may result from unequal distribution of chromosomes and spindle dysfunction. bonciu et al. (2018) asserted that nucleoplasmic bridges originate from dicentric chromosomes or occur as a result of a faulty longitudinal breakdown of sister chromatids during anaphase. it has also been reported that anaphase and telophase bridges may have been the result of inversions (tabur and demir 2010 b). it is thought that humic acid alone or salt concentrations used in our study may have been caused to all these abnormalities mentioned above by triggering the stimulation/ inhibition of enzymes and proteins necessary for the normal cell division, by disturbing the spindle mechanism and by accelerating mitotic cycle. conclusion the mechanisms by which salinity inhibits growth are complex and controversial. moreover, these mechanisms may vary substantially according to factors, such as plant species, the developmental stage of the plant, the strength of the stress and duration of the treatment. unfortunately, a universal mechanism about this contradiction has not been established yet. although the causes of salinity have been characterized, our understanding of the mechanisms by which salinity prevents plant growth is still rather poor. this work may provide new conceptual tools for designing hypotheses of salt tolerance in plants. as a result, we have attempted 9role of humic acid against salt-induced cytotoxicity in hordeum vulgare l. to serve the filling of a gap in the literature by comparing their interactions between the mitotic activity and chromosome behaviors of humic acid under normal and salt stress conditions using barley seeds, an important model plant for molecular studies. in future studies, the investigation of the effects of humic acid on fundamental metabolic events such as nucleic acid metabolism, protein synthesis, and enzyme synthesis, which may be directly or indirectly effective on mitotic activity and chromosomal abnormalities will contribute to the clarification of mentioned mechanism. acknowledgements we would like to thank dr. dilek çavuşoğlu (sdu atabey vocational school, food business administration) for their help in shaping. in addition, we are grateful to the süleyman demirel university scientific research projects management unit for the financial support of this work. funding this project is supported as financial by süleyman demirel university scientific research projects management unit under grant project no. 3275-yl1-12. references anuradha s, rao ssr. 2001. effect of brassinosteroids on salinity stress induced inhibition of seed germination and seedling growth of rice (oryza sativa l.). plant growth regul. 33(2): 151–153. bonciu e, firbas p, fontanetti cs, wusheng j, karaismailoğlu mc, liu d, menicucci f, pesnya ds, popescu a, romanovsky av, schiff s, ślusarczyk j, souza cp, srivastava a, sutan a, papini a. 2018. an evaluation for the standardization of the allium cepa test as cytotoxicity and genotoxicity assay. caryologia. 71(3): 191-209. calvo p, nelson l, kloepper jw. 2014. agricultural uses of 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(apiaceae) kamini gautam1,2,*, ravinder raina1,3 cytotoxic and genotoxic activity of plantago major l. extracts amira ždralović, aner mesic, izet eminović, adisa parić* genetic diversity of rhododendron simsii planch. natural populations at different altitudes in wujiashan mountain (central china) shuzhen wang, yanyan luo, tao yang, yujia zhang, zhiliang li, weibin jin, yuanping fang* nonreduction via meiotic restitution and pollen heterogeneity may explain residual male fertility in triploid marine halophyte limonium algarvense (plumbaginaceae) sofia i. r. conceição1, ana sofia róis1,2, ana d. caperta1,* effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy yavar sharafi active chemical constituents of cynanchum viminale and its cytotoxic effects via apoptotic signs on allium cepa root meristematic cells neethu kannan bhagyanathan*, john ernest thoppil clastogenic and cytotoxic effects of aerial parts’ aqueous extract of synedrella nodiflora (l.) gaertn. on wistar rat bone marrow cells saumabha chatterjee1,2, sanjib ray2* cytogenetics of accanthopus velikensis (piller et mitterpacher, 1783) (tenebrionidae: helopini) dirim şendoğan1, beril gündoğan1, maxim v. nabozhenko2,3, bekir keskin1, nurşen alpagut keskin1,* chromosome number and genome size diversity in five solanaceae genera amanda teixeira mesquita1,*, marìa victoria romero-da cruz1, ana luisa sousa azevedo2, eliana regina forni-martins1 caryologia. international journal of cytology, cytosystematics and cytogenetics 72(2): 69-79, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-232 citation: a. vangelisti, g. usai, f. mascagni, l. natali, t. giordani, a. cavallini (2019) a whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of populus trichocarpa l. subjected to different stresses. caryologia 72(2): 69-79. doi: 10.13128/cayologia-232 published: december 5, 2019 copyright: © 2019 a. vangelisti, g. usai, f. mascagni, l. natali, t. giordani, a. cavallini. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. a whole genome analysis of long-terminalrepeat retrotransposon transcription in leaves of populus trichocarpa l. subjected to different stresses alberto vangelisti#, gabriele usai#, flavia mascagni#, lucia natali, tommaso giordani*, andrea cavallini department of agriculture, food and environment, university of pisa, via del borghetto 80, i-56124 pisa, italy # these authors contributed equally to this study * corresponding author: tommaso.giordani@unipi.it abstract. long terminal repeat retrotransposons have a main role in shaping the structure of plant genomes. we used available genomic resources to study as several factors affect the expression of long terminal repeat retrotransposons in populus trichocarpa. such factors included redundancy of a retrotransposon in the genome, chromosomal localization, “genotype” of the retrotransposon, and changes in the environment. overall, we identified and annotated 828 full-length retrotransposons, and analyzed their abundance in the genome. then, we measured their expression in leaves of plants subjected to several stresses (drought, cold, heat, and salt) as well as in control plants. our analyses showed that the expression of retrotransposons was generally low, especially that of abundant elements. the transcription of an element was found to be only slightly dependent on its chromosomal localization, rather it depended on the superfamily and the lineage to which the retrotransposon belonged. finally, some retrotransposons were specifically activated by different environmental stresses. keywords. ltr-retrotransposons, retrotransposon expression, retrotransposon abundance, illumina cdna libraries, populus trichocarpa. introduction transposable elements are mobile dna sequences, which are abundant and widespread in all eukaryotic genomes. they can change their position on chromosomes by a mechanism, called transposition, driven by enzymes encoded by the element itself. transposable elements can be divided between retrotransposons (res, class i) and dna transposons (class ii), according to their transposition mechanism (wicker et al. 2007). the transposition of res occurs through a “copy and paste” replicative mechanism that includes the transcription of an rna intermediate followed by its retro-transcription and insertion into the genome (wicker et al. 70 alberto vangelisti et al. 2007). this transposition mechanism has allowed res to become the largest portion of most eukaryotic genomes, often represented by many thousands of copies (sanmiguel et al. 1998; vicient et al. 1999). a retrotransposon can be classified as ltror not ltr-re, according to the presence of long terminal repeats (ltrs) at its ends. as for the ltr-res, the promoter elements, the polyadenylation signals and the expression enhancers are found in the ltrs. these domains regulate the transcription of the element (bennetzen 2000). in the coding portion of the ltr-res, gag and pol domains can be found. gag encodes virus-like particles, pol encodes the enzymes necessary to produce new cdna molecules from the re transcripts and to integrate them into new sites in the host genome (bennetzen 2000). other structural features involved in the re replication process include a primer binding site and a poly-purine tract (bennetzen 2000). the ltr-res are essentially subdivided into two superfamilies, gypsy and copia (wicker et al. 2007), according to the order of gene sequences within the pol domain. superfamilies, in turn, are distinguished into several lineages in relation to sequence conservation and structure (barghini et al. 2015a; usai et al. 2017; buti et al. 2018; mascagni et al. 2017; 2018a). the replicative activity of ltr-res can determine large variations in the genome structureof eukaryotic species (springer et al. 2009; vitte et al. 2014). among the effects of retrotransposition, besides determining changes in genome size, re-related structural variations can modify the regulation patterns of protein-encoding genes and, consequently, their activity, influencing the phenotype (slotkin and martienssen 2007; butelli et al. 2012; falchi et al. 2013; lisch 2013). the first phase of retrotransposition is represented by the transcription of the element. the re transcripts can be capped and polyadenylated or not. in the former case, transcripts should be destined to be translated into the enzymes for retrotransposition, in the latter case, transcripts should be reverse-transcribed (chang et al. 2013; meignin et al. 2003). transcription of res has been described in several plant species (grandbastien 2015). in some grass species ltr-res are poorly constitutively transcribed (vicient et al. 2001; ishiguro et al. 2014). in other species, for example in populus x canadensis, certain ltr-res are expressed constitutively, without apparent induction conditions (giordani et al. 2016). retrotransposition is completed when a new copy of the element is inserted into the genome. this has been reported for tnt1 and tto1 elements of nicotiana and for tos17 of rice, induced by tissue culture (grandbastien 1998). complete retrotransposition of a copia re has been described also in sunflower seedlings, grown under standard conditions (vukich et al. 2009). retrotransposition is generally limited by the host genome due to its potentially mutagenic action. a major mechanism to inactivate mobile elements involves the methylation of histones and cytosine residues with consequent silencing of chromatin (dieguez et al. 1998). post-transcriptional silencing by rna degradation also plays an important role in the epigenetic control of re activity (slotkin and martienssen 2007; lisch 2013; ito 2013). in recent years, many studies have been carried on the ltr-res of the genus populus and in particular on p. trichocarpa, which is considered a model species for forest trees. the p. trichocarpa genome was the first genome to have been sequenced for a forest species (tuskan et al. 2006) and has been recently updated (zeng et al. 2017). this species has a relatively small genome (550 mbp) and res cover approximately 176 mbp (32% of the genome), with a prevalence of gypsy over copia re sequences (tuskan et al. 2006). populus trichocarpa res have been identified and annotated according to their superfamily and lineage, and ltr-re genomic abundance and age of insertion were analyzed as well (natali et al. 2015; mascagni et al. 2018b). p. trichocarpa ltrres have been also used as a reference for several analyses related to the repetitive component in other species of the genus populus (giordani et al. 2016; usai et al. 2017). the transcription of res is only the first step for retrotransposition and insertion of new copies of the element in the genome. for this reason, analyses on ltrre activity should include searching for new insertion events. however, an overall study of factors potentially able to influence the transcription of these elements is not yet available for poplar. we therefore decided to perform a meta-analysis of ltr-re expression by using publicly available genomic dna and cdna libraries obtained from leaves of plants cultivated under standard conditions or subjected to four types of abiotic stress (cold, drought, heat, and salt). the objectives of this work were to evaluate i) the expression level of res under standard and stress conditions; ii) the correlation between abundance of res and their expression level; iii) the possibility that different ltr-res are induced by different (and specific) stresses; iv) the possibility that the expression of a re is related to the “genotype” of the re itself, i.e., to the lineage to which it belongs; v) the possibility that the chromosomal localization of a re can influence its expression. 71genome analysis of long-terminal-repeat retrotransposon transcription in leaves of populus trichocarpa methods isolation of full-length ltr-res of p. trichocarpa putative full-length ltr-res were isolated from the gca_000002775.3 version (zeng et al. 2017) of the p. trichocarpa genome sequence (tuskan et al. 2006; slavov et al. 2012), deposited at the ncbi site (wgs project number aarh02, http://www.ncbi.nlm.nih.gov/ assembly/gcf_000002775.3). full-length ltr-res were isolated by using: i) ltrharvest (ellinghaus et al. 2008) with the following parameters: minlenltr=100, maxlenltr=6000, mindistltr=1500, maxdistltr=25000, mintsd=5, maxtsd=5, similar=85, vic=10, including the presence of tg and ca dinucleotides at 5’ and 3’-ends, respectively; ii) ltr-finder (xu et al. 2007), under default. a random sample of putative ltr-res (around 20% of the isolated elements) were manually validated using dotter (sonnhammer and durbin 1995) to verify the occurrence of the two ltrs, of dinucleotides tg and ca at the respective 5’ and 3’ ends, and of the tandem site duplications. all ltr-res were annotated by using blastn search against plant re datasets (barghini et al. 2015b; natali et al. 2015; usai et al. 2017; buti et al. 2018) and by using the domain search tool of repeatexplorer (novak et al. 2013). whenever possible, the fulllength ltr-res were identified as belonging to gypsy or copia superfamilies and to the respective lineages. a multi-fasta file with the sequences of identified full-length ltr-res is available at the sequence repository site of the department of agriculture, food and environment of the university of pisa (http://pgagl.agr. unipi.it/sequence-repository/). illumina cdna libraries collection the expression of ltr-res was analyzed using illumina cdna paired-end libraries publicly available at the ncbi sra (https://www.ncbi.nlm.nih.gov/sra/, bioproject accession prjeb19784) (filichkin et al. 2018). such cdna libraries were obtained from rnas from leaves of p. trichocarpa (clone nisqually 1) plants exposed to different stresses, i.e., heat, cold, drought, and salt. all cultivation conditions are described by filichkin et al. (2018). in brief, for heat stress, plants were treated at 39°c for 12 h (short treatment) or 7 days (prolonged treatment). for cold stress, plants were exposed to cycles of 4°c (night)/12°c (day) for 24 h (short treatment) or 7 days (prolonged treatment). for drought treatment, watering was withheld until soil moisture reached 0.1 m3/m3 and maintained at the level of 0.06 –0.1 m3/m3 for 5 days (short treatment) or for 12 days after water withholding (prolonged treatment). for salt stress, plants were irrigated with 100 mm nacl solution for 24 h (short treatment) or for 7 days (prolonged treatment). three replicate libraries were downloaded for each stress and control plants. illumina genomic dna sequences of the same clone of p. trichocarpa were retrieved from the ncbi sequence read archive (ncbi, washington, usa, https://www. ncbi.nlm.nih.gov/sra, sra id srr1801106). the quality of the cdna and genomic dna reads was checked using fastqc (v. 0.11.3) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and the overall quality was improved by removing illumina adapters and trimming the reads using trimmomatic (v. 0.38) (bolger et al., 2014) with different parameters for cdna (illuminaclip:2:30:10, slidingwindow:4:20, crop:96, headcrop:12 and minlen:90) and genomic dna (illuminaclip:2:30:10, slidi ngw i n dow:4:15, crop:85, m i n l en:85). organellar sequences were removed from the illumina libraries by mapping against a database of chloroplast genomes of poplar species (usai et al. 2017) using clcbio genomicworkbench (v. 9.5.3, clc-bio, aarhus, denmark) with the following parameters: mismatch cost 2, insertion cost 3, deletion cost 3, length fraction 0.5, similarity fraction 0.8. all matching reads were considered putatively belonging to organellar genomes and removed. estimation of retrotransposon expression and abundance in the genome the expression of ltr-res was measured mapping cdna sequence reads of control and cold-, drought-, heat-, or salt-exposed leaves onto the library of p. trichocarpa full-length ltr-res, using clc-bio genomic workbench with the following parameters: mismatch cost 1, deletion cost 1, insertion cost 1, similarity 0.9 and length fraction 0.9. the expression level of each sequence was calculated and converted both to mapped reads per million (mrpm) and to rpkm (mortazavi et al. 2008). ltr-res mapped by 1 to 10 reads per million of reads in at least one sample were considered as expressed (lu et al. 2013), those mapped by at least 10 reads per million were considered as highly expressed. expression values were compared, using baggerley’s test (baggerley et al. 2003), considering rpkm values in the short and prolonged stage of each treatment in comparison to control leaves. the weighted proportion fold changes between a treatment and controls were considered significant when the weight of a sample was at least two-fold higher or lower than another, with a false dis72 alberto vangelisti et al. covery rate (fdr; benjamini and hochberg, 1995) corrected p-value ≤ 0.05. in order to assess genomic abundance of res, genomic dna reads of p. trichocarpa were mapped onto reference retrotransposon domains library using clcbio genomics workbench with the same parameters described above. for each ltr-re the average coverage was calculated. the average coverage is the sum of the bases of the aligned parts of all the reads divided by the length of the reference sequence. localization of expressed res along the poplar genome each of the 19 linkage groups (lgs) of the currently available p. trichocarpa genome sequence (version gca_000002775.3, zeng et al. 2017) were subdivided into 3-mbp-long genome regions. then, in order to localize ltr-re sequences in the genome, the ltr-res were used for masking the 3-mbp-long fragments of the poplar genome using repeatmasker (http://www.repeatmasker.org) with the following parameters: s, no-is, no-low. masking was performed using i) all isolated full-length elements; ii) all chromovirus ltr-res; iii) a putative poplar centromeric sequence (islam-faridi et al. 2009; cossu et al. 2012); iv) all ltr-res expressed in control leaves (mapped by more than ten reads per million). the number of masked bases was then counted for each of the 3 mbp fragment using an in-house perl script. results identification of full-length ltr-res of p. trichocarpa the full-length ltr-res used in this study were isolated from the updated genome sequence of p. trichocarpa (zeng et al. 2017), by performing a complete genome scan with ltrharvest and ltrfinder. besides using these tools with stringent parameters, a sample of isolated elements were manually validated at structural level and all were confirmed as ltr-res. the dataset includes 828 full-length ltr-res. table 1 reports the number of ltr-res belonging to the gypsy and copia superfamilies, subdivided according to the lineage to which they belong, i.e. athila, ogre and chromovirus for gypsy elements and ale (distinguished into alei and aleii), angela, bianca, ivana/oryco, sire and tar/tork for copia elements. for each lineage the mean average coverage is also reported, calculated after mapping elements with illumina gdna reads, which represents the mean abundance of that lineage in the p. trichocarpa genome. transcription of ltr-res the expression of 828 full-length ltr-res was measured by mapping the elements with illumina cdna reads obtained from leaves of plants of p. trichocarpa cultivated in standard conditions (controls) and under different stress (drought, heat, cold, or salt). in the control leaves, only 0.47% of the cdna reads mapped the library, hence, in general, ltr-res are barely expressed (fig. 1). the expression level of ltr-res decreased with stress, in the decreasing order drought-cold-heat-salt (fig. 1). no significant difference was observed between short and prolonged treatments, with the exception of cold treatment, where expression decreased reduced in prolonged exposition. according to lu et al. (2013), we considered as expressed those ltr-res mapped by more than one read per million. the number of expressed ltr-res in controls and in drought-, cold-, heatand salt-exposed leaves is reported in fig. 2. the number of ltr-res expressed in drought-treated leaves is similar to that of control leaves. on the contrary, this number is strongly reduced after the other treatments (fig. 2). however, the table 1. number and mean average coverage of full-length ltr-res collected in the p. trichocarpa genome (version gca_000002775.3) and separated according to their superfamily and lineage. super-family lineage nr. of elements mean average coverage copia alei 42 14.04 aleii 122 22.33 angela 2 64.13 bianca 1 28.24 ivana/oryco 104 19.06 sire 7 42.11 tar/tork 90 17.45 total 368 19.89 gypsy athila 126 57.37 chromovirus 174 40.20 ogre 67 41.16 unknown 50 13.46 total 417 42.34 unknown 43 22.65 total 828 31.34 73genome analysis of long-terminal-repeat retrotransposon transcription in leaves of populus trichocarpa number of expressed ltr-res increased after prolonged salt treatment. the relationship between the abundance of a retrotransposon in the genome and its expression in another analysis we measured the relationship between the abundance of a ltr-re in the genome and the corresponding expression level. such data are reported for control leaves in fig. 3. it can be observed that abundant ltr-res (average coverage > 100) are lowly expressed. similar results were also found in leaves of plants exposed to different stresses (not reported). since assessing the expression of a ltr-re is based on the occurrence of ltr-re sequences in cdna libraries, one might ascribe such occurrence to genomic dna contamination. actually, since the most abundant ltrres resulted slightly or even not expressed, contamination by genomic dna in the cdna libraries can be largely ruled out. influence of chromosomal localization on retrotransposons expression in order to verify whether active ltr-res were localized at specific chromosomal sites, we aligned ltr-re sequences, highly expressed (mrpm > 10) in the control leaves, to the genome of p. trichocarpa (fig. 4). for comparison, we separately aligned the genome with all the 828 ltr-res; furthermore we determined the putative position of the centromere on each linkage group (lg) by aligning a tandemly repeated centromeric sequence of p. trichocarpa (islam-faridi et al. 2009) and chromovirus elements (which preferentially localize at centromeres, neumann et al. 2011). the chromosomal profiles of all the ltr-res and highly expressed ltrres were substantially similar (fig. 4). in some cases, minor peaks in the general ltr-res profiles were apparently absent in the expressed ltr-res profiles, suggesting that elements at those loci were generally inactive. fig. 1. total number of mapped reads (per million of reads) on the 828 full-length ltr-res of p. trichocarpa, in leaves of control and stress-exposed plants. stresses included cold, drought, heat and salt treatments, for short and prolonged times. the differences between control and each treatment and between short and prolonged stress treatments were significant at p<0.001 (***), p<0.01 (**), p<0.05 (*), or not significant (n.s.) according to tukey’s test. fig. 2. number of expressed (mrpm > 1) ltr-res in leaves of control and stress-exposed plants. stresses included cold, drought, heat and salt treatments, for short and prolonged times. 74 alberto vangelisti et al. only two peaks (within lg i and lg xix) were apparently more evident in the expressed ltr-res profiles, indicating that res at these loci were particularly active. in general, it can be observed that peaks in putative centromere positions were less evident in the expressed ltr-re profiles, suggesting that centromere ltr-res were less active than elements lying at other loci (fig. 4). influence of the superfamily/lineage of the retrotransposon on its expression in order to assess whether the expression of ltrres was related to the superfamily/lineage to which the element belonged, ltr-res were subdivided into lineages and separated among highly expressed (i.e., mapped by more than 10 reads per million), expressed (1-10 mapped reads per million) and not expressed (less than 1 mapped read per million). gypsy elements resulted more expressed than copia, in fact 48 out of 417 gypsy res (11.5%) were expressed, compared to 16 out of 368 copia res (4.4%). in general, most lineages showed low percentages of highly expressed or expressed ltr-res (fig. 5). however, for two copia lineages (sire and tar/tork) and one gypsy lineage (ogre) the majority of ltr-res resulted highly expressed or expressed (fig. 5). in particular, the ogre lineage showed the highest percentage of expressed elements. diffused ltr-re expression was also observed for those elements belonging to the gypsy superfamily, but for which the lineage could not be determined. fig. 3. relationship between average coverage and rpkm for each of 828 full-length ltr-res of p. trichocarpa. fig. 4. percentage of aligned nucleotides along p. trichocarpa lgs using all the full-length ltr-res expressed in control leaves, all isolated full-length ltr-res, all isolated chromovirus res and a putative centromeric sequence (in black). red arrows indicate the putative position of the centromeres. black arrows indicate minor peaks which are especially evident in the expressed ltr-res profiles or in the profiles of all ltr-res. fig. 5. percentages of highly expressed (mrpm > 10), expressed (mrpm ranging from 1 to 10) and unexpressed (mrpm < 1) ltrres, distinguished among ltr-re superfamilies and lineages. 75genome analysis of long-terminal-repeat retrotransposon transcription in leaves of populus trichocarpa stress-specific induction of retrotransposons expression most ltr-res which were expressed in control leaves were also expressed in leaves of stress exposed plants. of 313 ltr-res expressed (mrpm > 1) in controls and/or in different stresses, only 4 (1.3%) were expressed only in controls and 81 (25.9%) were specifically activated by one or more stresses. figure 6 reports the number of ltr-res expressed (i.e. with more than one mapped read per million) after different stresses (in both short and prolonged treatments). of 264 ltrres expressed during one or more stresses, 87 (33.0%) were active under each stress. fifty-six elements (21.2%) were specifically active during drought treatments, 30 (11.4%) during salt treatments and 35 (13.3%) during both drought and salt stresses, indicating that these treatments were the most effective in inducing ltrre expression. on the contrary, cold and heat stresses induced only a limited number of ltr-res (fig. 6). we also analysed differential expression of ltrres during the different stresses compared to the controls. considering only the 72 highly expressed elements (mrpm > 10), 70 showed differential expression (fold change > 2, fdr-corrected p < 0.05) in at least one treatment (fig. 7). no elements were differentially expressed along all treatments. in most cases, the same ltr-re was under-expressed (blue in fig. 7) or unaffected (white in fig. 7) during the different stresses. only 7 ltr-res were induced (red in fig. 7) or unaffected. thirteen elements were repressed by certain treatments, activated by other, or unaffected (blue, red, or white in fig. 7). discussion availability of the updated sequence of the p. trichocarpa genome and of genomic dna and cdna libraries obtained from plants of the same genotype and subjected to different treatments, allowed us to evaluate several factors which can influence the expression of ltr-res in this species. in general, our data confirmed that the expression of retrotransposons is generally limited: only 72 out of 828 ltr-res were mapped by more than ten reads per million. the transcription of res have been reported in tissues and organs of many plant species (grandbastien 2015), related to biotic and abiotic stresses or even without apparent induction. in rice, sunflower, citrus sinensis, and even in poplars certain ltr-res are actively transcribed (rico-cabanas and martínez-izquierdo fig. 6. venn diagram of expressed (mrpm > 1) ltr-res in the four stresses used in these experiments (d = drought; h = heat; c = cold; s = salt). results of short and prolonged treatments were cumulated for each stress. fig. 7. differential expression (fold change > 2, fdr-corrected p < 0.05) of ltr-res after short (s) or prolonged (p) treatments with different stresses compared to controls. blue cells refer to underexpression, red cells to over-expression, white cells indicate no effect of the treatment in comparison to control. 76 alberto vangelisti et al. 2007; vukich et al. 2009; gao et al. 2015; giordani et al. 2016). however, the majority of ltr-res are barely expressed (vicient et al. 2001; ishiguro et al. 2014; vangelisti et al. 2019). in some cases, specif ic ltr-r e sublineages have been shown to be activated and possibly overexpressed by different culture conditions, as tissue culture (kashkush et al. 2003; liu et al. 2004), wounding, methyl jasmonate and fungal elicitors (takeda et al. 1999), various phytohormones and cold stress (he et al. 2010, 2012), heat stress (ito et al. 2013). hormones, and biotic/abiotic stresses induced a general ltr-re activation in pine (voronova et al. 2014; fan et al. 2014) and in sunflower (vangelisti et al. 2019). in the present study, as in all previous works, the same treatment up-regulated certain ltr-res and repressed or unaffected other elements. in p. trichocarpa, the overall re expression level was higher in leaves of control plants than in those of plants exposed to different stresses, suggesting that plants responded to stresses increasing defence mechanisms related to res. this is different from what found by vangelisti et al (2019) in roots of sunflower: in this species, the expression level of ltr-res remained substantially very low but it slightly increased after different stresses. although the generally low level of ltr-res expression, more than 40 elements showed a significant activity (more than 10 mapped reads x million), either in controls and stressed plants, suggesting that they are not silenced and hence may still have mutagenic potential, if retrotranscription and insertion in new sites would occur after expression. the comparison, for each ltr-re, of the abundance in the genome and its expression in leaves of control or stressed plants, showed that most expressed elements are generally lowly abundant. such lack of correlation between ltr-re abundance and transcription is not surprising: other studies showed that the more an element is repeated the more it is recognized by the rna silencing machinery (meyers et al. 2001; yamazaki et al. 2001; lisch 2009). low levels of transcription of repeated sequences are often attributed to dna contamination of rna samples. the low expression level of most abundant ltr-res suggested also that the occurrence of retrotransposon-related reads in the cdna libraries was not due to dna contamination. genome localization of highly expressed (mrpm > 10) ltr-res indicated that, in poplar, the expression of an element is only slightly related to its chromosomal localization, because the profiles of expressed ltr-res parallels those of all ltr-res. however, we observed a few specific chromosome regions showing differences between profiles of all the ltr-res and expressed ltrres, suggesting that some regions are specifically activated or repressed. in species with much larger genomes than poplar, as the sunflower, ltr-re expression was observed in specific genomic regions, relatively distant from putative centromeres, and preferentially located at chromosome ends (mascagni et al. 2019). concerning the relationship between expression and superfamily/lineage of the elements, our results showed that expression of gypsy res was higher than expression of copia elements. at lineage level, ogre ltr-res were by far the most transcribed elements. among copia lineages, the most expressed were sire and tar/tork, indicating that, besides chromosomal localization and genome abundance, also the “genotype” of the ltr-re may play a role in its activation. our results confirmed what previously shown in other studies, since many of the ltr-res expressed in other species are actually of the copia superfamily (ma et al. 2008). in the case of tobacco, both tnt1 and tto1 (which are induced by tissue culture) belong to the tar/tork lineage (neumann et al. 2019). gypsy ltr-re induction was reported in cotton (hawkins et al. 2006), one of the families analyzed in that study belonged to the ogre lineage. it can be concluded that, probably, different ltr-re lineages are specifically activated in different species. it can be assumed that young ltr-res are more prone to be expressed than older elements, probably because the host needs time to develop defence mechanisms against new elements. ogre and tar/tork elements are the youngest ltr-res in p. trichocarpa (mascagni et al. 2018b): this could explain why these two lineages showed the highest percentages of expressed elements. although most ltr-res were expressed at the same level in plants subjected to different treatments, two stresses (salt and drought) specifically induced a number of ltr-res. no elements were always induced or always repressed by every stress. in some cases, the same element was up-regulated by one stress and repressed by another, probably because of the occurrence, within the ltrs, of cis-regulatory motifs recognized in specific stresses, as those identified in the ltr of the hacre1 element of sunflower (buti et al. 2009). in conclusion, this study outlines a general picture of ltr-re activity in leaves of poplar plants treated with different stresses. results allowed us to have a global insight on the features that affect ltr-re expression. since ltr-re expression 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siljak-yakovlev3,* assessment of cytotoxicity and mutagenicity of insecticide demond ec25 in allium cepa and ames test arzu özkara cytogenetic effects of fulvic acid on allium cepa l. root tip meristem cells özlem sultan aslantürk evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of allium test ioan sarac1, elena bonciu2,*, monica butnariu1, irina petrescu1, emilian madosa1 fluorescence in situ hybridisation study of micronuclei in c3a cells following exposure to elf-magnetic fields luc verschaeve1,2,*, roel antonissen1, ans baeyens3, anne vral3, annemarie maes1 phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities nicoleta anca şuţan1,*, irina fierăscu2, radu fierăscu2, deliu ionica1, liliana cristina soare1 telomeric heterochromatin and meiotic recombination in three species of coleoptera (dorcadion olympicum ganglebauer, stephanorrhina princeps oberthür and macraspis tristis laporte) anne-marie dutrillaux, bernard dutrillaux* a whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of populus trichocarpa l. subjected to different stresses alberto vangelisti#, gabriele usai#, flavia mascagni#, lucia natali, tommaso giordani*, andrea cavallini differences in c-band patterns between the japanese house mice (mus musculus) in hokkaido and eastern honshu hikari myoshu, masahiro a. iwasa* karyotypic description and repetitive dna chromosome mapping of melipona interrupta latreille, 1811 (hymenoptera: meliponini) natália martins travenzoli1, ingrid cândido de oliveira barbosa2, gislene almeida carvalho-zilse2, tânia maria fernandes salomão3, denilce meneses lopes1,* caryologia. international journal of cytology, cytosystematics and cytogenetics 73(3): 65-70, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-203 caryologia international journal of cytology, cytosystematics and cytogenetics citation: f. farsi, h.e. eroğlu, j. nozari, v. hosseininaveh (2020) karyotype analysis of trichogramma embryophagum htg. (hymenoptera: trichogrammatidae) using a new method and estimate its karyotype symmetry. caryologia 73(3): 65-70. doi: 10.13128/ caryologia-203 received: april 03, 2019 accepted: june 02, 2020 published: december 31, 2020 copyright: © 2020 f. farsi, h.e. eroğlu, j. nozari, v. hosseininaveh. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyotype analysis of trichogramma embryophagum htg. (hymenoptera: trichogrammatidae) using a new method and estimate its karyotype symmetry fatemeh farsi1, halil erhan eroğlu2, jamasb nozari1,*, vahid hosseininaveh1 1 department of plant protection, college of agriculture and natural resources, university of tehran, karaj, iran 2 yozgat bozok university, faculty of science and art, department of biology, yozgat, turkey * corresponding author. e-mail: nozari@ut.ac.ir abstract. different methods of chromosome preparation for insect are now being used across the world. well-spread chromosomes with explicit morphology, in addition to no cell wall debris are required for karyotype investigations. cytogenetic knowledge in trichogramma is extremely limited. in the article, chromosome characters, karyotype and monoploid ideogram of trichogramma embryophagum htg. (hymenoptera: trichogrammatidae) were evaluated using a new method. also, karyotype symmetry/ asymmetry of this species was calculated for the first time as one of the trichogramma species. our results showed that the diploid chromosome number of the wasp was 2n = 10. the karyotype formula was 6m + 4a. the symmetry/asymmetry index value was 1.8. the new method resulted in higher quality metaphase plates spread and provided an ideal karyomorphology for this parasitoid which has small chromosomes. keywords: ideogram, karyotype, 8-hydroxyquinoline, symmetric karyotype. introduction a high-quality chromosome preparation is critical for cytogenetic studies of hymenoptera parasitoids. in the majority of parasitoid species, complications are caused by the minute size of the insect (baldanza et al. 1993), the small size of their chromosomes (paladino et al. 2013), and low number of chromosomes, 2n = 6 found in brachymeria intermedia (chalcididae) and encarsia protransvena (aphelinidae) (hung 1986; baldanza et al. 1999) which make their cytogenetic studies limited. although modern molecular techniques such as fluorescent in situ hybridization (fish) have already taken been on an important role (yalcin and kulduk 2018), they have never been able to be a complement replacement for classical methods. some disadvantages including being costly and difficult (due to requiring probes and fluo66 fatemeh farsi, halil erhan eroğlu, jamasb nozari, vahid hosseininaveh rescent microscope), requiring highly-skilled cytogeneticists, and the inability to reveal chromosome inversions (dinizo et al. 2017) have caused cytogenetics to show a significant tendency towards using classical methods. squash is a well-known and oldest method for flattening metaphase plate in insects (kocak and okutaner 2017). however, difficulty in achieving well-spread, distorted, and stretched chromosome are the main disadvantages of this method (chirino et al. 2014; kocak and okutaner 2017). during squashing, producing wellspread plates are often not desirable due to the concentration of cells in a small area. over time, spread (hotplate spread) method conducted by various researchers flourished (bressa et al. 2009; sadilek et al. 2016). its major disadvantage is the attendance of cellular debris around the chromosomes (chirino et al. 2014), making their morphology unpleasant. considering the mentioned constraints, there is always a need for a simple and efficient method for the preparation of metaphase plates in parasitoids. despite the vast amount of research on insect karyotype, only about 500 species of parasitoid wasps have available data (gokhman 2009). among these, only more than 10 are related to trichogramma. so far, 239 species of trichogramma have been reported throughout the world (khan and yousuf 2017). there are eleven species of this parasitoid in iran (nazeri et al. 2015) that trichogramma embryophagum htg. is one of them. t. embryophagum distributed in some of regions of iran, including the provinces of west and east azerbaijan, yazd, khorasan (poorjavad et al. 2011). cytogenetic studies and karyotype investigation have not yet been done on this species. because of the substantial position that trichogramma occupy in the biological control programs (parra 2009), investigations on their chromosome details have provided precious information for the understanding various perspectives such as mechanisms of sex determination, evaluation of sex chromosome, existence of accessory chromosomes and phylogenetic relationships. an evaluation of existing literature shows that different methods was used in karyotype studies of trichogramma. studies about trichogramma were derived from routine chromosomal preparation methods, including the use of various percentages of colchicine as pretreatment, staining by different colors, and then squashed (hung 1982; liu and xiong 1998). interestingly, no banding staining on the trichogramma species has ever been reported. molecular cytogenetic including florescence in situ hybridization (fish) has been applied for only the two species of t. kaykai pinto & stouthamer (van vugt et al. 2005) and t. pretiosum riley (gokhman et al. 2017). it should be noted that the problems described for each of these methods also exist for trichogramma karyotype studies. the minute size and lifestyle of trichogramma species also intensify these problems (manickavasagam 1991; hung 1982). in most studies, only the haploid number of these species has been raised, and the chromosomal details including the length of the arms that could provide many comparisons were not possible (hung 1982; laurent et al. 1998). one parameter that can be obtained from the chromosomal information is karyotype symmetry/asymmetry (peruzzi and eroğlu 2013). karyotype symmetry/asymmetry is obtained based on the availability of chromosome detail, including relative chromosome size and position of centromere. until now, this parameter which can help in the evaluation of enterspecies relationship seldom has calculated in class of insect. in the cytogenetic studies, species that are more similar in the terms of chromosomal parameters such as karyotype symmetry/asymmetry will have more affinity. in this research, the karyotype, ideogram, and chromosomal detail of t. embryophagum were investigated for the first time with the introduction of a new method considering all steps of hymenoptera parasitoids chromosome preparation. these chromosome analyses were complemented by an estimation of its karyotype symmetry as one of the species of trichogrammatidae. material and method insect t. embryophagum htg. individuals originated from parasitized eggs of carob moth, ectomyelois ceratoniae, (zeller) (lepidoptera: pyralidae), were collected on pomegranate in the center of iran (yazd region, 32.1006° n, 54.4342° e). the wasps were reared on ephestia kuehniella zeller (lepidoptera; pyralidae) eggs in a climate-controlled chamber at 25 ± 1 °c, 70 ± 5% relative humidity, l16: d8 photoperiod. e. kuehniella eggs were obtained from a culture maintained in the biosystematics laboratory at the university of tehran, karaj, iran. preparation chromosome sample from egg host were dissected out in physiological solution for ephestia (glaser, 1917, cited by lockwood 1961). two kinds of pre-treatment were used, including hypotonic and other is combined it. first, samples were transferred into a drop of 0.075 m kcl for 8 minutes [0.075 m kcl: 0.5592 g kcl/100 ml redistill. 67karyotype analysis of trichogramma embryophagum h2o] on a shaker at 20 °c and 30 rpm. after wise, the samples were excised and pretreated in solution mixture of 8-hydroxiquinoline (0.002 w.v): colchicine (0.05 w.v) contains low concentration of dimethyl sulfoxide (dmso) at about 4° c for 30 minutes on shaker 30 rpm; and were washed in hypotonic solution (nacl 0.9% + kcl 0.042% + cacl2 0.025% in distilled water) for 3 times. then they were fix in freshly prepared carnoy s̀ fixative (6:3:1 ethanol: chloroform: acetic acid) for 20-30 minutes. they were transferred on a clean slide into a drop (5-10μl) of 60% acetic acid and their head was cut off from other parts of the body with tungsten needles, after a while, 5-10μl of 60 % acetic acid was added again. the slides were put on a heating plate at 45 °c until the acetic acid almost evaporates. water was removed bypassing the slides through an ethanol series (70 %, 80 %, 96 %; for 30 seconds each of it, respectively) and the samples were let air-dried. the slides were stained immediately with 5 % giemsa in phosphate buffer (ph 6.8). microscopic photograph and analysis the chromosome slides were examined under olympus bx53 microscope and chromosomes were photographed using an olympus dp72 camera. chromosome measurements were made on 10 metaphase plates by application of karyotype software (altınordu et al. 2016). arm rations, average lengths, and centromeric index were calculated and then were classified according to levan et al. (1964) considering their centromere position. the karyotype symmetry/asymmetry was calculated. the formula for symmetry/asymmetry index is given below. s/ai = (1 × m) + (2 × sm) + (3 × a) + (4 × t) / 2n (eroğlu 2015). eroğlu (2015) reported the new classification model from full symmetric to full asymmetric with five different types. the chromosomes, are all metacentric, form full symmetric karyotype; unlike those, are all telocentric, form full asymmetric karyotype. result the karyotype of t. embryophagum htg. contained three pairs of large metacentric and two considerably smaller pairs of acrocentric chromosomes. two of the metacentric chromosomes were of similar size whereas the third pair was close to submetacentric. thus, the diploid chromosome number was 2n = 10, and the karyotype formula was 6m + 4a (figure 1a, b). the detailed chromosomal data are given in table 1, and monoploid ideogram is given in figure 2. in t. embryophagum, haploid chromosome length and mean chromosome length are 6.38 and 1.28 μm. the rates of relative length and centromeric index range from 9.40 to 28.06 and from 18.33 to 48.04, respectively. the symmetry/asymmetry index value is 1.8. discussion in this research, the main issues were to have a regular cell layer without overlapping and the absence of qualitative damages to the morphology of chromosomes while allowing the possible spread of chromofigure 1. a, b) mitotic metaphase plate of t. embryophagum. giemsa staining. magnification 100x. 68 fatemeh farsi, halil erhan eroğlu, jamasb nozari, vahid hosseininaveh somes within the cell. th e method explained here has aimed at achieving both goals. th e chromosome structure had a high resolution, which is related to how the chromosomes was prepared. th e process of karyotyping is completed through some steps such as pretreatment, fi xation and staining. on both sides of the cell, outside and inside, the concentration of water is the same, and potassium and sodium ions are the most osmotically solutions inside and outside of the cell, respectively (leaf 1973). in this case, the cell is in an isotonic state. th e kcl changed the osmosis of the cell. pre-treatment by kcl aff ects both cellular swelling and better chromosomes spread. about kcl, hypotonic rate and duration of treatment are important. rupturing the cell membrane and the damages of chromosome (earley 1975) can be caused by the lack of attention to these cases. our result indicated that the hypotonic rate and duration of kcl reported here overcome protoplast damage. sadilek et al. (2016) stated that kcl causes the osmosis of the cell due to receiving additional water that resulted in a larger cell. th is process eff ects more identifi ed of the chromosome. th e next step aft er kcl was using combined pretreatment. colchicine is a substance that aff ects the metaphase stage of the dividing cell. guo et al. (2018) stated that colchicine increased the yield of the metaphase plate. th e 8-hydroxyquinoline is eff ective in prolonging the metaphase and further compression of the chromosomes. it is noteworthy that, until the present time, this substance has not been used as pre-treatment in insect karyotype studies but has been used extensively in plant karyotype. this substance can maintain the shape of the chromosome during the preparation process. th is feature has been used in some plant studies (fernandez et al. 2009; zarifi and güloğlu 2016). we found that 8-hydroxyquinoline prevented the distortion and stretch of the chromosome, which are the main drawbacks of squash and spreading methods. use of colchicine alone showed each chromosome clumped exceedingly and their centromeres were not distinctly in our pre-tests while using 8-hydroxyquinoline alone, the metaphase plates were few. as a result, a combination of two substances as pretreatment improved chromosome spreading. ma et al. (1996) reported that the effects of these materials together make this as improvement whereas colchicine depolymerized microtubules, 8-hydroxyquinoline decreased the rate of progression among mitotic stages and also resulted in a disorderliness in chromosome movement. in combined pretreatment, dimethyl sulfoxide (dmso) aff ected the better penetration of compounds of pretreatment into the cell. dmso has been reported as an eff ective penetration enhancer (gurtovenko and anwar 2007; williams and barry 2012). notman et al. (2006) stated that dmso induces pores in the membrane. another study indicated that dmso concentration is critical (gurtovenko and anwar 2007). we used a low concentration of dmso in our method. in this concentration, dmso induces membrane thinning (gurtovenko and anwar 2007).th e approach used in the present study focused on providing a quick and easy method for those insects which have small chromosomes, especially trichogramma wasps. th e advantages of the new method are as follows: 1. in the present method, the chromosomes are readily accessible through morphological features, including the centromere position. according to this, the evaluation of all of the parameters such as karyotype symmetry/asymmetry can be available. 2. higher quality metaphase plates spread which is achieved through the use of a combined pre-treatment which its results are an ideal karyomorphology. table 1. th e detailed measurement data of chromosome pairs of trichogramma embryophagum. pair l (μm) s (μm) l + s (μm) rl (%) l / s ci (%) type 1 0.93 0.86 1.79 28.06 1.08 48.04 m 2 0.91 0.73 1.64 25.71 1.25 44.51 m 3 1.02 0.61 1.63 25.55 1.67 37.42 m 4 0.58 0.14 0.72 11.29 4.14 19.44 a 5 0.49 0.11 0.60 9.40 4.45 18.33 a abbreviations: long arm length (l), short arm length (s), total chromosome length (l + s), relative length (rl), arm ratio (l/s), centromeric index (ci), metacentric (m), acrocentric (a). figure 2. ideogram of t. embryophagum. chromosomes are numbered from the longest (1) to shortest (5). 69karyotype analysis of trichogramma embryophagum 3. providing a single layer of cell and decreasing the overlapping cells, therefore, a better selection of metaphase plates is possible. the trichogrammatidae species were less likely to be karyotypically studies in comparison with other parasitoid families. the karyotype t. embryophagum htg. consists of 10 chromosomes (n= 5, 2n = 10). members of this family showed 10 chromosomes previously published (hung 1982; van vugt et al. 2003; gokhman et al. 2017), although there is an exception, t. kaykai, which has been diagnosed with one b chromosome that is termed psr (from paternal sex ratio) (stouthamer et al. 2001). the metaphase images indicated that chromosomes are metacentric (first, second and third chromosome pairs) and acrocentric (fourth and fifth chromosome pairs). according to these chromosome types, the s/ai value is 1.8, and the karyotype is symmetric type in t. embryophagum. karyotype symmetry/asymmetry is one of the cheapest and most popular parameters that can be obtained from cytogenetic studies (peruzzi and eroğlu 2013). the symmetric karyotype is characterized by an excess of metacentric, submetacentric chromosomes. in t. embryophagum due to two pairs of acrocentric chromosomes, s/ai value is close to 2.0 (between symmetric and asymmetric). gokhman (2009) expressed that two trends have occurred in evolution of karyotype in parasite hymenoptera. first one is decreasing in chromosome number, and the other one is karyotype dissymmetrisation, which happened as a result of the proportion acrocentric in a chromosome set. despite the widespread use of various methods that are adopted to calculate the symmetry/asymmetry karyotype in plants, this parameter has not been taken into consideration in other organisms (eroğlu 2015). so far, karyotype symmetry/asymmetry has been seldom calculated in the class of insects and other species of parasitoids. our result showed for the first time that in one of the species of trichogramma wasps, the karyotype is symmetric type according to a new method. karyotype symmetry/ asymmetry is applied on one hand to determination evolutionary relationship, and on the other hand, to compare different levels of the taxonomy (eroğlu 2015). in the case of trichogrammatidae, information obtained due to the lack of other information’s in this case is not possible to assess the issue mentioned above in this time but our results can be used as a basis for future studies in the above fields. in conclusion, the described method is cost-effective and technically easier to make for the resolution of chromosome details. the morphological characteristics of each chromosome are better observed. also, no special equipment is required as compared to molecular methods. according to the described method preparation of chromosome detail, karyotype and also ideogram of t. embryophagum were provided for the first time. we adduce as other species of trichogramma are also evaluated in which the diploid number is 2n = 10. the karyotype is a symmetric type. acknowledgments our grateful thanks go to prof. frantisek marec and dr. ehssan torabi for their help. references altınordu f, peruzzi l, yu y, he xj. 2016. a tool for the analysis of chromosomes: karyotype. taxon. 65: 586-592. baldanza f, odierna g, viggiani g. 1993. a new method for studying chromosomes of parasitic hymenoptera, used on encarsia berlesei (howard) (hymenoptera: aphelinidae). boll lab ent agr filippo 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firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-720 citation: j.-x. wang, y.-j. cao, y.-c. han, s.-. zhou, k. liu (2019) karyotype analysis of a natural lycoris double-flowered hybrid. caryologia 72(2): 3-7. doi: 10.13128/caryologia-720 published: december 5, 2019 copyright: © 2019 j.-x. wang, y.-j. cao, y.-c. han, s.-. zhou, k. liu. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyotype analysis of a natural lycoris doubleflowered hybrid jin-xia wang1, yuan-jin cao1, yu-chun han1, shou-biao zhou1,2, kun liu1,* 1 anhui provincial key laboratory of the conservation and exploitation of biological resources, college of life sciences, anhui normal university, wuhu, anhui, china 2 anhui provincial engineering laboratory of water and soil pollution control and remediation, college of environmental science and engineering, anhui normal university, wuhu, anhui, china * corresponding author: hudixiao@126.com abstract. a putative natural double-flowered hybrid in lycoris herb. was found on mt. zhangjiajie in hunan, china. the putative natural hybrid had a chromosome number of 2n = 18 and was karyotypically formulated as 2n = 4m + 6st + 5t + 3t. the karyotype of the putative natural hybrid was classified as 2b type according to the degree of asymmetry and stebbins’ criteria. according to the gross morphology, phenology and karyotype of the putative natural hybrid, it was suggested that this taxon was probably from the natural hybridization between l. aurea and l. radiata. keywords. karyotype, lycoris herb., natural hybrid. introduction the genus lycoris herb., a small group of the family amaryllidaceae, comprises approximately 20 species, distributed in warm temperate and subtropical zones of east asia, with a few extending to northern indochina and nepal (liu and hsu 1989; hsu et al. 1994; shi et al. 2006). the lycoris species are very popular bulb flowers characterized by their plentiful colors and beautiful and varied shapes (hsu et al. 1994; zhou et al. 2007). the hybridization frequently happens in the genus lycoris and causes a number of the presently established hybrid taxa, such as l. houdyshelii traub, l. straminea  lindl., l. squamigera maxim., l. incarnata comes ex c. sprenger and l. × hubeiensis k. liu (kurita 1987; hsu et al. 1994; shi et al. 2006; meng et al. 2018). during our field investigations of the wild populations of lycoris in hunan province, china, a mixed population of three taxa, namely l. aurea  (l’her.) herb., l. radiata (l’her.) herb., and a putative natural hybrid was found. the putative natural double-flowered hybrids attracted our more attention, which were discovered for the first time in lycoris in the wild. to illustrate the origin of the natural double-flowered hybrids, some bulbs were successfully transplanted and cultivated in the experimental garden, together 4 jin-xia wang et al. with their putative parents. the karyotypes of the three lycoris taxa were analyzed in this study, indicating that the double-flowered taxon might be from the hybridization between l. aurea and l. radiata. materials and methods the plants were collected from mt. zhangjiajie, hunan, china (110º17’e, 29º19’n), and then transplanted to the experimental garden of anhui normal university, wuhu, china. the flowers of each taxon were shown in fig. 1. for chromosome observation, actively growing root tips were pretreated in p-dichlorobenzene solution at 4 °c for 5 h before they were fixed in carnoy i (glacial acetic acid : absolute ethanol = 1:3) at 4 °c for 20 h. then they were macerated in 1 mol l-1 hydrochloric acid at 60 °c for 3 minutes, stained in phenol-fuchsin for 20 h, and squashed in 45% acetic acid. the karyotype formula was based on the measurements of metaphase chromosomes taken from photographs. for each taxon, measurements were taken from at least five well-spread metaphase cells in no fewer than three different individuals. for the description of karyotypes, the symbols had been adapted according to levan et al. (1964): m for median-centromeric chromosome with arm ratio of 1.01-1.70; st for subterminalcentromeric chromosome with arm ratio of 3.01-7.00; t for terminal-centromeric chromosome with arm ratio of over 7.00; t for terminal-centromeric chromosome with no short arm. karyotypes were classified on the basis of their degrees of asymmetry according to stebbins (1971) and li and chen (1985). the intrachromosomal asymmetry index (a1) and interchromosomal asymmetry index (a2) were calculated using romero zarco’ equations (1986). results 1. the putative natural lycoris double-f lowered hybrid (table 1, 2; figure 2a, 2d) – the chromosomes were counted to be 2n = 18, consisting of 4 large median-centromeric (m), 6 subterminal-centromeric (st), 5 terminal-centromeric (t) and 3 terminal-centromeric (t). the karyotype was formulated as 2n = 4m + 6st + 5t + 3t. the average length of chromosome complement was 122.3 μm. the ratio of the length of the largest chromosome to that of the smallest was 3.39, and the proportion of chromosomes with arm ratio >2:1 was 77.8%. the karyotype was therefore of 2b type according to the degree of asymmetry and the chromosomes ranged from fig 1. the flowers of the three taxa. a: the putative natural hybrid; b: l. aurea; c: l. radiata. 5karyotype analysis of a natural lycoris double-flowered hybrid 3.36~11.38 in relative length. the values of a1 and a2 were 0.72 and 0.50, respectively. 2. lycoris aurea (table 1, 3; figure 2b, 2e) – the chromosome number was 2n = 14, consisting of 8 large median-centromeric (m) and 6 terminal-centromeric (t). the karyotype formula was 2n = 8m + 6t. the average length of chromosome complement was 138.6 μm. the ratio of the length of the largest chromosome to that of the smallest was 3.32, and the proportion of chromosomes with arm ratio >2:1 was 42.9%. the karyotype type was 2b. the relative lengths of all chromosomes ranged from 3.49 to 11.59. the values of a1 and a2 were 0.47 and 0.44, respectively. table 1. karyotype characteristics of the studied lycoris taxa. species karyotypic formula stebbins’ type a1 a2 tcl (μm) hybrid 2n = 4m + 6st + 5t + 3t 2b 0.72 0.50 122.3 l. aurea 2n = 8m + 6t 2b 0.47 0.44 138.6 l. radiata 2n = 12st + 10t 4a 0.86 0.10 111.2 a1: intrachromosomal asymmetry index, a2: interchromosomal asymmetry index, tcl: average length of total chromosome complement. table 2. measurements of somatic chromosomes of the putative natural hybrid. no. relative length arm ratio type ll sl tl 1 5.71 5.67 11.38 1.01 m 2 5.81 5.38 11.19 1.08 m 3 6.11 4.65 10.76 1.31 m 4 4.39 4.09 8.48 1.07 m 5 4.63 0.37 5.00 12.51 t 6 4.63 0.33 4.96 14.03 t 7 3.97 0.65 4.62 6.11 st 8 4.53 0.00 4.53 ∞ t 9 4.49 0.00 4.49 ∞ t 10 3.90 0.46 4.36 8.48 t 11 4.34 0.00 4.34 ∞ t 12 3.47 0.54 4.01 6.43 st 13 3.61 0.31 3.92 11.65 t 14 3.39 0.49 3.88 6.92 st 15 3.22 0.46 3.68 7.00 st 16 2.99 0.60 3.59 4.98 st 17 2.99 0.46 3.45 6.50 st 18 3.03 0.33 3.36 9.18 t note: ll, relative length of long arm; sl, relative length of short arm; tl, total relative length; ll + sl = tl. the same is below. fig 2. the metaphase chromosome morphology and karyotypes of the putative natural hybrid, lycoris aurea and l. radiata. a, d: the putative natural hybrid; b, e: l. aurea; c, f: l. radiata. scale bar = 10 μm. table 3. measurements of somatic chromosomes of l. aurea. no. relative length arm ratio type ll sl tl 1 5.81 5.76 11.57 1.01 m 2 5.56 5.38 10.94 1.03 m 3 5.52 5.34 10.86 1.03 m 4 5.47 4.93 10.40 1.11 m 5 4.75 4.13 8.88 1.15 m 6 4.39 4.26 8.65 1.03 m 7 4.13 3.90 8.03 1.06 m 8 3.99 3.41 7.40 1.17 m 9 4.93 0.00 4.93 ∞ t 10 3.81 0.00 3.81 ∞ t 11 3.77 0.00 3.77 ∞ t 12 3.68 0.00 3.68 ∞ t 13 3.59 0.00 3.59 ∞ t 14 3.49 0.00 3.49 ∞ t 6 jin-xia wang et al. 3. lycoris radiata (table 1, 4; figure 2c, 2f) – the chromosome number of this species was 2n = 22. it consisted of 12 subterminal-centromeric (st) and 10 terminal-centromeric (t). the karyotype was formulated as 2n = 12st + 10t. the average length of chromosome complement was 111.2 μm. the ratio of the length of the largest chromosome to that of the smallest was 1.43, and the proportion of chromosomes with arm ratio >2:1 was 100%. the karyotype type was 4a. the relative lengths of all chromosomes ranged from 3.56 to 5.10. the values of a1 and a2 were 0.86 and 0.10, respectively. discussion the karyotype of the putative natural double-flowered hybrid is formulated as 2n = 18 = 4m + 6st + 5t + 3t. lycoris aurea in this study has a chromosome number of 2n = 14, in agreement with other reports (liu and hsu 1989; hsu et al. 1994). l. radiata has a chromosome number of 2n = 22, similar to some previous studies (shao et al. 1994; zhou et al. 2007; liu et al. 2016). based on the values of the two indices, a1 and a2, l. radiata had the largest intrachromosomal asymmetry and the smallest interchromosomal asymmetry among the three taxa. considering the sympatric distribution of l. aurea and l. radiata, it was supposed that this putative natural hybrid might be a diploid between l. aurea, which produced the gamete having 4m + 3t, and l. radiata with areduced gamete having 6st + 5t. according to our observation, the leaf of this putative natural hybrid emerged in september, the same as the putative parents. the sizes of leaf blade and bulb of this putative natural hybrid were intermediate between its two putative parents. so far, some lycoris species were confirmed to be hybrids by karyotype or molecular sequence analysis, such as l. houdyshelii, l. straminea, and l. incarnata (hsu et al. 1994; shi et al. 2006; liu et al. 2011), and these hybrids had the similar flower characteristics with their putative parents. here, a putative natural double-flowered hybrid with no pistil and stamen was discovered and its karyotype was described for the first time. based on the karyotype analysis, further molecular study was needed to uncover the origin of this natural hybrid and distinguish the paternal donor from the maternal donor. judging from the absence of seed, pistil and stamen, the putative natural hybrid could be sexually sterile. the lycoris species all had considerable ornamental value. compared with the normal lycoris species, the doubleflowered hybrid had more ornamental value. because of the lack of seed, it was needed to propagate the doubleflowered hybrid for the large application in landscaping in future by tissue culture and quick propagation technology. acknowledgments we thank the anonymous reviewers for helpful comments that improved the paper. funding this work was financially funded by the national natural science foundation of china (31400291) and collaborative innovation center of recovery and reconstruction of degraded ecosystem in wanjiang city belt, anhui province; 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0.1 mmol/l for oleuropein) have been separately applied to female and male populations of d. melanogaster for the control and application groups. in both female and male populations, it has been observed that zinc oxide/titanium dioxide has decreased the lifespan and oleuropein has increased the lifespan according to the control group, depending on the concentration. these findings demonstrate the beneficial effect of oleuropein, suggested as a protective role in the prevention of zinc oxide/titanium dioxide induced developmental toxicity. keywords: drosophila melanogaster, lifespan, oleuropein, toxicity, zinc oxide/titanium dioxide nanoparticles. introduction nanotechnology has a great potential for applications in many fields advancements in nanotechnology are increasing rapidly (karahalil 2013). so, this technology is appearing as a new research field that is investigating the potential risks of nanoparticles (nps) on human and environmental health (landsiedel et al. 2009). in this respect, many reports have demonstrated various effects of nps exposure on different animals, plants, and microorganisms; depending on their species, growth conditions, nps type, and exposure concentrations. also, the in vitro and in vivo studies using different experimental models indicate that nanoparticles may cause genotoxic effects that involve oxidative stress and inflammation (kang et al. 2008; xu et al. 2009; petković et al. 2011). however, the mechanisms involved in nps-induced toxicity have not been clearly explained and are poorly studied in vivo. 128 tuğba atıcı, deniz altun çolak metal nps are the most widely used among nanoparticles. titanium dioxide (tio2) and zinc oxide (zno) nps are of special concern since they used in food products, plastics, paper, drugs, cosmetics, sunscreens, paints, and medical materials and are two of the fastest-growing product categories in the nanotechnology industry (yeber et al. 2000). as a result of this widespread use, increased environmental release and a higher potential for human exposure will appear. the data from several in vitro studies demonstrate that tio2nps cause various adverse effects at the cellular level, such as oxidative stress and dna damage (wang et al. 2007). the aging process is generally described as the progressive decline of homeostatic maintenance functions and physiological fitness (sohal et al. 2002). drosophila or fruit fly is commonly used for studying in the aging model system because the genetic background, developmental processes, and some more aspects are well known. they are also highly similar to mammalians in terms of genetic structure (çakır and bozcuk 2000). the production of free radicals and other oxidants produced during aerobic respiration is the most reason for mechanistic explanations for aging links (barja 2002). oxidative stress is supposed to be an important mechanism underlying nanoparticle toxicity. thus, it is believed that nanoparticle toxicity can be prevented by antioxidants (nel et al. 2006; mocan et al. 2010). from this point of view, the effects of zinc oxide/titanium dioxide (znotio2) nanocomposite on the lifespan of d. melanogaster and the protective role of oleuropein (ole, olive leaf extract), a strong antioxidant, on these effects. materials and methods experimental organism drosophila melanogaster (diptera; drosophilidae) oregon r strain was reared in standard drosophila medium (sdm) containing 15 g sucrose, 17 g cornmeal, 3 g agar-agar, and 9 g yeast in 360 ml distilled water within environmental chamber maintained at 25±1  °c and 40-60% relative humidity in darkness. further addition of 1 ml propionic acid as a preservative/fungicide was done. the flies used in the experiments were at the same age (1-3 days) and the females were virgins. chemicals and solutions oleuropein (98% pure; st. louis, mo, usa) and titanium dioxide (99% pure, anatase, steinheim, germany) were purchased from sigma aldrich. zno-tio2 (zinc oxide-titanium dioxide) nanoparticles were synthesized in the chemistry laboratory of black sea technical university. zno was loaded into the tio2 photocatalyst with a 1% ratio. tio2 catalyst (10 g) and water (10 ml) were mixed to have a slurry. zno (0.3355 g) was added to the slurry and calcined at 400 °c for 6 h. after this process, it was cooled in a desiccator and stored in a closed dark bottle. for the stock solutions, znotio2 nps powder was dispersed in deionized water. then, this solution was vortexed for 20 seconds, and sonicated for 30 min in an ultrasonic bath (sonorex, bandelin electronic, berlin, germany) at a frequency of 60 khz, to ensure uniform suspension of nps. finally, the nanoparticles concentrations were prepared by diluting the stock solution. characterization of znotio2nps znotio2nps size distribution and morpholog y were represented by scanning electron microscope (sem, (jeol jsm 5600 lv, tokyo, japan) at the magnification of 100×. the hydrodynamic diameter was characterized by a master sizer (malvern, zetasizer ver. 7.02, malvern instruments ltd, worcestershire, uk) using the dynamic light scatter (dls) technique. lifespan assay the lifespan experiments were studied separately in female and male populations. for this purpose, about 100 individuals were collected from among the same aged (1-3 days) female and male flies which were not mated and obtained from the pupa. the gathered individuals were then put into the empty culture bottles and left hungry for 2 hours before znotio2 and ole applications. for the application, two layers of blotting papers were placed into each culture vial; znotio2 and ole in different concentrations (0.005, 0.1, 0.5, and 1 g/l for znotio2; 0.1 mmol/l for ole) were absorbed into these papers. afterward, the flies were put into these application vials and were left for 2 hours. after 2 hours, the individuals (separately female and male flies) were placed into the culture vials containing only sdm as 25 × 25. the experiments for both the control and application groups were started at the same time. all the vials were kept in appropriate thermal cabins. during the experiments, food was replaced with fresh food twice a week. the number of individuals was controlled both at the beginning and the end of each application day and the dead individuals were counted. the application was carried out until the last individual died. the experiments were repeated three times. 129th e role of oleuropein against nanocomposite toxicity in fruit fl y: evidence for lifespan extension statistical analyses th e obtained data were analyzed with spss version 16.0 (statistical package for the social sciences soft ware, spss, chicago, il). th e mean lifespan values of the control and application groups were subjected to duncan’s one-way range test (p<0.05). results characterization of znotio2nps th e size of the nanoparticles is an important parameter that determines activity in biomedical applications. dls is an analytical method that estimates the hydrodynamic diameter while sem is used for the estimation of the actual diameter of nanoparticles. dls studies revealed that the hydrodynamic diameter of znotio2nps was 42.5±1.2 nm. th e morphological characterization of znotio2nps was performed by sem to visualize the actual particle size and the overall size distribution (figure 1). sem image indicates that the nanoparticles formed aggregates of diff erent sizes and these aggregations have a porous structure. lifespan assay in this study, it was obser ved that znotio2, depending on the concentration, has decreased the lifespan of the male and female population according to the control group. it was also determined that ole has increased the lifespan according to the control group. th e maximum female lifespan of the control and application groups was observed for 78 days while the maximum male lifespan belonging to the control and application groups was 76 days, respectively. th e diff erence between the control and application groups is not statistically signifi cant (p>0.05) (table 1). in the female population applied with znotio2, the maximum lifespan for the lowest concentration (0.005 g/l) was 70 days however for the highest concentration (1 g/l) the maximum lifespan was 54 days. also, in the 0.1 and 0.5 g/l znotio2 application groups, the female maximum lifespan was 68 and 62 days, respectively (figure 2). according to results, in the male population applied with znotio2, the maximum lifespan for the lowest concentration (0.005 g/l) was 71 days however for the highest concentration (1 g/l) the maximum lifespan was 51 days. also, in the 0.1 and 0.5 g/l znotio2 applicafigure 1. sem images of znotio2nps in dry form. table 1. th e longevity of male and female populations of d. melanogaster experimental group (no) female number maximum lifespan mean lifespan probability level male number maximum lifespan mean lifespan probability level control (1) 100 72 56.58±1.10 1-2* 4-7* 5-6* 100 74 55.79±1.12 1-2* 3-4* 3-7* 4-5* 4-7* 5-6* ole (0.1 mmol/l)(2) 100 78 57.64±1.23 100 76 55.51±1.17 znotio2 5 mg/l(3) 100 70 49.48±1.45 100 71 44.12±1.84 0.1 g/l(4) 100 68 45.17±1.59 100 66 41.03±1.85 0.5 g/l(5) 100 62 35.41±1.29 100 60 39.11±1.70 1 g/l(6) 100 54 34.19±1.65 100 51 35.50±1.45 znotio2 +ole (1 g/l+0.1 mmol/l)(7) 100 64 43.77±1.79 100 62 43.80±1.69 *th e mean diff erence is not signifi cant at the 0.05 level. 130 tuğba atıcı, deniz altun çolak tion groups, the male maximum lifespan was 66 and 60 days, respectively (figure 3). also in every group, it is determined that female individuals live longer in comparison to male individuals (table 1). discussion in recent years, studies about the toxic risks of nps are increasing (landsiedel et al. 2009; yilmaz öztürk 2019). first reports about the toxicity of some nanoparticles show that they can affect biological systems at the organ, tissue, cellular, subcellular, and protein levels (braydich-stolle et al. 2009; khajavi et al. 2019). in vivo toxicity studies have demonstrated that inhalation of tio2 nps causes pulmonary inflammation in rats and mice (bermudez et al. 2004) and tio2 nps induce dna damage and genetic instability in mice (trouiller et al. 2009). reeves et al. (2008) showed oxidative stressrelated effects, including inf lammation, cytotoxicity, and genomic instability, either alone or in the presence of uva irradiation, in mammalian studies. also, zinc oxide nanoparticles have been reported to be cytotoxic and exhibited strong protein adsorption abilities (horie et al. 2009). toxicity of zno nps on human bronchial epithelial cells was investigated and suggested that oxidative stress is a mechanism of toxicity (heng et al. 2010). the production of free radicals has been supposed to be one of the primary mechanisms of nps toxicity (nel et al. 2006; yang et al. 2009). it may result in oxidative stress, inf lammation, and consequent damage to proteins, membranes, and dna (bhabra et al. 2009; hu et al. 2009). thus, in our study, we investigated the toxic effects of znotio2 nanocomposite on the lifespan of d. melanogaster and the protective role of oleuropein (ole), a strong antioxidant. oleuropein is the major phenolic constituent of olive leaves (olea europaea) and is also present in the fruit and oil (van acker et al. 1998). many studies demonstrated that ole has an antiinflammatory activities (carluccio et al. 2003), free-radical scavenging properties (le tutour and guedon 1992; manna et al. 1997) and inhibit the growth of different tumor cell types (hamdi and castellon 2005). in our preliminary study, it was determined that znotio2nps were relatively increased levels of total  oxidant status (tos) and was decreased level of total  antioxidant capacity (tac) compared to the control group. but, in a znotio2+ole group, it was observed that vice versa (çolak et al. 2016). conclusion these experiments provide evidence that znotio2 nanocomposite can shorten the lifespan of drosophila which is partially or completely prevented by oleuropein. this means oxidative stress is the major contributor to znotio2 toxicity. as a result, although the emerging technologies and their opportunities are desirable to be implemented into daily life, before their applications, regulations should be defined to protect human health and the environment. acknowledgments this study was supported by the erzincan binali yı ldırım universit y [gra nt number bap-fen-a 090614-0088]. references barja g. 2002. rate of generation of oxidative stressrelated damage and animal longevity. free radic biol med. 33(9):1167-1172. bermudez e. mangum jb. wong ba, asgharian b, hext pm, warheit db, everitt ji. 2004. pulmonary 0 20 40 60 80 100 120 1 8 15 22 29 36 43 50 54 62 64 68 70 72 78 control 0.1 mmol /l ole 5 mg/l znotio2 0.1 g/l znotio2 0.5 g/l znotio2 1 g/l znotio2 1 g/l znotio2+0.1 mm ol/l ole ♀ age (days) % s u rv iv or sh ip 0 20 40 60 80 100 120 1 8 15 22 29 36 43 51 54 60 62 66 71 74 76 control 0.1 mmol /l ole 5 mg/l znotio2 0.1 g/l znotio2 0.5 g/l znotio2 1 g/l znotio2 1 g/l znotio2+0.1 mm ol/l ole % s u rv iv or sh ip age (days) ♂ figure 2. exposure to znotio2 and ole in female adult d. melanogaster leads to lifespan alteration. figure 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zhang h, xi z. 2009. comparative study of cytotoxicity, oxidative stress and genotoxicity induced by four typical nanomaterials: the role of particle size, shape and composition. j appl toxicol. 29(1):69-78. yeber mc, rodríguez j, freer j, durán n, mansilla hd. 2000. photocatalytic degradation of cellulose bleaching effluent by supported tio2 and zno. chemosphere. 41(8):1193-1197. yilmaz öztürk b. 2019. intracellular and extracellular green synthesis of silver nanoparticles using desmodesmus sp.: their antibacterial and antifungal effects. caryologia 72(1): 29-43. caryologia. international journal of cytology, cytosystematics and cytogenetics 72(4): 51-60, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-390 citation: f. farahani, a. sedighzadegan, m. sheidai, f. koohdar (2019) population genetic studies in ziziphus jujuba mill.: multiple molecular markers (issr, srap, its, cp-dna). caryologia 72(4): 51-60. doi: 10.13128/caryologia-390 published: december 23, 2019 copyright: © 2019 f. farahani, a. sedighzadegan, m. sheidai, f. koohdar. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. population genetic studies in ziziphus jujuba mill.: multiple molecular markers (issr, srap, its, cp-dna) farah farahani1, atieh sedighzadegan2, masoud sheidai2, fahimeh koohdar2 1 department of microbiology, qom branch, islamic azad university, qom, iran 2 faculty of life sciences and biotechnology, shahid beheshti university, tehran, iran *corresponding authors: farahfarahani2000@yahoo.com, asedighzadegan@yahoo.com, msheidai@yahoo.com, f_koohdar@yahoo.com abstract. ziziphus jujuba (jujube) is an important horticultural crop with medicinal value. it is under cultivation in many areas of iran and also grows as wild in several geographical populations throughout the country. we have no information on genetic variability and population structure of this important plant species in our country. therefore, the aim of the present study was to perform genetic fingerprinting of 13 geographical populations of jujuba for the first time and provide data on population genetic structure, admixture versus genetic fragmentation of this important crop. we used multilocus molecular markers (issrs and sraps) for genetic fingerprinting and also compared the results with bioinformatics investigation results we did on jujuba cultivars by using nuclear r-dna and chloroplast inter-genetic cp-dna sequences. genetic diversity parameters and amova test as well as ivanno test support some kind of genetic distinctness of the jujuba populations studied. we found that cp-dna inter-genic sequences can also discriminate jujuba cultivars as efficient as multilocus molecular markers and therefore, a multiple molecular approaches may be used for genetic fingerprinting of jujuba. the present study revealed good level of genetic diversity among wild/ uncultivated populations of jujuba which can be used in conservation and breeding of this important horticultural crop plant within the country. as this crop has several wild geographical populations throughout the country, we plan to continue our quest to investigate many more populations in nearby future and try to utilize cp-dna inter-genic sequences along with multilocus molecular markers for genetic discrimination of wild populations. keyword. cp-dna, issr, its, srap, ziziphus jujube. introduction the genus ziziphus mill. belongs to the buckthorn family rhamnaceae. it is contains about 40 species that are deciduous evergreen trees or shrubs distributed in the tropical and subtropical regions of the world (sing et al. 2007). the wide geographical and climatic distribution makes it interesting 52 farah farahani et al. for genetic diversity investigations and gene pool identification. south and southeast asia is the center of both evolution and distribution of the genus ziziphus (sing et al. 2007). tow fossil species are known for ziziphus in eocene era (us govt. printing office 1982). ziziphus species are of medicinal value and are known to be self-incompatible and have synchronous protandrous dichogamy and produce viable inter-specific hybrids (asatryan and tel-zur, 2013). among ziziphus species, few are well known like: z. jujuba (jujuba), and z. spina-christi (l.)desf. that grow in south-western asia, z. lotus in mediterranean region, ber (z. mauritiana), that is found in western africa to india and z. joazeiro mill. that grows in the caatinga of brazil (gupta et al. 2004; jiang et al. 2007; vahedi et al. 2008). traditional use of jujuba dates back 2,500 years ago in original chinese material medical records. the fruit, seed, and bark of jujuba are also described in korean, indian, and japanese traditional writings. they are used to alleviate stress and insomnia and as appetite stimulants, digestive aids, anti-arrhythmic, and contraceptives. the sweet smell of the fruit is said to make teenagers fall in love. the fruit is eaten fresh or dried and made into candy; tea, syrup, and wine are also made from the berries (gupta et al. 2004; jiang et al. 2007; vahedi et al. 2008). the fruit is energy-rich because of the large amount of sugar it contains. it is cultivated and eaten fresh, dry, and in jam. it is also added as a base in meals and in the manufacture of candy. the leaves can be either deciduous or evergreen depending on species, and are aromatic. the seeds, fruit, and bark of jujuba have been used in traditional medicine for anxiety and insomnia, and as an appetite stimulant or digestive aid. experiments in animals support the presence of anxiolytic and sedative properties. however, clinical trials are lacking (gupta et al. 2004; jiang et al. 2007; vahedi et al. 2008). some specific saponins, as well as ethyl acetate and water extracts of the fruit and bark, have explored the potential cytotoxicity of jujuba. apoptosis and differential cell cycle arrest are suggested to be responsible for the dosedependent reduction in cell viability. activity against certain human cancer cell lines has been demonstrated in vitro (lee et al. 2004; huang et al. 2007; vahedi et al. 2008). jujuba is one of the important horticultural crops in iran and about with annual production of 4980 kg that is about 14.7% of total cold region fruit production (34000 tones) (hosseinpour et al. 2016). it has been cultivated in several regions of the country and also is grown wild in several areas throughout iran. different molecular markers have been used for population genetic investigation and phylogenetic studies in ziziphus species. for example, islam and simmons (2006) performed an intra-generic classification of 19 ziziphus species by using morphological characteristics and nuclear rdna internal transcribed spacers, 26s rdna, and the plastid trnl-f intergenic spacer. similarly, the genetic relationships between different z. jujuba cultivars and/ or wild jujuba individuals was studied by using random amplified polymorphic dna (rapd), amplified fragment length polymorphisms (aflp), sequence-related amplified polymorphisms (srap), simple sequence repeats (ssr), inter-simple sequence repeats (issr), and chloroplast microsatellite (cp-ssr) markers (see for example, peng et al. 2000; liu et al. 2005; wang et al. 2007; singh et al. 2007; wang et al. 2014; zhang et al. 2014; huang et al. 2015). population genetic study is an important step for genetic evaluation of medicinally important species as it provides insight on the genetic structure, genetic diversity and gene flow versus genetic fragmentation of these plant species. it also produces data on the number of potential gene pools for conservation and breeding strategies for the studied taxa (sheidai et al. 2013, 2014, 2016). the aims of present study are: 1produce data on population genetic structure of ziziphus jujuba of iran for the first time and 2investigate the discrimination power of issr and srap molecular markers in ziziphus jujuba populations and compare them with sequencing data like nuclear r-dna sequences (its = internal transcribed spacer dna) and chloroplast gene sequences. we used issr (inter simple sequence repeats) and sr ap (sequence related amplif ied polymorphism) molecular markers, as these markers are very useful tool to detect genetic polymorphism, are inexpensive and readily adaptable technique for routine germplasm fingerprinting and evaluation of genetic relationship between accessions or genotypes and construction of genetic linkage maps (sheidai et al. 2013, 2014, 2016). moreover, srap markers target the open reading frames (orfs). material and methods plant materials in total 130 plants were studied in 13 geographical populations of ziziphus jujuba (table 1). ten plants were randomly selected in each population and used for molecular studied (issr and srap). 53population genetic studies in ziziphus jujuba mill.: multiple molecular markers (issr, srap, its, cp-dna) table 1. ziziphus jujuba population in issr and srap studies. province locality longitude latitude 1 qom kalaghneshin 50. 2536 ° 34.4122° 2 qom ghaziolia 50.2850° 34.3222° 3 qom dolatabad 50.3032° 34.1258° 4 qom jafarieh 50.3429° 34.4722° 5 qom khalajestan 50.3844° 34.2852° 6 markazi aveh 50.2523° 34.4732° 7 markazi delijan 50.4102° 33.5926° 8 markazi saveh 50.2124° 35.0117° 9 esfahan kashan niasar 51.0856° 33.5822° 10 esfahan koohpayeh 52.2623° 32.4249° 11 esfahan shahreza 51.5200° 32.0032° 12 esfahan dehaghan 51.3916° 31.5612° 13 esfahan ardestan 52.2238° 33.232.07° dna extraction for molecular studies, the fresh leaves were randomly collected from 53 randomly selected plants in the studied area and were dried in silica gel powder. the genomic dna was extracted using ctab-activated charcoal protocol (križ man et al. 2006). the extraction procedure was based on activated charcoal and poly vinyl pyrrolidone (pvp) for binding of polyphenolics during extraction and under mild extraction and precipitation conditions. this promoted high-molecular-weight dna isolation without interfering contaminants. quality of extracted dna was examined by running on 0.8% agarose gel. issr assay ten issr primers, ubc 807, ubc 810, ubc 811, ubc 834, cag(ga)7, (ca)7ac, (ca)7at, (ca)7gt (ga)9a, and (ga)9t, commercialized by the university of british columbia, were used. pcr reactions were performed in a 25-μl volume containing 10 mm tris-hcl buff er at ph 8, 50 mm kcl, 1.5 mm mgcl2 , 0.2 mm of each dntp (bioron, germany), 0.2 μm of a single primer, 20 ng of genomic dna, and 3 u of taq dna polymerase (bioron). amplification reactions were performed in a techne thermocycler (germany) with the following program: 5 min for initial denaturation step at 94 °c, 30 s at 94 °c, 1 min at 52 °c, and 1 min at 72 °c. th e reaction was completed by a final extension step of 7 min at 72 °c. the amplification products were visualized by running on 2% agarose gel, followed by ethidium bromide staining. the fragments size was estimated by using a 100-bp molecular size ladder (fermentas, germany). the experiment was replicated 3 times and constant issr bands were used for further analyses. srap assay five sequences related amplified polymorphism (srap) primer pairs including forward primers: me1, me2, me3, me4, me5 and reverse primers: em1, em2, em3, em4, em5 were used (feng et al. 2014). pcr reactions were carried in a 25μl volume containing 10 mm tris-hcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 ng genomic dna and 1 u of taq dna polymerase (bioron, germany). the amplifications, reactions were performed in techne thermocycler (germany) with the following program: 5min initial denaturation step 94°c, followed by five cycles of 94°c for 1min, 35°c for 45 sec, and 72°c for 1 min; followed by 35 cycles of 94°c for 1min, 50°c for 45 sec, and itc for 1 min; followed by 7 min at 72°c. the amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated by using a 100 bp molecular size ladder (fermentas, germany). its and cp-dna inter-genic sequences analyses cp-dna and nuclear-dna its sequences of 11 jujuba cultivars were obtained from ncbi(national center for bioinformatic information) and used to differentiate the studied cultivars. the cultivars accession numbers have been provided in tables 2 and 3. data analyses the issr and srap bands obtained were treated as binary characters and coded accordingly (presence = 1, absence = 0). the number of private bands versus common bands was determined. genetic diversity parameters like: the percentage of allelic polymorphism, allele diversity (weising, 2005), nei’s gene diversity (he), and shannon information index (i) (weising, 2005), were determined. we used genalex 6.4 for these analyses (peakall and smouse 2006). the nei genetic distance (weising 2005) was determined among the studied populations and was used for the grouping of the genotypes. genetic differentiation of the studied populations was studied by amova with 1000 permutations as performed in genalex 6.4 (peakall and smouse 2006). 54 farah farahani et al. table 2. the accession numbers of taxa in cp-dna studies. no species accession number 1 ziziphus jujuba hg765030.1 2 ziziphus jujuba hg765029.1 3 ziziphus jujuba hg765028.1 4 ziziphus jujuba gq435353.1 5 ziziphus jujuba eu075109.1 table 3. the accession numbers of taxa in its studies. no species accession number 1 ziziphus jujuba dq146578.1 2 ziziphus jujuba dq146577.1 3 ziziphus jujuba dq146576.1 4 ziziphus jujuba dq146575.1 5 ziziphus jujuba dq146574.1 6 ziziphus jujuba dq146573.1 7 ziziphus jujuba fj593183.1 8 ziziphus jujuba eu075088.1 9 ziziphus jujuba kf241298.1 10 ziziphus jujuba kf241297.1 11 ziziphus jujuba kf186458.1 the mantel test (podani 2000) was performed to study the association between genetic distance and geographical distance of the studied populations. we also used mantel test to investigate the agreement of results between issr and srap data. past ver. 3.14 (hammer et al. 2001). genetic structure of the populations was studied by model-based clustering as performed by structure software ver. 2.3 (pritchard et al. 2000). we used the admixture ancestry model under the correlated allele frequency model. a markov chain monte carlo simulation was run 20 times for each value of k (1-13) after a burn-in period of 105 . data were scored as dominant markers and analysis followed the method suggested by falush et al. (2007). for the optimal value of k in the studied populations we used the structure harvester website (earl and von holdt 2012) to perform the evanno method (evanno et al. 2005). the choice of the most likely number of clusters (k) was carried out by calculating an ad hoc statistic δk based on the rate of change in the log probability of data between successive k values, as described by evanno et al. (2005). for its and cp-dna the sequences were aligned by muscle program as implemented in mega 7. nj and maximum likelihood phylogenetic trees were constructed by mega7 software (tamura et al. 2012). kimura distance was determined for jujuba cultivars based on its and cp-dna sequences by mega ver.7. results issr assay we obtained 40 issr bands (loci) in total (table 4). the highest number of bands (27 bands) occurred in population 9 (neyasar), followed by population 7 (delijan) (23 bands). some of the populations had private bands with population 9 having the highest number (6 private bands). few common bands occurred in the population too. these are shared alleles among these populations. genetic diversity parameters determined in z. jujuba populations are presented in table 5. the percentage of genetic polymorphisma obtained ranged from 7.50 in population 2 (ghazi-olya) to 52.50 in population 7 (delijan). a good level of genetic polymorphism (37.50%) also occurred in three populations 3, 4, and 5 (doolatabad, jafariyeh, and dastjerd, respectively). the same populations had higher value of gene diversity (he). amova revealed that these populations differ significantly in their genetic content (phipt = 0.54, p = 0.001). amova identified that 72% of total genetic variability occurred among populations while, 28% of genetic variability was due to within population difference. paired-sample amova also produced significant difference among the studied populations. nj clustering (figure 1) revealed that most of the samples in the studied populations are grouped together and are almost separated from the other populations (for example, samples in populations 1, 2, 7, 8, 9, 11, 12, and 13). nei, genetic distance and genetic identity determined among ziziphus jujuba populations (table 6) revealed that genetic similarity among populations ranged from 0.58 between populations 9 and 13, to 0.93 between populations 3 and 5. table 4. details of issr bands obtained in the studied populations of ziziphus jujuba (populations numbers are according to table 1). population 1 2 3 4 5 6 7 8 9 10 11 12 13 no. bands 21 14 20 21 18 16 23 10 27 16 14 15 15 no. private bands. 0 1 0 0 0 1 2 0 6 0 0 0 1 no. lcomm bands (<=25%) 1 0 1 1 0 0 1 0 3 1 0 0 1 no. lcomm bands (<=50%) 5 2 4 3 2 2 6 2 6 3 4 3 1 55population genetic studies in ziziphus jujuba mill.: multiple molecular markers (issr, srap, its, cp-dna) mantel test between geographical distance and genetic distance produced signif icant correlation (p<0.01). therefore, with increase in geographical distance, genetic difference of the populations increased and isolation by distance (ibd) occurred in z. jujuba populations studied. the genetic structure of the studied populations and degree of gene flow/ or shared common alleles were determined by structure analysis. the structure plot (figure 2) revealed presence of different allele combinations (differently coloured segments) in the z. jujuba populations. however, some degree of shared common alleles was observed between populations 3 and 4, and to lesser extent population 5. similarly, populations 10 and 11 had genetic similarity. the other populations had unique allele combinations (specific coloured segment) as well as some degree of shared alleles. evanno test produced optimal number of genetic group k = 8. therefore, 13 studied ziziphus jujuba populations studied could be grouped in 8 genetic groups. srap markers assay we obtained 42 srap bands (loci) in total (table 7). the highest number of bands (26 bands) occurred in population 13, while the lowest number of srap bands occurred in population 4 (14 bands). populations 1, 4, 8 and 13 had private bands. few common bands occurred in the population too. these are shared alleles among the studied populations. genetic diversity parameters determined based on srap molecular markers in z. jujuba populations are presented in table 8. the percentage of genetic polymorphisma obtained ranged from 7.14 in population 8 to 38.10 in populations 3 and 13. these two populations had higher value of gene diversity (he). amova revealed that the studied ziziphus jujuba populations differ significantly in their genetic content (phipt = 0.65, p = 0.001). amova identified that 66% of total genetic variability occurred among populations while, 34% of genetic variability was due to within population difference. paired-sample amova also produced significant difference among the studied populations. nj distance clustering (figure 3) revealed that most of the samples in the studied populations are grouped together and are almost separated from the other populations (for example, samples in populations 1, 9, 12 and 13). this indicates that srap molecular markers can efficiently differentiate jujube populations and may be used in germplasm diversity evaluation. pcoa plot of the studied populations (figure 4) obtained after 99 permutations, almost separated the studied populations in two major groups (with populations 1 and 9 somewhere in the middle). the populations 2-7 formed the first group, while populations 8, 10-13, comprised the second group. therefore, zizphus jujuba populations can be genetically discriminated by issr markers. table 5. genetic variability parameters determined in ziziphus jujube populations based on issr markers (pop ulations numbers are according to table 1). pop na ne i he uhe %p pop 1 0.800 10157 0.146 0.097 0.116 %27.50 pop 2 0.425 1.045 0.041 0.027 0.033 %7.50 pop 3 0.875 1.251 0.209 0.142 0.157 %27.50 pop 4 0.900 1.283 0.232 0.154 0.176 37.50% pop 5 0.825 1.302 0.231 0.161 0.179 %37.50 pop 6 0.575 1.101 0.092 0.061 0.070 %17.50 pop 7 1.100 1.352 0.292 0.189 0.220 %52.50 pop 8 0.425 1.125 0.102 0.070 0.080 %17.50 pop 9 0.975 1.194 0.169 0.113 0.136 %30 pop 10 0.550 1.077 0.072 0.047 0.052 %15 pop 11 0.525 1.131 0.105 0.072 0.080 %17.50 pop 12 0.600 1.136 0.123 0.082 0.098 %22.50 pop 13 0.550 1.115 0.097 0.065 0.075 %17.50 n = no. plants, na = no. alleles, ne = no. effective alleles, i = shanon information index, he = nei gene diversity, uhe = unbiased gene diversity, %p = percentage of genetic polymorphism figure 1. nj dendrogram of ziziphus jujuba specimens showing genetic differences of the studied populations. 56 farah farahani et al. structure plot of srap molecular markers (figure 5) revealed more detailed information on the genetic affinity of the studied populations. it also revealed the presence of specific allele combinations (differently coloured segments) versus available common shared alleles (similarly coloured segments) in these populations. for example, close affinity between populations 1 and 9 that were identified by pcoa plot seems to be due to some low degree of shared common alleles between these populations. the same is true for the other studied populations. evanno test produced delta k = 2 as the optimal genetic groups. therefore, the studied jujuba populations can be differentiated in two broader and distinct genetic groups. the populations 1-7 form the first group, while populations 8-13 comprise the second group. mantel test performed between issr and srap data produced significant correlation (p = 0005). therefore, both types of molecular markers efficiently differentiate jujuba populations and also show similar genetic grouping. similarly, mantel test produced significant correlation (p = 0.001) between the studied molecular markers with geographical distance of the populations. therefore, with increase in geographical distance among jujube populations, the genetic difference of these populations also increases. this indicates the occurrence of ibd (isolation by distance) in the studied jujuba populations. table 6. nei, genetic distance and genetic identity (populations numbers are according to table1). pop id 1 2 3 4 5 6 7 8 9 10 11 12 13 1 **** 0.7857 0.7781 0.7202 0.7947 0.7194 0.8011 0.7576 0.8117 0.7012 0.7697 0.7462 0.7660 2 0.2412 **** 0.7929 0.7385 0.8074 0.6631 0.7905 0.7260 0.6737 0.7130 0.6715 0.6947 0.7035 3 0.2509 0.2321 **** 0.9339 0.9511 0.8575 0.9064 0.8043 0.7280 0.7418 0.7362 0.8516 0.8704 4 0.3282 0.3031 0.0684 **** 0.9309 0.8262 0.8844 0.7837 0.6696 0.7370 0.6891 0.8023 0.7803 5 0.2298 0.2139 0.0502 0.0716 **** 0.8507 0.9397 0.8183 0.6888 0.7431 0.7417 0.8282 0.8274 6 0.3294 0.4109 0.1537 0.1909 0.1617 **** 0.8230 0.7832 0.5932 0.7619 0.7230 0.8882 0.8224 7 0.2218 0.2351 0.0982 0.1228 0.0622 0.1948 **** 0.8516 0.6906 0.7853 0.8219 0.8283 0.8054 8 0.2776 0.3202 0.2178 0.2437 0.2006 0.2443 0.1606 **** 0.6428 0.7891 0.7644 0.7943 0.7376 9 0.2086 0.3950 0.3175 0.4011 0.3728 0.5222 0.3703 0.4419 **** 0.5813 0.6721 0.6473 0.7684 10 0.3549 0.3383 0.2986 0.3052 0.2970 0.2719 0.2416 0.2369 0.5425 **** 0.8698 0.7940 0.7124 11 0.2617 0.3983 0.3062 0.3724 0.2988 0.3244 0.1961 0.2686 0.3973 0.1395 **** 0.8327 0.6940 12 0.2928 0.3643 0.1606 0.2203 0.1886 0.1186 0.1883 0.2303 0.4350 0.2307 0.1831 **** 0.8511 13 0.2666 0.3517 0.1388 0.2481 0.1895 0.1956 0.2164 0.3043 0.2634 0.3392 0.3653 0.1613 **** table 7. details of srap bands obtained in the studied populations of ziziphus jujuba (populations numbers are according to table 1). population pop1 pop2 pop3 pop4 pop5 pop6 pop7 pop8 pop9 pop10 pop11 pop12 pop13 no. bands 21 21 20 14 18 17 21 16 17 20 19 20 26 no. bands freq. >= 5% 21 21 20 14 18 17 21 16 17 20 19 20 26 no. private bands 1 0 0 1 0 0 0 1 1 0 0 0 2 no. lcomm bands (<=25%) 3 0 0 0 2 1 1 0 2 1 1 1 1 no. lcomm bands (<=50%) 8 7 7 4 5 5 8 1 5 6 7 8 11 figure 2. structure plot of ziziphus jujuba populations studied (populations numbers are according to table1). table 8. genetic distance among jujube cultivars based on cp-dna psba sequences (populations numbers are according to table 2). 1 2 3 4 2 0 3 0 0 4 0.58 0.58 0.58 5 0.58 0.58 0.58 0 57population genetic studies in ziziphus jujuba mill.: multiple molecular markers (issr, srap, its, cp-dna) its and cpdna sequences nuclear r-dna (its) and chloroplast inter-genic region of trnh-psba sequence data were obtained for few jujuba cultivars. phylogenetic tree based on these sequences (figures 6 and 7) differentiated the studied cultivars in three clusters with high bootstrap values. therefore, we can also apply these sequence-based molecular markers in future studies to investigate jujuba cultivar discrimination, the methods that have not been utilized in genetic finger printing of this important horticultural plant species. pair-wise genetic distances in the studied jujube cultivars are provided in tables 9 and 10. in case of trnhpsba, we obtained the mean genetic distance of 0.58 which is comparable to the genetic distance obtained for issr and srap molecular markers. however, in case of its sequences, we obtained much lower genetic distance value (0.003-0.007). this is probably due to much more conservative nature of its sequences compared to that of cp-dna inter-genic sequences. therefore, we may suggest using cp-dna inter-genetic sequences for future table 9. genetic distance among jujube cultivars based on nuclear dna (its sequences) (populations numbers are according to table 2). 1 2 3 4 5 6 7 8 9 10 2 0 3 0 0 4 0 0.003 0.003 5 0 0.003 0.003 0 6 0 0.003 0.003 0 0 7 0 0.003 0.003 0 0 0 8 0 0.007 0.007 0.003 0.003 0.003 0.003 9 0 0.007 0.007 0.003 0.0037 0.003 0.003 0 10 0 0.007 0.007 0.003 0.0037 0.003 0.003 0 0 11 0 0.007 0.007 0.0037 0.0037 0.003 0.003 0 0 0 figure 3. nj dendrogram of the studied ziziphus jujube populations based on srap molecular markers. (populations 1-13 are according to table 1). figure 4. pcoa plot of ziziphus jujube populations based on srap molecular markers. (populations 1-13 are according to table 1). figure 5. top: structure plot of ziziphus jujuba populations based on srap data. bottom: structure plot based on k = 2 (populations 1-13 are according to table 1). 58 farah farahani et al. genetic finger printing of jujube cultivars and populations, but also keeping in mind that using multilocus molecular markers (issrs and sraps) are more costbenefit approaches. discussion population genetic study provides valuable information on genetic structure of plants, the stratification versus gene flow among the species populations, genetic divergence of the populations, etc. (sheidai et al. 2014). these information have different applications, and from pure understanding of biology of the species to conservation of endangered species, choosing of proper parents for hybridization and breeding and phylogeography and mechanism of invasion (freeland et al. 2011). ziziphus jububa is of wide spread in our country and it has several medicinal applications (vahedi et al. 2008), however we had no information on its genetic structure. the present study revealed interesting data about its genetic variability, and genetic stratification of this medicinally important species in the country. assessment of the genetic variation within collections of ziziphus jujuba genetic resources is crucial for the effective conservation and utilization of these resources in breeding programs, and could be dramatically enhanced by using molecular genotyping tools. the present study revealed that multilocus molecular markers like issrs and sraps are powerful technique for the assessment of genetic variability among ziziphus jujuba collections. moreover, we can also use cp-dna inter-genic sequences for genetic finger printing and discriminating jujuba cultivars and populations. for figure 7. maximum parsimony phylogenetic tree of jujube cultivars based on trnh-psba sequences (numbers above branches are bootstrap value). figure 6. maximum parsimony phylogenetic tree of jujube cultivars based on its sequences (numbers above branches are bootstrap value). table 9. genetic distance among jujube cultivars based on cp-dna psba sequences (populations numbers are according to table 2). 1 2 3 4 2 0 3 0 0 4 0.58 0.58 0.58 5 0.58 0.58 0.58 0 table 10. genetic distance among jujube cultivars based on nuclear dna (its sequences) (populations numbers are according to table 2). 1 2 3 4 5 6 7 8 9 10 2 0 3 0 0 4 0 0.003 0.003 5 0 0.003 0.003 0 6 0 0.003 0.003 0 0 7 0 0.003 0.003 0 0 0 8 0 0.007 0.007 0.003 0.003 0.003 0.003 9 0 0.007 0.007 0.003 0.0037 0.003 0.003 0 10 0 0.007 0.007 0.003 0.0037 0.003 0.003 0 0 11 0 0.007 0.007 0.0037 0.0037 0.003 0.003 0 0 0 59population genetic studies in ziziphus jujuba mill.: multiple molecular markers (issr, srap, its, cp-dna) grouping of the cultivars we can also utilizenuclear r-dns sequences. we obtained about 40 bands for either of issr and srap molecular markers and almost good level of genetic variability within each population (ranging from 17 to 35%). these markers have good discriminating power to differentiated jujuba populations. cp-dna inter-genic sequences also revealed high degree of genetic difference among jujuba cultivars (0.58). saleh et al. (2016), studied genetic diversity in populations of ziziphus spina-christi (l.) willd. by using 11 issr markers and reported the occurrence of 105 scorable loci, of which 93.4% were found to be polymorphic. they obtained genetic diversity value of 0.26, and total genetic diversity ht = 0.266, as well as intra-population genetic diversity, hs = 0.22. these values are in good agreement with genetic variability obtained here by both multilocus molecular markers (issrs and sraps) as well as cp-dna sequences. the genetic variability within the studied populations is of fundamental importance in the continuity of a species as it is used to bring about the necessary adaptation to the cope with changes in the environment (sheidai et al. 2013, 2014). this is particularly expected in ziziphus jujuba as it forms several geographical populations throughout the country. degree of genetic variability within a species is highly correlated with its reproductive mode, the higher degree of open pollination/ cross breeding brings about higher level of genetic variability in the studied taxon (freeland et al. 2011). ziziphus jujuba is a self-incompatible species (asatryan and tel-zur 2013) and therefore, moderate genetic variability in these populations may be related to the open pollination nature of this species. amova revealed significant genetic difference among the studied populations of jujube, while ivanno test identified 8 genetic groups within these populations. moreover, mantel test showed positive significant correlation between genetic distance and geographical distance. all these data support some kind of genetic distinctness of the jujuba populations studied. different mechanisms like isolation, drift, founder effects and local selection may act to bring about among population differentiation (jolivet and bernasconi 2007; sheidai et al. 2014). in conclusion, the present study revealed good level of genetic diversity among wild/ uncultivated populations of jujube 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cytology, cytosystematics and cytogenetics 73(3): 121-126, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-578 caryologia international journal of cytology, cytosystematics and cytogenetics citation: j.m. rodríguez-domínguez, e. tapia-campos, r. barba-gonzalez (2020) physical mapping of 45s and 5s rdna in two sprekelia formosissima cytotypes (amaryllidaceae) through fluorescent in situ hybridization (fish). caryologia 73(3): 121-126. doi: 10.13128/caryologia-578 received: july 31, 2019 accepted: july 14, 2020 published: december 31, 2020 copyright: © 2020 j.m. rodríguezdomínguez, e. tapia-campos, r. barba-gonzalez. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. physical mapping of 45s and 5s rdna in two sprekelia formosissima cytotypes (amaryllidaceae) through fluorescent in situ hybridization (fish) josé manuel rodríguez-domínguez, ernesto tapia-campos, rodrigo barba-gonzalez* unidad de biotecnología vegetal, centro de investigación y asistencia en tecnología y diseño del estado de jalisco, guadalajara, jalisco, méxico *corresponding author. e-mail: rbarba@ciatej.mx abstract. chromosome number and position of rdna were studied in plants of sprekelia formosissima (amaryllidaceae) collected in two populations with different ploidy level (2n=2x=60 and 2n=5x=150). the 5s and 45s rrna loci were localized and physically mapped using two-color fluorescence in situ hybridization probes. the diploid (2n=2x=60) cytotype showed four loci for the 45s rdna in two chromosome pairs (11 and 25) in telomeric position. the 5s rdna was present in six loci of three homologous chromosome pairs (3, 13 and 19) in subtelomeric and telomeric positions. the chromosomes of the pentaploid cytotype (2n=5x=150) showed five loci for the 45s rdna in telomeric position and five loci for the 5s rdna in subtelomeric position. the karyotypic formula is 13m + 16sm + 1 st and the karyotype symmetry/asymmetry index is tf % = 34.67, ask % = 65.32 and syi % = 54.81, concluding that it is an asymmetric karyotype, bimodal with one distinctively large pair of chromosomes (10.42 µm) and a gradual decrease in the size of the other chromosome pairs, from the longest of 6.84 µm, to the shortest of 2.61 µm. keywords: bulbous genus, karyogram, karyotypic formula, ornamental, plant chromosomes, ploidy level. introduction sprekelia heist, is a bulbous genus of the monocotyledonous family amaryllidaceae, it is a perennial, herbaceous monotypic genus commonly known as aztec lily or jacobean lily represented by the sole species sprekelia formosissima (flory 1977; sánchez 1979); it is distributed through mexico and guatemala (lópez-ferrari and espejo-serna 2002). its scarlet flowers with curved petals have made of it an exceptional ornamental pot plant. the technique of fluorescent in situ hybridization (fish) has become a very useful tool for the detection of specific dna in the genome of organisms. in this technique, the genes encoding the 5s ribosomal rna (rdna 122 josé manuel rodríguez-domínguez, ernesto tapia-campos, rodrigo barba-gonzalez 5s) and the 18s-5.8s-26s ribosomal rna (45s rdna) are commonly used as markers for the physical mapping of plant chromosomes due to the high number of their repeating units, specific position on chromosomes and highly conserved sequences (liu and davis 2011). the 45s ribosomal rna genes are clustered in tandem arrays of repeating units of the genes 18s, 5.8s and 26s, internal transcribed spacer sequences (its) and external nontranscribed spacer sequences (nts), with an approximate size of 7.5-18.5 kb in plants (mizuochi et al. 2007). the 5s ribosomal rna genes are also found in tandem repeats with an approximate size of 0.2-0.9 kb, with a highly conserved region (120 bp long) separated by a nts sequence (specht et al. 1997). physical mapping of rdna has been performed in many plants such as: maize (li and arumuganathan 2001), orchids (cabral et al. 2006), crotalaria juncea (mondin et al. 2007), agave (robert et al. 2008; gomez-rodriguez et al. 2013), asparagus (deng et al. 2012), lilium (lim et al. 2001; hwang et al. 2015), tigridia pavonia (arroyo-martínez et al. 2018), among many others, however, there is little work in plants of the amaryllidaceae family using these modern techniques of molecular cytogenetics. the objective of this study was the physical mapping of rdna 45s and 5s in two cytotypes (diploid and pentaploid) of sprekelia formosissima. material and methods plant material sprekelia formosissima bulbs were collected from two different populations and maintained in an in vivo collection at the centro de investigación y asistencia en tecnología y diseño del estado de jalisco a.c. (ciatej). two cytotypes were found, a diploid population (2n=2x=60) from the road villa-corona-cocula, and a pentaploid population (2n=5x=150) from a private collection in zapopan, both in the state of jalisco, méxico. the bulbs were placed in a container with moist substrate composed by a mixture of peat moss and vermiculite (7:3) for the bulbs to produce roots. sampling was performed in ten bulbs of each of the two populations. chromosome spreads obtaining metaphasic chromosomes was performed as described by rodríguez-domínguez et al. (2017), in brief, the root apex were pretreated in a saturated solution of α-bromonaphthalene (0.1%) for 48 h at 4 °c and fixed in a methanol:acetic acid (3:1) solution for 24 h at 4 °c. afterwards, the root apex were washed with milli-q water, macerated in an enzyme mixture containing each 0.2% (w/v) of pectolyase y23, rs cellulase and cytohelicase in 10 mm citrate buffer (ph 4.5) and incubated at 37 °c for 3 h. later, a cell suspension was obtained by vortexing, followed by washing with distilled water, centrifuging at 10,000 rpm for 45 s, washed with methanol, then, centrifuged at 11,000 rpm during 30 s. the cell pellet was resuspended in 100 µl methanol. finally, in a fume hood, the slides were covered with a layer of pure acetic acid and 10 µl of cell suspension were added to each slide. the slides were immediately inclined at an angle of 45 degrees, and two drops of pure acetic acid were added; the slides were exposed (facing down) to vapor from a water bath at 55 °c for 5 s, finally, a drop of pure acetic acid was added and allowed to air dry. dna probes two different clones were utilized as probe, clones pta71 and pta794 which contains the ecori fragment of 45s and 5s ribosomal dna from wheat respectively (gerlach and bedbrook 1979; gerlach and dyer 1980). the bacteria were cultured for 24 hours under constant agitation (225 rpm) at 37 °c in lb medium supplemented with 0.1 mg/ml ampicillin. isolation of wheat ribosomal dna was performed using the kit high pure plasmid isolation kit (roche®) according to the supplier’s instructions. in brief, 4 ml of the inoculated culture medium were transferred to 15 ml tubes and centrifuged at 9,000 rpm for 1 minute, the supernatant was discarded; the bacterial pellet was resuspended in 250 µl of suspension buffer and transferred to a 1.5 ml tube. 250 µl of lysis buffer were added and mixed by inversion, then incubated for 5 min and 350 µl of binding buffer were added and incubated for 5 min on ice; followed by centrifugation at 13,000 rpm for 10 min. the supernatant was transferred to a filtration membrane and centrifuged at 13,000 rpm for 1 minute; the membrane was transferred to a new tube and 500 µl of washing buffer i was added and centrifuged at 13,000 rpm for 1 minute. the column was transferred to a new tube and 700 µl of washing buffer ii was added; centrifuged at 13,000 rpm for 1 minute. the flowthrough liquid was discarded and the column was centrifuged at 13,000 for 1 minute to dry the membrane, which was transferred to a new 1.5 ml tube and 100 µl of elution buffer was added and centrifuged at 13,000 rpm for 1 minute. 123physical mapping of 45s and 5s rdna in two sprekelia formosissima cytotypes through fish labeling the probes fluorescent in situ hybridization (fish) was performed using two different probes (45s and 5s wheat rdna) which were direct labelled as follows: 1 µg of rdna in 12 µl mq water, 4 µl nick translation mix (roche®) and 4 µl of 5x fluorophore mix (5 µl of each 2.5 mm datp, dctp, dgtp, 3.4 µl 2.5 mm dttp, 4 µl 1 mm labeled dutp and 27.6 µl mq water) were incubated at 15 °c for 90 min. reaction was stopped by adding 1 µl of 0.5 m edta (ph 8.0) and heating at 65 °c for 10 min. 45s rdna was labeled with tetramethylrhodamine-5-dutp and 5s rdna with fluorescein-12-dutp. fluorescent in situ hybridization slides with metaphase chromosomes were incubated at 37 °c overnight, the next day, each slide was incubated in 200 μl rnase a (100 μg/ml) in 2xssc at 37 °c for 1 h and washed three times for 5 min each in 2x ssc, incubated in 0.01 m hcl for 2 min. 200 μl pepsin (5 μg/ml) were added and incubated at 37 °c for 10 min, washed in mq water for 2 min and in 2x ssc for 5 min each. the slides were incubated in 4% paraformaldehyde for 10 min and washed three times in 2x ssc for 5 min each. the slides were dehydrated in 70%, 90% and absolute ethanol series for 3 min each and airdried. hybridization followed using a mixture consisting of 20x ssc, 50% formamide, 10% sodium dextran sulphate, 10% sds and 25-50 ng of each probe. the dna was denatured by heating the hybridization mixture at 70 °c for 10 min and then placed on ice for at least 10 min. for each slide, 40 μl of hybridization mixture was used. the preparations were denatured at 80 °c for 10 min and incubated overnight in a humid chamber at 37 °c. after overnight hybridization, the slides were washed three times in 2x ssc at 37 °c for 5 min each, in the last wash temperature was increased to 42 °c and the slides were then washed three times in 0.1x ssc at 42 °c each followed by three washes in 2x ssc at rt for 5 min each. chromosomes were counterstained with 1μg/ ml dapi (4,6-diamidino-2-phenylindole) and a drop of vectashield anti-fade (vector laboratories) was added for its examination under a leica dmra2 microscope equipped with epi-fluorescent illumination, filter sets of dapi, fitc and cy3. images were captured by a evolution qei (media cybernetics) monochrome digital camera and processed with image-pro plus software. the dapi images were sharpened with a 7x7 high gauss spatial filter. dapi, fitc and tetramethylrhodamine fluorescence were pseudo-colored with their respective dye tint for the fish analyses. karyotype analysis karyotype analysis was based on at least 10 highquality metaphase plates with almost similar degree of chromatin condensation, chromosome measurements were made using the freeware software micromeasure (reeves 2001) vers 3.3. the following measurements of each pair of chromosomes were made: chromosome length, long arm (l), short arm (s), arm ratio (l/s). the chromosomes were assorted into different categories based on arm’s ratio arranged according to levan et al. (1964) and the karyogram was performed; the chromosome homology was assigned according to similarities in length, morphology and centromere position. the number of homologous chromosomes was assigned sequentially according to the reduction of the chromosomal length and in base to centromere position. three different methods of evaluating karyotype asymmetry were used: the tf% (huziwara, 1962), the ask% (arano, 1963) and the syi (greilhuber and speta, 1976; venora et al. 2002). results the physical distribution of the 45s and 5s rdna were investigated by fluorescent in situ hybridization (fish) (figures 1 to 3) in two cytotypes of sprekelia formosissima, a diploid (2n=2x=60) and a pentaploid (2n=5x=150). the diploid (2n=2x=60) cytotype showed figure 1. fluorescent in situ hybridization of wheat 45s (red signals, arrowheads) and 5s rdna (green signals, arrows) in a diploid sprekelia formosissima (2n=2x=60) cytotype. the chromosomes were counterstained with dapi. bar=10µm. 124 josé manuel rodríguez-domínguez, ernesto tapia-campos, rodrigo barba-gonzalez four loci for the 45s rdna in two chromosome pairs (11 and 25) in telomeric position. the 5s rdna was present in six loci of three homologous chromosome pairs (3, 13 and 19) in subtelomeric and telomeric positions (figures 1 and 3). chromosome pair 1 was identified due to its unmistakable large size. the chromosomes of the pentaploid cytotypes (2n=5x=150) showed five loci for the 45s rdna in telomeric position and five loci for the 5s rdna in subtelomeric position (figure 2). the hybridization loci for each probe was found always in different chromosomes. thirty chromosome pairs (60 chromosomes in total) were identified in the diploid cytotype of s. formosissima and were arranged based on arm’s ratio according to levan et al. (1964) in the karyogram shown in figure 3. the karyotypic formula was 13m + 16sm + 1 st with predominance of m and sm chromosomes. the longest per shortest chromosome ratio (l/s) ranges from 1.00 to 5.12. the karyotype symmetry/asymmetry index is tf % = 34.67, ask % = 65.32 and syi % = 54.81, concluding that it is an asymmetric karyotype, bimodal figure 2. fluorescent in situ hybridization of wheat 45s (red signals, arrowheads) and 5s rdna (green signals, arrows) in a pentaploid sprekelia formosissima (2n=5x=150) cytotype. the chromosomes were counterstained with dapi. bar=10µm. figure 3. karyogram of diploid sprekelia formosissima. the wheat 45s rdna hybridization loci (red signal) are present in chromosome pairs 11 and 25, while the wheat 5s rdna loci (green signal) are present in chromosome pairs 3, 13 and 19. table 1. chromosome parameters of sprekelia formosissima chromosome pair no. total length (µm) long arm (l) (µm) short arm (s) (µm) arm ratio l/s centromeric position 1 10.42 5.40 5.02 1.08 m 2 5.02 3.10 1.92 1.61 m 3 3.98 2.29 1.69 1.36 m 4 3.86 2.22 1.64 1.35 m 5 3.60 1.96 1.64 1.20 m 6 3.41 1.95 1.46 1.34 m 7 3.26 1.71 1.55 1.10 m 8 3.24 1.89 1.35 1.40 m 9 3.19 1.81 1.38 1.31 m 10 3.14 1.89 1.25 1.51 m 11 3.06 1.55 1.51 1.03 m 12 2.87 1.57 1.30 1.21 m 13 2.61 1.36 1.25 1.09 m 14 6.84 4.77 2.07 2.30 sm 15 6.46 4.70 1.76 2.67 sm 16 6.15 4.25 1.90 2.24 sm 17 6.08 4.46 1.62 2.75 sm 18 6.04 4.3 1.74 2.47 sm 19 5.50 4.12 1.38 2.99 sm 20 5.43 4.07 1.36 2.99 sm 21 5.22 3.77 1.45 2.60 sm 22 5.16 3.63 1.53 2.37 sm 23 5.14 3.63 1.51 2.40 sm 24 5.04 3.32 1.72 1.93 sm 25 4.91 3.19 1.72 1.85 sm 26 4.61 3.15 1.46 2.16 sm 27 4.31 2.92 1.39 2.10 sm 28 4.16 2.68 1.48 1.81 sm 29 3.68 2.50 1.18 2.12 sm 30 6.84 5.41 1.43 3.78 st 125physical mapping of 45s and 5s rdna in two sprekelia formosissima cytotypes through fish with one distinctively large pair of chromosomes (10.42 µm) and a gradual decrease in the size of the other chromosome pairs, from the longest of 6.84 µm, to the shortest of 2.61 µm. (table 1). discussion according to roa and guerra (2012), the most frequent loci number of 45s rdna in angiosperms is two or four per diploid karyotype and they are generally located in terminal regions of the chromosomes, found more frequently in the short arms. in the cytotypes of s. formosissima analyzed in this work, both signals (45s and 5s rdna) were detected in the short arms of the chromosomes. even though the localization sites and the number of copies of the 5s and 45s loci present variation, it is probably not related to the ploidy level as has been suggested in some studies (weiss-schneeweiss and schneeweiss 2013). this coincides with our results where four 45s rdna sites and six 5s rdna sites were detected in diploid plants, while five 45s rdna sites and five 5s rdna sites were detected in the pentaploid cytotype. there may be a different number of signals in species that have the same ploidy level, for example, garcía (2015) reported four 45s sites and nine 5s sites for diploid sprekelia howardii lehmiller, while in our work only four 45s sites were detected and six 5s sites for diploids analyzed. differences in signal sizes were also detected even when they were in homologous chromosomes, which may be due to the existence of different copy numbers of the rdna genes in each chromosome. there was a difference regarding the relation of the number of signals of rdna in the cytotypes analyzed in this study; in the diploid cytotype there was less 45s signals with respect to 5s signals, while a similar number of rdna signals were detected in the pentaploid cytotype. based on the chromosome number of the diploid cytotype (2n=60) it can be considered as a polyploid due to its high chromosome number (stebbins 1971), with this in mind, the lower 45s rdna loci could be explained since these are generally more prone to experience rapid homogenization, silencing, and loss of loci, especially in polyploids (clarkson et al. 2005; kovařík et al. 2005; weiss-schneeweiss et al. 2008; kotseruba et al. 2010). according the symmetry/asymmetry indexes used, the analyzes established that it is an asymmetric karyotype, however due to the predominance of metacentric and submetacentric chromosomes, it was impossible to differentiate them only by their size, for this reason the number of individual chromosomes identified was relatively low. in s. formosissima it was observed that the number of signals in the pentaploid cytotype does not correspond to a multiple of the number of signals present in the diploid cytotype, so it is concluded that at least for the wheat rdna probes used in this work, the ploidy level of s. formosissima is not related to the number of observed signals. the cytotype where the greatest number of chromosomes could be identified was the diploid s. formosissima (2n=2x=60), where two chromosomal pairs were identified with the 45s rdna probe, three pairs with 5s rdna probe and pair 1 due to its unmistakable large size (figure 3), so a total of six chromosomal pairs could be identified. acknowledgements the authors are grateful to the consejo nacional de ciencia y tecnología (conacyt-mexico), projects: cb-2012-01-183591, cb-2015-01-258866 and plantecc 314926, whose funding made this work possible. references arano h. 1963. cytological studies in subfamily carduoideae (compositae) of japan. ix. the karyotype analysis and phylogenic considerations on pertya and ainsliaea. bot mag tokyo. 76: 32-39. arroyo-martínez ha, arzate-fernández am, barbagonzález r, piña-escutia jl. 2018. karyotype analysis and physical mapping of the 5s and 45s rdna genes in tigridiapavonia var. dulce (iridaceae). caryologia. 71(1): 1-6. cabral js, felix lp, guerra m. 2006. heterochromatin diversity and its co-localization with 5s and 45s rdna sites in chromosomes of four maxillaria species (orchidaceae). genet mol biol. 29(4): 659-664. clarkson jj, lim ky, kovařík a, chase mw, knapp s, leitch ar. 2005. long-term genome diploidization in allopolyploid nicotiana section repandae (solanaceae). new phytol. 168(1): 241-252. deng cl, qin ry, wang nn, cao y, gao j, gao wj, lu ld. 2012. karyotype of asparagus by physical mapping of 45s and 5s rdna by fish. j genet. 91: 209-212. flory ws. 1977. overview of chromosomal evolution in the amaryllidaceae. nucleus. 20: 70-88. garcía bn. 2015. systematics and evolution of amaryllidaceae tribe hippeastreae (asparagales) [dissertation]. gainesville (fl): university of florida. gerlach wl, bedbrook jr. 1979. cloning and characterization of ribosomal rna genes from wheat and barley. nucleic acids res. 7(7): 1869-1885. 126 josé manuel rodríguez-domínguez, ernesto tapia-campos, rodrigo barba-gonzalez gerlach wl, dyer ta. 1980. sequence organization of the repeating units in the nucleus of wheat which contain 5s rrna genes. nucleic acids res. 8(21): 4851-4865. gomez-rodriguez vm, rodriguez-garay b, palomino g, martínez j, barba-gonzalez r. 2013. physical mapping of 5s and 18s ribosomal dna in three species of agave (asparagales, asparagaceae). comp cytogenet. 7(3): 191-203. greilhuber j, speta f. 1976. c-banded karyotypes in the scilla hohenackeri group, s. persica and puschkinia (liliaceae). plant syst evol. 126: 149-188. huziwara y. 1962. karyotype analysis in some genera of compositae. viii. further studies on the chromosome of aster. am j bot. 49: 116-119. hwang yj, song cm, younis a, kim ck, kang yi, lim kb. 2015. morphological characterization under different ecological habitats and physical mapping of 5s and 45s rdna in lilium distichum with fluorescence in situ hybridization. rev chil hist nat. 88(1): 8. kotseruba v, pistrick k, blattner fr, kumke k, weiss o, rutten t, fuchs j, endo t, nasuda s, ghukasyan a, houben a. 2010. the evolution of the hexaploid grass zingeria kochii (mez) tzvel. 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p. 124-154. venora g, blangiforti s, ruffini castiglione m, pignone d, losavio f, cremonini r, 2002. chromatin organisation and computer aided karyotyping of triticum durum desf. cv timilia. caryologia. 55: 91-98. weiss-schneeweiss h, tremetsberger k, schneeweiss gm, parker js, stuessy tf. 2008. karyotype diversification and evolution in diploid and polyploid south american hypochaeris (asteraceae) inferred from rdna localization and genetic fingerprint data. ann bot. 101(7): 909-918. weiss-schneeweiss h, schneeweiss gm. 2013. karyotype diversity and evolutionary trends in angiosperms. in greilhuber j, dolezel j, wendel j, editors. plant genome diversity vol. 2. vienna: springer; p. 209230. caryologia. international journal of cytology, cytosystematics and cytogenetics 72(3): 63-73, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-258 citation: y. sharafi (2019) effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy. caryologia 72(3): 63-73. doi: 10.13128/cayologia-258 published: december 13, 2019 copyright: © 2019 y. sharafi. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy yavar sharafi department of horticultural sciences, faculty of agriculture, shahed university, tehran, iran e-mail: y.sharafi@shahed.ac.ir abstract. zinc is classically the second abundant moveable metal in plants after iron and represented in all enzyme classes. zinc generally contributes in the biosynthesis of iaa and ga3 phytohormones which play the major role in fertilization and fruit set. zinc deficiency leads to reduction in leaf and shoot size, photosynthesis and finally decreases fruit set. foliar zinc spray was shown to be efficient and fast for improving zinc deficiency in fruit trees. in this research the effects of zinc solution by (0, 3000 and 5000 mg. l-1) were studied on pollen penetration to the pistil and ovary in the four apple cultivars crosses which included “golden delicious”, “red delicious”, “gala” and “fuji”. spraying was done on the shoots two weeks before blooming in the spring. pollen penetration was studied using fluorescent microscopy technique 72 and 120 hours after field pollination. results revealed that the effects of zinc, crosses and their interaction were significant on pollen germination on the stigma and tube penetration into the primary, middle and beginning of the ovaries and the highest pollen germination on the stigma (43.5%) was observed in the cross (♀golden delicious × gala♂), in 3000 mg. l-1 of zinc 120 hours after pollination and highest pollen tube penetration into the ovary (12/88%) was observed in this cross, respectively. finally, it was shown that fluorescence microscopy is an accurate technique for nutrition assay in pollination and fruit set. the foliar application of zinc increased pollen germination and pollen tube growth in all of the crosses. keywords. fruit set, micronutrient, ovary, pollination, pollen tube growth. introduction pollen can be used advantageously by breeders, geneticists, physiologists, germplasm supervisors and growers (dafni and frimage 2000; sharafi et al. 2017; sedgley 1990). in higher plants, pollen grains carry the male gamete on the female part of a flower and play a vital role in breeding program and assist in successful fruit set (dafni et al. 2012; hisock and allen 2008; sedgley 1990). generally high crop yield is dependent on viable pollen grains (dafni et al. 2005). pollen fertility and viability have dominant prominence in natural and artificial hybridization programs (sedgley 1990). 64 yavar sharafi zinc (zn) is an essential micronutrient in plants and has a vital role in cell division, nucleic acid metabolism, protein synthesis, photosynthesis, carbohydrate metabolism, and phytohormones regulation (broadley et al. 2007; chen et al. 2008). zn directly contributes in the biological synthesis of auxin (iaa) and gibberellin (ga3) which have utmost roles in the plant growth, pollination, fertilization and fruit set in fruit trees (broadley et al. 2007). also, zinc plays a major role as a cofactor in the structure and function of more than 300 enzymes in plants, such as cu/zn superoxide (cu/znsod), carbonic anhydrase (ca), and sorbitol dehydrogenase (sdh). zinc toxicity in plants is far less widespread than zn deficiency (andreini and bertini 2012; moghadam et al. 2013; nosarszewski et al. 2004; weinthal et al. 2010). it was reported that about 30% of the agricultural soils in the world show zn deficiency and zinc is the most common micronutrient deficient, mostly in highph soils (alloway 2008). fruit trees which grown in such soils encounter zn deficiency and show both poor growth and yield quantity and quality. on the other hand, apple (malus domestica l.) commercial cultivars are selfincompatible and therefore, need to be planted along with cross-compatible pollinizer that generates sufficient favorable pollen (hegedus et al. 2012; losada and herrero 2014; sedgley 1990; ramirez and davenpor 2013). on the contrary, apple trees are highly susceptive to zn deficiency (alloway 2008). zn deficiency decreases leaf and shoot size and reduces photosynthetic rates, and thus influences the apple yield and quality (yan et al. 2010). zinc deficiency in apple trees is observed as small leaves, late opening of flower and leaf buds, chlorosis between the lateral veins of the leaves, and terminal dieback (marshner 2011; macdonal 2000; sedgley 1990;). in many fruit trees foliar applications of zn have been effectively used to promote tree vigor, fruit set, and yield (wojcik 2007). some researchers (golzer and grant 2006; qin 1996; song et al. 2015; song et al. 2016; yadav et al. 2013; zhang et al. 2014) have reported that in the most fruit trees; foliar applications of zn on mature leaves is unsuccessful and does not provide significant zn to new leaves produced after spray application or in the following spring. the best time for zn foliar application is nearly after fruit harvest in the autumn or immediately after pistillate flower senescence followed by two weeks later. zhang et al (2013 and 2016) reported that zinc sulfate spray before bud break increases the activity of carbohydrate metabolic enzymes and regulates endogenous hormone levels in fruits of fuji apple cultivar. solar and stampar (2001) reported that the yield of hazelnut trees was highest in the treatment with 2000 mg.kg–1 b + 2000 mg.l–1 zn and lowest in the treatment with 1000 mg.l–1 b + 1000 mg.l–1 zn. hipps and davies (2000) reported that foliar application of zn after blooming could increase the zn concentration of apple fruit; and spraying zn on leaves in autumn notably improves the flower zn content in the coming year. also, foliar zn application promotes pollination and cell division. moreover, foliar application of zn was shown to be effective and fast for improving zn decreasing symptoms in many plants (sanchez and righetti 2002). various studies in palm, citrus and apple showed that foliar application of zn can significantly increase the zn concentration, fruit yield and quality (karimi et al. 2017; keshavarz et al. 2011; rodríguez et al. 2005; neilsen et al. 2004; neilsen et al. 2005 khayyat et al. 2007; zhang et al. 2013). boron, iron and zinc foliar applications have been observed to have a positive effect on chlorophyll contents in b, fe and zn deficient plants. pollination is one of the most critical stages in the life cycle of a flowering plant, involving a complex series of cell-cell interactions that constitute the pollen-pistil interaction (dafni et al. 2005; dafni et al. 2012; hisock and allen 2008). in order for fertilization, pollen must first establish molecular compatibility with the stigma and then germinate to produce a pollen tube that penetrates the stigma and grows through the transmitting tissue of the style to locate on the ovule within the ovary (radonic et al. 2017). initiation and successful completion of this sequence of events depends upon the stigma and style providing the exact requirements for pollen germination and sustained growth and guidance of the pollen tube through the pistil and ovary. the pollen must therefore be programmed to respond appropriately at every step of this interaction. (losada and herrero 2014; dafni et al. 1998; nepi and franchi 2000; sedgley 1990; shivanna 2003; rodriguez-riano and dafni 2000). zn deficiency can have a marked effect on pollination by affecting pollen production, pollen physiology, floral anatomy, and fruit set (usenik and stampar 2002). it has been demonstrated that the first action of stigma is to hook the pollen grains on its surface. for this mechanism receptive stigma must have an adhesive surface. pollen-stigma interaction is instituted after adhesion of pollen grains on the stigmatic head and multifold incidents occur. the first step is the hydration of the pollen grain and release of wall proteins that bind to receptors on the stigma surface (radunic et al. 2017; yellof and hunt 2005). also, f lorescence microscopy technique accomplished to study pollen tube growth after field and laboratory-controlled pollination and used to identify the 65effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy selfand cross-(in) compatibility of cultivars, effective pollination period (epp), and effects of pollen types on fruit set (altagic et al. 2012; kubitscheck 2017). furthermore, it has not been used for the investigation of the effects of macro and micro nutrients on the pollen germination and tube grow especially in the apple cultivars. in spite of numerous researches on the response of deficient fruit trees to zn foliar application, there is no enough information directly appropriate to apple trees. it could be said that because of the five partitions of the apple flower stigma and style; pollen penetration assays in the style have not been used for the screening of nutritional elements on the flower buds (mularczykoliwa et al. 2017; sheffield et al. 2005). the objective of this study was to assess the effects of foliar application of zn on the apple trees two weeks before bud break in the spring in some crosses by florescence microscopy technique. materials and methods plant materials, zn treatments, crosses and pollination this research was carried out on four apple cultivars which included “golden delicious”, “red delicious”, “gala” and “fuji”. all of the trees were 12-yearold on em126 rootstocks and foliar sprayed by znso4 as the zn source (0, 3000 and 5000 mg. l-1) two weeks before bud break in the spring. in the beginning volume of each treatment was calculated based on the fruit trees number and then znso4 was dissolved in distilled water and so sprayed with sprayer on the shoots. spraying was done in the in the morning (7-8 o clock), when the sky was cloudy and the weather moisture was 70%. crosses among the cultivars (six crosses) were programmed as ♀red delicious × golden delicious♂, ♀gala× fuji♂, ♀red delicious × fuji♂, ♀red delicious × gala♂, ♀golden delicious × fuji♂ and ♀golden delicious × gala♂. for each cross four repeats in all direction of the trees were considered and, in each repeat, at least 2 branches with 60 – 100 flower buds were labeled in winter. selected female cultivar’s flower buds were bagged at ‘balloon’ stage to prevent the entrance of foreign pollens on the closed pistils. pollens were collected from the male cultivar flower buds and maintained in freezer until usage in the field pollination time. pollen germination was tested in an in vitro medium before field application on the pistils. selected female cultivar’s flowers were pollinated with selected male parent pollen when the pistils were acceptable for pollens and repeated after 24 hours to increase the pollination accuracy. florescence microscopy assessment based on the apple trees epp; 72-120 h is enough for the pollen tubes to reach the ovary hence, pollinated flowers were collected at 72 and 120 h after pollination, sliced and fixed in acid faa solution (5 % v/v formaldehyde (40%), 90 % v/v alcohol ethylic 70% and 5 % acetic acid glacial 96 %). after rinsing with water two to three times, pistils were cleared in 16% naoh at room temperature for three days. they were then rinsed in water and stained with 0.1% aniline blue in 0.1% k2hpo4. each part of the pistil was placed on a microscope slide with 10% glycerol and squashed under a glass cover slip. the number of pollen tubes and the rate of pollen tube growth in the different parts of the style were measured using fluorescence microscopy (olympus ax70). in each pistil the number of germinated pollen grains on the stigma, the number of pollen tubes in the upper and middle parts of the style and in the beginning of the ovary were determined by a fluorescent microscope (fig 1). pollen germination percentage was determined by dividing the number of germinated pollen grains by the total number of pollen on the stigma and expressed as a percentage and normalized by angular transformation. the mean of the pollen tube number was calculated as the average number of pollen tubes in different parts of 10 pistils at least. due to the five partitions of the apple flower stigma and style; the mean of the 5 parts was evaluated for each of them. experimental design and data analysis the experiment was carried out as a factorial based on completely randomized design with three factors fig 1. florescence microscopy photographed from pollens of red delicious germinated on the golden delicious stigma and pollen tubes penetrated to the upper part of the style. 66 yavar sharafi including zn in three levels (0, 3000 and 5000 mg. l-1), time (72 and 120 hrs.’ after pollination) and six crosses in five replications (at least ten styles per cross). the data was analyzed using spss (24) software. mean values were analyzed by duncan’s multiple range test. results the analysis of variance showed that the crosses of four cultivars “golden delicious”, “red delicious”, “gala” and “fuji” had a significant effect on pollen germination percentage on the stigma and pollen tube number which penetrated into the upper and middle parts of the of style and also in the beginning of the ovary at p≤0.01 respectively. also, zn concentration and time independently affected the pollen germination percentage on the stigma, the number of pollen tubes which penetrated to the upper and the middle parts of the style and the primary part of the ovary significantly at p≤0.01 (table 1). the interaction among zn concentration × crosses was significant on the pollen germination on the stigma, penetration of pollen tubes in the upper and middle parts of the style and so the beginning of the ovaries at p≤0.01 (table 1), but the interaction of crosses × time was not significant on the studied traits (table 1). however, three ways interaction among the time × crosses × zn concentration was not significant on the pollen germination percentage on the stigma, the number of pollen tubes that penetrated into the middle and the end of the style and the number of pollen tubes in the beginning of the ovary (table 1). based on our findings, the foliar application of zn on the apple trees enhanced the growth of pollen tubes toward the ovary. in addition, the use of zn increased the pollen germination on the stigma. about 85 to 90% of pollen germination on the stigma occurred 48 hours after pollination in pollen which was treated by zn (data not shown). the highest pollen germination percentage on the stigma (43.5%) was observed in the cross (♀golden delicious × gala♂), in 3000 mg. l-1 of zn 120 hours after pollination and the highest pollen tube penetration into the ovary (12/88%) was observed in this cross, respectively. these results may be related to the zn positive effects on the cell division in pollen tube followed by elongation lead to arrival to the ovaries (fig. 2). in comparison with 3000 mg. l-1 and 5000 mg. l-1 concentration of the zn, pollen germination rate decreased significantly and showed a toxic effect on pollen. in the crosses which both of pollen parents style parents and were treated by 5000 mg. l-1 zn, pollen germination on the stigma was decreased. this could be related to the toxic effect of zn. the concentration of 3000 mg l-1 of zn has a positive effect on germination percentage and penetration of pollen tubes, and has been effective in maintaining and integrating the membrane of pollen cells. in this research pollen tubes which penetrated into the style and ovary was also affected. it appeared that the use of zn on both the male and female parents led to increase in the pollen germination on the stigma in the in-vivo condition. however, in trees treated with 5000 mg. l-1 zn, the number of pollen tubes and the swelling of the tip of the tubes were significantly reduced; this was in accordance with some researchers reports regarding the negative effects of zn on the vital phenomenon in high concentration (marschner, 2011; sedgley, 1990; sharafi et al., 2017; zhang et al., 2013; zhang et al., 2016) based on the results of figure. 1, the highest pollen germination percentage was observed in the cross ♀ red delicious × gala ♂ with 43.51% and the highest pollen tube penetration in the cross ♀red delicious × golden delicious with 19.27% respectively (fig. 1). table 1. analysis of variance of the effect of time, crosses and zn on the pollen germination on the stigma and tube penetration to upper and middle part of the style and so beginning of the ovary. beginning of ovary middle of style upper of style stigma df sources of variation 9.01 ns 12.10 ns **55.07 **149.64 5 cross **149.03 **207.02 **239.06 **4120.07 2 zn concentration **765.05 **601.04 **974.05 **2245.44 1 time 7.01 ns 15.00 ns 10.01 ns 55.97ns 5 cross × time **32.04 **02.48 96.50** 83.02 ** 10 cross × zn *35.07 *52.06 38.07 * **327.71 2 zn ×time 14.01 ns 38.07 ns 19.01 ns 29.01ns 10 cross × zn ×time 9.04 14.01 13.04 27.03 ns 144 error 179 total ns= non significant, * = significant at p ≤0.05, ** = significant at p ≤0.01. 67effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy results were showed that pollens of golden delicious on the red delicious lead to increase the ovule fertilization and finally fruit set among all of the studied crosses (data not shown). the results of figure 3 showed that the interaction of zn and crosses significantly affected the pollen tube penetration to the beginning of the styles in all of the six crosses. the highest (20.09%) and lowest (15.2%) pollen tube penetration to the beginning of the styles was observed in the cross ♀ red delicious × golden delicious ♂ in the 3000 mg. l-1 zn and not treated crosses (control) respectively and thus, pollen tub penetration to the beginning of the styles was decreased in 5000 mg. l-1 zn while it was higher than the controls in all of the crosses. it may be connected with the positive effects of zn on the apple pollen. the positive effects of zn in high concentration were reported in the most of the plants. the highest (18.08%) and lowest (13%) pollen tube penetration to the middle part of the styles was observed fed f fe f f fe abc a ae be abcd ab bf af bf bf cf bf 0 5 10 15 20 25 30 35 40 45 50 ♀ red × g alla ♂ ♀ red × gol den ♂ ♀ gol den × fuj i ♂ ♀ gal la × fuji ♂ ♀ red × f uji ♂ ♀ gol den × gal la ♂ po lle n ge rm in at io n % crosses control 3000 mg.l-1 zinc 5000 mg.l -1zinc dcb dcb d d dc abcd abc a cbd abcd abc ab bcd a abc abc bcd ab 0 5 10 15 20 25 ♀ red × g alla ♂ ♀ red × gol den ♂ ♀ gol den × fuj i ♂ ♀ gal la × fuji ♂ ♀ red × f uji ♂ ♀ gol den × gal la ♂ po lle n tu be n um be r % crosses control 3000 mg.l-1 zinc 5000 mg.l -1zinc fig 2. the interaction between zn concentration and crosses on the pollen germination on the stigma in different crosses. means in each column, followed by similar letter (s) are not significantly different at 1% probability level. fig 3. the interactions between zn concentration and crosses on the pollen tube penetration into the beginning of the style in different crosses. means in each column, followed by similar letter (s) are not significantly different at 1% probability level. 68 yavar sharafi in the cross ♀ red delicious × fuji ♂ in the 3000 mg. l-1 zn and not treated crosses (control) respectively and thus, pollen tube penetration to the middle part of the styles was decreased in 5000 mg. l-1 zn while it was higher than the controls in all of the crosses (figure 4). also, the results of figure 5 showed that the highest (12.88%) and lowest (10.28%) pollen tube penetration to the middle part of the styles was observed in the cross ♀ red delicious × golden delicious ♂ in the 3000 mg. l-1 zn and not treated crosses (control) ♀golden delicious × gala ♂ respectively and thus, pollen tub penetration to the middle part of the styles was decreased in 5000 mg. l-1 zn while it was higher than the controls in all of the crosses. based on the results shown in figures 6, 7, 8 and 9 the interaction of zn concentration and the time after pollination significantly affected the pollen germination on the stigma, tube number in the upper and middle parts of the style and also in the beginning of the ovary in all of the crosses. maximum (34.28%) and minimum cd d cd d d bcdbcd abc cd bcd a abcd bcd abc abcd ab bcd abcd 0 5 10 15 20 25 ♀ red × g alla ♂ ♀ red × gol den ♂ ♀ gol den × fuj i ♂ ♀ gal la × fuji ♂ ♀ red × f uji ♂ ♀ gol den × gal la ♂ po lle n tu be n um be r % ه crosses control 3000 mg.l-1 zinc 5000 mg.l -1zinc c c bc bc c bc abc ab bc bc abc abcbc ab abc a bc bc 0 2 4 6 8 10 12 14 16 ♀ red × g alla ♂ ♀ red × g old en ♂ ♀ go lde n × fuj i ♂ ♀ gal la × fuji ♂ ♀ red × f uji ♂ ♀ go lde n × gal la � po lle n tu be n um be r % crosses control 3000 mg.l-1 zinc 5000 mg.l -1zinc fig. 4. the interactions between zn concentration and crosses on the pollen tube penetration into the middle part of the style in different crosses. means in each column, followed by similar letter (s) are not significantly different at 1% probability level. fig. 5. the interactions between zn concentration and crosses on the pollen tube penetration into the beginning of the ovary in different crosses. means in each column, followed by similar letter (s) are not significantly different at 1% probability level. 69effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy (14.2%) pollen germination on the stigma was observed in the 3000 mg. l-1 zn and not treated crosses (control) respectively and thus, pollen germination on the stigma was decreased in 5000 mg. l-1 zn while it was higher than the controls in all of the crosses (figure 6). ma ximum (24.73%) and minimum (9.9%) pollen tube penetration to the beginning of the style was observed in the 3000 mg. l-1 zn and not treated crosses (control) respectively and thus, tube penetration to the beginning of the style was decreased in 5000 mg. l-1 zn while it was higher than the controls in all of the crosses (figure 7). maximum (26.48%) and minimum (8.63%) pollen tube penetration to the middle of the style was observed in the 3000 mg. l-1 zn and not treated crosses (control) respectively and thus, tube penetration to the middle of c b c ab a ab 0 5 10 15 20 25 30 35 40 control 3000 mg.l 5000 mg.l p ol le n g er m in at io n % zn concentration 72 hour 120 hour c b c b a b 0 5 10 15 20 25 30 control 3000 mg.l 5000 mg.l p ol le n t u b e n u m b er % zn concentration 72 hour 120 hour d b b c a a 0 5 10 15 20 25 30 35 control 3000 mg .l 5000 mg .l po lle n tu be n um be r % zn concentration 72 hour 120 hour d b bc a a 0 5 10 15 20 25 control 3000 mg.l 5000 mg. l p ol le n t u b e n u m b er % zn concentration 72 hour 120 hour fig. 9. the interactions between time and zn concentration on the pollen tube penetration percentage on the beginning of the ovary. means in each column, followed by similar letter (s) are not significantly different at 1% probability level. fig. 6. the interactions between time and zn concentration on the pollen germination percentage on the stigma. means in each column, followed by similar letter (s) are not significantly different at 1% probability level. fig. 7. the interactions between time and zn concentration on the pollen tube number in the beginning of style. means in each column, followed by similar letter (s) are not significantly different at 1% probability level. fig. 8. the interactions between time and zn concentration on the pollen tube penetration percentage on the middle part of style. means in each column, followed by similar letter (s) are not significantly different at 1% probability level. 70 yavar sharafi the style was decreased in 5000 mg. l-1 zn while it was higher than the controls in all of the crosses (figure 8). however, maximum (19.8%) and minimum (5.47%) pollen tube penetration to the beginning of the ovary was observed in the 3000 mg. l-1 zn and not treated crosses (control) respectively and thus, tube penetration to the beginning of the ovary was decreased in 5000 mg. l-1 zn while it was higher than the controls in all of the crosses (figure 9). in all of the crosses the highest pollen tube number in the beginning of the style were observed 120 hr after pollination. this phenomenon demonstrated that pollen germination and tube growth were increased followed by the time which may be related to the nutrition case in the style. however, there was a significant difference between the interaction of time and zn concentration on the pollen tube number in the middle part of the style and in the beginning of the ovary respectively (figures 8 and 9). maximum pollen tube numbers in the middle part of the style and in the beginning of the ovary were observed 120 hr after pollination. correllation between zn and pollen germination and penetration to different parts of the style and ovary is showen in table 2. there was a significant positive correlation between zn concentrations and germination percentage of pollen on the stigma and the tube number which penetrated to the upper and middle parts of the style and also to the beginning of the ovary respectively (table 2).the correlation between pollen tubes penetration to the upper (./68) and middle of the style (./85) and the beginning of the ovaries was positive (table 2). discussion in this research the pollen tube penetration percentage into the styles and ovaries increased by zn application two weeks before bud break especially in 3000 mg. l-1. in this study a large number of pollen grains germinated on the stigma exudate and formed callose, indicating good growth of pollen tubes by zn treatment in apple crosses. however, few of the pollen tubes were observed to penetrate to the style. the average number of pollen tubes was only slightly higher in some crosses. in addition, 120 h after pollination, the average pollen tube length and growth rate were slightly higher in all of the crosses. previous studies have suggested that self-pollen tubes can grow slower or have higher rates of abrasion than cross-pollen tubes (golzer and grant 2006; qin 1996; song et al. 2015; song et al. 2016; yadav et al. 2013; zhang et al. 2014; sedgley 1990). in accordance with our results pandey et al, (2006); observed that zinc is critically required for pollen function and fertilization in lentil. also, neilsen et al (2005) observsd that postbloom humic-and fulvic-based zinc sprays can improve apple zinc nutrition also neilsen et al (2004) reported positive effects of zinc and boron in fertigated high density apple orchards. keshavarz et al (2011); reported that foliar application of zinc and boron improves walnut vegetative and reproductive growth. they demonstrated the first report of the benefit of foliar b and zn on pollen germination in walnut trees. there was clear positive effect of b and zn applied as individual foliar applications and a synergistic effect when applied in combination on walnut yield and quality parameters. fei et al (2016); studied enzyme activities, and expression of zn/iron-regulated transporter-like protein (zip) family genes in the mild, moderate, and severe zn deficiency in (citrus sinensis l. osbeck). they reported that the expression of the zip family genes, zip1, zip3, and zip4, was promoted by zn deficiencies. however, chlorophyll contents and net photosynthetic rate decreased with reduction in zn contents reduction. also, comparison of severe zn-deficient and normal leaves revealed increased significant activities of peroxidase (pod) and catalase (cat, but significantly reduced zn-containing enzymes such as cu/zn superoxide dismutase (cu/zn-sod). in plants, about half of the zip genes could be induced under zn deficiency, while zip1-4 genes seem to be involved in plant zn transport. zinc/iron-regulated transporter-like proteins (zips) play a key role for zn uptake in plants and currently, over 100 zip family members have been recognized in diverse plant species (andreini and bertini 2012; moghadam et al. 2013; nosarszewski et al. 2004; weinthal et al. 2010). table 2. pearson correlation coefficients for the effect of the zn on the pollen germination percentage on the stigma and pollen tube penetration to the upper and middle parts of the style and so the beginning of the ovary. correlation stigma level upper of style middle of style beginning of ovary stigma level 1 upper of style 0.59ns 1 middle of style 0.45ns **0.79 1 beginning of ovary **0.57 **0.68 0.85** 1 ns= non significant, * = significant at p ≤0.05, ** = significant at p ≤0.01 71effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy furthermore, zn is involved in various cellular processes, including photosynthesis, nucleic acid and lipid metabolism, protein synthesis, detoxification of reactive oxygen species (ros), and membrane stability (broadley et al. 2007). the main role of zn is involvement in important procedures of cell division and gene expression associated with nucleic acid and protein metabolism. zinc play an essential role in processes leading to dna synthesis, and dna polymerase contains firmly bound zinc. these roles help to explain the nature of some of the symptoms characteristic of zn deficiency in fruit trees, for example, ‘rosetting’ and shoot tip dieback (broadley et al. 2007; andreini and bertini 2012; moghadam et al. 2013; nosarszewski et al. 2004; weinthal et al. 2010). zinc deficiency leads to malfunctioning of some enzymes, for example, alkaline phosphatase carbonic anhydrase. it could lead to decreased starch formation, accumulation of amino acids, and synthesis of auxin via impaired tryptophan synthesis. it decreases carbonate dehydrogenase activity which has affected the balance of carbon dioxide and carbon acid and thus has indirectly influenced the rate of photosynthesis. zinc deficiency has also been found to promote formation of abscisic acid causing premature abscission of leaves and flower buds (andreini and bertini 2012; moghadam et al. 2013; nosarszewski et al. 2004; weinthal et al. 2010). hence, zinc is important in dna and rna metabolism and protein synthesis and thus, maintains the structural integrity of biomembranes. more than 1,200 protein molecules (zn metalloprotein) have been identified including a large number of ‘zinc-finger’containing proteins and transcription factors, oxidoreductases and hydrolytic enzymes such as metalloproteases. furthermore, zn is a mechanical factor of ribosomes and thus vital for their structural integrity. it plays a major role in carbohydrate metabolism by regulating key enzymes, fructose 1,6-bisphosphatase and aldolase. synthesis of auxin, and indole acetic acid, is particularly impaired under zn deficiency (broadley et al. 2007; andreini and bertini 2012; moghadam et al. 2013; nosarszewski et al. 2004; weinthal et al. 2010). finally by comparing the effects of the contols with 3000 and 5000 mg. l-1 zn in figures 1, 2, 3, 4, 5, 6 ,7 and 8 it was demomonstrated that pollen germination and tube penetration to the style and ovary increased until 3000 mg. l-1 zn but, in 5000 mg. l-1 mentioned traits were decreased respectively. this could be interpreted that by increasing the zn concentration it may show toxic effects of pollen tube cell growth. however, in this study, 5000 mg. l-1 did not show toxic effect because it showed positive effects on pollen germination and tube penetration to the style and ovary in comparison with the crosses which were not treated with zn. conclusion “it was concluded that f luorescence microscopy technique is a very accurate method for assay of nutrient effects on pollen germination and tube penetration to the pistils in fruit trees in compared with fruit set percentage studies. in this research the mentioned method was used for assay of zn effects in four apple cultivars crosses which included “golden delicious”, “red delicious”, “gala” and “fuji”. results showed that the highest pollen germination percentage on the stigma (43.5%) and penetration 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(apiaceae) kamini gautam1,2,*, ravinder raina1,3 cytotoxic and genotoxic activity of plantago major l. extracts amira ždralović, aner mesic, izet eminović, adisa parić* genetic diversity of rhododendron simsii planch. natural populations at different altitudes in wujiashan mountain (central china) shuzhen wang, yanyan luo, tao yang, yujia zhang, zhiliang li, weibin jin, yuanping fang* nonreduction via meiotic restitution and pollen heterogeneity may explain residual male fertility in triploid marine halophyte limonium algarvense (plumbaginaceae) sofia i. r. conceição1, ana sofia róis1,2, ana d. caperta1,* effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy yavar sharafi active chemical constituents of cynanchum viminale and its cytotoxic effects via apoptotic signs on allium cepa root meristematic cells neethu kannan bhagyanathan*, john ernest thoppil clastogenic and cytotoxic effects of aerial parts’ aqueous extract of synedrella nodiflora (l.) gaertn. on wistar rat bone marrow cells saumabha chatterjee1,2, sanjib ray2* cytogenetics of accanthopus velikensis (piller et mitterpacher, 1783) (tenebrionidae: helopini) dirim şendoğan1, beril gündoğan1, maxim v. nabozhenko2,3, bekir keskin1, nurşen alpagut keskin1,* chromosome number and genome size diversity in five solanaceae genera amanda teixeira mesquita1,*, marìa victoria romero-da cruz1, ana luisa sousa azevedo2, eliana regina forni-martins1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(1): 13-22, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-550 caryologia international journal of cytology, cytosystematics and cytogenetics citation: n. sepahian, z. noormohammadi, m. sheidai, h.-r. zamanizadeh (2021) authentication, genetic fingerprinting and assessing relatedness of rice (oryza sativa) genotypes by ssr molecular markers. caryologia 74(1): 13-22. doi: 10.36253/caryologia-550 received: july 16, 2019 accepted: june 30, 2020 published: july 20, 2021 copyright: © 2021 n. sepahian, z. noormohammadi, m. sheidai, h.-r. zamanizadeh. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. authentication, genetic fingerprinting and assessing relatedness of rice (oryza sativa) genotypes by ssr molecular markers neda sepahian1, zahra noormohammadi1,*, masoud sheidai2, hamidreza zamanizadeh3 1 department of biology, science and research branch, islamic azad university, tehran, iran 2 faculty of biological sciences and technology, shahid beheshti university, tehran, iran 3 department of plant pathology, agriculture and natural resources faculty, science and research branch, islamic azad university, tehran, iran *corresponding author. e-mail:marjannm@yahoo.com; z-nouri@srbiau.ac.ir abstract. rice (oryza sativa l.), is a staple food and cash crop in many countries and studies on geneticstructure and differentiation patterns of rice land races along with the cultivated rice, provide important data for future rice breeding. therefore, the aims of present investigation were 1-to study the genetic diversity present withiniranian rice genotypes, 2-to study genetic relatedness of these rice genotypes, and 3-to providebarcoding of the rice genotypes based on ssr molecular markers and produce data for rice varieties authentication. in total, 201 rice samples originated from 10 geographical regions of iran were studied in this project. all rice samples underwent fragment analysis in every 64 ssr loci and different clustering and ordination methods performed. in general four major clusters were formed. both landraces as well as rice cultivars were distributed in different clusters due to their genetic difference. structure analysis of the studied genotypes followed by evanno test produced the optimal number of genetic groups k = 2. the mean nm = 13.6, for the studied genotypes indicates that a high degree of gene flow/ancestral common alleles are present in the rice genotypes studied. mantel test indicated a significant positive association between genetic distance and geographic distance of the rice genotype studied and presence of an overall isolation by distance (ibd) model of differentiation across the geographical regions of iran. overall, the significant genetic difference observed between rice landraces and rice cultivars ofthe country may be used in future hybridization and breeding of rice in the country. the landracerice genotypes may contain useful genes to be transferred to the popular rice cultivars. moreover, ssr loci that can differentiate rice genotypes are identified and can be used in rice cultivars authentication. keywords: barcoding, genotyping, genetic affinity, microsatellite, rice. introduction rice (oryzasatavia) is a diploid annual grass (2n = 24) of the family poaceae, which is an important food crop with the highest production after 14 neda sepahian, zahra noormohammadi, masoud sheidai, hamid-reza zamanizadeh sugarcane and maize. though it was originated in china, nowadays has several wild and related genotypes, many landraces and cultivated forms throughout the world (henga et al. 2018). successful breeding strategies for rice, requires a deep knowledge on the genetic diversity of rice cultivars, landraces and related genotypes within each country. rice is an important food and cash crop in iran, with several landraces and cultivars that are grown and cultivated in different regions of the country. we have however, limited data available on genetic structure and genetic diversity of iranian rice (see for example, nasabi et al. 2012). rice plant has suffered great genetic diversity reduction (about 80%), from that of the wild ancestor during the domestication as well as local artificial selection processes. this genetic erosion in the high-yielding rice varieties, results in disease susceptibility, and the loss of suitable genes (cuiet al. 2017). by contrast, the rice landrace, is a local variety which becomes adapted to the natural and cultural environment in which it grows. landrace populations contain relatively high level of genetic variability compared to the cultivated rice, and therefore provide a valuable source of potentially useful genes for rice breeding (cui et al. 2017). therefore, studies on genetic structure and differentiation patterns of rice landraces along with the cultivated rice, provide important data for future rice breeding (nethra et al. 2016, henga et al. 2018). rice varieties are among the most important human food resources. different rice varieties have specific agronomic characteristics, cooking properties, local adaptation, marketing demands, as well as ideas and pest resistance. some of the varieties are aromatic for example, thai fragrant rice, vietnamese fragrant rice, basmati rice, etc. and therefore, authentication of rice is of immediate importance in the rice industry (nethra et al. 2016, henga et al. 2018). genetic markers are very useful in managing germ plasm, investigating the genetic variability orgenetic finger printing of crop plants including rice (nethra et al. 2016, henga et al. 2018). molecular finger printing and genetic purity assessment of rice genotypes is vital for seed certification related to genotype distinctness, and seeds uniformity (henga et al. 2018). therefore, the aims of present investigation were: 1) to study the genetic diversity present within iranian rice genotypes, 2) to study genetic relatedness of these rice genotypes, and 3) to provide barcoding of the rice genotypes based on ssr molecular markers and produce data for rice varieties authentication. ssr markers are composed of tandem repeated nucleotides with 2-6 bp length, which can be amplified using the unique flanking region for primers annealing. these molecular markers are highly reproducible and polymorphic, and have been used a sideal marker in rice varieties genotyping. ssr markers can be utilized for paternity analysis, population genetics investigation, construction of high-density genome maps, germ plasm evaluation as well as marker-assisted selection (ma et al. 2011; henga et al. 2018). materials and methods samples in total, 201 rice samples were studied in this project. details of the samples obtained and their sources are as follows: one hundred and twenty-one rice samples in the form of panicles were received from iran rice research institute of iran (rrii). twenty-three rice samples were kindly provided by iran food and drug administration (ifad) and iranian rice importers association (iria), cooperatively. thirty-five rice seed samples were dedicated from the international rice research institute (irri), philippines and finally twenty-two parboiled rice grain samples were collected from the market (table s1). dna isolation and pcr amplification the genetic material was extracted using the qiaampdneasy mini kit (qiagen, germany) that works based on silica gel membrane technology which allowed an efficient recovery ofcomplete dna from plant tissues. dna was extracted from each of the samples between 3-5 times. all pcr amplification runs were performed using an abi simpliamp system (life technologies, usa). each amplification reaction contained 1x reaction buffer, 0.1-0.4 μm of each primer (table s2); 1 u taq dna polymerase (sinaclon, iran), 1.5-3 mm mgcl2, 0.200.25mm each datp, dctp, dgtp, and dttp (sinaclon, iran). either 2 or 5μ ldna was added to 15 or 18 μl prepared master mixes. all pcr reactions were performed based on tables s3 and s4. gel electrophoresis amplified dna fragments were electrophoresed on 2% agarose gels containing safe dyes and 1xtae buffer 15authentication, genetic fingerprinting and assessing relatedness of rice (oryza sativa) genotypes by ssr molecular markers was used for this purpose, and bands then were visualized by uv transillumination system. fragment analysis using qiaxcel all 201 rice samples underwent fragment analysis in every 64 ssr loci. qiaxcel fragment analyzer (qiagen, germany) was used for these verifications. qiaxcel dna high resolution dna kit with an accuracy of 3–5 bp was used to run the samples in capillaries which are filled with agel-matrix with a proprietary linear polymer with ethidium bromide intercalating dye. the qiaxcel screen gel® software which is been employed by qiaxcel advanced capillary electrophoresis system was used to estimate the size of each fragment and to do the interpretations. data analyses the ssr bands obtained were treated as binary characters and coded accordingly (presence = 1, absence = 0). the grouping of the rice genotypes were done by using different clustering and ordination methods (podani 2000). for clustering, we used nei and li distance as well as jaccard similarity index (podani 2000). these analyses were performed by past version 2.17 (hammer et al. 2012). we investigated the genetic structure of the rice samples by model-based clustering, based on the admixture ancestry model under the correlated allele frequency model, as performed by structure software ver. 2.3 (pritchard et al. 2000). data were scored as dominant markers and analysis followed the methods uggested by falush et al. (2007). the markov chain monte carlo simulation was run 20 times for each value of k (1-4) for 20 iterations after a burn-in period of 105. the structure results were followed by evanno method (evanno et al. 2005), as peformed by structure harvester online tool (earl and vonholdt 2012). the groups identified by evanno method were subjected to amova analysis to reveal the genetic differentiation of these samples. this was done by amova with 1000 permutations as performed in genalex 6.4 (peakall and smouse 2006). we also used multi-dimensional scaling (mds) method to investigate genetic distinctness of these groups as performed in past version 2.17 (hammer et al. 2012). rice samples studied were from 10 geographical regions of the country. we therefore, investigated thegenetic variability in these regions by estimating different genetic diversity parameters as determined in genalex 6.4 (peakall & smouse 2006). moreover, the mantel test (podani 2000) was performed to study association between genetic distance and geographical distance of the studied populations. results grouping of the rice genotypes studied by different clustering methods produced similar results, therefore, only ward dendrogram is presented (fig. 1). in general four major clusters were formed. the first major cluster is comprised of two sub-clusters. mostly cultivated rice genotypes form the first sub-cluster, while landraces and cultivars together comprised the second sub-cluster. the other major clusters were also formed by mixture of landraces and cultivated rice genotypes. it is interesting to see that both landraces as well as rice cultivars were distributed in different clusters due to their genetic difference. this indicates the presence of genetic diversity in iranian rice genotypes. structure analysis of the studied genotypes followed by evanno test produced the optimal number of genetic groups k = 2. structure plot based on k = 2 (fig. 2), placed the studied genotypes into genetic groups. therefore, based on both clustering and bayesian figure 1. ward dendrogram of rice genotypes based on ssr markers placing genotypes in four major clusters. (bluecolor = landrace, redcolor = cultivars; the cultivars number as in table 1). 16 neda sepahian, zahra noormohammadi, masoud sheidai, hamid-reza zamanizadeh approaches, the rice genotypes studied contain a good level of genetic diversity. we then randomly selected some of the rice genotypes from the two genetic groups identified by structure for further analyses. amova revealed significant genetic difference between the two groups (phipt = 0.30. p = 0.01). the fst value of 0.6 by structure analysis also supported amova in showing genetic difference of the genotypes. mds plot of these selected genotypes almost separated the two groups (fig. 3), indicating their genetic difference. moreover, spatial distribution of the genotypes within each group shows genetic variability within either groups. therefore, we have both among group genetic difference, as well as with in group genetic variability. the mean nm = 13.6, for the studied genotypes indicates that a high degree of gene flow/ancestral common alleles are present in the rice genotypes studied. genetic variability and geography of the rice genotypes the studied rice genotypes, were placed on 10 geographical groups (table s1). total number of ssr bands and private bands are provided in table 1. the highest number of ssr bands occurred in populations 1 and 2 (mazandaran and gilan, respectively). most of the studied geographical regions contained private bands, with the highest number in populations 1, 2, and 8 (mazandaran, gilan, and philipine, respectively). these private ssr bands, are specific bands occurred during rice varieties genetic differentiation. genetic diversity analyses of these populations are presented in table 2. two geographical regions of mazandaran (pop1), and gilan (pop2), contain the highest number of rice genotypes, as rice is mostly cultivated in northern iran. these regions had the highest value for genetic polymorphism (71 and 60%, respectively), followed by khuzestan (37%). however, the mean value for neigene diversity, shanon information index(i), and the number of effective alleles (ne) were almost close to each other in most of the geographical populations. amova produced significant difference among the studied geographical populations (phipt = 0.035, p = 0.02). it revealed that 3% of total genetic variance is due to among populations figure 2. q-plot of structure analysis grouping the rice genotypes in two major groups, based on k = 2. figure 3. mds plot of selected rice genotypes. 17authentication, genetic fingerprinting and assessing relatedness of rice (oryza sativa) genotypes by ssr molecular markers genetic difference, while 97% is due to within population genetic variability. these results indicate that we have a great deal of genetic diversity both within and among geographical populations. paired-sample amova (table 3), revealed that populations 4, 5, 7, 8, and 9, differed significantly with the others tudied populations. in spite of significant fst/phi-st values between most of the geographic populations, these populations have a high genetic similarity (>0.92, table 4). this is due to extensive common shared alleles within rice genotypes studied. mantel test with 999 permutations performed between genetic distance and geographical distance of the rice genotypes, produced significant association (r = 0.21, p = 0.01, fig. 4).this result indicates that with increase in geographical distance of rice genotypes, they become genetically differentiated. ward clustering was performed on the studied geography, after removing philippine, unknown, and ilam (single genotype) samples and combining to neighbor and closely placed provinces of mazandaran and gilans amples (fig. 5). ward dendrogram obtained (fig. 5), revealed that the rice samples in different geographical regions form a separate cluster due to their genetic difference. these genotypes were placed intwo major clusters. regions 1 and 2, comprised the first cluster, while regions 3-6 formed the second major cluster. the geographical region 1 (combined gilan and mazandaran begins), has 5 sub-clusters which indicate high within region genetic variability. these results suggest that, crossing of the rice samples in the two major clusters, may result in new genotypes to be evaluated in the field condition. table 1. details of ssr bands in geographical populations of rice genotypes. population pop1 pop2 pop3 pop4 pop5 pop6 pop7 pop8 pop9 pop10 no. bands 179 152 59 9 52 24 9 94 9 25 no. bandsfreq.>=5% 69 56 59 9 52 24 9 94 9 25 no.privatebands 39 25 2 1 6 1 0 16 0 0 no. lcomm bands(<=25%) 32 28 12 1 11 4 1 21 1 5 no. lcomm bands(<=50%) 80 75 39 3 35 17 7 57 6 20 populations 1-10 are: 1) mazandaran, 2) gilan, 3) unknown, 4) golestan, 5) isfahan, 6) khuzestan, 7) fars, 8) philipine, 9) boushehr, 10) ilam. table 2. genetic diversity parameters determined in geographical populations with rice genotypes. pop n na ne i he uhe %p pop1 47.000 1.421 1.040 0.084 0.037 0.037 71.03% pop2 44.000 1.206 1.039 0.078 0.036 0.036 60.32% pop3 8.000 0.468 1.042 0.065 0.035 0.037 23.41% pop4 2.000 0.036 1.000 0.000 0.000 0.000 0.00% pop5 7.000 0.413 1.043 0.063 0.035 0.037 20.63% pop6 3.000 0.187 1.041 0.044 0.028 0.034 9.13% pop7 2.000 0.036 1.000 0.000 0.000 0.000 0.00% pop8 13.000 0.746 1.043 0.076 0.038 0.039 37.30% pop9 2.000 0.036 1.000 0.000 0.000 0.000 0.00% pop10 3.000 0.198 1.051 0.051 0.033 0.039 9.92% populations 1-10 are: 1) mazandaran, 2) gilan, 3) unknown, 4) golestan, 5) isfahan, 6) khuzestan, 7) fars, 8) philipine, 9) boushehr, 10) ilam. table 3. paired-sample amova among geographical populations studied. pop1 pop2 pop3 pop4 pop5 pop6 pop7 pop8 pop9 pop10 0.000 0.108 0.480 0.001 0.495 0.178 0.001 0.266 0.004 0.300 pop1 0.004 0.000 0.144 0.002 0.267 0.079 0.002 0.088 0.003 0.153 pop2 0.000 0.010 0.000 0.026 0.502 0.097 0.013 0.497 0.024 0.123 pop3 0.187 0.219 0.277 0.000 0.016 0.143 0.001 0.011 0.001 0.001 pop4 0.000 0.006 0.000 0.216 0.000 0.243 0.033 0.473 0.067 0.022 pop5 0.018 0.034 0.039 0.393 0.023 0.000 0.083 0.467 0.151 0.227 pop6 0.193 0.207 0.268 1.000 0.204 0.458 0.000 0.016 0.001 0.117 pop7 0.004 0.009 0.000 0.203 0.000 0.000 0.180 0.000 0.015 0.123 pop8 0.167 0.191 0.210 1.000 0.249 0.393 1.000 0.167 0.000 0.093 pop9 0.009 0.022 0.042 0.413 0.085 0.077 0.433 0.033 0.287 0.000 pop10 populations 1-10 are: 1) mazandaran, 2) gilan, 3) unknown, 4) golestan, 5) isfahan, 6) khuzestan, 7) fars, 8) philipine, 9) boushehr, 10) ilam. 18 neda sepahian, zahra noormohammadi, masoud sheidai, hamid-reza zamanizadeh rice genotypes ssr barcoding based on allele frequency analysis, the following ssr alleles are specific in the studied rice genotypes. this ssr barcode scan be used in rice cultivars authentication (table s5). conclusion the present study revealed genetic variability both within and among rice varieties cultivated indifferentgeographical regions of iran. these cultivars differed genetically from each other. moreover, structure analysis divided these cultivars and landraces in two major genetic groups. we observed an extensive degree of genetic admixture possibly due to gene flow and gene exchange among the studied rice genotypes. in a similar investigation, wang et al. (2018), recognized, several geographical subpopulations and reported nucleotide polymorphisms, small indels and structural variations that result in withinand between-population variation. they also noticed a complex patterns of introgression in domestication genes. we observed a high degree of genetic similarity ranging from 0.91 to 0.99 in the studied rice genotypes of the country. however, nethra et al. (2016), used 58ssr markers for rice finger printing and noticed a moderate genetic polymorphism that ranged from 0.01 to 0.35 with the mean value = 0.23. they reported genetic similarity coefficient ranging from 0.65 to 0.92with the mean value = 0.314. we noticed significant genetic difference between rice landraces and rice cultivars of the country. these table 4. nei genetic identity versus genetic distance among rice geographical populations studied. pop id 1 2 3 4 5 6 7 8 9 10 1 **** 0.9995 0.9986 0.9655 0.9984 0.9930 0.9651 0.9989 0.9665 0.9952 2 0.0005 **** 0.9983 0.9646 0.9982 0.9927 0.9653 0.9989 0.9662 0.9951 3 0.0014 0.0017 **** 0.9624 0.9974 0.9914 0.9629 0.9979 0.9662 0.9936 4 0.0351 0.0360 0.0384 **** 0.9660 0.9642 0.9365 0.9643 0.9286 0.9596 5 0.0016 0.0018 0.0026 0.0346 **** 0.9922 0.9666 0.9980 0.9641 0.9923 6 0.0071 0.0073 0.0086 0.0365 0.0079 **** 0.9561 0.9930 0.9606 0.9880 7 0.0355 0.0353 0.0378 0.0656 0.0339 0.0448 **** 0.9656 0.9286 0.9581 8 0.0011 0.0011 0.0021 0.0363 0.0020 0.0070 0.0350 **** 0.9663 0.9939 9 0.0340 0.0344 0.0344 0.0741 0.0365 0.0402 0.0741 0.0343 **** 0.9679 10 0.0048 0.0049 0.0064 0.0413 0.0077 0.0121 0.0428 0.0061 0.0327 **** nei’s genetic identity (above diagonal) and genetic distance (below diagonal). figure 4. mantel test between genetic and geographic distance of rice genotypes showing positive significant association. (populations 1-10 are: 1) mazandaran, 2) gilan, 3) unknown, 4)golestan, 5) isfahan, 6) khuzestan, 7) fars, 8) philipine, 9) boushehr, 10) ilam). 19authentication, genetic fingerprinting and assessing relatedness of rice (oryza sativa) genotypes by ssr molecular markers genetic difference may be used in future hybridization and breeding of rice in the country. the landrace rice genotypes may contain useful genes to be transferred to the popular rice cultivars. leeet al. (2015) estimated genetic diversity in korean rice landraces, by using ssr markers and reported the polymorphism information content (pic) ranging from 0.11 to 0.93, and average observed heterozygosity ranging from 0.12 to 0.39. these landraces were divided in two major genetic groups by structure analysis, while clustering divided them in three genetic groups. landrace is a geographically or ecologically distinctive population, which differ genetically from each other and also differ from rice cultivars. therefore, rice breeders must pay specific attention to these ecological variants which may new suitable traits for rice improvement. these genotypes are shown to be excellent sources of genes for novel alleles (mccouch et al.1997; jackson 1999; guevarra et al. 2001). for example, rao et al. (2018), used ssr markers for association mapping, and identified 12 genomic regions for yield and yield associated traits under low nitrogen. somnath et al. (2016), studied the genetic structure of 64 hill rice landraces in india, by using microsatellite markers. these landrace genotypes were separated in two groups: umte (large-grained, late maturing) and tening (small-grained,early maturing).the kernel length and plant height were the main discriminatory characters between these cultivar groups. they showed high genetic diversity within rice genotypes. the genetic variability of rice landraces in brazil was investigated by ssr markers (borba et al. 2009). the study was performed in 417 landraces collected in 1986, 1987 and 2003. these researchers noticed that the number of landraces with long and thin grain type increased in the evaluated period, probably due to market demand. moreover, the genetic variability increased during this period and that, most of the landraces were grouped according to the year of collection. therefore, it was suggested that the selection performed by farmers are the most probable factor responsible for increasing landracegenetic variability, during the evaluated period. amova in present study revealed a higher degree of genetic variability within geographical populations (97%) and a lower degree, though significant difference, among the regions (3%). in a similar study by using ssrs, rao et al. (2018), also reported 9.66% genetic variation among the subgroups and 90.34% of variation within these subgroups. pusadeea et al. (2019) studied the population genetic structure of a single variety of landrace rice, buechofigure 5. ward dendrogram of the geographical regions 1-6 are: 1) combined mazandaran and gilan samples, 2) golestan, 3) isfahan, 4) khuzestan, 5) fars, and 6) boushehr. 20 neda sepahian, zahra noormohammadi, masoud sheidai, hamid-reza zamanizadeh mee, cultivated by karen people of thailand by using ssr markers. they observed high level of genetic variation within the studied villages despite predominant inbreeding in this crop. buechomee rice showed significant genetic differentiation among karen villages for both molecular content and genetically determined traits such as flowering time. similarly, sabori et al. (2008), compared iranian rice genotypes, including landrace, improved cultivars, and few exotic cultivars for their salinity tolerance at seedling stage and to determine tolerance indices, based on biomass, genotypic code and na+/k+ ratio. the characteristics studied were root and shoot length, root and shoot dry weight, na+ and k+ concentrations, and thegenetic score of the genotypes. these characters showed the variable degree of heritability. genetic score under salinity stress showed that tarom-mahalli, gharib, shahpasand mazandaranand ahlami-tarom with more biological yield root and shoot lengths, and low na+/k+ ratio were tolerant. we obser ved sig nif ica nt posit ive associat ion between genetic distance and geographic distance of the rice genotype studied and presence of an overall isolation by distance (ibd) model of differentiation across the geographical regions of iran. a similar observation was reported by pusadeea et al. (2019) while studying the population genetic structure of landrace rice, buechomee that is cultivated by karen people. therefore, they concluded that landraces serve as reservoirs of genetic variation which is influenced by natural processes such as selection and drift, and by the agriculture practices of local farmers. this is also supported by investigationperformed by wang et al. (2016). they used ssr markers and reported that the genetic diversity parameters were significantly higher in landraces under on-farm conservation compared to those under ex-situ conservation, in 12 villages of guizhou, yunnan and guangxi provinces of china.therefore, rice landraces under on-farm conservation programs had a higher genetic diversity compared to that of ex-situ conservation. this is affected by local on farm cultivation and onservation practice. previous genetic studies in iranian rice genotypes by different molecular markers also indicated significant difference among the studied genotypes, including landraces and the cultivars. for instance, nasabi et al. (2012), studied the genetic diversity in 20 iranians rice (oryza sativa l.) varieties using ssr markers linked to the genes controlling drought tolerance. they observed significant differences between varieties and drought resistance index. they reported total number of alleles of 142 with an average of 7.47 allele per locus. the average value of pic was0.817, and rice genotypes were divided into 6 groups. similarly, abootalebi et al. (2014), used ssr markers in 50 rice genotypes. they reported significant genetic difference among these genotypes which were divided in two genetic groups by neighbor-net networking and structure analysis. moreover, tabkhkar et al. (2012), studied the genetic diversity in 48 rice genotypes by using ssr molecular markers that were tightly linked to major qtls controlling three major components of rice cooking and eating quality (i.e. amylose content, gelatinization temperature and gel consistency). they reported the presence of a good level of genetic diversity in rice genotypes studied and the mean nei’s gene diversity = 0.72. cluster analysis divided the genotypes into four groups and separated the landrace cultivars with good cooking and eating quality (based on iranian taste) from others. in conclusion, the present investigation indicated genetic variability both within and among rice genotypes cultivated and grown indifferent geographical regions of iran. moreover, ssr loci that can differentiate rice genotypes are identified and can be used in rice cultivars authentication. list of abbreviations isolation by distance (ibd) iran rice research institute of iran (rrii) iran food and drug administration (ifad) iranian rice importers association (iria) international rice research institute(irri) authors’ contributions ns collected the samples and performed the pcr tests, qiaxcel data normalization and qiaxcelscreengel® software analyses. zn conceptualization of the project and data analyzed and interpreted. msh data analyzed and interpreted. hrz was co-advisor regards to plant biology. all authors were contributors in writing the manuscript and read and approved the finalmanuscript. acknowledgements we would like to thank parsian bio tech laboratory’s general manager mr. hessam shabani and lab personnel ms. fatemeh jamshidi for their collaboration. we 21authentication, genetic fingerprinting and assessing relatedness of rice (oryza sativa) genotypes by ssr molecular markers also thank iran rice research institute of iran (rrii), iran food and drug administration (ifad), iranian rice importers association (iria)and the international rice research institute (irri), philippines for providing rice samples. references abootalebi, s., fotookian, m.h., zeinolabedini, m. 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(2016). influence of ethnic traditional cultures on genetic diversity of rice landraces under on-farm conservation in southwest. chin journal of ethnobiology and ethnomedicine 12:51. doi:10.1186/s13002-016-0120-0. caryologia. international journal of cytology, cytosystematics and cytogenetics 73(2): 81-88, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-580 citation: t.b. jha, p.s. saha, s. jha (2020) a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding. caryologia 73(2): 81-88. doi: 10.13128/caryologia-580 received: august 1, 2019 accepted: march 12, 2020 published: july 31, 2020 copyright: © 2020 t.b. baran jha, p.s. saha, s. jha. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding timir baran jha1,*, partha sarathi saha2, sumita jha2 1 department of botany, maulana azad college, rafi ahmed kidwi road, kolkata 700113, west bengal, india 2 cas, department of botany, university of calcutta, 35, ballygunge circular road, kolkata-700019, west bengal, india *corresponding author. e-mail: tbjha2000@yahoo.co.in abstract. sesamum indicum commonly known as ‘sesame’, ‘til’ or ‘gingli’ is an ageold high valued oil crop. with distinct seed and floral diversity and no detailed chromosomal analysis is available on indian landraces of s. indicum (2n= 26). the present study demonstrates standardization of enzymatic maceration and air drying (ema) method of chromosome preparation and comparative karyometric analysis in four indian landraces of s indicum. all the landraces were characterized by very small chromosomes, length ranging from 1.24 ± 0.02 to 2.87 ± 0.09 µm. the emagiemsa based karyotype analysis revealed nine pairs of chromosomes with nearly median primary constriction, three pairs were submedian and a single satellite pair in each of the studied landrace. the cma staining of sesamum chromosomes revealed the presence of distinct cma positive (cma+ve) signals in all the studied landraces. the black seeded til (bt) and white seeded til (wt) were characterized by six chromosomes with distal cma+ve signal on short arm, while the dark brown seeded til (dbt) showed ten chromosomes with distal cma+ve signal on short arm. the light brown seeded til (lbt) was characterized by eight chromosomes with distal cma+ve signal on short arm. the results obtained from the scatter plot of a1 versus a2 and pca analysis provide a strong relationship with that of the fluorochrome banding analysis. the present research offers an explicit karyo-morphometric characterization of four indian landraces of s. indicum for the first time. keywords: fluorochrome banding, karyotype, sesame, sesamum indicum, small chromosomes, til. introduction sesamum indicum l. commonly known as ‘sesame’, ‘til’ or ‘gingli’ is an age-old high valued oil crop. as per the index kewensis the genus belongs to the family pedaliaceae and comprises 36 species. however, s. indicum is the only cultivated species of this genus (nayar and mehra 1970). sesame seeds are also known as the ‘queen of the oil seeds’ and the first oil known to be 82 timir baran jha, partha sarathi saha, sumita jha consumed by human (bedigian and harlan 1986). beneficial effects exhibited by sesame as antioxidant, antimicrobial, anti-inflammatory, antidiabetic, anticancer on human health has recently renewed the interest in this crop (amoo et al. 2017, zhang et al. 2013). the species was domesticated in india long back (bedigian 2003; 2010) and now ranked first in production and export of this crop (iopepc kharif 2017). cultivated s. indicum has highly variable genotypes and distinct differences have been noted in floral and seed colour morphology within the cultivated landraces (raghavan et al. 2010). chromosome analysis has played an important role in genetics and plant breeding for conservation of genetic diversity and improvement of crops. it is felt that chromosome analysis still provides foundational pieces of genomic information (soltis 2014) and considered “the quickest, cheapest, and easiest way to get any substantial information about the genome of a species which is not possible by any other methods” (guerra 2008). chromosome analysis in this cultivated species (2n=26) was reported long back by morinaga et al. (1929), raghavan and krishnamurthy (1947) and kobayashi (1949). raghavan and krishnamurthy (1947) reported that all 13 small chromosome pairs have terminal constrictions, while mukherjee (1959) reported presence of five types of somatic chromosomes. however, kobayashi (1949; 1991) in his analyses noted five pairs of median and eight pairs of sub median chromosomes. zhang et al. (2013) reported three pairs median, eight pairs sub median and two pairs sub-terminal chromosomes in s. indicum cv. yuzhi 11. it appears from the earlier reports that karyometric analysis of indian sesame deserves priority as detailed chromosomal analysis is not available on s. indicum along with their important landraces. thus, the present communication for the first time details the standardization of enzymatic maceration and air drying (ema) method of chromosome preparation and comparative karyometric analysis using non-fluorescent giemsa and fluorescent dapi and cma stains in four distinct indian landraces of s indicum. materials and methods plant materials the present study included four indian landraces of s. indicum namely black seeded till (bt), dark brown seeded till (dbt), light brown seeded till (lbt) and white seeded till (wt). among these four landraces, seeds of bt, wt and lbt were collected from different parts of west bengal and dbt was collected from mangalore, karnataka. all the collected seeds were germinated, grown in earthen pots and maintained under natural environment. voucher specimens were prepared for all the collected samples. somatic chromosome preparation and karyo-morphometric analysis nearly 2025 seeds from each landrace were imbibed in water for overnight and germinated in dark on moist filter papers to harvest their root tips. a minimum of ten healthy root tips of each sample were pre-treated separately in saturated solution of p-dichlorobenzene (pdb) at 1416ºc for 45 hrs, fixed overnight in glacial acetic acid: methanol (1:3) and finally stored therein at 20 °c. enzymatic maceration and air-drying (ema) method was carried out following our earlier published protocol (jha and yamamoto 2012; jha et al. 2015; jha and saha 2017) with required modifications of enzyme digestion time (55 min 90 min). completely air-dried slides were stained with 2% giemsa solution (merck; germany) in 1/15th phosphate buffer solution (ph 6.8) for 10-30 min at room temperature. after 45 times washing with ddh2o, the slides were air dried, mounted with xylene and observed (a minimum of 20 well scattered metaphase plates for each landrace). they were examined and photographed under carl zeiss, axio. lab. a1 microscope fitted with ccd camera using axiovision l. e4 software. for karyo-morphometric analysis, different karyological parameters viz. length of long arm (l) and short arm (s), absolute chromosome length (cl), relative chromosome length (rl) and total diploid chromatin length (tcl) were used. five somatic metaphase plates were used for karyometric analysis as well as to prepare ideogram. the centromeric index (ci) was used to classify the chromosomes according to levan et al. (1964) [metacentric (m) (1.00–1.70), submetacentric (sm) (1.70–3.00), subtelocentric (st) (3.00–7.00) and telocentric (t) (7.00–α)]. the karyotype asymmetry was estimated using intra-chromosomal asymmetry index (a1) and inter-chromosomal asymmetry index (a2) (zarco 1986), asymmetric karyotypes percent (ask%), asymmetry index (ai) (paszko 2006), total form percent (tf%), coefficient of variation of chromosome length (cvcl), coefficient of variation of centromeric index (cvci), coefficient of variation of arm ratio (cvr) and categories of stebbins (1971). fluorochrome staining with dapi and cma giemsa stained slides of each landrace were destained with 70% methanol for 40 min and air dried. 83a comparative karyo-morphometric analysis of indian landraces of sesamum indicum dapi and cma staining was carried out separately following the protocol of kondo and hizume (1982) with required modifications. for dapi staining, slides were kept for 30 min in mcllvaine buffer and then stained with 0.1µg ml-1 solution of dapi for 1030 min, mounted in non-fluorescent glycerol and observed under carl zeiss axio lab a1 fluorescent microscope using carl zeiss dapi filter cassette. for cma staining, the same slides were de-stained air-dried and then kept in mcllvaine buffer for 30 min followed by mcilvaine buffer with 5mm mgcl2 for 10 mins. slides were stained with 0.1mg ml-1 solution cma for 3060 mins followed and rinsed with mcllvaine buffer containing 5mm mgcl2. finally, slides were mounted with non-fluorescent glycerol and kept for maturation at 40c for 72 hrs. cma stained chromosomes were observed under the abovementioned fluorescent microscope fitted with carl zeiss fitc filter cassette and signals were analyzed using software prog res 2.3.3. statistical analysis descriptive statistics including mean values were analyzed for all measured parameters and variability in the data was expressed as the mean ± standard deviation (s.d.). one-way analysis of variance (anova) was performed to detect significant differences (p ≤ 0.05) in the mean (rohlf 1998). duncan’s multiple range test (dmrt) was used for post hoc analyses using spss v 16.0 statistical package. to study the karyotypic relationships among the collected landraces of s. indicum, scatter diagram of a1 versus a2 was drawn following the descriptions of paszko (2006). in order to further clarify the chromosomal relationship between each of the studied landrace, principal components analysis (pca) was conducted according to mcvean (2009). in this study, nine karyological variables (a1, a2, tf%, ask%, cvcl, cvci, cvr, ai and tcl) were used to plot the principal components using the infostat version 2013d (free version). results in the present study, four landraces of s. indicum differing in seed coat colour viz., black seeded til (bt), dark brown seeded til (dbt), light brown seeded til (lbt) and white seeded til (wt) were used for karyotype analysis (fig. 1). nearly 99% seeds of each sesame landrace germinated within 36 days after imbibition. distinct diversity in the floral morphology pertaining to four different landraces of sesamum was noted. the length of the corolla was 1520 mm with characteristic pigmentations on the lower lip. the flowers in black seeded til (bt) showed intense purple pigmentation in lower lip of corolla while in other landraces (dbt, lbt and wt) the intensity of the pigmentation ranged from pale lavender/ purple to light pink to white respectively (fig. 1). figure 1. flower and seed morphology of four indian landraces of s. indicum. a & e) black seeded til; b & f ) dark brown seeded til; c & g) light brown seeded til; d & h) white seeded til. dotted arrows indicate pigmentation patterns in lower lip of the corolla. 84 timir baran jha, partha sarathi saha, sumita jha karyo-morphometric analysis standardization of enzymatic maceration of root tip cells at 37 °c is the most crucial step to obtain well scattered metaphase chromosomes. in the present study, enzymatic maceration of root tips of all the collected landraces was performed for 5590 min and finally the time was optimized to 8590 min to obtain cytoplasm free well scattered chromosomes. the giemsa staining was done for 20 min. for each landrace, at least 20 countable metaphase plates were studied to determine diploid chromosome number. somatic chromosome number of 2n= 26 was observed in all the studied landraces of s. indicum (fig. 2; table 1). all the landraces were characterized by small sized chromosomes ranging from 1.24 ± 0.02 to 2.87 ± 0.09 µm (fig. 2; table 1). a significant variation in the total chromatin length was observed among the studied accessions. black seeded til (bt) was characterized by highest total chromatin length (52.75 ± 0.24 µm), while the lowest (44.85 ± 0.35 µm) being found in dark brown seeded til (dbt). the detailed karyotype analysis revealed nine pairs of chromosomes with nearly median primary constriction, three pairs with sub median primary constrictions and a single satellite pair in each of the studied landraces (fig. 2). the ordering of satellite (sat) bearing pair was found to be constant (5th pair) in all the landraces having identical haploid karyotype formula: 3sm + 9m + 1sm.sat (fig. 2; table 1). in the present study, several karyo-morphometric variations were also noted among the studied landraces. low values of intra-chromosomal asymmetry index (a1) and inter-chromosomal asymmetry index (a2) were observed in all the studied landraces (table 2). asymmetric index (ai), the product of coefficient of variation in chromosome length (cvcl) and coefficient of variation in centromeric index (cvci) was found to be low (ranging from 1.464 to 1.964) in all studied accessions of sesame (table 2). whereas, ask% and tf% showed moderate values for all the four sesamum landraces (table 2). analysis of the karyotype asymmetric indices also revealed that all studied landraces except black seeded til (bt) belong to the group 2a of stebbins classification while black seeded til (bt) belongs to group 2b (table 2). fluorochrome banding analysis in the present study, fluorochrome staining of sesamum somatic chromosomes using dapi and cma was standardized for the first time. chromosomes were stained properly with dapi when incubated for 30 min, while for cma, staining time was optimized at 60 min. the cma staining of sesamum chromosomes revealed the presence of distinct cma positive (cma+ve) signals/ zones in all the studied landraces however, the number of chromosomes showing cma+ve signals varied among them (table 3). based on the cma signalling patterns, chromosomes were grouped into two basic types: type a [chromosomes with no cma+ve signals] and type b [chromosomes (including one pair of sat-bearing chromosomes) with distal cma+ve signal on short arm]. both the type a and b chromosomes were present in all the four landraces of sesamum while, the number of each type was found to be landrace specific (table 3). the black seeded til (bt) and white seeded til (wt) were characterized by six chromosomes with distal cma+ve signal on short arm (fig. 3b and 3k), while the dark brown seeded til (dbt) showed ten chromosomes with distal cma+ve signal on short arm (fig. 3e). the light brown seeded til (lbt) was characterized by eight chromosomes with distal cma+ve signal on short arm (fig. 3h). however, we could not detect dapi +ve/-ve signals on chromosomes of any of the landraces studied (table 3). figure 2. panel a: ema based giemsa stained mitotic metaphase plates of four indian landraces of s. indicum showing 2n= 26 chromosomes. a) black seeded til; b) dark brown seeded til; c) light brown seeded til; d) white seeded til. arrows indicate secondary constricted chromosomes. bar= 5 µm. panel b: comparative ideograms of the studied four landraces. also showing positive cma fluorescent band on respective chromosomes, bar= 1 µm. 85a comparative karyo-morphometric analysis of indian landraces of sesamum indicum cma+ signals in four studied landraces are incorporated in the idiogram (fig. 2) scatter plot and principal component (pca) analyses the scatter diagram of a1 versus a2 revealed that dark brown seeded til (dbt) and light brown seeded til (lbt) were placed close to each other, thereby forming a cluster, while the black seeded til (bt) and white seeded til (wt) were positioned away from the cluster (fig. 4). in the present study, pca was further conducted to clarify the karyotypic relationship between the landraces using different karyo-morphometric parameters. in this eigenvector-based multivariate analysis, the component 1 (pc1) was found to be 59.6% of the total variation whereas component 2 (pc2) was 33.4% (fig. 5). the obtained cophenetic correlation was 0.998, indicating a good fit between the eigenvalues and eigenvectors distance matrix. the pca plot (fig. 5) displayed the close positioning of dark brown seeded til (dbt) with light brown seeded til (lbt), which was similar to that of the scatter plot (fig. 4). on the other hand, the black seeded table 1. chromosome morphometric analysis of four indian landraces of s. indicum*. s. indicum landraces zygotic chromosome number (2n) length of longest chromosome (μm) length of shortest chromosome (μm) total chromatin length (μm) (mean ± s.d.) ordering no. of sat bearing pair karyotype formulae (n)absolute (mean ± s.d.) relative (mean ± s.d.) absolute (mean ± s.d.) relative (mean ± s.d.) black seeded til 26 2.87 ± 0.09b 5.44 ± 0.14b 1.36 ± 0.02 c 2.58 ± 0.05a 52.75 ± 0.24c 5th 3sm+9m+ 1sm.sat dark brown seeded til 26 2.20 ± 0.12a 4.90 ± 0.24a,b 1.24 ± 0.02a 2.77 ± 0.06b 44.85 ± 0.35a 5th 3sm+9m+ 1sm.sat light brown seeded til 26 2.15 ± 0.14a 4.76 ± 0.28a 1.28 ± 0.02a 2.84 ± 0.07b 45.20 ± 0.42a 5th 3sm+9m+ 1sm.sat white seeded til 26 2.62 ± 0.07b 5.42± 0.06b 1.33± 0.03b 2.76 ± 0.03b 48.29 ± 0.74b 5th 3sm+9m+ 1sm.sat *values followed by same letter are not significantly different according to duncan’s multiple range tests test (p=0.05). table 2. comparative karyometric analysis of four indian landraces of s. indicum*. s. indium landraces a1 a2 tf% ask% cvcl cvci cvr ai stebbin’s group black seeded til 0.603 0.004 37.061 62.293 22.496 8.732 15.639 1.964 2b dark brown seeded til 0.619 0.007 37.747 61.360 19.056 10.218 17.877 1.947 2a light brown seeded til 0.628 0.009 38.228 60.886 17.369 8.433 14.562 1.464 2a white seeded til 0.636 0.015 38.163 61.007 21.370 7.523 12.311 1.607 2a *values followed by same letter are not significantly different according to duncan’s multiple range tests test (p=0.05). a1: intra-chromosomal asymmetry index; a2: inter-chromosomal asymmetry index; tf%: total form percent; ask%: asymmetric karyotype percent; cvcl: coefficient of variation of chromosome length; cvci: coefficient of variation of centromeric index; cvr: coefficient of variation of arm ratio; ai: asymmetry index. table 3. fluorescent banding patterns in four indian landraces of s. indicum. s. indicum landraces maximum no. of chromosomes with cma+ve bands position of cma+ve bands in chromosome cma karyotypes (n) maximum no. of chromosomes with dapi+ve/vebands black seeded til 6 distal part of short arm 20a+6b nil dark brown seeded til 10 distal part of short arm 18a+8b nil light brown seeded til 8 distal part of short arm 18a+8b nil white seeded til 6 distal part of short arm 20a+6b nil 86 timir baran jha, partha sarathi saha, sumita jha til (bt) and white seeded til (wt) were located at a significant distance from each other (fig. 5). discussion the present study demonstrates a comprehensive karyo-morphometric analysis of four indian landraces of s. indicum based on giemsa, cma and dapi banding analysis. due to its characteristic life forms and immense nutritive value, s. indicum has attracted the attention of researchers and breeders to plan a successful conservation strategies and improvements in breeding programs. however, only a few reports are available on the cytogenetics of indian varieties of sesamum till date may be owing to very small size of the chromosomes (< 3 µm) and technical limitations (raghavan and krishnamurthy 1947; mukherjee 1959). in the present study, we have adopted the ema based chromosome analysis to obtain cytoplasm free well scattered metaphase chromosomes of the species. the combination of enzymatic maceration and air dying methods is a very useful technique to analyze chromosome morphology, constrictions and types of chromosomes in detail (fukui 1996). this method was instrumental in obtaining uniformly spread chromosomes against a cytoplasm free background in several crop species with small and medium sized chromosomes (kurata and omura 1978; moscone 1996; yamamoto 2007; jha 2014; jha and halder 2016; jha et al. 2017; jha and saha 2017; ghosh et al. 2018). the results obtained from the present analysis offers several insights into the karyological characterization of different s. indicum landraces viz. black seeded til (bt), dark brown seeded til (dbt), light brown seeded til (lbt) and white seeded til (wt). the diploid chromosome number (2n= 26) in all studied landraces figure 5. principal component analysis (pca) plot showing grouping of four indian landraces of s. indicum based on nine karyomorphometric variables. figure 3. somatic metaphase chromosomes (2n= 26) of four indian landraces of s. indicum stained with giemsa, cma followed by dapi. ac) black seeded til; df ) dark brown seeded til; gi) light brown seeded til; jl) white seeded til. bar= 5 µm. arrows indicate the chromosomes showing cma+ve signals/ zones when stained with cma fluorochrome. figure 4. scatter diagram of intra-chromosomal asymmetry index (a1) versus inter-chromosomal asymmetry index (a2) of four indian landraces of s. indicum. 87a comparative karyo-morphometric analysis of indian landraces of sesamum indicum of s. indicum is in agreement with the earlier reports (morinaga et al. 1929; kobayashi 1949; raghavan and krishnamurthy 1947). raghavan and krishnamurthy (1947) identified 13 pairs of somatic chromosomes with terminal constrictions in this species, while kobayashi (1991) classified five pairs of median and eight pairs of submedian chromosomes including one pair (10th pair) of sat-bearing chromosomes in s. indicum. in the present study, a significant variation in chromosome size (ranging from 1.24 μm2.87 μm) has been scored among the sesamum landraces. mukherjee (1959) reported two pairs of chromosomes having secondary constrictions in s. indicum, while the present ema based analysis clearly revealed the presence of nine pairs of chromosomes with nearly median primary constriction, three pairs were submedian and a single pair (5th pair) of sat-bearing chromosomes in all the studied landraces and which can be considered as the modal karyotype for indian sesame. in addition to the ema based giemsa staining, the fluorochrome banding patterns are documented here for the first time in four studied indian landraces of sesamum. the application of nucleotide specific fluorochromes i.e. gc-specific cma, at-specific dapi in chromosome analysis has been reported to be very expedient in proper karyological characterization of many plant species (schweizer 1976; moscone et al. 1996). in the present study, cma banding analysis provides a comprehensive cytogenetic characterization of four indian landraces of s. indicum. based on both karyomorphology and cma signalling patterns, distinct homologies could be established between the studied landraces. presently, we could not locate dapi+ve bands in any of the studied samples. however, the differences in distribution of cma+ve signals/ zones clearly delimit each of the studied landraces of sesamum. in the present study, all the collected landraces of s. indicum exhibited symmetrical karyotypes based on categories of stebbins (1971). however, the analyses of scatter diagram of a1 versus a2 and pca plot unambiguously delimit each of the studied landrace (fig. 4 and 5). pca is a true eigenvector-based multivariate analysis, which can be used to project samples onto a series of orthogonal axes and to statistically clarify the genetic relationship among the studied samples (mcvean 2009). the results obtained from scatter plot of a1 vs a2 and pca analysis provide a strong relationship with that of the fluorochrome (cma) banding analysis. the dark brown seeded til (dbt) and light brown seeded til (lbt) exhibited maximum cma+ve signals/ zones and appeared close to each other, while both the black seeded til (bt) and white seeded til (wt) characterized by minimum cma+ve signals (i.e. six chromosomes with distal cma+ve signal on short arm) positioned distantly in the scatter diagram of a1 versus a2 and pca plot. as a whole, the present study involving ema based giemsa staining techniques demonstrates an explicit karyo-morphometric characterization of four indian landraces of s. indicum for the first time. distinct landracespecific variation in the distribution of cma+ve signals/ zones in somatic chromosomes was also established in the species. the grouping of the studied landraces was also corroborated by the analysis of scatter diagram of a1 versus a2 and pca plot which revealed a strong relationship with that of the fluorochrome banding analysis. however, further studies employing in situ hybridization techniques like fish/ gish and dna barcode analysis are required for clarification of evolutionary processes within the particular species. in conclusion, the present karyo-morphometric analysis explicitly characterizes four important indian landraces of s. indicum for the first time. application of ema method of chromosome analysis followed by giemsa and fluorescent dye cma which targets gc rich constitutive heterochromatin regions on chromosomes has clearly demonstrated that the method may be used as useful tool to characterize and differentiate s. indicum at the varietal level. acknowledgements tbj acknowledges the principal and hod, dept of botany, maulana azad college kolkata for the facilities provided and his teacher late prof. arun kumar sharma for his inspiration. references amoo so, okorogbona aom, du plooy cp, venter sl. 2017. sesamum indicum. in: kuete v, editor. medicinal spices and vegetables from africa. therapeutic potential against metabolic, inflammatory, infectious and systemic diseases. cambridge, massachusetts, united states: academic press, pp. 549-579. bedigian d. 2003. evolution of sesame revisited: domestication, diversity and prospects. genet resour crop evol. 50:779-787. bedigian d. 2010. characterization of sesame (sesamum indicum l.) germplasm: a critique. genet resour crop evol. 57:641-647. bedigian d, harlan jr. 1986. evidence for cultivation of sesame in the ancient world. econ bot. 40:137-154. 88 timir baran jha, partha sarathi saha, sumita jha fukui k. 1996. plant chromosomes at mitosis. in: fukui k, nakayama s, editors. plant chromosomes. laboratory methods. boca raton, tokyo: crc press, pp. 1-18. ghosh i, bhowmick bk, jha s. 2018. cytogenetics of two indian varieties of momordica charantia l. 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(plantaginaceae) saeed mohsenzadeh*, masoud sheidai, fahimeh koohdar a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding timir baran jha1,*, partha sarathi saha2, sumita jha2 chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india harminder singh2, jaswant singh1,*, puneet kumar2, vijay kumar singhal1, bhupendra singh kholia2, lalit mohan tewari3 population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies raheleh tabaripour1,*, masoud sheidai1, seyed mehdi talebi2, zahra noormohammadi3 induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions girjesh kumar, shefali singh* karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 caryologia. international journal of cytology, cytosystematics and cytogenetics 72(1): 29-43, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-249 citation: b. yilmaz öztürk (2019) intracellular and extracellular green synthesis of silver nanoparticles using desmodesmus sp.: their antibacterial and antifungal effects. caryologia 72(1): 29-43. doi: 10.13128/cayologia-249 received: 21th april 2018 accepted: 20th november 2018 published: 10th may 2019 copyright: © 2019 b. yilmaz öztürk. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. intracellular and extracellular green synthesis of silver nanoparticles using desmodesmus sp.: their antibacterial and antifungal effects betül yılmaz öztürk eskişehir osmangazi university central research laboratory application and research center (arum), 26480 eskişehir, turkey e mail: byozturk@ogu.edu.tr. orcid: 0000-0002-1817-8240 abstract. in this study aim was to perform green synthesis of synthesis silver nanoparticles (lac-agnps, rae-agnps and bae-agnps) by using desmodesmus sp., intracellular and extracellular synthesis methods and to compare the obtained products with physicochemical characterization techniques. the structural, morphological and optical properties of the synthesized nanoparticles were characterized using uvvis spectroscopy, tem, sem-eds, ftir, dls and zeta potential. these results clearly show that silver nanoparticles (agnps) could be synthesized in different sizes and stabilities with various biological materials obtained from desmodesmus sp. lac-agnps had size of 10-30 nm, rae-agnps had size of 4-8 nm and bae-agnps had size of 3-6 nm. also, the antibacterial activity of silver nanoparticles synthesized as intracellular and extracellular showed a strong antibacterial effect against pathogens such as salmonella sp. and listeria monocytogenes. additionally, they have effective antifungal activity against candida parapsilosis. the broth microdilution method was used for examining antibacterial antifungal effect of synthesis agnps. the minimum inhibitory concentration against salmonella sp., listeria monocytogenesis and candida parapsilosis were recorded as 3,125 μl, 1,5625 µl and 0,78125 µl synthesis agnps, respectively. as a result, it has thought that different sizes of synthesis agnps may have a great potential for biomedical applications. keywords. green synthesis, nanoparticle, algae, antimicrobial, desmodesmus sp. introduction in nanotechnology studies, the most attract attention topics is the synthesis of nanoparticles (nps) with strong potency in different sizes and shapes or by using various variables. for example, metal nps. the area of use for metal nps are very broad. even silver nps alone are used in countless areas with optics, electronics, catalysis, home furnishings and extensive medical applications, and the annual production is estimated to be hundreds of tons worldwide (ge et al. 2014). 30 betül yılmaz öztürk synthesis of nanomaterials containing noble metal requires alternative strategies because of their high cost. green synthesis or green chemical synthesis leads the list of these strategies and here the target is a biological synthesis of nanomaterial and to obtain products with positive effects in terms of the environment (sondi et al. 2000). green synthesis has preferred because of the high costs of materials used in conventional chemical methods and due to the toxic substances released into the environment. because the toxic effect of nanoparticles is proven in many studies on aquatic organisms in particular. for example, studies on algae (dağlıoğlu and öztürk 2016; dağlıoğlu and öztürk 2018; öztürk and dağlıoğlu 2018) have shown that aquatic invertebrates (artemia salina) (dağlıoğlu et al. 2016a), terrestrial invertebrates, honey bee (apis mellifera) (özkan et al. 2016), aquatic plants (lemna minor and myriophyllum spicatum) (dağlıoğlu and türkiş 2017a, b). nevertheless, it more attracts the attention of researchers for the production of metal nanoparticles because of its low cost, environmental friendliness, and simple approach. in the green synthesis does not use reducing agents like sodium borohydride (nabh4) used in chemical synthesis (kozma et al. 2015). these reducing agents are both expensive and may produce oxidized boron species bound to nps after synthesis. as a result the commercially produced nps are not appropriate for biological applications. nps produced by green synthesis are of great importance in terms of environmentally friendly production processes and low costs. many organisms or a variety of extracts produced by them may be used in the green synthesis process bacteria (joerger et al. 2000), plant (khatami et al. 2018a), fungi (boroumand et al. 2015) and algae (singh et al. 2013). algae will have a great platform for products to be used for various purposes over the next few years has a long-term sustainable potential, especially in the production of food and liquid fuels (koothari et al. 2017). for this reason algae are commonly chosen for green synthesis because their structures are a rich source of biologically active compounds like chlorophyll, carotenoids, astaxanthin, phenol, flavonoid, protein, vitamin and minerals (faulker 2000). additionally, these phytochemical materials are each effective metal reducing agents and their structures contain agents ensuring that hydroxyl, carboxyl, and amino functional groups coat metal nps (annamalia and nallamuthu 2015). it has been supported by various literatures that nps synthesis can be carried out by using various algae. for example, kannan et al. (2013) performed agnp synthesis with extraction from chaetomorpha linum species. the researchers succeeded in synthesizing spherical nps with nearly 30 nm size without using synthetic reactives. barwal et al. (2011) used a chlamydomonas reinhardtii model and focused on understanding the role of a variety of cellular proteins in the synthesis and coating of silver nps. prasad et al. (2013) studied the extract of the brown algae cystophora moniliformis species and reported that the sizes of nps may change linked to temperature. studies mainly use marine macroalgae with insufficient numbers of studies about microalgae. it has been known for many years that silver (ag) is an antimicrobial agent and it draws much attention due to its application in fields such as colloidal ag, catalysis and water purification. it is reported that using agnp may purify drinking water (pradeep 2009). furthermore, the effect of ag nps size variability on antibacterial properties has been a matter of interest. as a result, researchers who have successfully achieved green synthesis have simultaneously assessed the antibacterial and antifungal effects on a variety of microorganisms (like bacteria and yeast) (govindaraju et al. 2009; rajeshkumar et al. 2014; salari et al. 2016; suriya et al. 2012). our aim is to use the microalgae desmodesmus sp., which can easily be produced in a laboratory environment, to compare synthesis using both intracellular and extracellular routes. for this, the differences between nps forming under the same conditions were determined using characterization techniques like uv-vis, tem, sem-eds, ftir, dls and zeta potential. at the same time, antibacterial effects of synthesized nps on bacterial strains of important food pathogens such as salmonella sp. and listeria monocytogenesis were investigated. additionally, the antifungal effect on the human pathogen of candida parapsilosis species was investigated. material and method algae culture the test organism used in our study, desmodesmus sp. (kr261937), was taken from the algae culture collection of selçuk university hydrobiology laboratory. algae taken from stock cultures were transferred to the fluid bg-11 medium (nano3, 15; k2hpo4, 0.4; mgso4·7h2o, 0.75; cacl2·2h2o, 0.36; citric acid, 0.06; iron(iii) ammonium citrate, 0.06; na2-edta, 0.01; na2co3, 0.2 g/l, 1 ml; trace elements solution, (h3bo3, 61; mnso4·h2o, 169; znso4 ·7h2o, 287; cuso4·5h2o, 2.5; (nh4)6mo7o24·4h2o, 12.5 mg/l) in accordance with the procedure stated in rippka (1988). the microalgae were transferred to 250 ml erlenmeyer flask and left to proliferate under sterile conditions. the cultures were 31intracellular and extracellular green synthesis of silver nanoparticles using desmodesmus sp. left under 3000 lux fluorescent light appropriate for photosynthesis for 12 hours light and 12 hours darkness, at 28±2 °c degrees, and 120 rpm for 15-20 days. when algae passed the log phase stage, cells were centrifuged at 1000 rpm and the biomass was obtained. all chemicals used in this study were analytical quality. intracellular agnp synthesis using live algae cells silver nitrate (agno3,  ≥99.0%; sigma-aldrich) was prepared in 100 mm stock solution. microalgae passing the logarithmic phase were counted with the automatic cell counting device (luna-ii™ automated cell counter, south). using luna ™ cell counting slides, counting and viability of cells were determined with trypan blue. afterwards, live algae cells (lac) were harvested by centrifugation at 4000 rpm for 20 minutes at 4 °c. the culture filtrate was removed and the pelleted biomass was washed with sterile deionized water to remove foreign absorbed material. the washing procedure was repeated five times and the washed biomass was brought to suspension again in distilled water. the suspension of algal biomass had 5mm agno3 added the from stock solution. in the control setup, agno3 was not added to the algal biomass. cultures were incubated at 28 °c under similar conditions to those stated above for 72 hours. intracellular ag nps (lac-agnps) synthesis from lac was completed. after the reaction, the biomass was separated by the centrifuge and stored at -20 °c until characterization. extracellular synthesis of agnp two different algal extracts were obtained using desmodesmus sp. for the extracellular synthesis of agnp. these extracts were raw algal extract (rae) and boiled algal extract (bae). preparation of raw and boiled algae extracts for the preparation of r ae extract, 3.0 g (wet weight) algal biomass was suspended in 20 ml of deionized water for 5 days. at the end of the 5th day, it was centrifuged at 7000 rpm for 20 minutes. the supernatant (raw algal extract) was separated from cells and prepared for extraction. to begin the reaction, the final concentration of 5 mm agno3 was added. after this procedure, continuous mixing began. later the mixing procedure, a colloidal structure was obtained and after this process repeated centrifugation was completed at 3500 rpm for 5 minutes. these centrifuge cycles were completed to purify agnp. extracellular ag nps (rae-agnps) synthesis from rae was completed. after the reaction, the biomass was separated by the centrifuge and stored at -20 °c until characterization. to prepare the bae extract, 3.0 g (wet weight) algal biomass was suspended in 20 ml deionized water and heated to 100 °c for 20 minutes in an erlenmeyer flask. after the boiling procedure, it was cooled and filtered with grade gf/a glass microfiber filters (whatman™, pore size: 1,6µm). the obtained filtrate had the final concentration of 5 mm agno3 added at room temperature to begin the reaction. after the procedure continuous mixing was completed. later the mixing procedure, a colloidal structure was obtained and after this process repeated centrifugation was completed at 3500 rpm for 5 minutes. these centrifuge cycles were completed to purify agnp. extracellular ag nps synthesis (bae-agnps) from boiled algal extract was completed. after the reaction had completed, the biomass was separated by the centrifuge and stored at -20 °c until characterization. characterization of agnps biological reduction of silver ions with the green synthesis route was observed by taking 3 ml aliquot samples at different time intervals. absorption measurements were made with a uv-vis (ae-s90-2d uv-vis spectrophotometer, china.) spectrophotometer between 190 and 1100 nm. transmission electron microscope (tem) images of the intracellular and extracellular synthesized agnps were obtained using tem (jeol jem  1220 brand, japan) working at 100 kv acceleration voltage. samples used to obtain tem micrographs were prepared by dropping on a carbon-coated copper grid and dried under a vacuum before investigation. with the aim of investigating the cells at the ultrastructural level, routine tem monitoring procedure was performed and samples were submerged in epoxy resin. thin sections were taken at 60 nm thickness with the aid of an ultramicrotome (leica ultracut uct, leica, germany) (ilknur et al. 2012; li et al. 2012; zhang et al. 2016). elemental analysis of samples was completed with sem (a jeol jsm-5600 lv brand, japan) device fitted with eds (e2v scientific instruments, united kingdom). algae cells were freeze-dried and algae biomass was obtained. dried biomass was prepared using the kbr pellet technique and atr technique. the surface chemistry of reduced ag samples and the biologically active portions of the live microalgal cells were analyzed to check for comparisons. the fourier transform mid-infra32 betül yılmaz öztürk red ftir spectra (perkin elmer spectrum 100 ftiratr unit, germany) were collected with conduction mode from 400–4,000 cm-1 with 0,4 cm-1 spatial resolution. dynamic light scattering (dls) and zeta potential are necessary parameters to define the size distribution, particle size, homogeneity and stability of lac-agnps, rae-agnps and bae-agnps. particle size and polydispersity index (pdi) were determined using the dynamic light scattering technique. dls studies of lac-agnps, rae-agnps and bae-agnps diluted in deionized water were measured by a malvern-zetasizer (nano-z590, united kingdom) device. the zeta potential is a measurement of the attraction or repulsion values between particles. particles with certain load attractions with opposite polarity within the suspension, as a result, a strong bond surface is formed on the surface of the loaded particle and then a surface extending outward from the loaded particle forms. the behavior of particles within polar fluids is determined not by electric load but by zeta values. zeta potential studies were measured with a malvern (sn: mal1064144, united kingdom) brand zeta sizer zs device. antimicrobial susceptibility testing of synthesis nps in this study with the aim of determining the antibacterial and antifungal susceptibility of lac-agnps, rae-agnps and bae-agnps, salmonella sp. (gram-) and listeria monocytogenesis (gram+) bacteria and candida parapsilosis yeast was used. to determine the minimal inhibition concentration, the broth microdilution method was taken as a basis. bacteria cells were left in nutrient broth medium at 37 °c for 1 night, while yeast cells were incubated for 1 night in a shaking incubator at 30 °c in yeast extract-peptone-dextrose (ypd; %1 yeast extract, %2 peptone %2 dextrose) medium and cultures were taken. the density of bacteria and fungal cells were set according to the mcfarland 0.5 standard. tests of minimal inhibitory concentration (mic) were made in accordance with the clsi (clinical laboratory standards institute) criteria m27-a8 for bacteria and m27-a2 for yeast (zgoda and porter 2001). the extraction containing lowest nps that inhibited bacterial and fungal development was determined as the volume mic value. during this process 40 minutes sonication was applied to obtain the lac-agnps. the 96-well plates (lp italiana spa) was added 100 µl rpmi 1640 and 100 µl the lac-agnps, rae-agnps and bae-agnps. later they were diluted with microdilution and left for 24 hours incubation. the plate with resazurin added had results assessed in parallel with the colour change. results the test organism desmodesmus sp. (kr261937) is in the chlorophyceae class and is a water alga generally forming colonies with an oval or shuttle-shaped body. additionally, it is a photosynthetic microalga with 6-10 horn-like protrusions on the body. in the colonial structure, generally twin cells are found. additionally, sequences of 4 colonial cells may be seen in low numbers. the count of the automatic cell counting device is given in table 1. in the current study when algal biomass (lac) was exposed to ag ions (ag+), the colour of the algal biomass changed from natural bright green to brown and compared with the control biomass, the ag+ ion was biologically transformed (ag metal accumulation) to ag0. during exposure the colour change began in the first 24 hours; however, the largest colour difference compared to the first day occurred after 72 hours. lac-agnps was researched with uv-vis for 72 hours (figure 1a). simultaneously rae and bae were treated with 5 mm agno3 and extracellular ag nps (rae-agnps and bae-agnps) the formation was researched with uvvis spectroscopy. the rae-agnps were colourless and had a high peak at 280-300 nm (figure 1 b). the rae exposed to ag displayed a peak at 420 nm especially after the 48 hours day on uv-vis spectral analysis and it was considered agnp formation had begun. it is known that the concentration of the reducing agent in the reaction mixture plays an important role in the formation of nucleation points and then controls the size of agnp; this may have caused less stable agnp in the rae (hiramutsu and osterloh 2004; jena et al. 2014). as a result, another experiment was made to increase the concentration of this type of material in the reaction mixture. the biomass was boiled in water to obtain more reducing and stabilizing agents. initially, the boiled extract had a light yellow colour, which transformed to brown when exposed to the ag nitrate solution procedure. the formation of this colour is due to stimulation of surface plasmon resonance (spr) effect and the reduction in agno3 and may indicate the formation of agnp. with the increase in the reaction duration, the colour of the reaction mixture turned a darker table 1. desmodesmus sp. cell counting and cell viability analysis report. total cell live cell dead cell viability 4,48x108 4,08x108 4,00x107 80,6 % *stain: trypan blue. 33intracellular and extracellular green synthesis of silver nanoparticles using desmodesmus sp. shade for up to 72 hours. the formation of bae-agnps were proven with the uv-vis spectrum showing the characteristic surface plasmon resonance (spr) band for agnps. figure 1 c shows the uv-vis spectra series recorded for the reaction mixture at a variety of time intervals. the bae showed a peak at 280-320 nm which may be linked to the presence of peptides. the bae exposed to ag displayed a peak at 420 nm especially after the 24 hours day on uv-vis spectral analysis and it was considered bae-agnps formation had begun. our uv-vis results show a steady increase in reduction of ag ions in lac up to 420 nm, then it stabilized and appeared to pass to a downward trend. this section is defined as the “surface plasmon resonance band” and is due to the stimulation of free electrons in the nps. the symmetric shape of the band is an indicator of the regular distribution of the spherical nps (travan et al. 2009). after intracellular synthesis, the exposed biomass had tem analysis was made to research the morphology fig. 1. examination of intracellular and extracellular synthesis of silver nanoparticles by uv-vis. a. lac-agnps; b. rae-agnps; c. baeagnps. 34 betül yılmaz öztürk and dimensions of the nps. tem images revealed the cells were nearly 4.5-5 µm (length) × 2-3 µm (width) size with an oval shape and with 6-9 colonial protrusions (figure 2a). fig. 2. morphological characterization of the silver nanoparticles. a, b, c intracellular synthesis, tem image of desmodesmus sp. cell mediated synthesized silver nanoparticles. d, e, f cellular localization of in intracellular silver nanoparticles, tem micrograph of thin section (~ 60 nm). np:nanoparticle, s starch, v vakuol, cm cytoplasmic membrane. 35intracellular and extracellular green synthesis of silver nanoparticles using desmodesmus sp. when the algae cells exposed to agno3 were investigated with tem on a grid with the dropping method, distributed metal nps were observed, as shown in figure 2 a, b and c. as seen at different magnifications of the metal nanoparticle shapes, the periphery of the cells is observed more clearly and homogeneously, while the interior sections were not fully identified due to a more compact and electron-dense zone observed (figure 2 a, b). when micrographs of lac-agnps at high magnifications are investigated, the spherical structures of the agnps are clearly observed. the intracellular lacagnps are in the range of 10-30 nm in size and appear to display a homogeneous distribution. in tem analysis, a section of 60 nm in thickness was taken from the cells in order to be able to see the lac-agnps. additionally, sections were taken of these cells to identify where the nps were localized (figure 2 d, e, f). according to the results of these sections, nps were localized especially in areas close to the cell membrane, while also in other regions of the cell, e.g., in areas close to starch storage areas (figure 2 f). the nanoparticle dimensions obtained after raeagnps were mean 4-8 nm (figure 3 a, b). nps synthesized in this manner were less stable according to both tem and zeta potential results. when morphology and size of nps are examined after extracellular synthesis, the nps produced by bae-agnps were mean 3-6 nm. these nps had a homogeneous distribution without aggregation or flocculation (figure 3 c, d). the reason for this is that some stabilizing agents in the algal extract were released by boiling and entered the reaction (mohseniazar et al. 2011; nithya and ragunathan, 2009). at the same time, control of dimension and structure may be associated with interactions between biocomponents like polysaccharides, proteins, polyphenols and phenolic compounds with metal atoms (shao et fig. 3. tem image of extracellular synthesized silver nanoparticles a, b. desmodesmus sp. cell rae mediated synthesized silver nanoparticles (rae-agnps); c, d. desmodesmus sp. cell bae mediated synthesized silver nanoparticles (bae-agnps). 36 betül yılmaz öztürk al. 2004). as a result, bae-agnps showed the nps had equal distribution. analysis through energy dispersive sem-eds spectrometers confirmed the presence of an elemental ag signal of the ag nps. recognition lines for the major emission energies for ag are displayed and these match with peaks in the spectrum, thus giving confidence that ag has been correctly identified. in this study, the synthesized agnps were subjected to elemental analysis with sem-eds, the ag element was observed in all three situations (lac-agnps, bae-agnps, rae-agnps) at the rate (table 2) (figure 4 a, b, c). the highest ag was identified in the bae-agnps groups (figure 4 b). when these results are considered, they provide information about the purity of the formed nps. extracts obtained with different methods (bae and rae) and live cell algae (lac) the agnps obtained from these extracts (lac-agnps, r ae-agnps and bae-agnps) had ftir analysis completed on these samples to define the functional groups of chemical components. the analysis results observed many peaks (transmittance a.u) at 3284, 2919, 2851, 2161, 2027, 2034, 1638, 1535, 1380, 1242, 1149, 1023, 812, 717, and 551. when vibrations of biomass exposed to ag are investigated, the dense broadband at 3400 cm-1 is vibrations from alcoholic, phenolic and carboxylic groups (figure 1 f). primarily in addition to hydroxyl groups equivalent to o-h strain, vibrations equivalent to primary and secondary amines and amides are shown with n-h strain were observed. the band at nearly 2920 cm-1 reflects the c-h strain of alkanes. the lack of absorption in this region shows hydrogen linked to aliphatic carbon is not present (rónavári et al. 2017). the band at 2851 cm-1 from the aldehyde group reflects c-h strain. the band at 2161 cm-1 is -s-cξn thiocyanate, while the bands at 2027 and 2034 cm-1 are -n=c=s isothiocyanate. this situation shows that cyanate, elemental carbon and thiocyanate may be found within total organic carbon. the peak at nearly 1,638 cm-1 is equivalent to c = c vibration of aromatic structures, with the peak at 1242 cm-1 equivalent to c-o strain of phenolic groups. the peak at 1380 cm-1 is no2 asymmetric strain of table 2. eds reported from biosynthesized agnps. agnps intensity (c/s) error 2-sig conc. units control 0.060 0.129 0.023 wt.% lac-agnps 130.34 7.217 34.385 wt.% raeagnps 130.01 7.209 20.975 wt.% baeagnps 293.68 10.837 54.920 wt.% *kv 20.0, takeoff angle 35.0°. elapsed livetime 10.0. conc: concentration. fig. 4. sem-eds spectrum recorded from biosynthesized agnps a. control group; b. lac-agnps; c. rae-agnps; d. bae-agnps. 37intracellular and extracellular green synthesis of silver nanoparticles using desmodesmus sp. alkyl groups, and the peak at 1535 cm-1 is equivalent to c = c strain of biphenol group. around 1145 cm-1 the c-n strain vibration of aromatic primary and secondary amines is observed. aromacity may be mentioned between 900-690 cm-1 (jena et al. 2014). the extracellular bae-agnps obtained with the boiling method may have bonded to amine groups, while the r ae-agnps may have bonded to alkyne groups. our ftir results comply well with the literature data, with the surface of agnps obtained from intracellular desmodesmus sp. coated with organic components found in the extract, as shown in figure 3 b. as a result, successful green synthesis is revealed with the soluble organic components or proteins able to bind to ag ions and reduce ag ions to form nps (jegadeeswaran et al. 2012) (see 800-2919 cm-1 region). particle size and zeta potential are very important parameters for green synthesized ag nps. the particle size of ag nps, especially, has a large effect on the antimicrobial properties. another important value for particle size is pdi. pdi reveals homogeneity (sharma et al. 2018). both intracellular and extracellular agnps were well distributed in colloidal solution and the mean size distribution of these particles were as follows; the mean particle distribution for lac-agnps were 10-30 nm, with this value 4-8 nm and 3-6 nm from rae-agnps and bae-agnps (figure 6 a, c, e). according to pdi values for the results, homogeneous particle size distribution within the solution was observed to be highest for lac-agnps. though different sizes were found during synthesis, higher numbers of small-scale nps were observed in terms of numbers (table 3). zeta potential values are obtained from the high repulsion and attraction forces between each nanoparticle and these values define particle stability. intracellular and extracellular agnps have high negative zeta potential values. the high negative value affects the push between the particles, thereby increasing the stability of the formulation (rao et al. 2013). in our study, the zeta potential of agnps were measured as -20.2 mv for lacagnps (figure 6 b), -19.9 mv for bae-agnps (figure 6 d) and -14.2 mv for rae-agnps (figure 6 f). all values for particle sizes, pdi and zeta potentials are shown in fig. 5. ft-ir spectrum for a. desmodesmus sp. control group; b. desmodesmus sp. formed lac-agnps. table 3. the particle size of silver nanoparticles, polydispersity index and zeta potential. particle size (nm) dls pdi zeta potential (mv) lac-agnps 20-40 0.445 -20.2 bae-agnps 10-15 0.452 -19.9 rae-agnps 10-20 0.613 -14.2 38 betül yılmaz öztürk table 3. the observed result contains some differences compared with the tem studies but is well-correlated. the differences may be due to the tendency of ag nps to agglomerate within an aqueous solution; thus the values obtained from dls will be higher than tem values (domingos et al. 2009). antimicrobial and antifungal effect of synthesis agnps lac-agnps, rae-agnps and bae-agnps have been tested on salmonella sp. (gram +) and listeria monocytogenesis (gram -), the most frequently encountered bacteria in food poisoning (cantero et al. 2018; ma et al. 2018). the mic values of the synthesized nps were calculated on these bacteria. it was initiated by adding 100 µl of synthesized nps so that the mic values could be found. the lac-agnps, rae-agnps and bae-agnps inhibited 3,125 μl of salmonella sp. thus, this value was determined as the mic value. there was a larger effect on listeria monocytogenesis compared to salmonella sp. with 1,5625 µl mic value determined. candida parapsilosis is a pathogenic yeast strain with fig. 6. size distribution by dynamic light scattering and zeta potential measurement of biyosenthesis silver nanoparticles a, b. lac-agnps; c, d. rae-agnps; e, f. bae-agnps. 39intracellular and extracellular green synthesis of silver nanoparticles using desmodesmus sp. high biofilm formation capacity (soldini et al. 2017). candida parapsilosis appeared to be more affected compared to bacteria and the mic value was determined as 0,78125 µl (the synthesized agnps were initially used in 100 µl). the lac-agnps, bae-agnps and rae-agnps obtained from desmodesmus sp. with different methods showed significant degree of effect with different mic values for bacteria and yeast. discussion agnp is used in a variety of applications like catalysis, biosensing, imaging and antibacterial activity. as result researchers in recent times have performed many studies to synthesize agnp more economically and easily. green synthesis is an alternative method developed to produce metal nps using natural compounds or plant components. the most important advantages of these methods are the lack of toxic material involved in the chemical synthesis and the lack of high costs. in addition to green synthesis and sustainable synthetic methods can decrease environmental pollution to some extent (khatami et al. 2018c) . another significant advantage is that nps may be synthesized in an easy and reliable manner using only live organisms (algae, bacteria, fungus and plant), without requiring reducing or stabilizing agents (tippayawat et al. 2016; khatami et al. 2018b). microorganisms such as bacteria and algae have proven to be well suited for nanoparticle synthesis, where particle size and morphology must be stabilized by different methods in green synthesis (metuku et al. 2014). algae or plants or their extracts, many prokaryotic organisms and biocompatible macromolecules are very important for nanoparticle synthesis (poulose et al. 2014). algae are unique due to the lipids, minerals and some vitamin wealth contained in these organisms (namvar et al. 2012; zuercher et al. 2006). additionally, polysaccharides, proteins, and polyphenols are known as functional food as a variety of bioactive material with some medical uses like in cancer, oxidative stress, inflammation, allergies, thrombosis and lipidemia (mahdavi et al. 2013). as a result, hydroxyl, carboxyl and amino functional groups are found among this phytochemical material that is capable of acting in a single step as both effective metals reducing agents and as capping agents. generally many natural essences in cell contents or released after extraction are biologically active compounds and may be responsible for the reduction of ag ions and stabilization of the obtained nps. the carbonyl groups in proteins have strong binding ability against metal nps and as a result, proteins may form a coating layer on the surface of agnps (dhand et al, 2016; vivek et al. 2012). this may prevent agglomeration and increase the stability of nps synthesized in aqueous media. in our study, uv-vis data from algal cells exposed to ag nitrate support the view that of ag + ions were taken into cells within 24 hours and l-agnps formed (peaked at 420 nm within 24 h.). as metal ions (ag+) are on the algal surface, they were identified to be held by electrostatic interaction between the functional groups with the negative load on the cell surface, and following this, the metal ions were reduced (barwal et al. 2011). in this way, high intracellular accumulation and formation of metal particles may be linked to several probable mechanisms in the process. these mechanisms vary according to type and metal accumulation occurs with two processes. the first is adsorption or biosorption at the cell surface independent of metabolism, while the second is metabolism-dependent absorption by organelles or cytoplasmic ligands (chakraborty et al. 2006). metal nps synthesized with green methods may produce colloids with different sizes, shapes, and distributions. if changing the method used provides better results, the green approach may be chosen. as a result of our study, the focus was on the biosynthesis of both intracellular and extracellular agnp using different extraction techniques with desmodesmus sp. microalgae and agnp production was successfully achieved. in this study during intracellular synthesis, algal cells have pores of 3-5 nm width ensuring passage of low molecular weight material like water, inorganic ions, gases and other small nutritional material required for growth and metabolism (wang and chen 2009). large molecules or macromolecules cannot pass these pores. similarly, in our study, the intracellular lac-agnps appeared to be larger than the size of the pores in the algal cell walls (10-30 nm) (figure 2 a, b, c). the bae-agnps and rae-agnps also had spherical shape without agglomeration. sections through the cells transversely and longitudinally observed nanoparticle formation distributed through intracellular cytoplasm, with larger size nps in some regions, e.g., in regions close to vacuoles. in previous research, it was reported that maximum metal accumulation occurred within the cy toplasm, periplasm, nucleus and pyrenoids of organisms like chlamydomonas (ag), chlorella (au, pd, ru, rh), klebsormidium flaccidum (au) and shewanella (au, pt) (barwal et al. 2011; dahoumane et al. 2012; luangpipat et al. 2011). ftir can be used to identify the type of functional groups and biomolecules that are responsible for capping and efficient stabilization of nps and qualitative and quantitative identification of the molecular structure 40 betül yılmaz öztürk of organic compounds in the nps structure (khatami et al. 2018d). in thi study, the results of ftir spectroscopic investigations showed the presence of organic particles especially in the 800-2919 cm-1 region. linked to this result, the protein building block of amino acids was confirmed as having strong binding ability to metals and formed a layer surrounding metal nps. they acted as a coating material preventing agglomeration and thus are considered to have ensured high stability of metal nps. these results confirm the presence of proteins or peptides with possible function as stabilizing agents for agnps (singhal et al. 2011). the extracellular r ae-agnps synthesized was found to be less stable and have a tendency to agglomerate compared to extracellular synthesized bae-agnps and intracellular synthesized l-agnps. additionally, the rae-agnps synthesis process was slower compared to the others. when bae was used, both high rates of bae-agnps biosynthesis occurred and the stability of these nps were higher. however, according to the uv results, l-agnps were more rapid biosynthesised than extracellular bae-agnps and rae-agnps. this situation may be related to macromolecules like protein or peptide released by boiling directly reducing ag+ ions. a study by jena et al. (2014) produced similar results. the researchers explained that in this situation the protein or peptide concentration has a vital role in agnp formation and stabilization. a similar study by barwal et al. (2011) prepared chlamydomonas cell extract treated with ag nitrate with both whole cell esence and proteinremoved extraction. when the results are compared, the protein-removed cell extraction was identified to synthesize larger sizes of agnp. the researchers explained that the protein concentration is directly correlated with particle formation rate and inversely correlated with particle size (barwal et al. 2011; jena et al. 2014). due to a variety of reasons in the antimicrobial mechanism, ag ions or salts only have limited use as an antimicrobial agent. however, the use of agnps may overcome these limitations. in biological systems (animal cell culture, plants, bacteria) the effect of agnps in different concentrations has been investigated (sobieh et al. 2016, khatami et al. 2018). for the use of ag against microorganisms in a variety of areas, it is important to prepare ag with appropriately priced methods and to know the antimicrobial effect mechanism to increase this effect (kim et al. 2007). green synthesized nps may expand these areas of use significantly. in this study, agnps may be a potential antibacterial and antifungal agent and could be prepared cost-effectively. because, desmodesmus sp., a green algae, could be a low-cost production house for intracellular and extracellular agnps synthesis because of the minimum growth regimens required for growth, such as water, sunlight and commercial fertilizers, and high biomass efficiency. conclusion in this study different sizes of agnp were successfully synthesized from desmodesmus sp. microalgae using both intracellular and extracellular green synthesis routes. in particular, intracellular uv results of lacagnps peaked at 420 nm within the first 24 hours. the purity of ag nps was confirmed with sem-eds. nanoparticle size and stability were determined according to dls and zeta potential results. agnps with optimized size were determined to have lethal potential against bacteria and yeast. thus this study is 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karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini). caryologia 73(2): 121-126. doi: 10.13128/caryologia-610 received: august 26, 2019 accepted: april 13, 2020 published: july 31, 2020 copyright: © 2020 r. monteiro do nascimento, a. freire carvalho, w. c. santana, a. barth, m. a. costa. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* 1 departamento de ciências biológicas, universidade estadual de santa cruz, ilhéus, ba, brazil 2 departamento de entomologia, universidade federal de viçosa, viçosa, mg, brazil 3 instituto federal de educação ciência e tecnologia de mato grosso, campus rondonópolis, brazil *corresponding author. e-mail: costama@uesc.br abstract. frieseomelitta (ihering, 1912) is a genus of stingless bees, distributed in the nearctic and neotropical regions. specimens can be found in forests, cerrado, caatinga and mountainous regions. this genus has 16 species, of which 13 are recorded in brazil. cytogenetics has contributed to evolutionary studies of some hymenoptera groups and although many frieseomelitta species have been described, few species have been studied cytogenetically. the present study aims to contribute to the knowledge of the karyotype diversity of this genus, seeking to understand the possible evolutionary mechanisms that occurred in the diversification of the karyotype of this genus. frieseomelitta portoi and frieseomelitta trichocerata and frieseomelitta doederleini showed diploid karyotypes with 2n = 30 chromosomes, similarly to all the species previously analyzed in the genus. unprecedentedly, frieseomelitta longipes showed 2n = 34. these results confirm that the frequent diploid number of 30 chromosomes is typical of this genus. the finding of 2n = 34 chromosomes in f. longipes comprises the first record of a diploid chromosome number different from 2n=30 in this group, which suggests that it can be the result of a recent chromosome change event. an interspecific comparative analysis was developed involving present and previous studies, as well as a discussion on the mechanisms involved in the karyotypic evolution in the genus. keywords: heterochromatin, karyotype evolution, centric fission, chromosome, variation. introduction frieseomelitta (ihering, 1912) is a neartic and neotropical genus of stingless bees, and occurs from mexico to southeast brazil (camargo and pedro, 2013). these bees present small to medium size, with body measuring about 10 mm (silveira et al., 2002). according to camargo and pedro (2013), 16 species are known to be valid in the genus frieseomelit122 renan monteiro do nascimento et al. ta, of which 13 are found in brazilian territory: frieseomelitta dispar (moure, 1950), frieseomelitta doederleini (friese, 1900), frieseomelitta flavicornis (fabricius, 1798), frieseomelitta francoi (moure, 1946), frieseomelitta freiremaiai (moure, 1963), frieseomelitta languida (moure, 1989), frieseomelitta longipes (smith, 1854), frieseomelitta meadewaldoi  (cockerell, 1915), frieseomelitta paranigra  (schwarz, 1940), frieseomelitta portoi (friese, 1900), frieseomelitta silvestrii (friese, 1902), frieseomelitta trichocerata (moure, 1988) and frieseomelitta varia (lepeletier, 1836). cy togenetic analysis has been a widely used tool in evolutionary and taxonomic studies in some groups of hymenoptera, however, for the genus frieseomelitta, the chromosomal analysis are hitherto restricted the species f. doederleini (kerr and silveira, 1972; tarelho, 1973; rocha et al., 2003; santos et al., 2018), f. languida (rocha et al., 2003), f. varia (kerr, 1969; kerr and silveira, 1972; tarelho, 1973; rocha et al., 2003; santos et al., 2018), f. dispar (carvalho and costa, 2011; santos et al., 2018), f. francoi (carvalho and costa, 2011; santos et al., 2018), frieseomelitta meadewaldoi and frieseomelitta sp. n. (santos et al., 2018). during the karyotype analyzes of several species of bees, kerr (1972a, b) recorded the haploid chromosome number n = 15 chromosomes in the species f. varia, f. doederleini and f. ghilianii. rocha et al. (2003) reported the same chromosome number and described the karyotype of the species f. doederleini, f. languida and f. varia as part of an analysis of different genera of stingless bees. carvalho & costa (2011) also described the karyotypes of f. dispar and f. francoi, and santos et al. (2018) developed a comparative analysis of the hybridization patterns of microsatellite dna probes in karyotypes of five species, f. dispar, f. doederleini e f. francoi, f. meadewaldoi, frieseomelitta sp. n. and f. varia. in addition, other karyotypic features were reported. in all these cases the chromosome number was consistently n = 15 or 2n = 30. these differ from the chromosome numbers determined for other less closely related genera, such as n = 9 for melipona, and n = 17, common to several of the other genera of stingless bees. however, groups closely related to frieseomelitta such as genus duckeola also have shown n = 15 chromosomes (kerr 1972a, b). the taxonomy and phylogeny of friesomelitta is still not well resolved, and there are some species with broad geographic distribution. in this context, the present study aimed to contribute to the knowledge of the karyotype diversity of this genus, including characterizing samples of new points in the distribution of species and new species. furthermost, we search for data that may aid in the taxonomic resolution of the group and in the understanding of the possible evolutionary mechanisms that occurred in the diversification of the karyotype of the group. material and methods we analyzed samples of four species from different localities of brazil, frieseomelitta doederleini, from the municipality of canavieiras, state of bahia, (15º 61 ‘s, 39º42’ w), frieseomelitta longipes, from the municipality of belém, state of pará, 1:30 ‘s, 48 ° 73’ w); frieseomelitta portoi, from the municipality of rio branco, state of acre (9º98 ‘s, 67º90’ w); and frieseomelitta aff. trichocerata, from the municipality of juína, state of mato grosso (11º52 ‘s, 60º50’ w). taxonomist of bees identified the collected specimens and adult specimens of each of the species were mounted on entomological pins and deposited in the entomological collection of the universidade estadual de santa cruz, ilheus, ba. the slide preparations were made from cells of the cerebral ganglia of specimens in the prepupa stage, according to the protocol described by imai et al. (1988). the prepared slides were stained conventionally with 3% giemsa/sorensens’s buffer and the selected metaphases were photographed on an olympus cx-41 microscope with attached olympus c7070 digital camera. staining with the base-specif ic f luorochromes 4,6-diamidino-2-phenylindole (dapi) and chromomycin a3 (cma3) to evidence the chromosomal regions rich in at (dapi) and cg (cma3), respectively, were performed according to schweizer (1980), with modifications proposed by guerra and souza (2002). coverslips were mounted on slides with antifading vectashield (vector laboratories, burlingame, usa). the images were captured on a leica dmra2 epifluorescence microscope using the leica im50 software (leica microsystems imaging solutions ltda, cambridge, uk). to allow comparison with previous studies, we followed chromosomal nomenclature proposed by imai (1991). (m) metacentric chromosome: the arms of approximately equal sizes and euchromatic, the heterochromatin restricted to the centromeric region; (a) acrocentric chromosome: centromeric region and short heterochromatic arms; (am) pseudoacrocentric chromosome: centromeric region, middle or long heterochromatic arms and short eucrotic arm. the karyograms were organized with the use of adobe photoshop® cs6 13.0x 64 software. from the karyotypes, the chromosome pairs, diploid (2n) and haploid (n) numbers and karyotype formulas (2k) were defined. 123karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) results and discussion the chromosome number found for the species f. doederleini, f. portoi and f. aff. trichocerata was 2n = 30 for females. in f. longipes, females showed 2n = 34 chromosomes (table 1, fig. 1). kerr and silveira, 1972 registered 2n = 30 chromosomes for f. doederleini and f. varia and rocha et al. (2003) found 2n = 30 chromosomes for f. doederleini, f. languida, f. varia. the same number was found by carvalho and costa (2011) for f. dispar and f. portoi. together these results indicate that the frequent diploid number of 30 chromosomes is characteristic of the genus. the finding of 2n = 34 chromosomes in f. longipes comprises the first record of a diploid chromosome number different from 30 in this genus. in the species analyzed here, the following karyotypic formulas were found: f. doederleini, 2k = 4m + 4a + 22am, f. portoi, 2k = 4m + 26a, f. aff. trichocerata, 2k = 6m + 20a + 4am, and f. longipes, 2k = 8m + 12a + 14am (tab. 1). the karyotypic formula observed in f. doederleini is similar to that cited by rocha et al., (2003) for another population of the same species, evidencing intraspecific karyotypic stability. the predominance of acrocentric and pseudoacrocentric (am which contains a long heterochromatic arm) chromosomes in karyotypes was consistent with that observed in previous studies for other species of the genus, such as f. dispar. f. francoi, f. languida, and f. varia (rocha et al., 2003; carvalho and costa, 2011). however, in f. portoi and f. aff. trichocerata was observed a reduced number of am chromosomes. the classical cytogenetics using conventional giemsa staining and c-banding allowed observing heterochromatin in studies of several meliponine genera such as friesella (mampumbu, 2002), leurotrigona (pompolo and campos, 1995), melipona (hoshiba, 1988; rocha & pompolo, 1998; rocha et al., 2002); nannotrigona (hoshiba & imai, 1993), partamona (costa et al., 1992; martins et al., 2012), plebeia (caixeiro & pompolo, 1999), tetragonisca (barth et al., ), trigona (hoshiba and imai, 1993; costa, 2004; domingues et al., 2005), among others. the distribution of heterochromatin has still been the focus of comparative studies in stingless bees (e.g. travenzoli et al., 2019), and for being variable table 1. available karyotype data of frieseomelitta species. 2n = diploid number, 2k = diploid karyotype formula, m = metacentric, a = acrocentric, am = pseudoacrocênctrico chromosomes. species sampling location 2n 2k references f. dispar ilhéus/ba 30 4m + 2m + 4a + 20am  carvalho & costa (2011) f. doederleini santana do seridó/rn canavieiras/ba 30 30 30 4m + 4a + 22am  4m + 4a + 22am kerr & silveira, (1972) rocha et al., (2003) present study f. francoi cairú/ba 30 4m + 2m + 4a + 20am  carvalho & costa (2011) f. languida divinópolis/mg 30 4m + 4a + 22am rocha et al., (2003) f. longipes belém/pa 34 8m + 12a + 14am present study f. portoi rio branco/ac 30 4m + 26a present study f. aff. trichocerata juína/mt 30 6m + 20a + 4am  present study f. varia divinópolis/mg 30 30 4m + 4a + 22am  kerr & silveira, (1972) rocha et al., (2003) figure 1. karyotypes of workers of frieseomelitta species stained with giemsa: (a) f. doederleini (b) f. portoi; (c) f. aff. trichocerata; (d) f. longipes. bar = 10um. 124 renan monteiro do nascimento et al. among genera and species is a useful cytological information for the characterization of species or populations of these bees. imai et al. (1988) have suggested that the tandem addition of terminal heterochromatin in chromosomes is a relatively rapid way of restoring telomeric stability after centric fission events, leading to the formation of am chromosomes. elimination of any excess heterochromatin addition may follow this addition by deletion mechanisms, as a tendency to reduce non-specific heterochromatin interactions of the long pseudoacrocentric chromosomes. the high content of heterochromatin found, however, contrasts with the numerical or even structural stability observed in frieseomelitta, especially considering species such as f. doederlaini, analyzed in different studies and localities of its geographic distribution. the cma3/dapi fluorochrome staining in f. doederleini, showed that the centromeric region of the metacentric, acrocentric and the heterochromatic arm of the pseudoacrocentric chromosomes are rich in at base pairs (dapi+). this same region was also weakly stained with cma3 (fig. 2a). these results were similar to the findings of rocha et al. (2003) for f. varia and this marking pattern may be related to the presence of a nucleolus organizing region. these regions are often labeled by cma3 in karyotypes of bees. nor banding or in situ hybridization of ribosomal probes are likely to confirm this location. in the f. portoi, cma3+/dapibands were identified in short heterochromatic arms in chromosomal pairs 1, 2, 3, 4, 5, 6, 8, 9, 11, 12 and 14. pairs 7, 10 and 13 were totally cma3-/dapi+ in this species (fig. 2b). frieseomelitta aff. trichocerata showed cma3+/dapibands in chromosomal pairs 1, 2, 3, 5, 7, 8, 10 and 11, and pairs 4, 6, 9, 12, 13, 14 and 15 were homogeneously cma3-/dapi+ (fig. 2c). we observed in the four species analyzed here that the first pair showed the cma3+/dapilabel on the short arm. although in f. longipes only one of the homologues was labeled by cma3, suggesting the presence of a heteromorphism, this labeling, which may be associated with the presence of nor, seems to be common in the genus. this can be confirmed in further analyzes that include new species. diverging from the other species, f. longipes had the long arms of pairs 6 and 11 and pair 14 strongly labeled cma3+/dapi(fig. 2d). the results obtained in the present analysis together with the previous karyotypic descriptions show that the karyotype differentiation in frieseomelitta mostly involved minor structural alterations such as heterochromatin gain and loss. however, numerical change occurred in the differentiation of f. longipes, possibly due to centric fission. since this is the only record of a chromosome number other than 2n = 30 in this genus, this is possibly a derived characteristic in this group. the lack of a better taxonomic definition and a more resolved and complete phylogeny for frieseomelitta leaves this question open. if a derived position f. longipes is found, the recent origin of this numerical difference will be confirmed. from the observation of the high frequency of the chromosome number 2n = 30 (n = 15) in the frieseomelitta, it is possible to suggest that this chromosome number is basal for this genus. the n = 15 chromosomes was also found in the species duckeola ghilianii kerr, 1972a, b; kerr and silveira, 1972). according to the meliponini phylogeny proposed by rasmussen and cameron (2010) duckeola is closely related to the genus frieseomelitta. however, without a more detailed phylogenetic assessment, the hypothesis that the 2n = 34 found in f. longipes, alternatively, represents the basal condition for this group can not be discarded, since this chromosome number is found in most genera of neotropical meliponini (rocha et al., 2003). more complete phylogenetic analyzes, including a representative sampling of friesemelitta diversity, however, would be necessary to better clarify these questions. acknowledgments this study was supported by cnpq (conselho nacional de desenvolvimento científico e tecnológico – process number 310178/2015-0). rmn and afc were figure 2. karyotypes 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(2019) karyotypic description and repetitive dna chromosome mapping of melipona interrupta latreille, 1811 (hymenoptera: meliponini). caryologia 72(2), 91-95. caryologia international journal of cytology, cytosystematics and cytogenetics volume 73, issue 2 2020 firenze university press the first molecular identification of egyptian miocene petrified dicot woods (egyptians’ dream becomes a reality) shaimaa s. sobieh*, mona h. darwish gene flow patterns reinforce the ecological plasticity of tropidurus hispidus (squamata: tropiduridae) fernanda ito, danielle j. gama-maia, diego m. a. brito, rodrigo a. torres* the technique of plant dna barcoding: potential application in floriculture antonio giovino1,*, federico martinelli2,*, anna perrone3 cytogenetic of brachyura (decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species alessio iannucci1, stefano cannicci1,2,*, zhongyang lin3, karen wy yuen3, claudio ciofi1, roscoe stanyon1, sara fratini1 comparison of the evolution of orchids with that of bats antonio lima-de-faria identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection mehdi zahravi1,*, panthea vosough-mohebbi2, mehdi changizi3, shahab khaghani1, zahra-sadat shobbar4 populations genetic study of the medicinal species plantago afra l. 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(cluster bean): reasons and repercussions girjesh kumar, shefali singh* karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(4): 21-28, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-641 caryologia international journal of cytology, cytosystematics and cytogenetics citation: halil erhan eroğlu, esra martin, ahmet kahraman, elif gezer aslan (2021) the new chromosomal data and karyotypic variations in genus salvia l. (lamiaceae): dysploidy, polyploidy and symmetrical karyotypes. caryologia 74(4): 21-28. doi: 10.36253/caryologia-641 received: october 01, 2019 accepted: september 24, 2021 published: march 08, 2022 copyright: © 2021 halil erhan eroğlu, esra martin, ahmet kahraman, elif gezer aslan. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid hee: 0000-0002-4509-4712 em: 0000-0002-5484-0676 ak: 0000-0002-9344-1993 ega: 0000-0003-4645-3892 the new chromosomal data and karyotypic variations in genus salvia l. (lamiaceae): dysploidy, polyploidy and symmetrical karyotypes halil erhan eroğlu1,*, esra martin2, ahmet kahraman3, elif gezer aslan4 1 department of biology, faculty of science and art, yozgat bozok university, yozgat, turkey 2 department of biotechnology, faculty of science, necmettin erbakan university, konya, turkey 3 department of biology, faculty of science and arts, uşak university, uşak, turkey 4 department of medical services and techniques, vocational school of health services, kırklareli university, kırklareli, turkey *corresponding author. e-mail: herhan.eroglu@bozok.edu.tr abstract. in this study, it was aimed to determine the chromosome number of 21 salvia l. species, to determine chromosome morphology, to reveal karyotype analysis in detail and to contribute to the cytotaxonomy of salvia. in this context, the results are as follows: (i) the first report for the number of chromosomes of ten species, namely s. corrugata vahl. (2n = 16), s. curviflora benth. (2n = 16), s. darcyi j.compton, s. greggii a.gray, s. longifolia nutt., s. vitifolia benth. (2n = 22), s. subrotunda a.st.-hil. ex benth. (2n = 44), s. oppositiflora ruiz & pav. (2n = 56), s. stolonifera benth. and s. atrocyanea epling (2n = 60); (ii) the karyotypic variations and new chromosome numbers different from previous reports for three species, namely s. cardiophylla benth. (2n = 36), s. cuspidata ruiz & pav. (2n = 44) and s. microphylla sessé & moc. (2n = 46); (iii) the same chromosome numbers from previous reports for eight species, namely s. campanulata wall. ex benth. (2n = 16), s. elegans vahl. (2n = 20), s. involucrata cav., s. mexicana sessé & moc. (2n = 22), s. apiana jeps., s. leucophylla greene, s. mellifera greene (2n = 30), and s. splendens ker gawl. (2n = 44); (iv) the detailed chromosome measurements and karyotype analyses for all species studied for the first time; (v) the symmetrical karyotypes for all studied species; (vi) the variations resulting from dysploidy or polyploidy and discussing their reasons. keywords: chromosomal alteration, karyotype asymmetry, sage, turkey. introduction the word salvia that means sage in turkish is derived from the latin salvare, which means protect and heal because of its medicinal properties. the genus salvia is placed in the family lamiaceae and is one of the largest 22 halil erhan eroğlu et al. genera of the family with nearly 1000 perennials, biennial or annual, often strongly aromatic species throughout the world (sheidai and alijanpoo 2011). this ratio corresponds to one quarter of the family. the salvia species usually spread in tropical and temperate regions of the world. the species are mostly distributed in three different regions: central and south america (about 500 species), west asia (about 200 species) and east asia (about 100 species) (walker and sytsma 2007). turkey, which has 98 species in terms of the diversity of salvia species, is an important gene center in asia (hedge 1982; kahraman et al. 2011). aboveground organs of salvia species have been used in cough, colds, teeth, stomach and abdominal pains and skin diseases since ancient times. most of salvia species are used as folk medicine because of their antioxidant, antidiabetic, antimicrobial, antitumor, antiplasmodial, antihypertensive and anti-inf lammatory properties (ulubelen 2003; kamatou et al. 2008; şenol et al. 2010). some salvia species have been reported to be used to prevent memory loss (perry et al. 1996). in addition, salvia species are frequently used in food, perfumery, cosmetics and pharmaceutical industries (chalchat et al. 1998; baylac and racine 2003). many salvia species are easily cultured frequently due to their aromatic nature; and because of their beautiful appearance, they are grown as decorative ornamental plants in parks and gardens (nakipoğlu 1993; marin et al. 1996). many karyological reports showed that salvia is a polybasic genus with diverse chromosome numbers in different regions of the world and the species are polyploid origins (sheidai and alijanpoo 2011). it was reported that the basic number is x = 16 for california species (epling et al. 1962); is x = 11 for species of russia and europe (patudin et al. 1975); is x = 7 for mediterranean species (afzal-rafii 1976). according to the chromosome databases, comprehensive chromosomal reports exist in genus salvia. due to the high number of species and samples, there may be some cytotaxonomic uncertainties. the purpose of this work is to contribute to the cytotaxonomy of salvia with the following questions: (1) the chromosome numbers of which species will be reported for the first time? (2) are there species with karyotypic variations and new chromosome numbers different from previous reports? (3) what are the detailed chromosome measurements and karyotype analysis results for all species? (4) what are the karyotype asymmetry states for all species? symmetrical or asymmetrical. (5) what are the chromosomal variations caused by polyploidy and dysploidy in genus salvia? (6) what are the possible causes of polyploidy, dysploidy, and symmetrical/asymmetrical karyotypes? materials and methods the seeds of the plants included in the study were provided by mr. robin middleton, who cultivated many salvia species in his personal botanical garden in england. identification and confirmation of the specimens were performed by the third author of this study. the cytogenetical study was conducted on root tips germinated on wet filter paper in petri dishes. after germination, the fresh root tip meristems were pretreated in α-mono-bromonaphthalene at 4°c for 16 hours, fixed in glacial acetic acid and absolute alcohol (1:3) at 4°c for 24 hours, deposited in 70% ethanol at 4°c, and then hydrolyzed in 1 n hcl at room temperature for 12 minutes. finally, they were squashed and stained in 2% aceto-orcein. permanent slides were prepared using standard liquid nitrogen method (altay et al. 2017; martin et al. 2019). karyotypes were determined using image analysis system (bs200pro) on a personal computer. 10 mitotic plates were assessed to determine the chromosome numbers. the following variables were measured: long arm (la), short arm (sa), total chromosome length (la + sa), arm ratio (la / sa), centromeric index [(sa / la + sa) × 100], total haploid length (thl), mean chromosome length (mcl), and relative length (rl%). centromere positions and karyotype formulae of 17 salvia species were determined. from the point of view of chromosome morphology, median (m, m), submedian (sm) and subtelocentric (st) chromosome pairs were observed (levan et al. 1964). as centromere positions of the other taxa (s. cardiophylla, s. cuspidata, s. oppositiflora, and s. atrocyanea) could not be determined, their total chromosome length and haploid chromosome length were measured. intrachromosomal asymmetry and interchromosomal asymmetry were determined with the parameters of mca (peruzzi and eroğlu 2013) and cvcl (paszko 2006), respectively. the intrachromosomal asymmetry increases by shifting of centromere position from the center to the end of the chromosome. in this case there is a transition from median/submedian chromosomes to subterminal/terminal chromosomes. the interchromosomal asymmetry depends on relative variation in chromosome length, namely it determines how different the chromosome lengths of a complement. finally, a scatter diagram was drawn between mca and cvcl. results chromosomal data chromosome records of 21 taxa are herein provided (figure 1), ten of which are reported for the first time, 23the new chromosomal data and karyotypic variations in genus salvia figure 1. mitotic metaphase chromosomes of salvia: (a) s. corrugata, (b) s. campanulata, (c) s. curviflora, (d) s. elegans, (e) s. darcyi, (f) s. greggii, (g) s. involucrata, (h) s. longifolia, (i) s. vitifolia, (j) s. mexicana, (k) s. apiana, (l) s. leucophylla, (m) s. mellifera, (n) s. cardiophylla, (o) s. cuspidata, (p) s. splendens, (r) s. subrotunda, (s) s. microphylla, (t) s. oppositiflora, (u) s. stolonifera, (v) s. atrocyanea (scale bar: 10 μm). 24 halil erhan eroğlu et al. three possess new chromosome numbers, and eight have the same results including previous reports. ten different chromosome numbers (2n = 16, 18, 20, 22, 30, 36, 44, 46, 56, and 60) are also detected (table 1). among the studied taxa, the smallest and the largest chromosome shapes are 0.53 μm in s. oppositiflora and 3.28 μm in s. campanulata, respectively. the smallest and the highest values of total haploid chromosome length are 9.12 μm in s. curviflora and 36.92 μm in s. stolonifera, respectively (table 2). in addition, the detailed chromosome measurements of all chromosome pairs are given in supplemental online material (supplementary tables 1–21). basic numbers and ploidy levels there are six basic chromosome numbers within salvia, namely x = 7 in only one species, x = 8 in two species, x = 9 in two species, x = 10 in six species, most common x = 11 in nine species, and x = 23 (probably dysploidy) in only one species. the ploidy levels are 2x (in 11 species), 3x (in three species), 4x (in four species), 6x (in two species), and 8x (in only one species) (table 2). the monoploid ideograms generated by the basic chromosome numbers are given in figure 2. karyotype formula and karyotype asymmetry 17 taxa possess median (m) and submedian (sm), whereas none subtelocentric (st) chromosomes and telocentric (t) chromosomes. due to the uncertainty of centromere positions, the karyotype formulae of four taxa are not given, namely s. cardiophylla, s. cuspidata, s. oppositiflora, and s. atrocyanea. four different formulae are observed, namely (1) m-m, (2) m, (3) m-sm, and (4) m-m-sm. the mca values for intrachromosomal asymmetry vary from 14.94 in s. curviflora to 26.01 in s. corrugata and are characterized by taxa with symmetric karyotypes consisting entirely of median and submedian chromosomes. the cvcl values for interchromosomal asymmetry vary from 10.73 in s. longifolia to 22.13 in s. mellifera (table 2). table 1. the chromosome counts of the investigated species in present and previous studies. species previous results references present results explanation n 2n 2n s. corrugata 16 first report s. campanulata 8 32 saggoo and bir 1986; hu et al. 2016 16 detailed measurements s. curviflora 18 first report s. elegans 10 cherian and kuriachan 1990 20 detailed measurements s. darcyi 22 first report s. greggii 22 first report s. involucrata 7 22 + 0-1b gill 1984; alberto et al. 2003 22 detailed measurements s. longifolia 22 first report s. vitifolia 22 first report s. mexicana 22 palomino et al. 1986 22 detailed measurements s. apiana 15, 16 32 carlson and stuart 1936; stewart 1939 30 detailed measurements s. leucophylla 24, 30 stewart 1939; epling et al. 1962 30 detailed measurements s. mellifera 30, 32 epling et al. 1962; stewart 1939 30 detailed measurements s. cardiophylla 44 + 0-1b alberto et al. 2003 36 new count s. cuspidata 22 alberto et al. 2003 44 new count s. splendens 8 32, 44 44 + 0-1b carlson and stuart 1936; haque and ghoshal 1980; gill 1984; alberto et al. 2003 44 detailed measurements s. subrotunda 44 first report s. microphylla 11 22 haque and ghoshal 1980; alberto et al. 2003 46 new count s. oppositiflora – – 56 first report s. stolonifera – – 60 first report s. atrocyanea – – 60 first report 25the new chromosomal data and karyotypic variations in genus salvia discussion table 1 shows the chromosome counts of the investigated species in present and previous studies. the chromosome numbers are the first report for ten species, namely s. corrugata (2n = 16), s. curviflora (2n = 16), s. darcyi, s. greggii, s. longifolia, s. vitifolia (2n = 22), s. subrotunda (2n = 44), s. oppositiflora (2n = 56), s. stolonifera and s. atrocyanea (2n = 60). the chromosome numbers are new counts different from previous reports for three species, namely s. cardiophylla (2n = 36), s. cuspidata (2n = 44) and s. microphylla (2n = 46). in literature, the chromosome numbers are 2n = 44 for s. cardiophylla, 2n = 22 for s. cuspidata and s. microphylla (haque and ghoshal 1980; alberto et al. 2003). the chromosome numbers of the other eight species are the same as the previous reports, namely s. campanulata (2n = 16), s. elegans (2n = 20), s. involucrata and s. mexicana (2n = 22), s. apiana, s. leucophylla, and s. mellifera (2n = 30) and s. splendens (2n = 44) (carlson and stuart 1936; epling et al. 1962; haque and ghoshal 1980; palomino et al. 1986; saggoo and bir 1986; cherian and kuriachan 1990; alberto et al. 2003). it is already known that genus salvia includes diploids and polyploids (carlson and stuart 1936; epling et al. 1962; haque and ghoshal 1980; gill 1984; alberto et al. 2003; hu et al. 2016). with chromosome data available at present, 11 species are diploids with 2n = 16, 18, 20, 22, and 46 (probably dysploidy) (c.52% of the species with available data) and 10 species are polyploids (c.48% of the species with available data). when previous and current chromosomal data are compared, four species, s. campanulata, s. cuspidata, s. splendens, and s. microphylla, show both diploid and polyploid status (c.19% of the species with available data). this suggests that intraspecific polyploidy may be common in genus salvia. the polyploid nature are demonstrated by the prevalence of cells with 2n = 30, 36, 44, 56, and 60 chromosomes in 10 species. polyploidy originates by autopolyploidy mechanism (genome duplication in a species) and allopolyploidy (genome duplication with hybridization between species) and has played a major role in the speciation and evolution of higher plants (demirci kayıran and özhatay 2017). the polyploidy possibly caused by glacial, climatic changes, altitude and high latitudes may have contributed to salvia speciatable 2. the karyological features of the studied salvia taxa; karyotype formula (kf), shortest chromosome length (sc), longest chromosome length (lc), relative length (rl), total haploid chromosome length (thl), mean chromosome length (mcl), centromeric index (ci), coefficient of variation of chromosome length (cvcl), mean centromeric asymmetry (mca), median point (m), median (m), submedian (sm). taxa kf sc (μm) lc (μm) rl (%) sc–lc thl (μm) mcl (μm) ci (min–max) cvcl mca s. corrugata 8m + 8sm 1.55 2.44 10.58–16.66 14.65 1.83 31.55–41.38 15.84 26.01 s. campanulata 10m + 6sm 1.92 3.28 9.41–16.08 20.40 2.55 36.16–45.97 17.29 22.14 s. curviflora 2m + 16m 0.72 1.35 7.89–14.80 9.12 1.01 38.89–50.00 20.05 14.94 s. elegans 20m 1.21 2.16 7.48–13.35 16.18 1.62 37.13–45.22 17.06 18.55 s. darcyi 18m + 4sm 1.17 1.84 7.49–11.78 15.62 1.42 27.17–48.51 12.91 16.30 s. greggii 2m + 14m + 6sm 0.84 1.60 6.87–13.08 12.23 1.11 32.71–50.00 20.28 19.59 s. involucrata 2m + 14m + 6sm 1.03 1.76 7.05–12.04 14.62 1.33 30.00–50.00 16.00 20.90 s. longifolia 2m + 14m + 6sm 1.09 1.60 7.14–10.48 15.26 1.39 26.25–50.00 10.73 22.59 s. vitifolia 14m + 8sm 1.22 2.21 6.66–12.06 18.33 1.67 31.15–43.95 17.33 21.48 s. mexicana 20m + 2sm 0.97 1.70 6.37–11.16 15.23 1.38 35.37–44.88 15.94 17.32 s. apiana 28m + 2sm 1.07 1.93 4.87–8.79 21.95 1.46 33.86–46.34 16.17 17.31 s. leucophylla 26m + 4sm 0.95 1.81 5.01–9.55 18.96 1.26 35.00–43.28 17.62 20.83 s. mellifera 26m + 4sm 0.97 2.17 4.44–9.92 21.87 1.46 34.75–45.89 22.13 18.26 s. cardiophylla 0.82 1.61 3.70–7.26 22.19 1.23 s. cuspidata 1.05 1.88 3.43–6.14 30.63 1.39 s. splendens 28m + 16sm 0.72 1.38 3.23–6.20 22.26 1.01 27.27–45.79 18.23 23.67 s. subrotunda 2m + 26m + 16sm 0.75 1.42 3.18–6.01 23.62 1.07 25.96–50.00 15.32 21.92 s. microphylla 34m + 12sm 0.78 1.89 2.49–6.03 31.36 1.36 31.53–46.56 19.53 21.43 s. oppositiflora 0.53 1.21 2.31–5.27 22.98 0.82 s. stolonifera 48m + 12sm 0.81 1.73 2.19–4.69 36.92 1.23 25.49–46.34 18.79 22.40 s. atrocyanea 0.62 1.51 2.23–5.43 27.80 0.93 26 halil erhan eroğlu et al. tion. although salvia is a polybasic genus with species of polyploid origin (sheidai and alijanpoo 2011), variations are observed resulting from dysploidy shows that different basic numbers with karyotypes that contain one or a few chromosomes more or less than that of the original, occur in a genus. s. microphylla has different basic number (x = 23) probably with dysploidy. these data indicate that the effects of dysploidy on the lineage diversification of salvia should be investigated further. in studied species, b-chromosomes, a special type of supernumerary chromosomes and are extra chromosomes other than basic a-chromosomes in diploid and polyploid species, have been reported. the karyotype formulae are 22 + 0-1b in s. involucrata and 44 + 0-1b in s. cardiophylla and s. splendens (alberto et al. 2003). we have not observed b-chromosomes. as a matter of fact, while b-chromosomes do not exist in some individuals of the same population, the others may have different numbers. when the number of b-chromosomes is small, they cannot have a visible effect on the phenotype and their presence can be determined only by cytological examinations. in case of high numbers, they have a negative effect on the development and fertility of plants (houben 2017). a symmetric karyotype contains a high proportion of median and submedian chromosomes, unlike an asymmetric karyotype has a high rate of subterminal and terminal chromosomes (peruzzi and eroğlu 2013). in intrachromosomal asymmetry, the most symmetrical and asymmetrical karyotype are s. curviflora and s. corrugata, respectively. the relatively higher asymmetric karyotypes than other species may have been caused by chromosomal structural changes as centric fission or centric fusion observed in especially polyploid and dysploidy species. in interchromosomal asymmetry, the most symmetrical and asymmetrical karyotype are s. longifolia and s. mellifera, respectively. the relatively higher asymmetric karyotypes than other species may be the result of chromosome rearrangements and may also result in bimodality observed in s. campanulata, s. splendens, and s. microphylla. in these species, the bimodal karyotypes may occur due to loss of chromosomal segments following polyploidy. the symmetric and asymmetric karyotypes are different between intrachromosomal asymmetry and interchromosomal asymmetry with very low positive correlation (r = 0.157) (figure 3). all studied salvia species contain only median and submedian chromosomes and are symmetrical as a common condition figure 2. ideograms of salvia: (a) s. corrugata, (b) s. campanulata, (c) s. curviflora, (d) s. elegans, (e) s. darcyi, (f) s. greggii, (g) s. involucrata, (h) s. longifolia, (i) s. vitifolia, (j) s. mexicana, (k) s. apiana, (l) s. leucophylla, (m) s. mellifera, (n) s. cardiophylla, (o) s. cuspidata, (p) s. splendens, (r) s. subrotunda, (s) s. microphylla, (t) s. oppositiflora, (u) s. stolonifera, (v) s. atrocyanea (scale bars: 1 μm). 27the new chromosomal data and karyotypic variations in genus salvia in genus salvia (sheidai and alijanpoo 2011; doğan et al. 2019). on the contrary, hu et al. (2016) reported that s. bulleyana diels, s. digitaloides diels and s. przewalskii maxim. had asymmetrical karyotypes. in this study, it was aimed to determine the chromosome number of 21 salvia species, to determine chromosome morphology, to reveal karyotype analysis in detail and to contribute to the cytotaxonomy of salvia. in this context, the results are as follows: (i) the first report for the number of chromosomes of ten species, (ii) the karyotypic variations and new chromosome numbers different from previous reports for three species, (iii) the detailed chromosome measurements and karyotype analyses for all species studied for the first time, (iv) the symmetrical karyotypes for all studied species, (v) the variations resulting from dysploidy or polyploidy and discussing their reasons. on the other hand, the genus salvia is one of the largest in the world with about 1000 species. the results of such studies provide important data supports for salvia cytotaxonomy. it is an important issue that combining all supporting data with further comparative studies and integrating them into morphological data. acknowledgments this work was supported by the [uşak university scientific research projects fund] under grant [number 2013/mf003]. references afzal-rafii z. 1976. etude cytotaxonomique et phylogenetique de quelques salvia de la region mediterrafigure 3. scatter diagram between mca and cvcl: (a) s. corrugata, (b) s. campanulata, (c) s. curviflora, (d) s. elegans, (e) s. darcyi, (f) s. greggii, (g) s. involucrata, (h) s. longifolia, (i) s. vitifolia, (j) s. mexicana, (k) s. apiana, (l) s. leucophylla, (m) s. mellifera, (n) s. splendens, (o) s. subrotunda, (p) s. microphylla, (r) s. stolonifera. 28 halil erhan eroğlu et al. neenne: groupe du salvia officinalis l. acta bot gall. 123:515–531. alberto cm, sanso am, xifreda cc. 2003. chromosomal studies in species of salvia (lamiaceae) from argentin. bot j linn soc. 141:483–490. altay d, eroğlu he, hamzaoğlu e, koç m. 2017. karyotype analysis of some taxa of dianthus section verruculosi (caryophyllaceae, sileneae). turk j bot. 41:367–374. carlson em, stuart bc. 1936. development of spores and gametophytes in certain new world species of salvia. new phytol. 35:1–49. demirci kayıran s, özhatay fn. 2017. a karyomorphological study on the genus muscari mill. growing in kahramanmaraş (turkey). turk j bot. 41:289–298. doğan h, eroğlu he, coşge şenkal b, cesur c, uskutoğlu t, özkan u, altay d. 2019. the detailed chromosome measurements and b-chromosomes in salvia virgata. acta biologica turcica. 32:23–26. epling c, lewis h, raven ph. 1962. chromosomes of salvia, section audibertia. aliso. 5:217–221. gill ls. 1984. the incidence of polyploidy in the westhimalayan labiatae. rev cytol biol vég bot. 7:5–16. haque ms, ghoshal kk. 1980. karyotypes and chromosome morphology in the genus salvia linn. cytologia. 45:627–640. hedge ic. 1982. salvia l. in: davis ph, editor. flora of turkey and the east aegean islands, vol. 7. edinburgh: edinburgh university press; p. 400–461. houben a. 2017. b chromosomes – a matter of chromosome drive. front plant sci. 8:1–6. hu gx, xiang cl, liu ed, dong hj, funamoto t. 2016. karyotypic study of eighteen taxa of salvia (lamiaceae) from china. caryologia. 69:50–57. kahraman a, doğan m, celep f. 2011. salvia siirtica (lamiaceae), a new endangered species from turkey. nord j bot. 29:397–401. kamatou gpp, makunga np, ramagola wpn, viljoen am. 2008. south african salvia species: a review of biological activities and phytochemistry. j ethnopharmacol. 119:667–672. levan a, freda k, sandberg aa. 1964. nomenclature for centromeric position on chromosomes. hereditas. 52:201–220. marin pd, duletic s, petkovic b. 1996. nutlet ornamentation in selected salvia l. species (lamiaceae). fl medit. 6:203–211. martin e, i̇çyer doğan g, karaman erkul s, eroğlu he. 2019. karyotype analyses of 25 turkish taxa of astragalus from the sections macrophyllium, hymenostegis, hymenocoleus, and anthylloidei (fabaceae). turk j bot. 43:232–242. nakipoğlu m. 1993. bazı adaçayı (salvia l.) türleri ve bu türlerin ekonomik önemi. dokuz eylül üniversitesi yayınları, eğitim fakültesi dergisi. 6:45–58. palomino g, mercado p, ramamoorthy tp. 1986. chromosomes of salvia subgenus calosphace (lamiaceae), a preliminary report. cytologia. 51:381–386. paszko b. 2006. a critical review and a new proposal of karyotype asymmetry indices. plant syst evol. 258:39–48. patudin av, yurtsev vn, pakaln da. 1975. chromosome number in some species of salvia l. 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(caprifoliaceae), using scot molecular markers fengzhen chen1, dongmei li2,* , mohsen farshadfar3 the new chromosomal data and karyotypic variations in genus salvia l. (lamiaceae): dysploidy, polyploidy and symmetrical karyotypes halil erhan eroğlu1,*, esra martin2, ahmet kahraman3, elif gezer aslan4 cytogenetic survey of eight ant species from the amazon rainforest luísa antônia campos barros1, gisele amaro teixeira2, paulo castro ferreira1, rodrigo batista lod1, linda inês silveira3, frédéric petitclerc4, jérôme orivel4, hilton jeferson alves cardoso de aguiar1,5,* molecular phylogeny and morphometric analyses in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f. neda atazadeh1,*, masoud sheidai1, farideh attar2, fahimeh koohdar1 a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) melika tabasi1, masoud sheidai1,*, fahimeh koohdar1, darab hassani2 genetic diversity and relationships among glaucium (papaveraceae) species by issr markers: a high value medicinal plant lu feng1,*, fariba noedoost2 morphometric analysis and genetic diversity in rindera (boraginaceae-cynoglosseae) using sequence related amplified polymorphism xixi yao1, haodong liu2,*, maede shahiri tabarestani3 biosystematics, fingerprinting and dna barcoding study of the genus lallemantia based on scot and remap markers fahimeh koohdar*, neda aram, masoud sheidai karyotype analysis in 21 plant families from the qinghai–tibetan plateau and its evolutionary implications ning zhou1,2, ai-gen fu3, guang-yan wang1,2,*, yong-ping yang1,2,* some molecular cytogenetic markers and classical chromosomal features of spilopelia chinensis (scopoli, 1786) and tachybaptus ruficollis (pallas, 1764) in thailand isara patawang1,*, sarawut kaewsri2, sitthisak jantarat3, praween supanuam4, sarun jumrusthanasan2, alongklod tanomtong5 centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates) simona ceraulo, vanessa milioto, francesca dumas* repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil marina souza cunha1,2,*,#, silvana melo1,3,#, filipe schitini salgado1,2, cidimar estevam assis1, jorge abdala dergam1,* karyomorphology of some crocus l. taxa from uşak province in turkey aykut yilmaz*, yudum yeltekin variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l. egizia falistocco1,*,+, gianpiero marconi1,+, lorenzo raggi1, daniele rosellini1, marilena ceccarelli2, emidio albertini1 caryologia. international journal of cytology, cytosystematics and cytogenetics 72(1): 23-28, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-248 citation: w. thongnetr, a. tanomtong, s. prasopsin, n. maneechot, k. pinthong, i. patawang (2019) cytogenetic study of the bent-toed gecko (reptilia, gekkonidae) in thailand; i: chromosomal classical features and nors characterization of cyrtodactylus kunyai and c. interdigitalis. caryologia 72(1): 23-28. doi: 10.13128/cayologia-248 received: 19th may 2018 accepted: 19th october 2018 published: 10th may 2019 copyright: © 2019 w. thongnetr, a. tanomtong, s. prasopsin, n. maneechot, k. pinthong, i. patawang. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. cytogenetic study of the bent-toed gecko (reptilia, gekkonidae) in thailand; i: chromosomal classical features and nors characterization of cyrtodactylus kunyai and c. interdigitalis weera thongnetr1,2, alongklod tanomtong1,3, suphat prasopsin4, nuntiya maneechot5, krit pinthong5, isara patawang6,7,* 1 department of biology, faculty of science, khon kaen university, muang, khon kaen, thailand 2 walai rukhavej botanical research institute, mahasarakham university, kantharawichai, maha sarakham, thailand 3 toxic substances in livestock and aquatic animals research group, khon kaen university, muang, khon kaen, thailand 4 research academic supports division, mahidol university, kanchanaburi campus, saiyok, kanchanaburi, thailand 5 department of fundamental science, faculty of science and technology, surindra rajabhat university, muang, surin, thailand 6 department of biology, faculty of science, chiang mai university, muang, chiang mai, thailand 7 center of excellence in bioresources for agriculture, industry and medicine, chiang mai university, muang, chiang mai, thailand * corresponding author: isara.p@cmu.ac.th abstract. this study analysed the karyotype of cyrtodactylus kunyai and c. interdigitalis from loei province in northeastern thailand. the metaphase and meiotic chromosome preparations were obtained by squash technique from bone marrow and testes, respectively. the chromosomes were stained by giemsa staining and ag-nor-banding techniques. the results showed diploid chromosome number (2n) of 40 for c. kunyai and 42 for c. interdigitalis. the chromosome types of metacentric, submetacentric, acrocentric and telocentric chromosomes were 8-4-0-28 and 4-2-4-32, respectively. the agnors banding technique provides the pair of nucleolar organizer regions (nors) of both two species at telomeric region of the long arm of the pair 12, metacentric type in c. kunyai and telocentric type in c. interdigitalis. there are no sex differences in karyotypes between males and females of both two species. we found that during metaphase i on meiosis of c. kunyai and c. interdigitalis, the homologous chromosomes showed synapsis of 20 and 21 bivalents, respectively. moreover, the meiotic phase on prophase ii exhibited 20 and 21 haploid chromosome number (n) as respective diploid species. their karyotype formulas is as follows: c. kunyai (2n = 40): lm2 + lsm4 + lt4 + mt6 + sm6 + st18, and c. interdigitalis (2n = 42): la4 + lt14 + mt2 + sm4 + ssm2 + st16. keywords. cyrtodactylus, cyrtodactylus kunyai, cyrtodactylus interdigitalis, karyotype stasis. 24 weera thongnetr et al. introduction the bent-toed gecko, genus cyrtodactylus gray 1827, belong to the class reptilia, order squamata, suborder lacertilia, and family gekkonidae. the cyrtodactylus is the most diverse gekkonid genus with ~250 species (uetz et al. 2018). the species distribution of cyrtodactylus found in mainland of asia, from boundary between middle east and india to southeast asia, archipelago of southeast asia and pacific to australia (shea et al. 2011; hartmann et al. 2016). in thailand, there are about 24 species of cyrtodactylus that were reported: including c. angularis, c. auribalteatus, c. brevipalmatus, c. chanhomeae, c. consobrinus, c. dumnuii, c. erythrops, c. feae, c. interdigitalis, c. intermedius, c. jarujini, c. lekaguli, c. macrotuberculatus, c. oldhami, c. papilionoides, c. peguensis, c. phuketensis, c. pulchellus, c. quadrivirgatus, c. sumonthai, c. surin, c. thirakhupti, c. tigroides, and c. variegatus (chuaynkern and chuaynkern 2012). among the gekkonidae population in thailand, the cyrtodactylus is the most diverse group than other gekkonid genera and there is continually new species discovered. karyological analyses in bent-toed gecko thus far has differentiated species based on mitotic metaphase chromosomal morphology while sporadic reports have based the species differentiation based on meiotic metaphase chromosomal morphology. thus the basic diploid number of the bent-toed gecko is in the range of 42-48 (ota et al. 1992). example of chromosome study of other gekkonid that have been reported such as; gekko: diploid number ranging from 38-42 and mostly 38 (ota 1989; shibaike et al. 2009; trifonov et al. 2011; patawang et al. 2014; patawang and tanomtong 2015a), hemidactylus: diploid distance from 40-56 and mostly 40 or 46 (de smet 1981; patawang and tanomtong 2015b), gehyra: mostly 44 (king 1984), ptychozoon: 2n = 34 and 42 (ota and hikida 1988), paroedura: diploid number ranging from 31-38 and mostly 36 (aprea et al. 2013; koubová et al. 2014), phelsuma: 2n = 36 (aprea et al. 1996), and dixonius: 2n = 42 (ota et al. 2001). most gekkonid chromosome complements consist of acrocentric or telocentric chromosomes which gradually decrease in size, and karyotype evolution within the group is accompanied by robertsonian fissions, fusions and pericentric inversions (gorman 1973). the report of king (1987) presented eight putative ancestral karyomorphs (2n = 32, 34, 36, 38, 40, 42, 44, and 46 all acrocentric or telocentric chromosomes) and assigned the genus cyrtodactylus to a group sharing an ancestral karyomorph consisting of 2n = 42 uni-armed chromosomes. in thailand, there are no studies of cyrtodactylus’s chromosome or karyotypic analyses. the present study of the cytogenetic of two cyrtodactylus provides the first report of both two species, overall on the conventional giemsa, ag-nor banding, and meiotic cell division. data provided here will increase our knowledge of cytogenetic information which can be used as a basis to comprehensively examine the taxonomy and evolutionary relationship of cyrtodactylus species and other gekkonid. materials and method five male and four female specimens of c. kunyai (figure 1a) and five male and six female of c. interdigitalis (figure 1b) were collected from puan phu sub-district, nong hin district, loei province, northeastern thailand. chromosome preparation was conducted by the squash technique, from bone marrow for mitotic cell and testis for male meiotic cell, and followed with colchicine-hy potonic-f i xation-air-dr y ing technique (patawang et al. 2014). the chromosomes were stained with 10% giemsa for 30 min and nors were identified through ag-nor staining (howell and black 1980; rooney 2001). the length of short arm (ls) and long arm (ll) chromosomes were measured and calculated for the length of total arm chromosomes (lt, lt = ls+ll). relative length (rl, rl = lt/∑lt) and centromeric index (ci, ci = ll/lt) were estimated. ci was also computed to classify the types of chromosomes (turpin and lejeune 1965; chaiyasut 1989). all parameters were used in karyotyping and idiograming. results and discussion mitotic chromosome features from giemsa staining karyomorphology of the c. kunyai and c. interdigitalis revealed that the diploid chromosome number (2n) fig. 1. general characteristic of male cyrtodactylus kunyai (a) and female c. interdigitalis (b) (scale bar = 2 cm). 25cytogenetic study of the bent-toed gecko (reptilia, gekkonidae) in thailand is 40 and 42, respectively. the karyotype of c. kunyai composed of 8 metacentric, 4 submetacentric, and 28 telocentric (table 1 and figures 2a-b), while the karyotype of c. interdigitalis comprised 4 metacentric, 2 submetacentric, 4 acrocentric, and 32 telocentric (table 2 and figures 2c-d). both two species exhibit no sex differences in karyotypes between males and females (figures 2a-d). chromosome sizes of c. kunyai have pairs 1st to 5th to be large, pairs 6th to 8th to be medium, and pairs 9th to 20th to be small (figure 3a). for the size of c. interdigitalis included large size in pairs 1st to 9th, medium size in the 10th, and small size in pairs 11th to 21st (figure 3b). the diploid number of c. kunyai (2n = 40) and c. interdigitalis (2n = 42), both showed difference and accordance with others cyrtodactylus that have been reported (table 3). however, overall of these karyotypes of c. kunyai and c. interdigitalis resemble to other cyrtodactylus and other gekkonid, which comprised many gradient mono-armed (telocentric) and few bi-armed chromosomes (metaor submetacentric). proximity of chromosome number and karyotype feature within genus cyrtodactylus represents a close evolutionary line in the group. nucleolar organizer region and meiotic cell characteristics the objective of the ag-nor banding technique is to detect nucleolar organizer regions (nors) which represent the location of genes that have a function in ribosome synthesis (18s and 28s ribosomal rna). the first cytogenetic study of c. kunyai and c. interdigitalis performed by ag-nor banding technique was obtained from this research. we found the clearly observable nors on the region adjacent to telomere of long arm of the metacentric chromosome pair 12th (figures 4a-b) for c. kunyai and on the region adjacent to telomere of long arm of the telocentric chromosome pair 12th (figures 5a-b) for c. interdigitalis. compared with other geckos, most showed two nors appearing near telomeric region of small bi-armed or small mono-armed chromosome. an example of the previous reports of the geckos’ nor fig. 2. conventionally stained somatic metaphase complement and karyotypes of male (a) and female (b) of cyrtodactylus kunyai, 2n = 40, and male (c) and female (d) of c. interdigitalis, 2n = 42, (scale bars = 10 μm). table 1. mean length of short arm chromosome (ls), long arm chromosome (ll), total chromosomes (lt), centromeric index (ci), relative length (rl) and standard deviation (sd) of ci and rl from 20 metaphases of male and female cyrtodactylus kunyai, 2n = 40. ch.p ls ll lt ci±sd rl±sd ch.s ch.t 1 4.306 6.392 10.698 0.597±0.005 0.106±0.005 l m 2 2.753 5.639 8.392 0.672±0.036 0.083±0.000 l sm 3 2.911 5.120 8.031 0.637±0.042 0.080±0.003 l sm 4 0.000 7.179 7.179 1.000±0.000 0.071±0.003 l t 5 0.000 6.838 6.838 1.000±0.000 0.068±0.003 l t 6 0.000 6.452 6.452 1.000±0.000 0.064±0.002 m t 7 0.000 5.837 5.837 1.000±0.000 0.058±0.003 m t 8 0.000 5.443 5.443 1.000±0.000 0.054±0.003 m t 9 0.000 5.210 5.210 1.000±0.000 0.052±0.002 s t 10 0.000 4.799 4.799 1.000±0.000 0.048±0.003 s t 11 0.000 4.208 4.208 1.000±0.000 0.042±0.004 s t 12* 1.946 2.147 4.093 0.525±0.017 0.041±0.003 s m 13 0.000 3.319 3.319 1.000±0.000 0.033±0.002 s t 14 0.000 3.300 3.300 1.000±0.000 0.033±0.003 s t 15 0.000 3.189 3.189 1.000±0.000 0.032±0.003 s t 16 1.570 1.580 3.150 0.547±0.030 0.035±0.003 s m 17 0.000 2.793 2.793 1.000±0.000 0.028±0.003 s t 18 0.000 2.654 2.654 1.000±0.000 0.026±0.002 s t 19 0.000 2.443 2.443 1.000±0.000 0.024±0.003 s t 20 1.136 1.156 2.292 0.552±0.020 0.025±0.002 s m abbreviations: ch.p, chromosome pair; ch.s, chromosome size; ch.t, chromosome type; *, nucleolar organizer region; l, large size; m, medium size; s, small size; m, metacentric; sm, submetacentric; t, telocentric. 26 weera thongnetr et al. localization included in the genus gehyra (king 1983), gekko (chen et al. 1986; shibaike et al. 2009; patawang et al. 2014), hemidactylus (patawang and tanomtong 2015b), and lepidodactylus (trifonov et al. 2015). these previous studies showed the nor appearing near terminal region of one homologous small chromosome. the present study on male meiotic cell division in c. kunyai and c. interdigitalis found the late interphase to early prophase that the cell of each species showed two signals of nucleolus by positive silver staining (figures 4c, 5c). the characteristics of two nucleolus structure at early phase of cell division supported the appearing of two nors on one homologous at metaphase chromosome in both two cyrtodactylus species. we found diplotene phase in meiotic cell of both two species (figures 4d, 5d), which showed synapsis between two of homologous and compacted chromosome. the metaphase i (meiosis i, reductional division) was found in two species, which can be defined as the 20 bivalents for c. kunyai (figure 4e) and 21 bivalents for c. interdigitalis (figure 5e). no metaphase i cells with partially paired bivalents, which are speculated to be male heteromorphic sex chromosomes in both two cyrtodactylus species. moreover, n = 20 in c. kunyai (figure 4f) and n = 21 in c. interdigitalis (figure 5f ) were found at metaphase ii (meiosis ii, equational division) of spermatid cells. form these results, behavior and number of chromosome in metaphase i and metaphase ii confirmed of each other’s accuracy and also verified the accuracy of diploid chromosome in somatic cells. overview of chromosomal feature of the two cyrtodactylus gekkonid chromosome that has been reported in the past, most species show the gradient karyotype, which comprising of many mono-armed chromosomes table 2. mean length of short arm chromosome (ls), long arm chromosome (ll), total chromosomes (lt), centromeric index (ci), relative length (rl) and standard deviation (sd) of ci and rl from 20 metaphases of male and female cyrtodactylus interdigitalis, 2n = 42. ch.p ls ll lt ci±sd rl±sd ch.s ch.t 1 2.715 6.857 9.572 0.716±0.012 0.090±0.005 l a 2 2.193 6.374 8.567 0.744±0.018 0.081±0.003 l a 3 0.000 8.136 8.136 1.000±0.000 0.077±0.004 l t 4 0.000 7.822 7.822 1.000±0.000 0.074±0.003 l t 5 0.000 7.505 7.505 1.000±0.000 0.071±0.005 l t 6 0.000 6.513 6.513 1.000±0.000 0.062±0.002 l t 7 0.000 6.066 6.066 1.000±0.000 0.057±0.003 l t 8 0.000 5.874 5.874 1.000±0.000 0.056±0.003 l t 9 0.000 5.677 5.677 1.000±0.000 0.054±0.002 l t 10 0.000 5.337 5.337 1.000±0.000 0.050±0.003 m t 11 0.000 4.539 4.539 1.000±0.000 0.043±0.004 s t 12* 0.000 4.031 4.031 1.000±0.000 0.038±0.003 s t 13 0.000 3.808 3.808 1.000±0.000 0.036±0.003 s t 14 1.824 1.830 3.654 0.501±0.027 0.035±0.004 s m 15 1.102 2.235 3.337 0.670±0.041 0.032±0.004 s sm 16 0.000 3.113 3.113 1.000±0.000 0.029±0.003 s t 17 0.000 2.856 2.856 1.000±0.000 0.027±0.003 s t 18 0.000 2.820 2.820 1.000±0.000 0.027±0.001 s t 19 1.399 1.410 2.809 0.502±0.038 0.027±0.003 s m 20 0.000 2.017 2.017 1.000±0.000 0.019±0.003 s t 21 0.000 1.712 1.712 1.000±0.000 0.016±0.002 s t abbreviations: ch.p, chromosome pair; ch.s, chromosome size; ch.t, chromosome type; *, nucleolar organizer region; l, large size; m, medium size; s, small size; m, metacentric; sm, submetacentric; a, acrocentric; t, telocentric. table 3. review of cytogenetic study of the genus cyrtodactylus. species 2n nf karyotype nor locality reference c. consobrinus 48 50 2bi-armed+46t sarawak, malaysia ota et al. (1992) c. interdigitalis 42 52 4m+2sm+4a+32t loei, thailand present study c. kunyai 40 52 8m+4sm+28t loei, thailand present study c. pubisulcus 42 44 2bi-armed+40t sarawak, malaysia ota et al. (1992) abbreviations: 2n, diploid chromosome; nf, fundamental number; nor, nucleolar organizer region; bi-armed, bi-armed chromosome; m, metacentric; sm, submetacentric; a, acrocentric; t, telocentric. fig. 3. standardized idiogram of cyrtodactylus kunyai (a) and c. interdigitalis (b) by conventional staining. 27cytogenetic study of the bent-toed gecko (reptilia, gekkonidae) in thailand and few bi-armed chromosomes. present results of c. kunyai and c. interdigitalis agree with chromosomal evolution line hypothesis within the gekkonid group. the karyotype of c. kunyai and c. interdigitalis showed the gradient of most telocentric while comprised of a few bi-armed chromosome, 12 chromosomes in c. kunyai and 10 chromosomes in c. interdigitalis. these features conform to the hypothesis of rearrangement from ancestral karyotype by robertsonian fissions, fusions or pericentric inversions (gorman 1973; king 1987). acknowledgements this research was financially supported by sharing from the research fund for supporting lecture to admit high potential student to study and research on his expert program year 2016, the toxic substances in livestock and aquatic animals research group, khon kaen university, and the royal thai government scholarship national science and technology development agency (nstda). the project was approved by the institute of animals for scientific purpose development of national research council of thailand (resolution u1-02740-2559). references aprea g, odierna g, capriglione t, caputo v, morescalchi a, olmo e. 1996. heterochromatin and nor distribution in the chromosomes of six gekkonid species of the genus phelsuma (squamata: gekkonidae). j afr zool. 110(5):341–349. aprea g, andreone f, fulgione d, petraccioli a, odierna g. 2013. chromosomal rearrangements occurred repeatedly and independently during species diversification in malagasy geckos, genus paroedura. afr zool. 48(1):96–108. chaiyasut k. 1989. cytogenetic and cytotaxonomy of genus zephyranthes. bangkok: department of botany, faculty of science, chulalongkorn university. 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[accessed 2018 march 15]. http://reptile-database.reptarium.cz/ search?search=cyrtodactylus&submit=search substantia an international journal of the history of chemistry vol. 2, n. 1 march 2018 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 75(3): 65-83, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-1774 caryologia international journal of cytology, cytosystematics and cytogenetics citation: denisa manolescu, georgiana uță, anca șuțan, cătălin ducu, alin din, sorin moga, denis negrea, andrei biță, ludovic bejenaru, cornelia bejenaru, speranța avram (2022). biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts and evaluation of their biosafety potential. caryologia 75(3): 65-83. doi: 10.36253/ caryologia-1774 received: august 02, 2022 accepted: september 11, 2022 published: april 5, 2023 copyright: © 2022 denisa manolescu, georgiana uță, anca șuțan, cătălin ducu, alin din, sorin moga, denis negrea, andrei biță, ludovic bejenaru, cornelia bejenaru, speranța avram. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts and evaluation of their biosafety potential denisa manolescu1,2, georgiana uță1,2,*, anca șuțan3, cătălin ducu1, alin din1, sorin moga1, denis negrea1, andrei biță4, ludovic bejenaru4, cornelia bejenaru5, speranța avram2 1 regional research and development center for innovative materials, products and processes from automotive industry, university of pitesti, 11 doaga street, 110440 pitesti, arges, romania 2 department of anatomy, animal physiology and biophysics, faculty of biology, university of bucharest, 91-95th independence street, ro-050095 bucharest, romania 3 department of natural sciences, faculty of science, physical education and informatics, university of pitesti, targu din vale street, 110040 pitesti, arges, romania 4 department of pharmacognosy & phytotherapy, faculty of pharmacy, university of medicine and pharmacy of craiova, 2 petru rareş street, 200349 craiova, dolj county, romania 5 department of pharmaceutical botany, faculty of pharmacy, university of medicine and pharmacy of craiova, 2 petru rareş street, 200349 craiova, dolj county, romania * corresponding author. e-mail: georgiana.uta@drd.unibuc.ro abstract. in this study we targeted the noble metal nanoparticles (mnps) biogenic synthesis capacity of two medicinal species with therapeutic potential, namely melissa officinalis l. (lemon balm) and salvia officinalis l. (sage), cultivated in romania. plant material was extracted by maceration, microwave assisted extraction (mae) and ultrasound assisted extraction (uae). bright field scanning transmission electron microscopy and energy dispersive x-ray spectroscopy (bfstem-eds) techniques were used in order to investigate particles shape, dispersion and chemical elemental analysis. the total polyphenol content for both simple extracts and nanostructured mixtures was determined using the folin-ciocalteu method and antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (dpph) method. identification and quantification of secondary metabolites of m. officinalis and s. officinalis were performed by ultra-high performance liquid chromatography (uhplc). the allium assay was used to evaluate the potential cytogenotoxic activity, for both simple and nanostructured phytochemical complexes, in the case of s. officinalis l. species being performed for the first time. spherical shaped mnps with diameters of about 20 nm were biosynthesised in lemon balm extracts. larger aunps were phytosynthesized in sage extract obtained by uae. compared to the simple extracts, the antioxidant capacity as well as the amount of total polyphenols in the nanostructured extracts decreased, substantiating the involvement of bioorganic material in the reduction of metal ions. low frequency of chromosomal aberrations corresponding to crude extracts and extracts supplemented with mnps, suggest the cytoprotective, antigenotoxic, and safe use of these plant species as potential therapeutic forms in various diseases. keywords: lemon balm, sage, metal nanoparticles, antioxidant, cytotoxicity. 66 denisa manolescu et al. introduction melissa officinalis l. (lemon balm) and salvia officinalis l. (sage) are representatives medicinal plant species family lamiaceae, whose remarkable therapeutic effects have been attested since ancient times (shakeri et al. 2016; ghorbani and esmaeilizadeh 2017). the bioactivity of natural compounds of m. officinalis has been noted especially for the treatment of neuropsychiatric disorders such as alzheimer’s and parkinson’s diseases, epilepsy, psychosis, depression or anxiety (gomes et al. 2009; shakeri et al. 2016; avram et al. 2017; udrea et al. 2018). as for s. officinalis, this plant species has been cultivated both as a medicinal plant, used therapeutically by humans for the treatment of various diseases such as gout, hyperglycemia, paralysis, rheumatism, cancer, bronchitis, and not least in the relief of symptoms of neurodegenerative diseases (garcia et al. 2016; šulniūtė et al. 2016), for decorative purposes (kintzios 2000) and food or spice (longaray delamare et al. 2007). however, the use of plant extracts in medicine is quite limited, mainly due to the inability of therapeutic plant compounds to penetrate the target, affected structures of organisms. this phenomenon occurs because of the large size of phytochemicals compared to the size of the target structures. it is now well known that due to the nanometric size of noble metal nanoparticles (mnps), biocompounds embedded in such “capsules” or attached to their surface show both higher bioavailability and stability (pandey et al. 2003). at present, the literature abounds with information on the advantages of using different types of metal nanoparticles synthesised using plant extracts in therapy, making phytosynthesis a promising and sustainable alternative to conventional chemical or physical methods (azeez et al. 2020; naikoo et al. 2021; shelembe et al. 2022). these nanostructured phytocomplexes are also only toxic at extremely high concentrations, doses which are not currently used for therapeutic purposes (badmus et al. 2022). the biosynthesis of mnps aims, along with the fragmentation of phytochemicals, to embed various therapeutic plant compounds, or even certain drugs, in nanosized “metal envelopes” that allow their penetration and release into all structures of the target organism (sun et al. 2008; kumari et al. 2010; parveen et al. 2012). in addition, in recent decades several technologies have emerged to deliver along with nps conventional drugs, recombinant proteins or even vaccines or nucleotides needed to treat cancer or other diseases (parveen et al. 2012). since the antibacterial and anti-inflammatory action of silver nanoparticles (agnps) due to ag ions is well known worldwide (kirsner et al. 2001; tripathy et al. 2008; yilmaz öztürk 2019), recently, the attention of researchers has been directed towards obtaining silver chloride nanoparticles (agclnps) which have been shown to exhibit identical or even improved properties compared to agnps (eugenio et al. 2018). moreover, agclnps have attracted considerable attention because they are easier to synthesize and also exhibit strong antimicrobial activity (hu et al. 2009). gold (au) nanorods have become some of the most important and commonly used materials in drug delivery and nanomedicine. the main reason for the use of aunps is to facilitate targeted drug transport, particularly in cancer therapy. to this end, a system using aunps conjugated with tumour necrosis factor (tnf) molecules has been designed, that has the effect of efficiently destroying only tumour cells while having low cytotoxicity to healthy cells (mocellin and nitti 2008; das et al. 2011). in addition, the pharmacological properties of lemon balm and sage are due to the content in secondary metabolites, such as polyphenols, alkaloids, triterpenes or sterols (jaimez ordaz et al. 2018; uță et al. 2021) which are usually found in quite low concentrations, their recovery in a higher concentration being a challenge. one of the most important factors affecting the quality of bioactive compounds obtained from plant sources is the extraction method, also considered as a sample preparation technique, playing a vital role on the overall yield and final result. the conventional extraction methods, e.g. maceration, soxhlet extraction, have been intensively used in recent decades (zhang et al. 2018), but they have a number of drawbacks like timeconsuming and use of a large amount of solvent. the latter not only increases process costs but is also associated with a negative environmental impact (sasidharan et al. 2011). therefore, numerous studies have aimed at developing efficient and environmentally friendly extraction techniques. among the extraction techniques that have been successfully applied in obtaining active phytocompounds are microwave-assisted extraction (mae) and ultrasound-assisted extraction (uae) (wang and weller 2006; grosso et al. 2015). studies in the literature highlight that the amount of polyphenols extracted from this medicinal plant varies depending on certain factors such as: extraction method, solvent range, solvent:plant ratio, temperature, extraction time (hernández et al. 2009; zhang et al. 2018), stages of its primary processing (drying, grinding), harvesting area of plant material (dent et al. 2017), and harvesting period (francik et al. 2020). 67biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts phytotherapy based on plant extracts has gained worldwide popularity because it is not addictive, it does not have harmful side effects and a high risk of toxicity like synthetic medicines, and last but not least it is cheap (avram et al. 2005; andrade et al. 2019; lin et al. 2019). thus, based on this information, this study was focused on the biogenic synthesis of noble metal nanoparticles, namely agclnps and aunps, using the medicinal species m. officinalis and s. officinalis, correlated with the determination of the optimal method for extracting the highest possible concentrations of phytocompounds from the plant species, the analysis of the antioxidant capacity of simple extracts and extracts supplemented with mnps, as well as in vivo testing of the cytotoxic activity of the extracts obtained and of nanostructured phytochemical complexes, in order to highlight the safety of these systems as potential therapeutic forms. materials and methods reagents and chemicals the 96.9% pharmaceutical ethyl alcohol used for the extraction processes was purchased from sc. coman product s.a.; 2,2-diphenyl-1-picrylhydrazyl (dpph), folin-ciocalteu reagent, 99.5% absolute ethyl alcohol, distilled water, na2co3 powder, gallic acid, trolox, glacial acetic acid, absolute ethyl alcohol, 1n hcl and orcein were purchased from carlo erba reagents s.a.s; acetonitrile, methanol and water were purchased from merck. reference compounds such as protocatechuic acid, ferulic acid, p-coumaric acid, caffeic acid were obtained from merck, while quercetin, rutin and rosmarinic acid were purchased from sigma-aldrich and chlorogenic acid was purchased from alfa aesar. the plant material aerial parts of m. officinalis, s. officinalis leaves and allium cepa l. bulbs, were collected from a local producer, arges county, in september 2020. the authentication of the plant material was performed by assoc. prof. ph.d. anca sutan, and kept in paper bags, protected from moisture and sunlight, until primary processing. primary processing of lemon balm and sage consisted of drying the plants in an oven at 40°c, a temperature designed not to denature the phenolic compounds of interest, and grinding the plant material in a retsch grindomix laboratory mill at the following parameters: 3 min pulse grinding at 4,000 rpm and 30 sec continuous grinding at 10,000 rpm (manolescu et al. 2022). extraction procedures the extraction of secondary metabolites was performed by maceration as classical method and two nonconventional methods, mae and uae, respectively. two different ratios of pharmaceutical ethyl alcohol and distilled water were used as solvent mixtures: 70:30 v/v and 50:50 v/v. for plant maceration, 1 g of dry sample, ground and weighed on an analytical balance to 4 decimal places, was immersed in 10 ml solvent (pharmaceutical ethyl alcohol:distilled water). maceration was carried out at room temperature, shielded from sunlight, for 7 days; the first 4 days with continuous stirring for 6 hours at 30 rpm on the biosan mini-rotator, and the next 3 days without stirring (dent 2015). the same binary solvents and the same 1:10 plant to solvent ratio were used for mae. initially the plant material was hydrated in the solvent for 1 hour and then subjected to microwave irradiation for 3, 5 and 10 minutes at a maximum power of 250 w. microwave-assisted extraction was performed using the neos-gr equipment, milestone. the final temperature range of the samples was between 54-78°c (dent 2015). uae was carried out using a hielscher up200st ultrasonic extraction system under a working amplitude equal to 80% of the maximum rated output power of the device. in order to avoid overheating of the experimental samples and possible destruction of phytocompounds we used a cooling system, extracts were obtained at temperatures below 45°c (dent 2015; žlabur et al. 2016). all experimental variants (table 1) were then centrifuged twice (10 min total time) at 6,000 rpm. the supernatants obtained were subjected to vacuum filtration through pall flex membrane filters qry:100; mm: 47 filter paper on a rocker model filtration system: vf6. pending analysis of total polyphenol content, antioxidant activity, hplc analysis, mnps synthesis and evaluation of cytogenotoxicity, samples were kept in glass vials at -18°c. determination of the total polyphenol content of the obtained plant extracts and nanostructured phytochemical complexes quantitative determination of polyphenolic structure compounds in the obtained extracts was carried out by the folin-ciocalteu spectrophotometric method (sutan et al. 2018). from each experimental variant, diluted beforehand until a dilution factor of 600 was reached, a volume of 500 µl extract was taken over which 2.5 ml folin-ciocalteu reagent 10% (aqueous mixture of phosphomolybdate and phosphotungstate) was 68 denisa manolescu et al. added. the tubes were kept at room temperature for 5 minutes and then 2 ml sodium carbonate solution (7.5%) was added. the tubes were shaken vigorously and kept in the dark at room temperature for 1 hour. total polyphenol content (tpc) analysis was performed on the ocean optics hr2000+ uv-vis spectrophotometer at 765 nm wavelength. distilled water was used as blank instead of extract. polyphenol concentration was expressed as mg gallic acid equivalent/g plant (mg gae/g) based on the calibration curve constructed for different concentrations of the etalon, i.e. for 7 points of concentrations from 10 to 70 μg/ml gallic acid (y = 0.0115x + 0.0094; r2 = 0.9995). tpc values, expressed in mg gallic acid equivalent/g plant were obtained according to the formula (phuyal et al. 2020): (1) where c is the concentration measured from the calibration curve, m is the dry plant mass and df is the dilution factor. the quantitative determination of compounds with polyphenolic structure in all phytochemical complexes supplemented with mnps was also carried out by the folin-ciocalteu spectrophotometric method, also used for simple extracts (sutan et al. 2018). determination of the trolox equivalent antioxidant capacity (teac) of simple extracts and nanostructured phytochemical complexes using the dpph method the free radical scavenging activity of the extracts and nanostructured mixtures was measured using the 2,2-diphenyl-1-picrylhydrazyl (dpph) method. the analysis was determined according to shimamura et al. (2014), but with some modifications. table 1. experimental variants used in this study. no sample code plant species extraction procedure pharmaceutical ethyl alcohol:distilled water ratio (v/v) extraction parameters 1 m_m_50 m. officinalis maceration 50:50 7 days; room temperature; in the dark 2 m_m_70 maceration 70:30 7 days; room temperature; in the dark 3 m_mae_3_50 mae 50:50 3 min; 250 w 4 m_mae_5_50 mae 50:50 5 min; 250 w 5 m_mae_10_50 mae 50:50 10 min; 250 w 6 m_mae_3_70 mae 70:30 3 min; 250 w 7 m_mae_5_70 mae 70:30 5 min; 250 w 8 m_mae_10_70 mae 70:30 10 min; 250 w 9 m_uae_3_50 uae 50:50 3 min; 80% amp 10 m_uae_5_50 uae 50:50 5 min; 80% amp 11 m_uae_10_50 uae 50:50 10 min; 80% amp 12 m_uae_3_70 uae 70:30 3 min; 80% amp 13 m_uae_5_70 uae 70:30 5 min; 80% amp 14 m_uae_10_70 uae 70:30 10 min; 80% amp 15 s_m_50 s. officinalis maceration 50:50 7 days; room temperature; in the dark 16 s_m_70 maceration 70:30 7 days; room temperature; in the dark 17 s_mae_3_50 mae 50:50 3 min; 250 w 18 s_mae_5_50 mae 50:50 5 min; 250 w 19 s_mae_10_50 mae 50:50 10 min; 250 w 20 s_mae_3_70 mae 70:30 3 min; 250 w 21 s_mae_5_70 mae 70:30 5 min; 250 w 22 s_mae_10_70 mae 70:30 10 min; 250 w 23 s_uae_3_50 uae 50:50 3 min; 80% amp 24 s_uae_5_50 uae 50:50 5 min; 80% amp 25 s_uae_10_50 uae 50:50 10 min; 80% amp 26 s_uae_3_70 uae 70:30 3 min; 80% amp 27 s_uae_5_70 uae 70:30 5 min; 80% amp 28 s_uae_10_70 uae 70:30 10 min; 80% amp 69biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts for the preparation of the standard, 2.50 mg trolox was weighed on a microbalance and placed in a 10 ml volumetric flask. 5 ml of 99.5% ethanol was added and sonicated for complete dissolution, after which the solvent was made up to 10 ml. this was the stock solution from which the dilution range of 10-70 µg/ml was prepared, which was necessary to carry out the calibration curve (y = 1.4028x + 2.4888; r2 = 0.9991). the dpph reagent used for both the standard and all experimental variants was prepared as follows: 3.2 mg of 2,2-diphenyl-1-picrylhydrazyl was placed in a 100 ml volumetric flask to which 50 ml of 99.5% ethanol was added; it was then sonicated and made up to 100 ml with solvent. the dpph solution was prepared fresh and stored at room temperature, protected from light. from all 28 experimental variants of simple extracts, only those samples with the highest polyphenol content for each extraction procedure were chosen to show dpph free radical scavenging activity and to assess the biogenic synthesis capacity of noble mnps. simple extracts were diluted to obtain 6 different concentrations (250 µg/ml; 500 µg/ml; 1,000 µg/ml; 5,000 µg/ml; 10,000 µg/ml; 100,000 µg/ml), and those supplemented with mnps were diluted to obtain 3 different concentrations (500 µg/ml; 1,000 µg/ml; 5,000 µg/ml). from each sample of different concentration, 250 µl simple extract/nanostructured mixture was taken over which 1,750 µl dpph was added. the systems were kept in the dark at room temperature for 30 minutes and then for each sample, the absorbance at 517 nm wavelength was read after 5 minutes of stabilization under uv influence. the inhibition percentage of dpph (%ip) was calculated according to the formula (adebiyi et al. 2017): (2) where a0 is the control sample absorbance and a1 is the sample absorbance. to determine the half maximum inhibitory concentration (ic50), two concentration points of each sample were selected for which the inhibition ratio had a value around 50% (one < 50% and one > 50%) and the regression curve (y = ax + b) was drawn. the ic50 value (sample concentration x) was calculated by replacing y by 50 (shimamura et al. 2014). the half maximum inhibitory concentration values are required for the determination of the antioxidant activity calculated in trolox equivalent according to the formula: (3) uhplc analysis. sample preparation a stock solution of 0.1 mg/ml from all standard compounds was prepared by dissolving 10 mg of each reference in 100 ml methanol. this stock solution was kept refrigerated at 4°c and used when needed. to obtain the solutions for the calibration curve the stock solution was diluted with a mixture of the first gradient line of the mobile phase. the dilution factors were 2000, 1000, 500, 250 and 100, respectively. uhplc-pda-ms analysis separation of polyphenols was carried out on a waters arc system coupled with a waters 2998 pda detector and a waters qda mass detector. the column used was a waters cortecs c18 (4.6 × 50 mm, 2.7 μm) eluting with solvent a (0.1% formic acid in water), solvent b (0.1% formic acid in methanol) and solvent c (0.1% formic acid in acetonitrile). solvent b was set at 1% during the entire separation. the gradient was as follows: 0–4 min 3%-14% c, 4–7.5 min 14% to 29% c, 7.5– 13 min 29% to 89% c, 13–15 min 89% to 3% c. the flow rate of the mobile phase was set at 1.0 ml/ min. the column temperature was equilibrated to 35°c. the injection volume was 5 μl. all samples were kept at 20°c during the entire analysis (velamuri et al. 2020). eluted compounds were analysed using a waters pda 2998 and a qda mass detector equipped with electrospray ionization (esi) source. capillary voltage was maintained at 0.8 kv, cone voltage was kept at 20 v and the mass spectra spectra were recorded in negative ion mode in the range 100–800 m/z. quantification was established in selected ion recording (sir) mode for each compound (as shown in table 2) using external calibration curves prepared for each standard. also, the retention times for all reference compounds are presented in table 3. biogenic synthesis of noble metal nanoparticles mediated by plant extracts for the biosynthesis of mnps using extracts of lemon balm and sage, the experimental variant that was found to contain the highest content of polyphenols was used from each extraction procedure, since literature data show that phytochemicals, especially polyphenols, present in plant extracts have the strongest reducing properties of silver and gold ions and also confer the highest stability of the nanoparticles (swilam and nematallah 2020). for the synthesis of agclnps, a 1mm 70 denisa manolescu et al. silver nitrate (agno3) solution obtained by weighing 16.98 mg agno3 salt was used as a precursor and made up to 100 ml volume with distilled water. tetrachloroauric acid (haucl4) solution of 1mm concentration, obtained by adding 35.80 mg haucl4 to 100 ml of distilled water, was the precursor for the phytosynthesis of gold nanoparticles. the simple extracts were then mixed with the specific precursors in volume ratios of 1:1 and incubated for 24 h at room temperature (25°c). the colour change of the extracts after the addition of the 2 types of precursor solutions was noticeable within the first 2 min, which was a first confirmation of the formation of noble mnps (sutan et al. 2019). bfstem-eds analysis of nanostructured phytochemical complexes bright field scanning transmission electron microscopy (bfstem) and energy dispersive x-ray spectroscopy (eds) were used to investigate particles size, shape and dispersion and perform chemical elemental analysis. these analyses were carried out using the fesemhitachi su8230 microscope. prior to these analyses, samples were homogenized for 1 minute in an ultrasonic bath (kerry guyson) for a better dispersion. then, one drop of each sample was spread on a copper grid with formvar and the grid was kept for 24 hours in the exicator to evaporate the solvent. coating cu grids with formvar film cu grids with thin formvar films are primarily used for transmission electron microscopy for sampling and analysis of ultra-thin sections (shields 1999). the formvar film also acts as a support for various suspensions or powders to be analysed by sem/ bfstem. to obtain cu grids with formvar film, the ~1% formvar solution in 1,2-dyclorethane was poured into a tall covered container. a glass microscope slide was cleaned with distilled water, but not insistently. the glass slide was inserted into the container with the formvar solution and left for 2-3 minutes. the glass slide was then removed from the formvar solution and drained, after which the slide was left at room temperature for a further 2-3 minutes after which the excess solution was removed. after drying the film, it was cut on the edge of the glass blade using a razor blade. a container was filled with clean distilled water and the glass blade was inserted at an angle of 45° to loosen the formvar film. the cu grids were placed face down over the formvar film on the surface of the water. the formvar grids were collected using another pre-cleaned glass slide, after which the grids were left at room temperature covered with the lid of a petri dish to dry and adhere the film to the cu grid (sherman 2014). in vivo testing of the cytogenotoxic activity of simple extracts and nanostructured phytochemical complexes root tip cells were obtained by placing bulbs of allium cepa l. with discoidal stem in contact with distilled water for 48 h, in the dark. the bulbs were transferred to table 2. calibration curve statistics of reference compounds. calibration curve standard fit type equation r2 protocatechuic acid quadratic (2nd order) y = -3.98e+003 x^2 + 1.41e+005 x + 5.19e+003 0.998221 chlorogenic acid y = -9.88e+003 x^2 + 1.40e+005 x + 1.83e+004 0.994078 caffeic acid y = -1.98e+004 x^2 + 3.63e+005 x + 2.73e+004 0.997099 p-coumaric acid y = -3.79e+003 x^2 + 1.01e+005 x + 2.14e+003 0.997983 ferulic acid y = -4.99e+002 x^2 + 2.01e+004 x 2.72e+002 0.998986 rutin y = -1.52e+003 x^2 + 7.64e+004 x + 4.40e+003 0.998255 rosmarinic acid y = -5.96e+001 x^2 + 5.35e+004 x + 3.51e+005 0.994586 quercetin y = -2.50e+004 x^2 + 7.79e+005 x 5.95e+001 0.999115 table 3. retention times for all reference compounds. peak no. compound name coding m/z retention time [min] 1 protocatechuic acid pro 153 1.667 2 chlorogenic acid chl 353 3.082 3 caffeic acid caf 179 3.332 4 p-coumaric acid cou 163 4.459 5 ferulic acid fer 193 5.152 6 rutin rut 609 5.580 7 rosmarinic acid ros 359 6.715 8 quercetin que 301 7.757 71biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts simple extracts and nanostructured mixtures (24 h). after 24 h the root tip meristematic cells were removed and subjected to fixation using farmer’s reagent (glacial acetic acid:absolute ethyl alcohol, 1:3 v/v) overnight and then transferred to 70º ethyl alcohol for long-term preservation. for each experimental variant a number of 5 roots were subjected to attenuated hydrolysis with 1n hcl for 18 minutes at 60ºc. the fixed and macerated roots were stained with 2% aceto-orcein solution for 15 minutes at 60ºc. from the stained meristematic tips, microscopic preparations were made by the squash technique. microscopic slides were analysed under the mshot trinocular ml 11-ii biological microscope at 400× magnification. microscopic analysis consisted of determining the number of cells at different stages of mitosis, the frequency of chromosomal and nuclear aberrations, based on approximately 3,000 cells per experimental sample. the mitotic index (mi) was determined as the percentage ratio of the number of cells in mitosis to the total number of cells analysed (tedesco and laughinghouse 2012). based on the total number of cells in mitosis, the percentage ratio of cells in prophase, metaphase, anaphase or telophase was determined. the frequency of chromosomal aberrations and nuclear abnormalities was determined by relating them to the appropriate stage of the cell cycle, i.e. mitosis. statistical interpretation of the results the results of the experimental analyses were expressed as mean values ± standard deviation (sd). oneway analysis of variance (anova) followed by šídák’s multiple comparisons test was used to analyse differences between mean values. a probability of p<0.0001 was considered highly significant. statistical analysis was performed using graphpad prism 9.0.0.0 software. for cytogenetic analysis, statistical processing of data was performed using ibm spss statistics 20 software. statistical significance and significant differences between variables were determined using analysis of variance (one way anova) and duncan’s test for multiple comparisons, respectively. values of p ≤ 0.05 were considered statistically significant. graphs and tables were compiled based on mean values ± standard error of several independent experiments. results and discussions determination of the total polyphenol content of the obtained plant extracts and nanostructured phytochemical complexes as can be seen in figure 1, the highest amount of polyphenols, i.e. 70.07±1.07 mg gae/g plant, was recorded for the lemon balm extracts obtained by microwave-assisted extraction technique, in solvent with a volume ratio of pharmaceutical ethyl alcohol and distilled water of 70:30, and after 10 minutes of microwave action on the extraction mixture. for macerates, the highest amount of total polyphenols (32.26±0.26 mg gae/g plant) was recorded for those obtained in the solvent with equal ratio of water and solvent. for the ultrasound-assisted extraction of aerial parts from lemon balm plants with solvent with a volume ratio of pharmaceutical ethyl alcohol and distilled water of 70:30, there is a directly proportional increase in the amount of total polyphenols with the time of ultrasound action, with the highest amount of these compounds (53.98±0.16 mg gae/g plant) obtained after 10 minutes of extraction. a variation from 2.816 to 7.796 mg/ml of phenolic compounds was reported by papoti et al. (2019) in aqueous preparations, respectively: infusion, decoction, maceration, ultrasound-assisted extraction. total polyphenols ranging from 18.17±0.04 to 64.17±0.52 mg gae/g dry plant was obtained by petkova et al. (2017) when infusions made from lemon balm plants cultivated in bulgaria were analysed. of the two sage extracts obtained by maceration, the highest polyphenol content was determined for the one with a ratio of 70:30 pharmaceutical ethyl alcohol:distilled water (v/v) (25.30±0.96 mg gae/g plant), data illustrated in figure 2. figure 1. total polyphenol content of m. officinalis extracts (data are expressed as mean ±sd values from independent triplicate experiments). 72 denisa manolescu et al. in contrast, pop et al. (2015) doubling the extraction time by maceration and using 80% etoh obtained lower tpc values, 19.49 mg gae/g dry plant, which shows that a prolonged extraction time may not always lead to a higher phytocompound concentration. this is also confirmed by the results obtained by osmić et al. (2019), who using a 40% aqueous ethanol solution and the same plant:solvent ratio used by us, obtained from the leaves of s. officinalis l. a polyphenol content of 137.11 mg gae/g in only 60 minutes of maceration at room temperature. however, there are experimental studies in which maceration resulted in much lower tpc values than the present study, namely 13.6±0.4 mg gae/g (proestos et al. 2005); 4.25 mg-5.95 mg gae/g (roby et al. 2013). moreover gîrd et al. (2014) using the same solvent, ethanol 70% managed to extract from one gram of sage leaves only a minimum tpc of 3.26 mg gae/g and a maximum of 6.32 mg gae/g. the extracts obtained using mae showed a total amount of polyphenolic compounds between 29.05±0.1639.88±1.19 mg gae/g plant, the maximum of 39.88±1.19 mg gae/g plant being obtained after irradiating the plant material with electromagnetic waves for 5 minutes, also at a higher concentration of alcohol, at a temperature of 75°c and a power of 250w. similar values were also recorded in the experimental study conducted by dragović-uzelac et al. (2012), where the range of tpc values obtained was between 31.7-47.0 mg rae/g, the maximum being determined in the sample irradiated for 9 minutes at a power of 500w. for the samples subjected to sonication, in order to reach a maximum tpc of 36.75±1.41 mg gae/ g plant, it was necessary to prolong the acoustic cavitation phenomenon to a maximum of 10 minutes, an alcohol concentration of 70% and a temperature of 45°c. this polyphenol content is much higher compared to pop et al. (2015) 19.06 mg gae/g plant and brindisi et al. (2021), 18.7-35.3 mg ca/g. in contrast, there are studies attesting the recovery of higher concentrations of polyphenolic compounds from sage, such as 67.75 mg gae/g (dent 2015); 99.03 mg gae/g (zeković et al. 2017); 61.3-143.6 mg gae/g (veličković et al. 2011). a possible explanation for the differences between the tpc values in the literature, and those obtained by us, except for the extraction technique and parameters, would be the time at which the plant material was harvested, farhat et al. (2014), demonstrating that the highest polyphenol content was recorded for sage plants harvested at the fruiting stage. the results could also be attributed to the drying protocol of the plants (hamrouni-sellami et al. 2012) as well as the geographic area of cultivation (farhat et al. 2014; dent et al. 2017). as can be seen in figure 3, the highest amount of phenolic compounds for lemon balm extracts supplemented with mnps was recorded for those obtained by mae technique, i.e. 41.741±0.052 mg gae/g plant for m_mae_10_70_agcl and 40.842±0.343 mg gae/g plant for m_mae_10_70_au. for the macerates, it can be seen that there are statistically insignificant differences between the tpc values of extracts and mixtures with agclnps and aunps. strongly statistically significant higher values were observed for extracts obtained by using uae without mnps comparing with the extracts supplemented with mnps, the tpc content ranging from 53.988±0.166 mg figure 3. total polyphenols content of simple extracts and nanostructured phytochemical complexes of m. officinalis (data are expressed as mean ± sd values from independent triplicate experiments and p values were calculated by one-way anova followed by šídák’s multiple comparisons test; ****p < 0.0001; *p = 0.0472; ns p =0.1058; 0.8170; 0.2920). figure 2. total polyphenol content of s. officinalis extracts (data are expressed as mean ±sd values from independent triplicate experiments). 73biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts gae/g plant to 17.360±0.069 mg gae/g plant for m_ uae_10_70_agcl and 14.932±0.044 mg gae/g plant for m_uae_10_70_au. a statistically significant decrease was also observed for extracts obtained by mae technique with agclnps (41.741±0.052 mg gae/g) and aunps(40.842±0.343 mg gae/g) compared to extracts without mnps (70.070±1.070 mg gae/g plant). it is important to highlight unlike lemon balm extracts enriched with mnps where the highest amount of polyphenols was obtained for extracts with agclnps, for sage, extracts with aunps were found to exhibit this characteristic (figure 4). moreover, in the case of nanostructured phytochemical complexes of sage, the maximum total amount of polyphenols was also recorded for the experimental variant using the extract obtained after microwave irradiation of the plant material, i.e. 13.04±0.26 mg/g gae for sample s_mae_5_70_70_au. however, there are no signif icant differences between the amount of polyphenols obtained for sage extracts with agclnps versus those with aunps, for these extracts enriched with mnps the amount of total polyphenols remained relatively constant regardless of the extraction technique used, with tpc values varying only from 8.88±0.2 mg gae/g plant for the agclnps macerate and 13.04±0.26 mg gae/g plant for the extract obtained by mae and with aunps. making a comparison between the tpc values of simple extracts and phytochemical complexes, in the case of both medicinal species it can be observed that following the phytosynthesis of mnps, the amount of polyphenols was decreased. an explanation for this is provided by the experimental study conducted by dzimitrowicz et al. (2016), which highlights that following the reduction reaction of metal ions by a plant extract, a large part of the compounds of polyphenolic nature are oxidized. determination of the trolox equivalent antioxidant capacity (teac) of simple extracts and nanostructured phytochemical complexes using the dpph method from figure 5 it can be seen that the best ability of the lemon balm extracts with mnps to behave as hydrogen atom or electron donors for the conversion of the purple free radical dpphto its reduced yellow form dpph-h was for extracts obtained by the mae technique, the teac values were 0.106±0.019 for m_mae _10_70_agcl and 0.053±0.003 for m_ mae _10_70_au. also, for these types of extracts a highly significant statistical difference is observed compared to extracts without metal nanoparticles whose teac values were 0.339±0.027. similar to the tpc values, the statistical differences of teac values are insignificant between macerates without mnps and those supplemented with mnps. a highly significant statistical difference can also be observed for the teac values of the extract obtained by ultrasound action on plant material without mnps (0.103±0.011) compared to extracts with agclnps (0.021±0.004) and aunps (0.019±0.004). for both simple extracts and systems consisting of sage extracts and mnps, the highest antioxidant activity was recorded for the experimental variant s_mae_5_70 (figure 6). at the opposite pole, the lowest antioxidant activities were recorded for the samples subjected to macerafigure 5. trolox equivalent antioxidant capacity of simple extract and nanostructured phytochemical complexes of m. officinalis (data are expressed as mean ± sd values from independent triplicate experiments and p values were calculated by one-way anova followed by šídák’s multiple comparisons test; ****p < 0.0001; ***p = 0.0003; ns p = 0.6144; 0.6707; 0.9953; 0.9814). figure 4. total polyphenols content of simple extracts and nanostructured phytochemical complexes of s. officinalis (data are expressed as mean ± sd values from independent triplicate experiments and p values were calculated by one-way anova followed by šídák’s multiple comparisons test; ****p < 0.0001; ns p = 0.1310; 0.1497; 0.4079). 74 denisa manolescu et al. tion and sonication processes. in contrast zeković et al. (2017), using other extraction parameters, demonstrate that their simple extracts obtained by the cavitating phenomenon show a slight improvement in antioxidant activity as opposed to those subjected to microwaving, but the difference is not a noticeable one. comparing the antioxidant activity of simple extracts with that of nanostructured phytochemical complexes, both m. officinalis l. and s. officinalis l. simple extracts have the highest free radical scavenging capacity, as they also have the highest concentrations of polyphenols. the results of the evaluation of the antioxidant capacity as well as the amount of total polyphenols in the nanostructured extracts obtained are in agreement with the existing data in the literature, data which show that the reduction in the amount of total polyphenols correlated with a decrease in the antioxidant activity of extracts with mnps compared to simple extracts is attributed to the fact that the bioorganic material, especially the polyphenols, participates in the reduction of metallic ions as well as in the formation of the coating halo that stabilizes the nanoparticles (csakvari et al, 2021; nayeri et al., 2021; siakavella et al., 2020). uhplc analysis to identify the compounds obtained by the 3 different extraction methods, 6 experimental variants were subjected to uhplc-pda-ms analysis (m_m_50; m_ mae_10_70; m_uae_10_70; s_m_70; s_mae_5_70; s_uae_10_70), namely those variants with the highest tpc. thus, according to the data presented in table 4, maceration of the aerial parts of lemon balm resulted in significant amounts of rosmarinic acid (227,120 μg/ ml), the most abundant compound in this medicinal species (kim et al. 2010). however, modern extraction methods have allowed the recover y of much higher concentrations of it. thus, uae revealed a quantity of 263.805 μg/ml of rosmarinic acid, and the mae met hod y ielded t he highest amount of rosmarinic acid, i.e. 1,266.89 μg/ml. similar value (17.03 mg/g) indicating a signif icant amounts of rosmarinic acid was obtained by caniova and brandsteterova (2001) using the liquid extraction technique (methanol and water in a volume ratio of 60:40) of m. of ficinalis l. plants grown in slovakia. the hig hest concent rat ions of protocatechuic acid and rutin were obtained from maceration of sage leaves. however, mae proved to be the optimal method to obtain the highest amounts of all other compounds. as mentioned above, the final temperature range of the samples subjected to sonication and microwaves, was in the case of uae between 37-45°c, and in mae between 54-75°c, the final temperature of 75°c being reached in sample s_mae _5_70. based on this result, we can also see that at higher temperatures, the solvent’s solubilizing power of dissolved substances increases due to the decrease in viscosity and surface tension, thus facilitating wetting and matrix penetration (paré et al. 1991; chen et al. 2006; hayat figure 6. trolox equivalent antioxidant capacity of simple extract and nanostructured phytochemical complexes of s. officinalis (data are expressed as mean ± sd values from independent triplicate experiments and p values were calculated by one-way anova followed by šídák’s multiple comparisons test; ****p < 0.0001; ns p = 0.1727; 0.1231; 0.6421; 0.9794; 0.9908). table 4. the amount of natural compounds (µg/ml) obtained from m. officinalis and s. officinalis by the three extraction techniques. sample/compound name pro chl caf cou fer rut ros que m_m_50 25.640 15.960 131.650 5.280 2.805 27.735 227.120 0.450 m_mae_10_70 34.675 49.190 77.585 3.530 1.400 31.645 1266.890 0.230 m_uae_10_70 24.245 39.795 40.010 2.245 1.000 28.265 263.805 0.190 s_m_70 35.930 12.125 27.845 3.465 6.895 30.295 502.540 0.000 s_mae_5_70 35.845 16.140 48.935 3.790 12.520 10.240 593.715 0.000 s_uae_10_70 9.560 11.610 44.045 3.035 6.450 26.195 687.260 0.225 75biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts et al. 2009). consistent with the results in table 4, it can be seen that depending on the compound targeted for extraction, one extraction technique may be more advantageous than another. thus, if in order to extract a maximum concentration of rosmarinic acid of 687.26 µg/ml from sage leaves, the phenomenon of acoustic cavitations is the most beneficial, the same cannot be said in the case of protocatechuic acid where the exposure of plant material to sound waves allowed the recovery of the lowest amounts of this phenolic acid compared to the other extraction techniques. similar results regarding the discrepancy between the values of recovered compounds from this medicinal species were also recorded in the study conducted by zeković et al. (2017). bfstem-eds analysis of nanostructured phytochemical complexes from the dimensional analysis on agclnps dispersion in bfstem at 1,000,000 magnifications for the lemon balm extracts, it is observed that all these extracts showed the ability to phytosynthesize spherical shaped mnps with diameters of about 20 nm, most of them having sizes just below 10 nm (5 nm, 6 nm, 7 nm and 9 nm). agclnps phytosynthesized in sage extracts are also spherical in shape, ranging in size from 5 mm to 20 mm. these aspects illustrated in figure 7. for the aunps phytosynthesized in all three types of extracts, a reduced dispersion was observed, being found as agglomerates, in which, aunps show sizes of about 10 nm. however, larger aunps were phytosynthesized in sage extract obtained by uae, figure 8 showing 3 agglomerates of aunps with sizes of 105 nm, 92 nm and 25 nm. the aggregation phenomenon of aunps may be due to the low concentration of the precursor salt (i.e. haucl4), but also to the ph of the solution or even the age of the plants (teimouri et al. 2018; boruah et al. 2021). eds analysis allowed us to superimpose the spectra generated for the formvar film-coated copper grids and those with nanostructured phytochemical complexes. in figure 9 it can be seen that agclnps and aunps are present only in these phytocomplexes. figure 7. dispersion dimensional analysis of agclnps for m_uae_10_70_agcl (a) and for s_m_70_agcl (b) in bfstem at 1,000,000 (x1000k) magnification. figure 8. dimensional analysis of aunps aggregates obtained in sage ethanolic extracts. 76 denisa manolescu et al. in vivo testing of the cytogenotoxic activity of simple extracts and nanostructured phytochemical complexes the allium cepa l. test is widely used to determine the benefits and especially the adverse effects of medicinal plants, which are increasingly used nowadays. this is because the test is a very good indicator of toxicity and mutagenicity (tedesco and laughinghouse 2012). in our study, the allium assay was used to evaluate the possible cytogenotoxicity of crude or supplemented extracts with mnps. while for m. officinalis l. there is some data on the use of this test, in the case of s. officinalis l. it is performed for the first time. the variation of the mi in onion root tip meristematic cells subjected to treatment with ethanolic extracts of melissa officinalis l. before and after mnps phytosynthesis is shown in figure 10. these results revealed that for m. of ficinalis l. extracts, the highest percentage value of the mi (11.126%) corresponded to the sample m_m_50. likewise, for the control, the highest mi was recorded for roots incubated in solvent with a ratio of 96% pharmaceutical ethyl alcohol and 50:50 distilled water (8.643%). phy tosynthesis of agclnps and aunps inhibited the mitostimulatory action of ethanolic extracts. thus, there was a statistically significant reduction in the percentage of dividing cells. the sample defined by the extracts supplemented with mnps, showed a mi values close to those determined for the corresponding concentrations of alcohols, except for the sample m_mae_10_70_au for which the frequency of dividing cells was significantly higher (9.613%). the inhibition of mitotic activity, in the experimental variant m_mae _10_70_agcl compared to controls, may be correlated with the ion imbalance that can be induced at the cellular level by the extracts tested (chakraborty et al., 2009). hsin et al. (2008) showed that agnps stimulate intracellular production of reactive oxygen species (ros), which stimulates cell cycle progression while causing oxidative stress at the dna level (carlson et al., 2008). statistical analysis of the results on the distribution of mitosis phases (figure 11) indicates a significant increase of prophase index that the decrease in for sample m_m_50_au compared to the control. however, a distribution of mitosis phases similar to the control variants was noted for sample m_mae_10_70_agcl. moreover, a significantly higher frecvency of metaphases were defining for thesamples m_uae_10_70_agcl and m_m_50_agcl, suggesting the interference ofagclnpfigure 9. superimposed eds spectra obtained for cu grid with formvar and m_mae_10_70_agcl with agclnps on cu grid with formvar (a) and cu grid with formvar and s_mae_5_70_au with aunps on cu grid with formvar (b). figure 10. mi (%) of simple extracts of m. officinalis and nanostructured phytochemical complexes (a–d: interpretation of the significance of the differences, by means of the duncan test, p < 0,05). 77biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts son the formation and/or functioning of the mitotic spindle. results of cy togenotoxicity analysis show that extracts of s. officinalis and those enriched with mnps induced statistically significant variation of mi compared to distilled water, and to the control with the equivalent concentration of pharmaceutical ethyl alcohol (figure 12). thus, treatment of onion root meristems with these samples resulted in a statistically significant increase of mi, with a maximum value of 10.01% for the experimental variant s_m_70_agcl, in contrast to control samples, which showed mitoinhibitory effects. the distribution of mitosis phases has been investigated and is shown in figure 13. the highest prophase frequency was observed in the s_m_70_au variant, which was associated with the lowest anaphase and telophase indices. the allium test allows the assessment of the toxic potential of substances both by significant variation of mi and by analysing the type and frequency of chromosomal aberrations. statistical interpretations of the results on the frequency of chromosomal aberrations (figure 14) such as vagrant chromosomes, laggard chromosomes, sticky chromosomes, metaphase chromosomes (c-mitosis), fragmented chromosomes, pole-topole metaphases, bridge cells or multipolar anaphases are presented in table 5 and table 6, as well as nuclear aberrations (figure 15) such as binucleated cells, budded nuclei, irregularly shaped nuclei, ghost cells and giant cells found in the allium cepa l. root tip meristematic cells exposed to the action of controls, ethanolic extracts of lemon balm and sage and mixtures with mnps for 24 hours. vagrant chromosomes represent the chromosomal aberrations found with a high frequency in all control samples but also in all experimental variants with the exception of those defined by s. officinalis macerate and ethanolic extracts of m. officinalis obtained by mae technique supplemented with agclnps and aunps. these types of chromosomal aberrations occur as a result of “weak spindle formations” (onwuamah et al. 2014). laggard chromosomes were observed in all samples defined by the ethanolic extracts of lemon balm and sage extracts obtained by using uae and in those with mnps, with the exception of sage extracts with aunps. it is believed that the formation of lagging chromosomes is the result of the disruption of the division spindle forfigure 12. mi (%) of simple extracts of s. officinalis and nanostructured phytochemical complexes (a–d: interpretation of the significance of the differences, by means of the duncan test, p < 0,05). figure 11. frequency of mitosis phases (%) in simple extracts of m. officinalis and nanostructured phytochemical complexes (a–c: interpretation of the significance of the differences, by means of the duncan test, p < 0,05). figure 13. frequency of mitosis phases (%) in simple extracts of s. officinalis and nanostructured phytochemical complexes (a–e: interpretation of the significance of the differences, by means of the duncan test, p < 0,05). 78 denisa manolescu et al. mation process under the action of toxic agents (haliem, 1990). the frequency of sticky chromosomes significantly decreased in cells treated with lemon balm ethanolic extracts supplemented with agclnps and aunps regardless of the extraction technique used. stickies may be the consequence of subchromatid bonds between chromosomes (liman et al., 2010). although present at a moderate frequency in the control variants, but also in some experimental variants, the frequency of cells with nucleic buds figure 14. chromosomal aberrations found in a. cepa root tip meristematic cells following treatments with simple extracts and with extracts supplemented with mnps (a – vagrant in etoh_50; b – laggards in m_uae_10_70_agcl; c – c-mitosis in m_uae_10_70_agcl; d – sticky chromosomes in m_mae_10_70_agcl; e – pole-to-pole metaphase in s_uae_10_70_au; f – cell with fragmented chromosomes in s_mae_5_70; g – bridges in s_mae_5_70_au; h – multi-polar anaphase cell in m_m_50). figure 15. nuclear aberrations found in a. cepa root tip meristematic cells following treatments with simple extracts and with extracts supplemented with mnps (a – binucleate cells in m_mae_10_70; b – nuclei buds in m_m_50_agcl; c – irregularly shaped nuclei in h2od; d – ghost cells in m_m_50_agcl; e – giant cell in etoh_70). 79 ta bl e 5. c hr om os om al a nd n uc le ar a be rr at io ns o bs er ve d in a . c ep a ro ot t ip m er is te m at ic c el ls t re at ed w ith e th an ol ic e xt ra ct s an d na no st ru ct ur ed p hy to ch em ic al c om pl ex es o f m . offi ci na lis . sa m pl e c el ls w ith va gr an t ch ro m os om es l ag ga rd ch ro m os om es c -m ito si s st ic ky ch ro m os om es po le -t opo le m et ap ha se c el ls w ith fr ag m en te d ch ro m os om es br id ge s m ul tipo la r an ap ha se c el ls bi nu cl ea te ce lls n uc le i b ud s ir re gu la rl y sh ap ed n uc le i g ho st c el ls g ia nt c el ls t o ta l h 2o d 3. 51 ±1 .7 6 ab 0. 00 ±0 .0 0 c 0. 00 ±0 .0 0 b 12 .8 3± 6. 43 a b 0. 00 ±0 .0 0 b 1. 85 ±1 .0 0 a 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 33 .6 9± 6. 06 a 0. 00 ±0 .0 0 b 0. 06 ±0 .0 6 a 32 .7 1± 5. 52 a et o h _5 0 36 .4 7± 19 .5 7 a 0. 00 ±0 .0 0 c 0. 00 ±0 .0 0 b 9. 44 ±3 .8 8 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 1. 11 ±1 .1 1 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 03 ±0 .0 3 b 0. 4± 0. 30 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 a 0. 98 ±0 .2 9 b et o h _7 0 14 .1 6± 3. 81 a b 0. 00 ±0 .0 0 c 0. 00 ±0 .0 0 b 4. 16 ±4 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 33 .8 2± 16 .5 9 a 0. 00 ±0 .0 0 b 0. 33 ±0 .3 3 a 32 .8 5± 15 .5 8 a m _m _5 0 10 .9 2± 1. 44 a b 0. 00 ±0 .0 0 c 0. 00 ±0 .0 0 b 13 .4 2± 4. 63 a b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 2. 75 ±1 .4 2 ab 8. 51 ±5 .9 6 a 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 4. 46 ±2 .8 4 b 0. 00 ±0 .0 0 b 0. 07 ±0 .0 7 a 4. 87 ±2 .6 9 b m _m a e_ 10 _7 0 27 .6 2± 19 .5 2 ab 0. 00 ±0 .0 0 c 0. 00 ±0 .0 0 b 12 .4 0± 8. 58 a b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 61 ±0 .6 0 b 1. 38 ±1 .3 0 b 0. 23 ±0 .1 5 a 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 36 ±0 .3 0 a 1. 26 ±0 .6 7 b m _u a e_ 10 _7 0 15 .0 7± 8. 15 a b 3. 05 ±1 .5 4 c 0. 00 ±0 .0 0 b 24 .3 0± 9. 30 a b 0. 23 ±0 .1 9 a 0. 00 ±0 .0 0 b 3. 72 ±2 .9 3 ab 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 09 ±0 .0 6 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 a 1. 28 ±0 .1 5 b m _m _5 0_ a gc l 6. 66 ±5 .0 0 ab 0. 00 ±0 .0 0 c 0. 00 ±0 .0 0 b 9. 44 ±3 .8 8 ab 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 2. 08 ±0 .6 3 a 0. 00 ±0 .0 0 b 1. 83 ±1 .8 3 a 0. 09 ±0 .0 9 a 3. 86 ±2 .1 5 b m _m a e_ 10 _7 0_ a gc l 0. 00 ±0 .0 0 a 0. 00 ±0 .0 0 c 0. 00 ±0 .0 0 b 33 .8 8± 9. 64 a 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 25 ±0 .1 2 b 0. 00 ±0 .0 0 b 0. 25 ±0 .2 0 b 0. 34 ±0 .2 1 a 1. 17 ±1 .1 5 b m _u a e_ 10 _7 0_ a gc l 16 .6 6± 9. 62 a b 12 .2 9± 7. 84 a b 47 .6 6± 12 .4 6 a 1. 33 ±1 .3 3 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 1. 66 ±1 .5 0 ab 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 08 ±0 .0 8 b 0. 20 ±0 .2 0 b 0. 00 ±0 .0 0 b 0. 12 ±0 .1 2 a 1. 90 ±0 .2 0 b m _m _5 0_ a u 5. 00 ±2 .8 8 ab 0. 74 ±0 .5 c 0. 00 ±0 .0 0 b 3. 88 ±3 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 2. 52 ±0 .9 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 a 2. 56 ±0 .8 9 b m _m a e_ 10 _7 0_ a u 11 .1 1± 7. 34 a b 1. 11 ±1 .1 1 c 0. 00 ±0 .0 0 b 11 .5 7± 8. 93 a b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 9. 24 ±5 .7 9 a 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 1. 06 ±0 .3 6 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 a 1. 32 ±0 .4 2 b m _u a e_ 10 _7 0_ a u 0. 00 ±0 .0 0 a 0. 00 ±0 .0 0 c 0. 00 ±0 .0 0 b 19 .2 3± 12 .4 2 ab 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 6. 33 ±4 .6 6 ab 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 00 ±0 .0 0 b 0. 75 ±0 .5 1 b 0. 00 ±0 .0 0 b 0. 02 ±0 .0 2 a 1. 36 ±0 .4 2 b ta bl e 6. c hr om os om al a nd n uc le ar a be rr at io ns o bs er ve d in m er is te m at ic r oo t ce lls o f a . c ep a tr ea te d w ith e th an ol ic e xt ra ct s an d na no st ru ct ur ed p hy to ch em ic al c om pl ex es o f s. o ffi ci na lis . sa m pl e m et ap ha se w ith v ag ra nt ch ro m os om es a na ph as e w ith v ag ra nt ch ro m os om es l ag ga rd ch ro m os om es st ic ky ch ro m os om es po le -t opo le m et ap ha se c el ls w ith fr ag m en te d ch ro m os om es br id ge s m ul tipo la r an ap ha se c el ls bi nu cl ea te d ce lls ir re gu la rl y sh ap ed n uc le i g ia nt c el ls el on ga te d nu cl eu s ce lls o th er ab er ra tio ns t o ta l e to h 70 6. 50 ±3 .7 1 ab 10 .8 3± 6. 51 a 0. 00 1. 67 ±1 .6 7 cd 0. 00 0. 00 0. 00 0. 00 0. 00 29 .6 9± 12 ,3 9 b 0. 38 ±0 .3 8 a 0. 00 0. 00 28 .9 5± 11 .8 2 a h 2o d 2. 78 ±2 .7 8 b 6. 67 ±6 .6 7 a 1. 33 ±1 .3 3 b 6. 67 ±3 .3 3 bc d 0. 00 0. 00 0. 00 0. 00 0. 00 28 .6 6± 4. 04 b 0. 06 ±0 .0 6 a 0. 00 0. 00 27 .7 5± 3. 64 a s_ m _7 0 0. 00 3. 24 ±1 .6 7 a 0. 00 0. 00 0. 00 0. 00 2. 72 ±1 .3 6 c 0. 00 0. 00 0. 00 0. 00 0. 00 0. 06 ±0 .0 6 a 0. 21 ±0 .1 0 b s_ m a e_ 5_ 70 14 .9 4± 5. 76 a 4. 76 ±4 .7 6 a 0. 00 3. 70 ±3 .7 0 cd 0. 00 20 .6 3± 9. 15 a 1. 85 ±1 .8 5 c 2. 38 ±2 .3 8 a 0. 09 ±0 .0 9 a 0. 00 0. 00 0. 00 0. 08 ±0 .0 4 a 1. 27 ±0 .2 5 b s_ u a e_ 10 _7 0 7. 10 ±5 .1 6 ab 0. 00 1. 04 ±1 .0 4 b 10 .9 5± 3. 43 a bc 0. 00 6. 94 ±4 .2 2 b 17 .6 5± 5. 71 a bc 2. 78 ±2 .7 8 a 0. 04 ±0 .0 4 a 0. 00 0. 00 1. 37 ±0 .7 3 a 0. 15 ±0 .1 0 a 2. 51 ±1 .2 1 b s_ m _7 0_ a gc l 0. 00 0. 00 0. 00 0. 00 6. 25 ±3 .6 8 ab 3. 89 ±3 .8 9 b 7. 5± 3. 82 a 10 .7 8± 3. 01 a bc 0. 00 16 .2 7± 12 .3 4 ab c 0. 00 0. 00 0. 12 ±0 .0 9 a 0. 98 ±0 .2 1 b s_ m a e_ 5_ 70 _a gc l 0. 00 0. 00 0. 00 4. 3± 2. 08 c 0. 00 0. 00 10 .4 8± 5. 79 a 0. 00 0. 00 10 .0 0± 5. 77 a bc 2. 38 ±2 .3 8 a 0. 00 0. 06 ±0 .0 3 a 4. 34 ±2 .0 4 b s_ u a e_ 10 _7 0_ a gc l 0. 00 0. 00 0. 00 49 .0 0± 16 .4 4 a 0. 33 ±0 .3 3 b 0. 67 ±0 .6 7 b 0. 00 3. 67 ±0 .8 8 cd 0. 33 ±0 .3 3 a 6. 00 ±0 .5 8 bc 0. 00 0. 00 0. 00 5. 59 ±1 .6 7 b s_ m _7 0_ a u 0. 43 ±0 .4 3 b 0. 00 0. 00 19 .4 7± 3. 88 a 0. 00 0. 00 21 .7 4± 3. 55 a bc 0. 00 0. 06 ±0 .0 6 a 0. 00 0. 00 0. 00 0. 00 2. 62 ±1 .5 1 b s_ m a e_ 5_ 70 _a u 4. 44 ±2 .4 2 ab 0. 00 0. 00 14 .9 3± 4. 90 a b 0. 00 0. 00 31 .1 1± 7. 47 a 0. 00 0. 00 0. 04 ±0 .0 4 a 0. 00 0. 00 0. 04 ±0 .0 4 a 1. 44 ±0 .2 6 b s_ u a e_ 10 _7 0_ a u 6. 35 ±6 .3 5 ab 0. 00 4. 52 ±2 .4 3 a 6. 77 ±4 .0 1 bc d 1. 19 ±1 .1 9 b 0. 00 24 .8 6± 13 .1 5 ab 0. 00 0. 00 0. 00 0. 00 0. 00 0. 05 ±0 .0 5 a 2. 94 ±0 .8 5 ab c 80 denisa manolescu et al. was null in all sage extracts and in lemon balm extracts supplemented with aunps, regardless of the extraction method used, suggesting the protective action of these mnps on the organization and function of nuclei in onion root cells. giant cells and cells with irregularly shaped nuclei characterized the control variants, being encountered with much decreased frequency in the experimental variants, including those with agclnps and aunps. conclusions our experimental study demonstrated that the two species of medicinal plants, namely m. officinalis l. and s. officinalis l., can be considered valuable sources of bioactive compounds, being likely to design novel functional products with different therapeutic properties. according to bfstem analysis the biogenic synthesis process of noble metal nanoparticles, was successfully carried out. the agclnps particle sizes under 10 nm were obtained in both lemon balm and sage extracts. irrespective of the species tested, a direct proportionality between the total content of polyphenols of the extracts and nanostructured mixtures and their antioxidant activities was noticed. the most efficient method for obtaining polyphenols with the highest antioxidant activity was found to be microwave-assisted extraction, both for extracts and nanostructured mixtures. mitosis was slightly inhibited in nanostructured phytochemical complexes of lemon balm compared to those of sage. the controls showed the highest frequency of chromosomal aberrations compared both to samples of simple extracts and extracts supplemented with mnps, suggesting the cytogenoprotective, antigenotoxic, and the safety of using these bioformulations as therapeutic alternatives. acknowledgements this work was supported by a grant of the romanian ministry of research, innovation and digitization, cncs– uefiscdi, project number pn-iii-p4-idpce-2020-0620, within pncdi iii and by a grant of the university of pitesti, project number 2242/28.02.2022 (internal competition for research projects, code: cipcs-2021). references adebiyi oe, olayemi fo, ning-hua t, guang-zhi z. 2017. in vitro antioxidant activity, total phenolic and flavonoid contents of ethanol extract of stem and leaf of grewia carpinifolia. 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n. 2016. ultrasound-assisted extraction of bioactive compounds from lemon balm and peppermint leaves. int agrophys. 30:95-104. caryologia international journal of cytology, cytosystematics and cytogenetics volume 75, issue 3 2022 firenze university press chromosome mapping of repetitive dnas in the picasso triggerfish (rhinecanthus aculeatus (linnaeus, 1758)) in family balistidae by classical and molecular cytogenetic techniques kamika sribenja1, alongklod tanomtong1, nuntaporn getlekha2,* chromosome number of some satureja species from turkey esra kavcı1, esra martin1, halil erhan eroğlu2,*, fatih serdar yıldırım3 l-ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations selma tabur1,*, nai̇me büyükkaya bayraktar2, serkan özmen1 characterization of the chromosomes of sotol (dasylirion cedrosanum trel.) using cytogenetic banding techniques kristel ramírez-matadamas1, elva irene cortés-gutiérrez2, sergio moreno-limón2, catalina garcía-vielma1,* contributions of species rineloricaria pentamaculata (loricariidae:loricariinae) in a karyoevolutionary context a cius¹, ca lorscheider2, lm barbosa¹, ac prizon¹, ch zawadzki3, la borin-carvalho¹, fe porto4, alb portela-castro1,4 cadmium induced genotoxicity and antioxidative defense system in lentil (lens culinaris medik.) genotype durre shahwar1,2,*, zeba khan3, mohammad yunus khalil ansari1 biogenic synthesis of noble metal nanoparticles using melissa officinalis l. and salvia officinalis l. extracts and evaluation of their biosafety potential denisa manolescu1,2, georgiana uță1,2,*, anca șuțan3, cătălin ducu1, alin din1, sorin moga1, denis negrea1, andrei biță4, ludovic bejenaru4, cornelia bejenaru5, speranța avram2 polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel (foeniculum vulgare mill.) egizia falistocco methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in pisum sativum l. sazada siddiqui*, sulaiman a. alrumman comparative study and genetic diversity in malva using srap molecular markers syamand ahmed qadir1, chnar hama noori meerza2, aryan mahmood faraj3, kawa khwarahm hamafaraj4, sherzad rasul abdalla tobakari5, sahar hussein hamarashid6,* nuclear dna 2c-values for 16 species from timor-leste increases taxonomical representation in tropical ferns and lycophytes inês da fonseca simão1, hermenegildo ribeiro da costa1,2,3, helena cristina correia de oliveira1,2, maria helena abreu silva1,2, paulo cardoso da silveira1,2,* nuclear dna content and comparative fish mapping of the 5s and 45s rdna in wild and cultivated populations of physalis peruviana l. marlon garcia paitan*, maricielo postillos-flores, luis rojas vasquez, maria siles vallejos, alberto lópez sotomayor identification of genetic regions associated with sex determination in date palm: a computational approach zahra noormohammadi1,*, masoud sheidai2, seyyed-samih marashi3, somayeh saboori1, neda moradi1, samaneh naftchi1, faezeh rostami1 comparative karyological analysis of some turkish cuscuta l. (convolvulaceae) neslihan taşar¹, i̇lhan kaya tekbudak2, i̇brahim demir3, mikail açar1,*, murat kürşat3 identifying potential adaptive snps within combined dna sequences in genus crocus l. (iridaceae family): a multiple analytical approach masoud sheidai1,*, mohammad mohebi anabat1, fahimeh koohdar1, zahra noormohammadi2 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 57-65, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-680 citation: s. mehri, h. shirafkanajirlou, i. kolbadi (2020) genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill.. caryologia 73(1): 57-65. doi: 10.13128/caryologia-680 received: july, 2019 accepted: october, 2019 published: may 8, 2020 copyright: © 2020 s. mehri, h. shirafkanajirlou, i. kolbadi. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi department of agronomy and plant breeding, parsabad moghan branch, islamic azad university, parsabad moghan, iran *corresponding author. e-mail: sh.mehri2000@gmail.com abstract. stellaria media l. vill., is known under the name of chickweed, it is an annual plant in the family caryophyllaceae. stellaria media is distributed in the all regions of iran and has been introduced to many habitats of the world. s. pallida is very similar to s. media. this plant is considered to be as a herbal remedy and is used in folk medicine. stellaria media is edible and nutritious. in the present study, we used morphological and issr data for this species. for this, 43 morphological characteristics, including 16 qualitative and 26 quantitative. amova and gst analyses showed that the populations of this species are genetically differentiated. nm analysis revealed very low value of genetic diversity among the studied population and mantel test indicated isolation by distance occurred among them. the present study showed that the studied populations of s. media are differentiated in morphological characteristics and genetic content. in general, species relationships obtained from morphological and molecular data were largely congruent. keywords. genetic diversity, issr, morphology, species relationship, stellaria media. introduction the family caryophyllaceae comprised about 81 genera and 2600 species (bittrich 1993; ullah et al. 2019a). stellaria l. (caryophyllaceae, alsinoideae) includes both annual and perennial herbaceous plants that are widely distributed in the temperate zones of europe and asia (lu and rabeler 2001; keshavarzi and esfandani –bozchaloyi, 2014a, 2014b; ullah et al. 2019b, 2019c) and about 120 species with worldwide distribution, mainly in the north temperate zone (morton 2005; ullah et al., 2018a, 2018b). in flora iranica this genus has 9 species and divided into 2 sections: sect. pseudalsine boiss. consist of one species s. alsinoides boiss. & buhse and sect. stellaria with six species: s. holostea l., s. persica boiss., s. graminea l., s. nemorum l., s. media (l.) vill., s. pallida (dumort.) pire (rechinger 1988). main center of diversification for stellaria is eurasia, with a center of distribution in the mountains of e. central asia. some species are also cosmopolitan (bittrich 1993; ullah et al. 2018c). 58 shahram mehri, hassan shirafkanajirlou, iman kolbadi there are limited chromosome records for stellaria in the world. basic chromosome numbers of x=10, 11, 12 and 13 have been reported for the genus (federov 1969; moore 1973; goldblatt 1981). stellaria media, chickweed, are annual and with slender stems, they have hairs on one side of the stem. the leaves are linear or oval, smooth or minutely, 13 to 17 × 1.5 to 7 mm. flowers are hermaphrodite and petals are white with 5 deeply. sepals prominently 4 to 6-nerved, 4 to 7 mm. stigmas are 3 and the stamens are 3. stellaria media common in waste places, open areas, lawns, meadows, and widely distributed to temperate regions of europe, asia and northern america. stellaria media is edible and nutritious and has a history of herbal use and medicinal properties. this species has been used as to soothe severe itchiness even where all other remedies have failed (slavokhotova et al. 2011). it is considered for rheumatic pains, skin diseases, and period pain as well as for bronchitis and arthritis (slavokhotova et al. 2011). stellaria media possess significant chemicals known as saponins, which can be cause saponin poisoning in cattle (haragan 1991). there are many studies which are on taxonomy, pollen morpholog y, phylogeny, seed micromorphology, anatomy, trichome and cytology of stellaria species (esfandani-bozchaloyi and keshavarzi 2014; keshavarzi and esfandani-bozchaloyi 2014 a, b; ullah et al. 2018a, 2018b, 2018c). however, genetic diversity of stellaria species have been reported in a few studies (verkleij et al. 1980; chinnappa and morton 1984), also outcrossing or inbreeding, genetic structure, genetic variability within/ between populations and ecological adaptation on stellaria of iran have not been investigated yet. according to ellis and burke (2007) genetic diversity are essential in the adaptability and survival of population, because it is as a way for adapt to changing environments in populations. the adapt of the population to the changing environment will depend on the presence of the genetic diversity. large populations have higher genetic diversity due to more to maintain genetic material and small populations have the loss of diversity which is called genetic drift. mating or inbreeding between individuals with similar genetic occur in small population sizes, thus decreasing genetic diversity and finally we have more common alleles. hence, the used of markers will depend on the type of the species. we have been used dna marker based techniques such as inter-simple sequence repeats (issrs), due to easy, highly reproducible, stable and useful in species delimitation, gene tagging, gene flow, breeding programs and evolutionary biology (ellis and burke 2007; esfandanibozchaloyi et al. 2018a, 2018b, 2018c). therefore, we studied morphological and molecular study of 11 geographical populations of s. media for the first time in iran. materials and methods morphological studies 85 plant sample were selected from eleven populations located in three provinces of iran. identification of species stellaria media were based on the descriptions provided by flora iranica (rechinger 1988). the sampling sites and herbarium number are provided in table 1, figure 1. vouchers were deposited at the herbarium of islamic azad university, science and research branch, tehran, iran (iauh). dna extraction fresh leaves of 85 individuals following a modified ctab protocol. the quality was checked on a 1 % agarose table 1. location addresses and ecological characters of the stellaria media population locality latitude longitude altitude (m) voucher no. 1 guilan, road to sangar 37°06’ 57” 49°11’06” 47 iauh 201600 2 guilan, bandar anzali, pine artificial woodland 37°27’34” 49°42’40” -25 iauh 201701 3 guilan, loleman 37°28’59” 49°33’45” -29 iauh 201702 4 guilan, siahkal, sangar 37°09’08” 49°55’02” 27 iauh 201603 5 guilan, gole rodbar river 37°10’05” 49°56’38” 15 iauh 201604 6 guilan , sheytan kouh hill side 37°12’04” 50°03’12” 9 iauh 201605 7 guilan , lahijan , highlands of sheytan kouh 37°11’52” 50°03’17” 159 iauh 201606 8 guilan, bandar anzali, road side 37°27’48” 49°22’30” -11 iauh 201707 9 mazandaran, chalos neamat abad 36°49’02” 50°52’20” -16 iauh 201608 10 mazandaran, shirodi ring road 36°51’10” 50°32’11” -18 iauh 201709 11 mazandaran, noshahr 36°35’04” 51°35’14” -20 iauh 201710 59genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. gel and spectrophotometry. a set of ten primers; (gg) 5gt, (aa) 7gt, (aaa) 5gt, ubc 234, (gg) 7at, (ag) 7g, ubc 825, ubc 823, (gg) 5t and (gc) 8gg were used for issr analysis. pcr were carried out in 25 μl reactions containing 20 ng of template dna, 0.3 mm dntps, 1μm primers, 1.0 μl of 20 ×pcr buffer (cinnagen, iran),1.8 mm of mgcl2, and 5 units of taq polymerase (cinnagen, iran). the amplif ication was carried out, with programmed as initial pre-denaturation at 95°c for 5 min followed by 36 cycles of denaturation at 94°c for 45 s, annealing at temperature (52-55°c) for 40 s, and extension at 72°c for 1min. a final 5 min extension at 72°c followed the completion of 38 cycles. karyological study for somatic chromosome study, the seeds were soaked for 24 hours in running water and germinated in the laboratory (ca. 21º-24º). the root tips were cut between 9-11 am and pretreated in 0.002m 8hydroxyquinoline (4hours) and fixed in a cold mixture of ethanol and acetic acid (3:1) for 24 hours. root tips were macerated in 1n hcl for 10 minutes (cold hydrolysis) at room temperature. the slides were staining in 2% feacetocarmin for 10 hours. data analyses morphological studies for morphological studies 43 morphological characters including 16 qualitative and 26 quantitative characters were studied following the protocols of (ashfaq et al. 2019; attar et al. 2019; gul et al. 2019a; gul et al. 2019b; kandemir et al. 2019; shah et al. 2018a, 2018b; zaman et al. 2019) (table 2). figure 1. distribution map of the studied populations. table 2. list of selected characters and their codes in morphological studies. no. characters numerical code 1 plant height mm 2 length of basal leaves mm 3 width of basal leaves mm 4 length of stem leaves mm 5 width of stem leaves mm 6 bract length mm 7 width bract mm 8 length pedicel mm 9 number of seeds per capsule 10 number of flowers per inflorescence 11 number of calyx 12 length calyx mm 13 width calyx mm 14 number of petal 15 petal length mm 16 petal width mm 17 cleft size of petals mm 18 inter node length mm 19 number of stamen 20 number of stigma 21 capsule length mm 22 seed length mm 23 seed width mm 24 cleft size of capsule mm 25 number suture capsules 26 veins number sepals 27 growth period 0-annual 1perennial 28 bract apex 0-acute 1narrow 2 absence 29 state of stem 0-unbranched 1 branched 30 state of stem strength 0-thin 1strong 31 hairs of stem 1-unilateral hair 2 multilateral hair 32 cross-section of stem 0-round1rectangular 2 elliptical 33 shape of basal leaves 0-linear 1linear lanceolate 34 basal leaves apex 0-acute 1narrow 35 basal leaves petiole 0-absence 1presence 36 hair of basal leaves petiole 0-absence 1presence 37 shape caulin leaves 0linear 1linear lanceolate 38 caulin leaves apex 0acute 1narrow 39 caulin leaves petiole 0-absence 1presence 40 hair of caulin leaves petiole 0-absence 1presence 41 hair of caulin leaves margin 0-absence 1presence 42 hair of caulin leaves lamina 0-absence 1presence 43 shape of bract 0-linear 1linear lanceolate 60 shahram mehri, hassan shirafkanajirlou, iman kolbadi morphological traits were standardized (mean = 0, variance = 1) and used to estimate euclidean distance for ordination analyses (podani 2000). pca (principal components analysis) biplot and mds (multidimensional scaling) were applied for grouping and identify the most variable morphological traits of among the populations (podani 2000). we used from past version 2.17 (hammer et al. 2012) for multivariate statistical analyses. molecular analyses issr bands scored as present (1) or absent (0). genetic polymorphism was determined by genetic diversity parameters: shannon information index (i), percentage of polymorphism, the number of effective alleles and nei’s gene diversity (h) (freeland et al. 2011). neighbor-net networking was used for nei’s genetic identity among studied populations (huson and bryant 2006; weising et al. 2005). we used from past ver. 2.17 (hammer et al. 2012), splitstree4 v4.13.1 (2013) and darwin ver. 5 (2012) software for analysis data. for amova (analysis of molecular variance) we used of genalex 6.4 software (peakall and smouse 2006; meirmans and van tienderen 2004) that was determined genetic differentiation of the species and nei,s gst analysis in genodive ver.2 (2013) (hedrick 2005; jost 2008) were used to revealed genetic distance of the species. first data were scored as dominant markers (issr) so we used from structure analysis for estimate the parameters that related to gene flow among studied population. burn-in = 10000, and 10 runs were performed for relationship between genetic structure and distance of geographical. maximum likelihood method and bayesian information criterion (bic) was studied by structure analysis (falush et al. 2007; evanno et al. 2005; meirmans 2012). gene flow was determined by calculating nm from gst by popgene ver. 1.32 (1997). (pritchard et al. 2000). results in this study 11 populations of stellaria media were selected from northern regions of iran. genetic diversity parameters revealed that the highest percent of genetic polymorphism (48.89%) and gene diversity (0.179) exist in guilan, bandar anzali, (population no.5), while the lowest amount of genetic polymorphism (13.33%) showed in population guilan, road side bandar anzali (no.8) table 3. amova test showed that, 40% of total genetic diversity was within population and 60% was among population. hedrick standardized fixation index makes of genetic distance among the studied populations. we have moderate level for amova produced after 999 permutations (g’st = 0.515, p = 0.001) and hedrick differentiation index (d-est = 0.331, p = 0.001). our results showed that the populations of s. media are differentiated from each other. populations, genetic affinity neighbor-net network and nj tree revealed identity results but here only neighbor-net network is discussed (figure 2). in the network showed that the populations 1 and 4, as well as populations 7 and 8 show are placed close to each other and they have closer genetic affinity. the populations 3 and 5, 6, 11 are differentiated from the other populations. the studied specimen in mds plot revealed that they were stay in different groups, which this results were in agreement with the amova results (figure 3). the relationship between altitude distance and genetic distance by mantel test after 5000 permutations makes significant in these populations (r = 0.38, p = 0.001). we have isolation in stellaria media occurred that we have low amount of gene flow due to geographically more distant of populations. populations genetic structure the result carried out on structure analyses by evanno test which makes a peak at k = 9 (figure 4). furtable 3. genetic diversity parameters in the studied populations. (n = number of samples, ne = number of effective alleles, i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations). pop n na ne i he uhe %p pop1 6.000 0.633 1.136 0.120 0.080 0.087 23.33% pop2 8.000 0.644 1.119 0.121 0.077 0.083 25.56% pop3 23.000 0.756 1.140 0.138 0.088 0.090 32.22% pop4 5.000 0.511 1.123 0.109 0.073 0.081 20.00% pop5 10.000 1.011 1.312 0.265 0.179 0.188 48.89% pop6 6.000 0.944 1.279 0.231 0.158 0.172 40.00% pop7 5.000 0.533 1.118 0.101 0.068 0.075 18.89% pop8 4.000 0.422 1.099 0.078 0.054 0.061 13.33% pop9 6.000 0.678 1.140 0.120 0.081 0.089 21.11% pop10 6.000 0.922 1.260 0.227 0.152 0.166 42.22% pop11 6.000 0.878 1.217 0.198 0.130 0.142 40.00% 61genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. thermore, structure analyses shown genetic identity between populations 1 and 4 (similarly colored), populations 7 and 8, like populations 9-10. but also it indicated genetic difference of populations 3 and 5 (differently colored), likes 6 and 11. the results of reticulogram (figure 5), indicated some of shared alleles that is based on the least square method among populations 10 and 4, 6 and between 7 and 4 and 10, also between 3, 11, 1 and 2 and 8. the mean nm = 0.29 that is very low level of genetic diversity and supports genetic stratification as showed by structure analyses and k-means. nm result agreed with population assignment test and cannot showed gene flow among these populations. in total ten issr primers produced 90 bands, fragment size ranged from 100 to 2800 bp. morphometric analyses anova test for 85 plant specimen were examined from 11 populations. our results indicated significant difference in compare with the studied populations (p < 0.05). ordination plot and other analyses produced similar result among populations (figure 6). our result revealed that among of the studied populations exist of morphological divergence and this divergence was due to quantitative traits. for example, length of stem leaves character separated population no. 9, but the populations 3 and 5 separated from the other populations due to character calyx length. figure 2. neighbor-net network of populations in s. media based on issr data. figure 3. mds plot of populations in s. media based on issr data. figure 4. structure plot of s. media populations based on k = 9 of issr data. figure 5. reticulogram of s. media populations based on least square method analysis of issr data. (population numbers are according to table 1. figure 6. pca plot of s. media populations based on morphological characters. 62 shahram mehri, hassan shirafkanajirlou, iman kolbadi we performed for both morphological and issr data a consensus tree (figure 7). it indicated that some population are differenced from other population based on both morphological and molecular characters. karyological characteristics in this study three populations of s. media show a tetraploid level, 2n=40 (figure 8a), six populations show a tetraploid level, 2n=42 (figure. 8b) and two populations show a tetraploid level, 2n=44 (figure. 8c) is in accordance with previous report (morton 2005; runemark 1996). there are high morphological variations in populations of s. media so that in some references subspecies have been defined for these taxa. the results show that such variations have chromosome number differences in iran as most morphological variations were considered from different parts of iran for this study. in s. media have been reported 2n=28, 36, 40, 42 and 44 from eurasia with 2n=40 predominating (federov 1974; löve and löve 1975; moore 1973). this species shows a high phenotypic plasticity and genotypic flexibility. discussion according to çalişkan (2012) genetic diversity provides information about adapt to changing environments, understanding of positive influence in the conservation of endangered species, hybridization and gene flow among the populations. this study evaluates on the use of inter simple sequence repeats markers for compare gene flow and relationships within the population of s. media in iran. verkleij, et al (1980) showed that amylases isoenzymes could be successfully applied to assess interpopulational variation in stellaria media. s. media has many medicinal properties and distributed in our country, however, we provided information on current taxonomic, molecular study and geographical distance. the present study indicated data about gene flow and genetic structure in some part of iran. chickweed can any time of the year at all germinate and flower. system pollination is mainly self-pollinating, but sometimes can occur cross-pollination by flies and insects. according to chater and heywood (1993) stellaria media widespread weedy species and it is the accepted name. there are three subspecies;1subsp. media, 2subsp. cupaniana and 3subsp. postii but some people showed that subsp. cupaniana (sinha 1965; scholte 1978) and subsp. postii (sinha 1965) should be included in s. neglecta. according to fedorov (1969) chromosome numbers that have been reported for s. media included 2n = 24, 28, 36, 38, 40, 42 and 44 from many parts of the world. however, chromosome numbers 2n =40, 42 and 44 are the most commonly reported and this species revealed a high degree of genotypic variation that is highly correlated with its reproductive (freeland et al. 2011; verkleij unpublished). figure 7. consensus tree of morphological and molecular data in s. media populations. figure 8. micrographs of chromosomes of root tips in studied species. a) s. media (2n=40), b) s. media (2n=42), c) s. media (2n=44). 63genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. s. media is annual, characterized by the presence of five sepals and petals which are usually bifid; (whitehead and sinha 1967). generally, within family caryophyllaceae diversity of morphological features makes taxa complicated to be delineated and identified (whitehead and sinha 1967). s. media is occurring on abandoned fields and commonly sensitive to disturbance of its habitat. between s. pallida and s. media there are crossing barrier and they are self-pollinating (peterson 1936), this happened due to presence of polyploidy in s. media (2n=40-44) while observed the diploidy of s. pallida (2n = 22) (scholte 1978; slatkin 1993; jolivet and bernasconi 2007). therefore, breeding systems plays role important in low level of gene flow in s. media (hutchison and templeton 1999; medrano and herrera 2008). our results provided that the seed morphologies of stellaria media and s. pallida are similar. seed coat cells are rounded polygonal and v-shaped margin. based on these characters, we decided that stellaria media could be differenced from s. pallida. seed coat morphology observed of 18 species of stellaria by chen (2010). they stated that there are differences between myosoton and stellaria. rani et al. 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cihangir alaca1, ali özdemir1, bahattın bozdağ2, canan özdemir2,* clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 73(2): 89-98, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-618 citation: h. singh, j. singh, p. kumar, v. kumar singhal, b. singh kholia, l. mohan tewari (2020) chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india. caryologia 73(2): 89-98. doi: 10.13128/caryologia-618 received: september 8, 2019 accepted: april 3, 2020 published: july 31, 2020 copyright: © 2020 h. singh, j. singh, p. kumar, v. kumar singhal, b. singh kholia, l. mohan tewari. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india harminder singh2, jaswant singh1,*, puneet kumar2, vijay kumar singhal1, bhupendra singh kholia2, lalit mohan tewari3 1 department of botany, punjabi university patiala, india, 147002 2 botanical survey of india, northern regional centre, dehradun, india, 248195 3 department of botany, d.s.b. campus, kumaun university, nainital, india, 263001 *corresponding author. e-mail: jaswant_rs@pbi.ac.in abstract. present cytological study records existing chromosome number diversity, their male meiotic course and pollen fertility analysis in the two wheatgrass species of tribe triticeae dumort. (poaceae) from western himalaya, india. agropyron thomsonii hook. f. is an endemic grass of alpine zones of western himalaya and elymus nutans griseb., a widely distributed grass in sub-alpine to glacial regions of himalaya. the gametophytic chromosome number count of n=21 (jadh ganga valley, uttarkashi) is a pioneer count for a. thomsonii. during the male meiotic course of a. thomsonii, 14.0416.29% and 2.97-4.17% pollen mother cells, respectively at prophase-i and metaphasei, observed to be involved in phenomenon of cytomixis. seven accessions of e. nutans collected from bhagirathi valley and jadh ganga valley of uttarkashi district, and pangi valley of chamba district, recorded with gametophytic chromosome number count of n=21 and record of 1b-chromosome in pun61958 is a new record for the species. in three accessions 5.56-9.41% and 2.5% pollen mother cells at prophase-i and metaphase-i, respectively were also noted with phenomenon of cytomixis. in addition to phenomenon of cytomixis, during meiotic course of both species pollen mother cells also depicted associated meiotic course irregularities viz. non-synchronous disjunction of bivalents, chromatin bridges, laggards, micronuclei in sporads and shrivelled microspores. these species are growing in cold climatic condition habitats. so, cold stress seems to be a preferential inductor for cytomixis and associated meiotic abnormalities in the gametic cells of stamens of a. thomsonii and e. nutans that ultimately leads to reduction in pollen fertility. keywords: himalayan grasses, cold stress, polyploidy, male meiosis, cytomixis and meiotic abnormalities, pollen sterility. introduction tribe triticeae dumort. (poaceae) includes annual and perennial grass taxa, having large-sized chromosomes, comprising a polyploid complex of 90 harminder singh et al. 2x, 4x, 6x, 8x, 10x and 12x ploidy levels with a uniform base number of x=7 (dewey 1984). the members of tribe triticeae have distribution to almost all floristic regions of the globe. in himalaya, agropyron gaertn. and elymus l. are prominent grass genera of temperate to alpine zone habitats, and an inflorescence is a spike that has one to many spikelets at each rachis node. earlier, the genus agropyron was represented by more than 33 species in himalaya along with adjoining regions of similar terrain (bor 1960), and the modern concept of agropyron restricts it to only those species having keeled glumes and/or pectinately arranged spikelets (cope 1982). so, the most of the species of agropyron are now assigned to genus elymus (melderis 1978; singh 1983; karthikeyan et al. 1989). at present in himalayan regions of india genus agropyron is represented by a single species, i.e. a. thomsonii hook. f. and genus elymus by 31 species (singhal et al. 2018b). a. thomsonii hook.f. [= e. thomsonii (hook. f.) melederis; = e. nayarii karthik.] and elymus nutans griseb. are perennial and caespitose wild grasses growing in the high altitudinal regions of himalaya (above 3000 m). former is an endemic grass to western himalaya (pusalkar and singh 2012) and later species have wider in distribution that is growing in different ecological habitats of bhutan, china, india (himalaya), iran, japan, mongolia, nepal, pakistan and russia (bor 1960; lu 1993; murti 2001; chen and zhu 2006; pusalkar and singh 2012; dvorský et al. 2018). scrutiny of published cytological data reveals that a. thomsonii is still unrecorded for its chromosome number. as germplasm of grasses remained in a central position towards the breeding programs and germplasm enhancement to ensure the food and fodder demands. so to make their programs successful, there is a need to have complete knowledge and understanding about the genetic diversity of available germplasm (kawano 2018). the present study is in a line of endeavour to explore the morphological diversity in the grasses from phytogeographically distinct and unexplored regions of western himalaya. so, this study aimed to record the exact chromosome number in a. thomsonii and e. nutans along with to comment on the behaviour of pollen mother cells during the male meiotic course, and pollen fertility. we also try to correlate prevalence of cold conditions in natural habitat and the possible cause of cytomixis, associated meiotic abnormalities and reduction in pollen fertility in the currently studied species. materials and methods wild plant accessions of a. thomsonii and e. nutans were collected for detailed male meiotic and pollen fertility studies from bhagirathi and jadh ganga valley, uttarkashi district, uttarakhand and pangi valley, chamba, himachal pradesh. young and unopened spikes, fixed in carnoy’s fixative (ethanol: chloroform: glacial acetic acid= 6: 3: 1). after 48 h, the materials were transferred to 70% ethanol and stored in a refrigerator. meiocyte preparations were made by squashing the developing anthers from the unopened florets in 1% acetocarmine. chromosome counts and meiotic course was studied from freshly prepared slides having pollen mother cells (pmcs)/ meiocytes at diakinesis, metaphase-i (m-i), anaphase-i (a-i) and telophases (t-i, t-ii). apparent pollen fertility was estimated through stainability tests by squashing the mature anthers in glycerol and 1% acetocarmine (1:1) mixture. well-filled pollen grains with completely stained nuclei and cytoplasm were scored as fertile/viable, while partially stained and shrivelled ones as sterile/ non-viable. good preparations of pmcs with well-spread bivalents/chromosomes, meiotic irregularities, and pollen grains selected for photomicrographs using nikon 80i eclipse and leica qwin digital imaging system. pollen size was measured through microscopy. meiotically analyzed accessions were identified by studying, in detail the floral characters and by consulting the floras viz. grasses of burma, ceylon, india and pakistan (bor 1960), flora of pakistan-poaceae (cope 1982), flora of cold deserts of western himalaya-monocotyledons (murti 2001) and flora of gangotri national park, western himalaya (pusalkar and singh 2012). identifications revalidated by comparing the specimens with the vouchers already submitted by taxonomists in the herbaria, botanical survey of india, northern circle, dehra dun (bsd) and available specimens, facilitated by other herbaria as online resource (global plants1). voucher specimens of cytologically examined accessions deposited in the herbarium, department of botany, punjabi university, patiala (pun) and herbarium botanical survey of india, northern circle, dehra dun (bsd). results in the present exploration study, cytological investigations made on two accessions of a. thomsonii gathered from the alpine meadows of jadh ganga valley and seven accessions of e. nutans from bhagirathi valley, jadh ganga valley and pangi valley. data regarding the name of taxon, sites of the collection with altitude, accession number (bsd, pun), gametic chromosome number, ploidy level, pollen fertility percentage tabulated in table 1. 1 global plants (https://plants.jstor.org/). 91chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii and elymus nutans two wild accessions of a. thomsonii collected from the glacial floristic area of nelong, jadh ganga valley, an eastern part of cold deserts of india. current meiotically analyzed accessions of a. thomsonii are of dwarf habit (plant height 28 cm; spike length 9.5 cm; spikelet length 1.5 cm), which are hexaploid (6x; x=7) in nature with gametophytic chromosome number count of n=21, that confirmed from the presence of 21 bivalents in pmcs at m-i (fig. 1a) and 21:21 chromosomes at a-i poles (fig. 1b). during the meiotic course, 14.04-16.29% prophasei pmcs (fig. 1d, e) and 2.97-4.17% metaphase-i pmcs (fig. 1f) observed with the phenomenon of cytomixis. in the majority of pmcs, chromatin migration occurred as deformed mass or partially deformed bivalents through cytoplasmic channels. partial/complete migration of chromatin material among neighbouring pmcs leads to formation of hypoploid, hyperploid and enucleated pmcs (fig. 1d, e). some pmcs observed in a state of pycnosis (fig. 1h), and in few instances nucleolus pioneered migration of chromatin material occurs that noted as a presence of additional nucleolus in diakinesis pmcs (fig. 1c). further during the meiotic course considerable number of pmcs at m-i, a-i/ii and t-i/ ii observed with associated meiotic abnormalities like, table 1. information on taxon, locality of collection with altitude, accession number/s, meiotic chromosome number, ploidy level, pollen fertility percentage of the cytologically investigated taxa. sr. no taxon locality with altitude (m) accession number/s meiotic chromosome number (n) ploidy level pollen fertility (%) 1. agropyron thomsonii hook. f. (= elymus nayarii karthik.) nelong i, uttarkashi, uttrakhand, 3450 pun 61593 21 6x 60 nelong cp, uttarkashi, uttrakhand, 3500 pun 61594 21 6x 65 2. elymus nutans griseb. sural, chamba, hinmachal pradesh, 3008 bsd 1181196 21 6x 97 nelong i, uttarkashi, uttrakhand, 3450 pun 61955 21 6x 95 nelong cp, uttarkashi, uttrakhand, 3500 pun 61956 21 6x 90 bhojwasa, uttarkashi, uttrakhand, 3700 pun 61022 21 6x 98 bhojwasa, uttarkashi, uttrakhand, 3750 pun 61015 21 6x 98 bhojwasa, uttarkashi, uttrakhand, 3800 pun 61957 21 6x 95 gaumukh, uttarkashi, uttrakhand, 3900 pun 61958 21+0-1b 6x 95 table 2. data on the percentage of pmcs involved in chromatin transfer, abnormal sporads and pollen size of agropyron thomsonii and elymus nutans. taxon accession number (pun) pmcs involved in cytomixis (%) out of the plate bivalents/ chromosomes (%) laggards/ chromatin bridges (%) sporads (%) pollen size (µm) dyads with tetrads with prophase-i m-i m-i a-i a-i/t-i a-ii/t-ii micronuclei micronuclei 1-2 shrivelled microspores agropyron thomsonii hook. f. 61593 14.04 (40/285) 2.97 (4/135) _ _ 12.30 (8/65) 15.00 (6/40) 10.00 (2/20) 10.29 (7/68) 4.41 (3/68) small: 29.31 x 29.31 medium: 36.80 x 36.80 large: 42.68 x 42.68 61594 16.29 (57/350) 4.17 (10/240) 6.25 (15/240) 8.57 (6/70) 17.14 (12/70) 14.29 (8/56) 13.64 (3/22) 8.89 (8/90) 6.67 (6/90) elymus nutans griseb. 61955 9.41 (8/85) 6.67 (4/60) small: 27.86 x 27.86 medium: 37.47 x 37.47 large: 41.32 x 41.32 61956 8.00 (6/75) 2.50 (2/80) 12.50 (10/80) 16.00 (8/50) 4.00 (2/50) 61958 5.56 (5/90) 11.43 (4/35) 92 harminder singh et al. figure 1. male meiosis in agropyron thomsonii: (a) m-i pmcs with 21 bivalents (b) a-i pmc with 21:21 chromosomes at each pole; (b) pmcs involved in chromatin migration at p i (arrowed); (c) a diakinesis pmc possessing relatively small sized additional nucleolus pmcs; (d) prophase-i pmcs depicting phenomenon of cytomixis (arrowed); (e) diakinesis pmcs with chromatin migration and depicting formation of hyperploid and enucleated pmc (arrowed); (f) m-i pmc involved in chromatin migration (arrowed); (g) a-i pmc with non-synchronous dysjunction of bivalents depicted by univalents and bivalent bridges (arrowed); (h) a pmc depicting abnormal spindle and state of pycnosis with fragments (arrowed); (i) a-i pmc with several laggards (arrowed); (j) early t-i pmc with dicentric bridge, fragments and laggards (arrowed); (k) t-ii pmc as dyad with on subunit with laggard (arrowed); (l) t-i pmcs with miocronuclei at one pole (arrowed); (m) dyad subunits with micronuclei (arrowed); (n) tetrad microspore subunits with micronuclei (arrowed); (o) a tetrad with two shrivelled microspore units (arrowed); (p, q) fully stained fertile, shrivelled and unstained as sterile pollen grains. scale bar=10μm. 93chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii and elymus nutans chromatin bridges (fig. 1g) and laggards/ fragments (fig. 1i, j, k, l), and micronuclei in sporads (figs. 1m, n), collectively depicting a syndrome of errors occurred during the meiotic course (table 2). during, the microsporogenesis two types of sporads were noted, first having all normal microspores and another type with shrivelled microspores (fig. 1o). these meiotic abnormalities during meiotic course have resulted in low pollen fertility (60-65%) and formation of heterogeneous sized pollen grains in these accessions (fig. 1p, q). seven accessions of e. nutans collected from subalpine and glacial vegetation zones of three valleys noted to have variable plant height in their natural habitat, viz. dwarf (pun 60482, 61956: plant height, 9-15 cm; spike length, 3-5.5 cm; spikelet length, 10-12.5 mm; awn length, 7-10 mm), intermediate (pun 61955: plant height, 25 cm; spike length, 8-8.5 cm; spikelet length, 9-10 mm; awn length, 15-17.5 mm) and tall (pun 61022, 61957-58, bsd 1181196: plant height, 50-65 cm; spike length, 15-18 cm; spikelet length 30-45 mm; awn length, 25-45 mm). these accessions unequivocally have a gametic chromosome number count of n=21, confirmed from the presence of 21 bivalents in pmcs at m-i (fig. 2a). 1b-chromosome was also observed in few pollen figure 2. male meiosis in elymus nutans: (a) m-i pmc with equal-sized 21 bivalents; (b) m-i pmc with 1b-chromosome associated with a-bivalent (arrowed); (c) m-i pmc with independent 1b-chromosome (arrowed); (d) m-i pmc with chromatin stickiness; (e) m-i pmc depicting the phenomenon of cytomixis (arrowed); (f) m-i pmc with migrated chromatin material as pycnotic mass (arrowed); (g) a-i pmc with non-synchronous dysjunction of bivalents (arrowed); (h) heterogeneous sized pollen grains (arrowed); (i, j) fully stained fertile, partially stained and unstained as sterile pollen grains. scale bar=10μm. 94 harminder singh et al. mother cells at metaphase-i (fig. 2b, c) of an accession scored from gaumukh. majority of the pollen mother cells during meiotic course observed with normal meiotic behaviour except some percentage of pollen mother cells of three accessions (pun 61955, 61956, 61958) noted with the phenomenon of cytomixis at diakinesis and m-i (fig. 2e), chromatin stickiness (fig. 2d), with migrated chromatin material masses (fig. 2f), late dysjunction of bivalents, chromatin bridges (fig. 2g), and formation of variable-sized pollen grains (fig. 2h) in these accessions (table 2). due to a low ratio of meiotically abnormal pollen mother cells to normal pollen mother cells in the studied accessions, recorded high percentage of pollen fertility (90-98%) (fig. 2i, j). discussion chromosome number and ploidy a. thomsonii is an endemic grass to western himalaya, and the analyzed gametophytic chromosome number count of 2n=42 is a first record for the species. in the case of e. nutans record of 1b-chromosome is also a first record in the species. earlier, the first chromosome number count of 2n=42 for e. nutans has recorded by gohil and koul (1985) from fotula, a cold desert region of ladakh, western himalaya, india and from sichuan and qinghai regions of china by liu (1985). reports of chromosome number, 2n=42 are also known from pakistan himalaya (salomon et al. 1988) and other distant regions of china (lu et al. 1990; lu 1993, 1994; lu and bothmer 1993; chen et al. 2009, 2013; dou et al. 2009, 2017; yan et al. 2009, 2010). as tribe triteceae has basic chromosome number, x=7 and species of genus agropyron have three ploidy levels (2n=2x, 4x, 6x) with only p genome, but the species of genus elymus have four ploidy levels (2n=2x, 4x, 6x, 8x) with genome constitution of ‘s/st’, ‘h’ and ‘y’ in different combinations (dewey 1984). so, a. thomsonii chromosomally exists at hexaploid ploidy level, and the genome is autopolyploid in nature. similarly, e. nutans is also a hexaploid species, but its genome constitution is of strict allopolyploid in nature (lu 1993, 1994). both the species are hexaploid and during the meiotic course in pollen mother cells, noted with formation of regular bivalents without any indication of multivalents, displaying the diploid like meiosis. the diploid like meiosis is probably the result of the selection of mutations in loci involved in chromosome pairing and chiasma formation facilitated by parental genome chromosomes (mcguire and dvořák 1982). analysis of synaptonemal-complex in allopolyploid grasses reveals the existence of diplodizing genetic system as in festuca spp. (thomas and humphreys 1991, thomas and thomas 1993), triticum and aegilops spp. (holm 1986, holm and wang 1988, cuñado et al. 1996 a, b, c, cuñado and santos 1999) that works through the restriction of synapsis to homologous chromosomes and suppression of crossing over among non-homologous chromosomes. cytomixis, meiotic abnormalities, and its consequent effects the abnormal meiotic course often leads to disturbances in microsporogenesis henceforth resulting in pollen malformation or sterility and furthermore negatively influence the reproductive success of the species in the wild (lattoo et al. 2006; kumar and singhal 2008; singhal and kumar 2008; kumar 2010). meiotic abnormalities in natural conditions act as agents of polyploidy in plants (mason and pires 2015). the deviation from the normal meiotic course may result in unreduced gamete formation. such male meiotic studies in wheat grasses (agropyron and elymus) can be of great importance in discovering wild relatives of cultivated crops. in the current study the phenomenon of cytomixis observed predominantly during the first meiotic division in both species. cytomixis is a natural phenomenon, involving transfer of chromatin material mainly in proximate meiocytes/cells of plants through cytomictic channels (mursalimov and deineko 2017). körnicke (1901) was the first one to observe cytomixis in meiocytes of crocus sativus. however it was gates (1911) who coined the term ‘cytomyxis’ (nowadays ‘cytomixis’) and was defined it as the chromatin extrusion process which is a natural part of meiosis. nowadays, cytologists based on the observations with modern tools employed in plant sciences consider it, as cell to cell communication (kravets et al. 2017); a biological process (sidorchuk et al. 2016) that takes place without any damage to migrated chromatin material (mursalimov et al. 2018) and as an additional putative genetic recombination process (mursalimov and deineko 2017). so, cytomixis is a natural meiotic aberration of potential evolutionary significance (singhal et al. 2018a). in present study, during male meiotic course of a. thomsonii, 14.04-16.29% and 2.97-4.17% pmcs at prophase-i and m-i, respectively depicted the phenomenon of chromatin transfer. similarly, 5.56-9.41% and 2.5% pmcs of e. nutans at prophase-i and m-i, respectively showed the phenomenon of chromatin transfer among proximate pollen mother cells. cytomixis was observed in the early stages of the first meiotic division only. high frequency of cytomixis during the first meiotic division results in high sterility and formation of heterogeneous sized pollen grains (de and sharma 1983; consolaro and pagli95chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii and elymus nutans arini 1995; de souza and pagliarini 1997; pierozzi and benatti 1998; singhal and kumar 2008). the chromatin transfer involves partial or complete migration of nuclear contents, which results into origin of hyper, hypoploid and enucleated meiocytes. also, few pmcs during chromatin transfer along with/without migrated chromatin material also acquire additional nucleoli (kumar 2010). kumar and singhal (2016) while enlisting 31 species possessing additional nucleoli suggested that in majority of the species with an additional nucleoli are resulted due to phenomenon of cytomixis. pmc’s at m-i, a-i/ ii and t-i/ii observed with other meiotic abnormalities, depict a syndrome of errors occurred during the meiotic course. differential extent and intensity of pairing/crossing over among non-sister chromatids of homologous chromosomes results to non-synchronous dysjunction of bivalents during meiosis-i, and formation of chromatin bridges occurs (kumar 2010). crossing over within paracentric inversion pairing loops or u-type exchange between non-sister chromatids during paring creates dicentric bivalent bridges and acentric fragments configurations observed at early t-i. formation of dicentric bridge and laggards/fragment as meiotic configurations during meiotic course appears as a meiotic syndrome that depicts reduced control over meiotic course (jones and brumpton 1971). presence of large number of laggards, possibly due to abnormal spindle, disturbed cytoskeleton and other cellular changes. these chromatin fragments/laggards lead to formation of micronuclei in sporads. as, abnormal chromosome segregation, presence of micronuclei and reduced pollen fertility results due to formation of multiple spindles at meiosis-i (vasek 1962). in jadh ganga valley, cold conditions are prevalent and both the accessions of a. thomsonii growing in this region are prone to extreme temperature fluctuations i.e. short warm days and long cold nights. male reproductive organs and their development are extremely sensitive to cold stress (liu et al. 2019). so, during the microsporogenesis, formation of shrivelled microspores noted in tetrads that may be due to cold stress-induced abnormal development mediated through malnutrition. cold stress disrupts stamen development and prominently interferes with tapetum programmed cell death, which is crucial for progression of normal meiotic course and development of microspores to pollens (oliver et al. 2005, 2007; sharma and nayyar 2014; liu et al. 2019). the phenomenon of cytomixis and associated meiotic abnormalities in a. thomsonii and e. nutans seems to be due to low-temperature stress conditions that are prevalent in the region and have potential to alter the expression of certain alleles controlling the vital steps of meiosis. previous cytological studies of bedi (1990), bellucci et al. (2003), malallah and attia (2003), kumar et al. (2010, 2011, 2014, 2017), singhal and kumar (2008, 2010) and mandal et al. (2013) are also in the view that cytomixis is under direct control of genetic factors. pollen development is a complex process regulated at different genetic levels. mutants showing abnormal pollen development can be of beneficial help in understanding the process of pollen development (sheila 1993). cy tomixis, coupled with associated meiotic abnormalities, leads to the formation of genetically variable pollen grains, affecting pollen size and fertility. pollen size variation depends upon the extent of chromatin material, or amount of dna (stebbins 1971) possessed/ lost by meiocytes during chromatin transfer, and presently somewhat pollen size variation was observed. effects of cytomixis on meiotic course, pollen size, and pollen fertility have previously been reported in grasses viz. agropyron cristatum (bauchan et al. 1987), alopecurus arundinaceus (koul 1990), brachiaria humidicola (boldrini et al. 2006), elymus semicostatus (singhal et al. 2018c), urochloa panicoides (basavaiah and murthy 1987), and many other flowering plants viz. vicia faba (bhat et al. 2006), nicotiana tabacum (mursalimov and deineko 2011), chlorophytum borivilianum (mandal and nandi 2017), anchusa spp. (keshavarzi et al. 2017), thalictrum cultratum (kumar et al. 2017) and clematis ladakhiana (khan et al. 2018). in the end, it may be summarized that individuals of a. thomsonii and e. nutans of cold desert habitat are of dwarf habit and whereas of other alpine regions of western himalaya are taller. respectively, both the species unequivocally noted with chromosome number count of 2n=42 and 2n=42+0-1b with 6x ploidy level are pioneer counts for the species. in natural habitats of these species cold climatic conditions are prevalent and seem these species are differentially affected by cold stress, which is a potential inducer for abnormal meiotic course and sporads. the phenomenon of cytomixis and associated meiotic abnormalities observed in pollen mother cells of a. thomsonii and e. nutans affects the pollen size and pollen fertility in the species. acknowledgments the authors wish to thank the university grants commission (ugc), new delhi for financial support under drs, sap-i, ii, iii, asist program and dsa-i schemes; department of biotechnology (dbt), new delhi under dbt-ipls project [bt/pr4548/ inf/22/146/2012]; for award of ugc-bsr-fellowship [award letter no. 15610/research/03/06/2015] and sci96 harminder singh et al. ence and engineering research board-department of science and technology (serb-dst) start-up research grant (young scientists) [vide serb sanction no. sb/ ys/ls-182/2014 dated 8/9/2015]. thanks are also due to the head, department of botany, punjabi university patiala, and director, botanical survey of india, kolkata and head of office, nrc, dehradun, for providing necessary laboratory, herbarium (pun, bsd), internet facilities and co-ordinator, university sophisticated instrumentation centre for kind permission to use imaging system facility. references basavaiah, murthy tcs. 1987. cytomixis in pollen mother cells of urochloa panicoides p. beauv. 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(plantaginaceae) saeed mohsenzadeh*, masoud sheidai, fahimeh koohdar a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding timir baran jha1,*, partha sarathi saha2, sumita jha2 chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india harminder singh2, jaswant singh1,*, puneet kumar2, vijay kumar singhal1, bhupendra singh kholia2, lalit mohan tewari3 population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies raheleh tabaripour1,*, masoud sheidai1, seyed mehdi talebi2, zahra noormohammadi3 induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions girjesh kumar, shefali singh* karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 caryologia. international journal of cytology, cytosystematics and cytogenetics 72(3): 97-103, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-771 citation: d. şendoğan, b gündoğan yağbasan, m.v. nabozhenko, b. keskin, n. alpagut keskin (2019) cytogenetics of accanthopus velikensis (piller et mitterpacher, 1783) (tenebrionidae: helopini). caryologia 72(3): 97-103. doi: 10.13128/caryologia-771 published: december 13, 2019 copyright: © 2019 d. şendoğan, b gündoğan yağbasan, m.v. nabozhenko, b. keskin, n. alpagut keskin. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. cytogenetics of accanthopus velikensis (piller et mitterpacher, 1783) (tenebrionidae: helopini) dirim şendoğan1, beril gündoğan1, maxim v. nabozhenko2,3, bekir keskin1, nurşen alpagut keskin1,* 1 faculty of science, department of zoology, section of biology, ege university, i̇zmir, turkey 2 precaspian institute of biological resources of the daghestan federal research centre of the russian academy of sciences, makhachkala, russia, 3 dagestan state university, makhachkala, russia *corresponding author: nursen.alpagut@ege.edu.tr abstract. the karyotype and cytogenetic features of darkling beetle accanthopus velikensis were analysed using conventional and differential staining. the diploid number was determined as 2n = 20 and the presence of xyp sex determination system was observed with dapi and silver staining as well as conventional staining. although a single nucleolar material was observed in prophase i nuclei, multiple argyrophilic signals in diakinesis-metaphase i plates makes it difficult to determine the exact nor location. both conventionally and differentially stained plates showed that heterochromatin is mostly concentrated on centromeric regions of a. velikensis chromosomes. obvious telomeric signals on some rod shaped bivalents as well as the x chromosome were also detected with agno3 and dapi staining. although presented karyotype of a. velikensis resemble to those of other helopini members and follows the common patterns of tenebrionid karyotypes, slight differences in chromosome morphologies, nors and the heterochromatin distribution were detected. our specimens also showed a unique haplotype for coi sequences with an 84-83% sequence similarity to database sequences for tenebrionidae. keywords. karyotype, nor, coi, dna barcoding, helopini, tenebrionidae. introduction accanthopus dejean, 1821 (= enoplopus solier, 1848) is a small tenebrionid genus with two lichen-feeding species, a. velikensis and a. reitteri (brenske, 1884) distributed in southern and partly central europe and occurring in fagus, abies and quercus forests. although the genus is considered to be included in the tribe helopini since lacordaire (1859), several additional taxonomic placements have been also proposed. historically, the genus has been placed in either a separate tribe (enoplopites – solier 1848; reitter 1917) or different subtribes in helopini (i.e. enoplopina – reitter 1922; nabozhenko 2018; cylindrinotina – ardoin 1958; helopina – nabozhenko 2008; naboz98 dirim şendoğan et al. henko and löbl 2008). ardoin (1958) also suggested erecting a separate tribe within the subfamily tenebrioninae for this genus. the genus accanthopus has unusual external and internal structures, some of which support its position in the tribe helopini. several structures including the inner prothoracic skeleton, ovipositor, defensive glands female genital tubes are typical for helopini (tschinkel & doyen 1980, nabozhenko, 2005). on the other hand, the genus possess numerous non-helopine characters, such as very wide and spherical body, mentum with sexual dimorphism, profemora with strong and large acute tooth dorsally, strongly widened epipleura, very short and wide metaventrite, structures of mesonotum, metendosternite, aedeagus (ardoin 1958), male inner sternite viii and lobes of gastral spicula. therefore, the position of accanthopus in relation to other helopini and tenebrioninae lineages needs to be tested with additional data sets and integrated with molecular phylogenetic analyses. the cytogenetic data among tenebrionids have covered only about 1% of the species diversity (guenin 1950, 1951a,b; smith 1952; smith and virkki 1978; yadav et al. 1980; petitpierre et al. 1991; juan and petitpierre 1991a; holecová et al. 2008; blackmon and jeffery 2015; gregory 2016). in general, most of the species present a karyotype with 2n = 20, but the diploid number ranges from 2n = 14 to 2n = 38 in tenebrionidae (juan and petitpierre 1991a; pons 2004; holecová et al. 2008; lira-neto et al. 2012). based on available data, main karyological patterns in tenebrionid beetles were noticed in chromosome morphology, sex determining systems and distribution of heterochromatin (juan and petitpierre 1990; petitpierre et al. 1991; juan and petitpierre 1991a, 1991b; juan et al. 1993; bruvo-madaric et al. 2007; şendoğan and alpagut keskin 2016). although chromosomal data are available for several representatives of subfamilies allecullinae, diaperinae, lagriinae, pimelinae, and tenebrioninae, even basic information is scarce or totally lacking for other subfamilies (juan and petitpierre 1991a; blackmon and jeffery 2015). the chromosomes of accanthopus have not yet been studied. furthermore, cytogenetic data concerning the tribe helopini are only known for some nesotes allard, 1876, euboeus boieldieu, 1865 (=probaticus seidlitz, 1896), nalassus mulsant, 1854 and turkonalassus keskin et al., 2017 species (juan and petitpierre 1986, 1989, 1991a, 1991b; palmer and petitpierre 1997; şendoğan and alpagut keskin 2016). considering the limited cytogenetics information, the increase of chromosomal data may provide valuable phylogenetic signals about tenebrionid diversity. in this study, the mitotic and meiotic chromosomes of both sexes of a. velikensis were analysed using conventional and differential staining methods, with the aim of providing new data that will improve the knowledge on tenebrionidae cytogenetics. we also sequenced the mt coi gene, for genetic identification of our a. velikensis specimens and barcoding of presented karyotype for further phylogenetic analysis. materials and methods specimens accanthopus velikensis specimens were collected from pınarhisar, kırklareli (41o46’02” n/27o37’51” e, 835 m). adult beetles were collected on the trunks of trees at night when they are active. chromosome analysis mitotic and meiotic chromosomes of 9 male and one female specimens were analysed using conventional and differential staining. chromosome spreads were prepared from male and female gonads following the microspreading (chandley et al. 1994) or splashing (murakami and imai 1974) methods with some modifications (şendoğan and alpagut keskin 2016). the slides were stained with 4% giemsa for 20 minutes for conventional staining. silver impregnation technique of patkin and sorokin (1983) was performed to figure out the position of nor regions. chromosome spreads were examined and photographed with zeiss axio scope a1 light microscope using zen software. the chromosomal measurements were obtained using the levan plugin (sakamoto and zacaro 2009) and the karyotype was created with the chias plugin (kato et al. 2011) of the program image j (rasband 1997-2015). heterochromatin distribution patterns were visualized with fluoroshielddapi (sigma) specific to at-rich chromosomal regions under olympus bx50 fluorescent microscope. mt coi barcoding genomic dna was obtained from the thorax of the specimens using the promega 96-well plate kit according to the manufacturer’s instructions. the mitochondrial cytochrome oxidase i (coi) gene was amplified using the primers jerryten and patten (papadopoulou et al. 2009) for genetic identification of a. velikensis specimens and barcoding of the karyotype. pcr products 99cytogenetics of accanthopus velikensis (piller et mitterpacher, 1783) (tenebrionidae: helopini) were purified and then sequenced in both directions. sequencher 5.0 software was used to assemble and edit sequence chromatograms (gene codes, ann arbor, mi) and the coi sequences were submitted to genbank for accession numbers. we performed a haplotype analysis using dnasp v.5.10.1 (rozas et al. 2017) and a blast search for all our sequences, in order to compare them with sequences deposited in genbank. results we amplified the partial 829 bp sequences of cytochrome oxidase gene. our specimens showed a unique haplotype for coi sequences with an 84-83% sequence similarity to database sequences for tenebrionidae. the cytogenetic analyses of spermatogonial and oogonial metaphase plates of accanthopus velikensis revealed the diploid number to be 2n = 20, consisting of 2 pairs of metacentric and 7 pairs of submetacentric chromosomes (figure 1-2, table 1). while in male metaphase plates a minute subtelocentric y and a small submetacentric x chromosome appear as a heteromorphic pair (figure 2b), no heteromorphism was observed among female metaphase plates (figure 1a). x and y chromosomes are the smallest elements of the a. velikensis karyotype with the lengths of 2.434 µm and 0.759 µm, respectively (table 1). the observation of male metaphase i plates determined meioformula as 9 + xyp. the heteromorphic pair that composed the xyp was clearly observed in both conventionally and differentially stained male metaphase i plates (figure 3a-c). in diplotene/diakinesis of a. velikensis, 7 rod-shaped (terminal chiasma), and 3 ring-shaped (two terminal chiasmata) bivalents were observed (figure 3d, 4d). in diakinesis/metaphase i; most of the homologous chromosomes fig. 1. (a) oogonial metaphase (b) spermatogonial metaphase of accanthopus velikensis. red and black arrows show x and minute y chromosomes respectively. bars = 5μm. fig. 2. male karyotype and idiogram of a. velikensis 2n = 20. bar = 5μm. table 1. chromosome morphologies and measurements of accanthopus velikensis. ci: centromere index, rl: relative length. (centromere positions were determined according to levan et al. 1964). chromosome length (μm) %rl ci morphology 1 4.999 13.7 45 m 2 4.466 12.2 28 sm 3 4.336 11.9 38 sm 4 3.771 10.3 28 sm 5 3.771 10.4 36 sm 6 3.553 9.8 30 sm 7 2.955 8.1 39 sm 8 2.782 7.6 44 sm 9 2.608 7.2 45 m x 2.434 6.7 39 sm y 0.759 2.1 23 st fig. 3. xyp sex bivalents and heterochromatin in (a-c) metaphase i, (a) giemsa (b) silver nitrate (c) dapi staining (d) diplotene-diakinesis (dapi staining) arrows show xyp sex bivalents, asterisk show heterochromatin. arrowheads indicate telomeric signals on some of the rod shaped bivalents bars = 5μm. 100 dirim şendoğan et al. formed rod shaped bivalents and 1-2 cross-bivalents were also observed due to interstitial chiasma (figure 3a–c). in prophase nuclei, silver nitrate staining revealed the existence of a single impregnated mass of nucleolar material (fig 4a). additionally, obvious signals in the telomeric and pericentromeric regions of some autosomal pairs as well as xyp, were also observed in both silver nitrate and giemsa stained diakinesis-metaphase i plates (figure 4b-d). giemsa staining of prophase nuclei indicated that all chromosomes of a. velikensis showed dark heterochromatic blocks mainly located in centromeric and pericentromeric regions (figure 4d). also with silver nitrate (figure 4b-c) and dapi staining (figure 3d, 4a) rich telomeric and interstitial signals were observed in the large arms of most of the chromosomes. in metaphase ii plates, while some haploid sets seemed to be n = 9 due to minute y chromosome not being detectable (figure 4b) the plates with the x chromosome showed the normal haploid number 10 (figure 4a). discussion due to predominant occurrence of the diploid number 2n=20 and parachute configuration of sex bivalents in the studied species, tenebrionid beetles considered as karyologically conservative group (juan and petitpierre 1988; 1991a; juan et al., 1989; palmer and petitpierre 1997; pons 2004). on the other hand, variations in sex chromosomes, nors and heterochromatin distribution in spite of the shared modal number reveal that intrachromosomal rearrangements have played a major role in tenebrionid karyotype divergence (juan et al. 1990; almeida et al. 2000). the extent of diploid numbers between 14-38 within the family suggests that interchromosomal rearrangements such as robertsonian processes or polyploidy could have also involved in karyotype evolution (juan and petitpierre 1991a; petitpierre et al., 1991; almeida et al. 2000; pons 2004; holecova ́ et al. 2008; lira-neto et al. 2012; goll et al. 2013). we showed that the karyotype of a. velikensis consists of 10 pairs of chromosomes (2n=20, xyp) which are mostly submetacentric (figure 1, 2, 3 a-c). this formula (n=10, xyp) was reported for other helopini species as well i.e. nesotes (juan and petitpierre 1986, 1989, 1991a), nalassus and turkonalassus (şendoğan and alpagut keskin 2016). despite this general resemblance, presence of mostly submetacentric chromosomes slightly differentiate a. velikensis karyotype from other helopini possessing predominantly metacentric chromosomes. furthermore, relative lengths of sex bivalents are obviously different in present karyotypes of helopini. while x chromosomes of a. velikensis and n. plebejus (küster, 1850) show similar relative lengths (6.9 % and 6.55 % respectively), t. bozdagus (keskin et nabozhenko, 2010) have clearly larger x (13.74 % of total complement) which has a conspicuous secondary constriction on the long arm. however, diploid numbers reported for the other helopine genera nesotes (2n=20, xyp) and euboeus (2n=20, xy) are based only on male metaphase i plates (juan and petitpierre 1986, 1989, 1991a, 1991b), and do not allow detailed comparison of chromosome morphologies. studies on differential patterns of karyotypes in tenebrionidae and some other coleopteran families revealed the occurrence of heterochromatic blocks in mainly pericentromeric regions and autosomal or sex fig. 4. nors and heterochromatin in (a) prophase i nuclei (silver and dapi staining), (b) diplotene-diakinesis, (c-d) pachytene after silver (b-c) and giemsa staining (d). red asterisk indicate xyp and the presence of obvious signal in the long arm telomeric region of submetacentric x, black asterisks show differentially stained chromosome regions. bars = 5μm (b-c possess the same scaling). fig. 5. (a.-b) metaphase ii plates (a) haploid set with x chromosome (b) haploid set with minute y chromosome which cannot be seen bar = 5μm. 101cytogenetics of accanthopus velikensis (piller et mitterpacher, 1783) (tenebrionidae: helopini) chromosomal location of nors (juan and petitpierre 1989; juan et al. 1993; pons 2004; rozek et al. 2004; schneider et al. 2007; holecová et al. 2008; karagyan et al. 2012; goll et al 2013; şendoğan and alpagut keskin 2016). the presence of heterochromatin blocks on pericentromeric regions of a. velikensis chromosomes was demonstrated with both agno3 and dapi staining (figure 3d, 4). additionally, telomeric signals on some rod shaped bivalents as well as the x chromosome (fig 3d, 4 b-d) were detected. our results showed that even a single nor site was present in prophase i nuclei (figure 4a), chromosomes in diakinesis-metaphase i plates gave multiple signals (figure 4b-d). therefore, further testing of exact nor locations with rdna-fish probes is required to determine whether these signals are directly associated with nors or a result of heterochromatin condensation. in conclusion, karyotype of a. velikensis resemble those of other helopini members and follows the common patterns of tenebrionid karyotypes with slight differences in chromosome morphologies, nors and heterochromatin distribution. to truly understand these specific patterns of a. velikensis karyotype, comparative molecular cytogenetic studies with related taxa is required. in order to broaden the knowledge on the chromosomal evolution of tribe helopini and assess the situation/position of a. velikensis within the tribe, cytogenetic studies should be combined with molecular phylogenetic analyses as well. acknowledgements we are sincerely grateful to members of molecular cytogenetic lab in faculty of medicine-ege university for their help for fluorescent microscopy. references almeida mc, zacaro aa, cella dm. 2000. cytogenetic analysis of epicauta atomaria (meloidae) and 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geographical distribution and karyotype of nannospalax ehrenbergi (nehring 1898) (rodentia, spalacidae) in iraq. caryologia 72(4): 79-83. doi: 10.13128/caryologia-318 published: december 23, 2019 copyright: © 2019 z.a. hamad, a. kaya, y. coşkun. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. geographical distribution and karyotype of nannospalax ehrenbergi (nehring 1898) (rodentia, spalacidae) in iraq zaitoon ahmed hamad1, alaettin kaya2, yüksel coşkun2,* 1 dicle university institute of science biology section, diyarbakır /turkey 2 dicle university science faculty, department of biology, diyarbakır/turkey *corresponding author: yukselc@dicle.edu.tr abstract. this paper concerns the karyological analysis of fourteen mole rats collected in four different localities of north-iraq (kurdistan region). the result showed that they belong to the following cytotypes of nannospalax ehrenbergi: «duhok-bardarash population» 2n = 52, nf = 76, and nfa = 72 and «arbil-sulaimania-kirkuk populations» 2n = 52, nf = 80 and nfa = 76. the karyotypes of the duhok population are similar to those from mosul, but the arbil-sulaimania-kirkuk populations’ karyotype represents a new chromosomal form. their distribution extends from north iraq to sulaimania. keywords. rodentia, spalacidae, nannospalax ehrenbergi, karyology, iraq. 1. introduction scientific research on mammals in iraq is scarce in the country and requires special attention in order to determine the mammalian fauna of iraq. amr (2009), garstecki & amr (2011) noted that the mammalian fauna of iraq consists of 74 species, including insectivores (6), bats (15), and carnivores (19) as well as extinct species such as the leopard (panthera pardus), artiodactyls (8). rodents constituted the largest mammalian group in iraq with 25 species. recently, an updated checklist of the mammals of iraq was published by al-sheikhly et al. (2015). the checklist takes into account 93 mammalian species of iraq and listed the mole rats under the name nannospalax ehrenbergi. the palearctic rodent blind mole rats (rodentia: spalacidae) are subterranean mammals and the chromosomally diverse and they are difficult to distinguish based on phenotype, whose phylogenetic relationships are problematic, resulting in taxonomic uncertainties at every level from species to higher taxa (savic & nevo 1990; musser & carleton 2005). fossil, morphological, chromosomal and molecular evidence suggest that spalacidae have two distinct genera spalax and nannospalax (topachevski 1969, lyapunova et al. 1974, hadid et al. 2012). morphologically nannospalax differs from spalax 80 zaitoon ahmed hamad, alaettin kaya, yüksel coşkun by the presence of supracondyloid foramina and two longitudinal ridges anterior surface of the upper incisors (topachevski 1969). karyologically, nannospalax has both low diploid (2n) and fundamental (nf) numbers and acrocentric chromosomes (lyapunova et al. 1974). the species nannospalax ehrenbergi is the south eastern representative of the genus – initially described by nehring (1898) on specimens, who were collected from yafa-israel – also occurs in the middle east, egypt, and southeast anatolia of turkey (lay & nadler 1972; musser & carleton 2005; coşkun et al. 2006). nannospalax ehrenbergi exhibits great diversity in both diploid number of chromosomes (2n= 48-62) and the number of chromosome arms (nf= 62-90) (wahrman et al. 1969; ivanitskaya et al. 1997; 1998; coşkun et al. 2006 and reference therein). the distribution of nannospalax ehrenbergi in iraq has been known mainly from morphological studies, which have not been extensive (cheesman 1920; reed 1958; harrison 1956; hatt 1959; turnbull & reed 1974; harrison & bates 1991). recently, spalacids from the hawraman mountains were identified as spalax leucodon by lahony et al. (2013). the old records and distribution of the species in iraq were previously summarized in detail by coşkun et al. (2012). the cytogenetic information, which was available for this mole rat (n. ehrenbergi) and the existing data, were restricted to conventional stained karyotypes or reports of the diploid chromosome number (coşkun et al. 2012; 2014). the geographical distribution and karyological peculiarities have not yet been documented in detail. the aim of the present work is to verify the distribution and the karyotype characteristics of several nannospalax populations from iraq to fill the gap in our knowledge about karyological forms as well as their distributional areas in the north of iraq. 2. material and methods the territory of iraq is lies between latitudes 29° to 38° n and longitudes 39° to 49° e and the landscape includes high mountains in the north (kurdistan), desert, arid lands and sandy steppes in the western and south-western plateau (al-badiyah), and the mesopotamian marshlands in the southern alluvial plain (zohayr 1973). the study was conducted on four populations of blind mole rats from duhokbardarash, arbil-new arbil, sulaimania-mughagh and kirkuk-shwan in the kurdistan province of iraq (fig. 1). in total, fourteen specimens (4 males, 10 females) of blind mole rats were studied. the sampled localities, the number of individuals analyzed, and karyological results are presented in table 1. direct chromosome preparations were made from bone marrow (hsu 1969) and about 25-30 metaphase cells, which were well stained, and whose chromosomes were separately examined. the diploid number of chromosomes (2n), the number of autosomal arms (nfa), the total number of chromosomal arms (nf), and the sex chromosomes were determined from photos of the metaphase plates according to the centromere position. the karyotype preparations and animals examined were deposited in the department of biology, the faculty of sciences at dicle university. 3. results and discussion morphological peculiarities of the mole rats of iraq were documented in detail by coşkun et al. (2016). they conclude that morphologically all studied populations in north iraq show great similarities and can be morphologically classified as nannospalax ehrenbergi. the approximate geographic area of each chromosomal form is shown in fig. 1. figure 1. sampling localities and geographical distribution of chromosomal forms of nannospalax ehrenbergi in the kurdistan region-iraq (∗: old records) 1near mosul (cheesman 1920); 2near sulaimania (bate 1930); 3sarsank (hatt 1959); 4jarmo, chemchamal valley (reed 1958); 5ser ‘amadia and tinn (harrison 1956); 6jarmo, palegawra cave (turnbull and reed 1974); 7-al-jurn (coşkun et al. 2012); 8al-jurn (coşkun et al. 2014); 9kirkuk-shwan (coşkun et al. 2014);10sulaimaniamughagh (coşkun et al. 2014); 11al-jurn (coşkun et al. 2016); (▲: this study) 12duhok-bardarash; 13arbil-new arbil; 14kirkukshwan; 15sulaimaniamughagh. 81geographical distribution and karyotype of nannospalax ehrenbergi (nehring 1898) (rodentia, spalacidae) in iraq 3.1. duhok population the karyotype of individuals from duhok (bardarash locality) was 2n = 52, nf = 76, nfa =72, which consists of 11 pairs of metacentric/submetacentric autosomes, and 14 pairs of acrocentric autosomes. the x chromosomes were large metacentrics (fig. 2a). this cytotype is similar to that observed in the previously studied individuals ascribed to nannospalax ehrenbergi from al-jurn (mosul) by coşkun et al. (2012). mole rats of this locality (duhok populations) inhabit the north of the great zab river (tab.1). 3.2. arbil population the samples from arbil (new arbil), kirkuk (shwan; 50 km north kirkuk) and sulaimania (mughagh; 55 km west sulaimania) possessed karyotypes of 2n=52, nf=80, nfa=76 and consists of 13 pairs meta /submetacentric 12 pairs acrocentric autosomes. the x chromosome was large metacentrics and the y chromosome was small acrocentric (fig. 2b and tab. 1). the karyotypes of these three populations which is newly described here, are similar with each other’s and they are located on the south side of the great zab river,in iraq. according to gromov & baranova (1981), spalacidae has two distinct genera, nannospalax and spalax, and turkish spalacids belong to the genus nannospalax. iraqi populations also belong to the genus nannospalax. reed (1958), hatt (1959), turnbull & reed (1974), harrison & bates (1991), lahony et al. (2013) have stated that mole rat samples in all iraq are s. leucodon but our results show that all samples across iraq are n. ehrenbergi. mole rat, belonging to the n. ehrenbergi exhibits two chromosomal forms that are widely distributed across north iraq. one chromosomal form is 2n= 52 and nf= 76, nfa= 72. this chromosomal form (duhok populations) is found north of the great zab river and is similar to the mosul-al jurn (coşkun et al. 2012) and turkish diyarbakır (coşkun et al, 2006) populations. the other form, 2n= 52 and nf= 80, nfa= 76 (arbil, kirkuk-sulaimania populations) in the south of the great zab river is a new chromosomal form that has not been previously described. each of the karyotype forms exhibits an allopatric distribution, separated mostly by the great zab river or some ecological barriers, which may limit their dispersal (fig. 2). chromosomal differences are frequently associated with taxonomic differences at the species level (patton & sherwood 1983). chromosomal change has been implicated as a primary isolating mechanism in speciation. chromosomal divergence is considered an indication of speciation events (nevo et al. 2001). this study filled the gaps in the knowledge of distribution of blind mole rat chromosomal forms in the north of iraq. according to the results n.ehrenbergi are distributed in all parts of north iraq, and it forms a potential species complex of n. ehrenbergi. figure 2. the karyotype nannospalax ehrenbergi: a. duhok-bardarash population, b. arbil population. (a: karyotype, b: metaphase plate). table 1. the localities of samples that chromosomal analysis was performed in iraq (n: sample size,2n: diploid chromosome numbers, nf: chromosomal arm numbers, nfa: autosomal arm number, m: metacentrics, sm: submetacentrics, a: acrocentric). locations n 2n autosomes nf nfa gonosomes reference city town village m/sm a x y kirkuk shwan --4♀ 52 13 12 80 76 sm . th is s tu dy sulaimania dukan mughagh 2♂, 2♀ 52 13 12 80 76 sm a arbil newarbil ---2♂, 3♀ 52 13 12 80 76 sm a duhok bardarash zamzamok 1♀ 52 11 14 76 72 sm mosul al jurn ---3♂ 52 11 14 76 72 sm a coşkun et al. (2012) 82 zaitoon ahmed hamad, alaettin kaya, yüksel coşkun in order to fully understand the distribution and karyology of blind mole rats in iraq, we need more information on hybrid zones in the territory, population structure and population size. there is a real necessity to establish long-term cytogenetic studies for this rodent. it is indeed very important to pay more attention to the role of natural barriers such as the great zab river and other ecological factors on speciation of iraqi mole rats. acknowledgement we would like to thank ms yazgülü zeybek of the bergische university wuppertal for english editing. our gratitude and thanks are also extended to the anonymous reviewers for their valuable comments and input which greatly improved the manuscript. this study was supported by the dicle university research fund (bap, project number 15 ff 010). statement of conflict of interest the authors declare that there is no any conflict ofinterests regarding the publication of this article. references al-sheikhly o. f., m. k. haba, f. barbanera, g. csorba, and d. l harrison 2015. checklist of the mammals of iraq (chordata: mammalia). bonn zool. bull., 64 (1): 33–58. amr z. 2009. mammals of iraq. publication no.ni-0209-002, nature iraq.sulaimania, kurdistan iraq. bate d. m. a. 1930. animal remains from dark cave, hazar merd. -bull. amer. school prehist. res. 6: 38–41. cheesman r.e. 1920. report on the mammals of mesopotamia collected by members of the mesopotamian expeditionary force, 1915 to 1919. j. bombay nat. hist. soc. 27: 323–346. coşkuny., s. ulutürk, and g. yürümez 2006. chromosomal diversity in mole-rats of the species nannospalaxehrenbergi(rodentia: spalacidae) from south anatolia, turkey. mamm. bio. z. saugetierkd. 71(4): 244–250. coşkun y., a. namee, a. kaya, and z. i. f. rahemo 2012. karyotype of nannospalax ehrenbergi (nehring 1898) (rodentia: spalacidae) in the mosul province, iraq. hystrix, it. j. mamm. 23(2): 72-75. coşkun y., n. aşan baydemir, a. kaya and a. karöz 2014.nucleolar organizer region distribution in nannospalax ehrenbergi (nehring, 1898) (rodentia: spalacidae) from iraq. turk. j. zool. 38: 250253. coşkun y., hamad z. a. and kaya a. 2016. morphological properties of nannospalax (rodentia: spalacidae) distributed in north-iraq. hacettepe j. bio. & chem. 44 (2): 173-179. garstecki, t. and z. amr 2011.biodiversity and ecosystem management in the iraqi marshlands – iucn screening study on potential world heritage nomination. amman, jordan. gromov i. m., and g. i. baranova1981. catalogue of mammals in ussr.nauka, leningrad. hadid, y., németh, a., snir, s., pavlíček, t., csorba, g., kázmér, m., major, á.,mezhzherin, s., rusin, m., çoşkun, y. and nevo, e., 2012. is evolution of blind mole rats determined by climate oscillations? 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(ed.), comp. mamm. cyt. springer verlag. new york: 30–48. zohary m. 1973. geobotanical foundations of the middle east. fischer verlage, stuttgart.v,1 substantia an international journal of the history of chemistry vol. 2, n. 1 march 2018 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 73(2): 63-72, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-752 citation: p. vosough-mohebbi, m. zahravi, m. changizi, s. khaghani, z.-s. shobbar (2020) identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection. caryologia 73(2): 63-72. doi: 10.13128/caryologia-752 received: december 3, 2019 accepted: april 3, 2020 published: july 31, 2020 copyright: © 2020 p. vosoughmohebbi, m. zahravi, m. changizi, s. khaghani, z.-s. shobbar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection panthea vosough-mohebbi1, mehdi zahravi2,*, mehdi changizi3, shahab khaghani1, zahra-sadat shobbar4 1 department of agronomy and plant breeding, arak branch, islamic azad university, arak, iran 2 seed and plant improvement institute, agricultural research, education and extension organization (areeo), karaj, iran 3 department of agriculture, arak branch, islamic azad university, arak, iran 4 department of system biology, agricultural biotechnology research institute, agricultural research, education and extension organization (areeo), karaj, iran corresponding author: email: mzahravi@yahoo.com abstract. bread wheat (triticum aestivum l.) is the most widely grown crop worldwide. powdery mildew caused by fungal pathogen blumeria graminis is one of the most devastating diseases of wheat. the present study aimed to identify differentially expressed genes and investigate their expression in response to b. graminis in susceptible (bolani) and resistant (kc2306) wheat genotypes, using publicly  available microarray data set and qrt-pcr analysis. a total of 5760 and 5315 probe sets were detected which 5427 and 4630 by adjusted p-value < 0.05 and 168 and 144 genes based on e-value < 1 × 10–5 cut-off were differentially expressed in susceptible and resistant wheat genotypes, respectively. among exclusively up regulated genes in the resistant genotype 12 hpi compared to its control, fifteen potential genes that may be responsible for b. graminis inoculation resistance were detected. the results of real time pcr for the candidate genes showed that the genes were upregulated in the resistant genotype 12 hpi compared to its control, which validated the results of microarray analysis. the bzip, erf, and arf1 genes may play an important role in b. graminis resistance. the powdery mildew responsive genes identified in the present study will give us a better understanding on molecular mechanisms involved in b. graminis resistance in different wheat genotypes. keywords: wheat, genotype, powdery mildew, microarray, qrt-pcr. introduction bread wheat (triticum aestivum l, aabbdd 2n = 42) is the most widely grown crop in the world, belongs to poaceae family (chen s et al. 2018). it occupying 17% of all the cultivated land and providing approximately 20% of globally consumed calories (gill et al. 2004; vetch et al. 2019). in 64 panthea vosough-mohebbi et al. iran, more than 39% of all cultivated lands belongs to wheat, as the most important human food (abdollahi 2008). there are many biotic and abiotic stresses which affecting the quality and quantity of crops in the country (sheikh beig goharrizi et al. 2016; sheikh-mohamadi et al. 2018; sanjari et al., 2019). powdery mildew caused by fungal pathogen blumeria graminis is one of the most devastating diseases of wheat, occurring in regions with cool and humid climate that particularly is very conducive for the development of this disease (chang et al. 2019; liu n et al. 2019). iran is one of the most important primary centers of the wheat distribution, thereby it has one of the richest wheat germplasm worldwide. therefore, the wild wheat relatives in the area could be a source of novel resistance genes to be transferred into wheat cultivars (pour-aboughadareh et al. 2018). also, this rich wheat germplasm can be used as a valuable material for better understanding of the molecular mechanisms involved in the wheat-pathogen interaction (brunner et al. 2012). traditional breeding has been used to transmitting resistant genes to susceptible wheat cultivars, by this method some powdery mildew resistant genes were discovered (xin et al. 2012). traditional breeding is based on the phenotype, therefore less information can derived and some traits, such as disease resistance, cannot be observed easily (chen h et al. 2014). the microarray technology provides us a lot of information about the genes. recognizing new genes and analyzing their expression in response to powdery mildew will provide a valuable molecular information for enhancing disease resistance in the plant and microarray analysis could provide a plethora of gene expression profiles (xin et al. 2011). microarray has been widely used to detect the pathogen-resistant genes in wheat responding to different plant diseases. li et al. (2018) identified 36 lr39/41-resistance related differentially expressed genes at 48 h post inoculation (hpi) in leaf rust resistant and susceptible wheat isogenic lines. foley et al. (2016) investigated the differentially expressed wheat genes in response to the rhizoctonia solani isolate (ag8) and identified a significant number of genes involved in reactive oxygen species production and redox regulation. erayman et al. (2015) examined the early response to fusarium head blight in moderately susceptible and susceptible wheat cultivars at 12 hpi using microarray technology. the authors reported that 3668 genes were differentially expressed at least in one time comparison, which the majority of them were associated with disease response and the gene expression mechanism. putative transcription factor (tf) genes constitute 7% of all plant genes, they are proteins which play a major role in gene expression regulation (yazdani et al., 2020). xin et al. (2012) examined the leaves transcriptomes before and after b. graminis inoculation in a susceptible and its near-isogenic wheat line and compared the result of microarray with qrtpcr analyses. since iran has one of the richest wheat germplasm in the world however, limited studies has performed regarding pathogen resistant genes in the germplasm. therefore the present study aimed to identify differentially expressed genes and investigate their expression in response to b. graminis in both susceptible and resistant wheat genotypes, using publicly  available microarray data set and qrt-pcr analysis. materials and methods plant material and growth condition two susceptible (bolani) and resistant (kc2306) wheat genotypes to powdery mildew, were obtained from the national plant gene-bank, karaj, iran. the seeds of these two genotypes were planted in 10 cm diameter pots, at greenhouse condition. at the first fully expanded leaf stage, the plants inoculated with a pathotype (b. graminis) collected from moghan, ardabil, iran (a local pathotype). inoculation was performed by dusting or brushing conidia from neighboring sporulating susceptible seedlings onto the test seedlings. leaf samples of each genotype were collected at 0 and 12 hpi. the samples were stored in liquid nitrogen for qrt-pcr analysis. in silico powdery mildew gene expression survey in the present study we used a publicly  available microarray data set published by xin et al. (2011), available at geo (http://www.ncbi.nlm.nih.gov/gds/) with gse27320 accession number. the authors used a susceptible wheat cultivar ‘8866’ and it’s near isogenic line with single powdery mildew resistance gene. the differentially expressed sequences were homology searched against wheat transcription factor (tf) database (http:// planttfdb.cbi.pku.edu.cn/), and then blast analysis with a strict cutoff e-value <1 × 10–5 was performed. venn diagram was generated to find the differentially expressed genes between the two genotypes and the tfs which up-regulated in the resistant genotype were selected. to confirm the authenticity of the selected tfs, pfam proteins family database (https://pfam.xfam. org/) was used. also, the hierarchical cluster algorithm was performed on the differentially expressed genes among the samples. 65identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection rna extraction and qrt-pcr analysis in order to extract the total rna of the samples, trizol reagent (invitrogen, usa), was used. in addition, total rna was treated using rnase-free dnase (geneall, korea) and reversely transcribed to double-stranded cdnas using oligo (dt)18 primers by cdna synthesis kit (takara, japan). oligo software was used to design gene-specific primers for 15 selected genes (table 1). the qrt-pcr was performed on a bio-rad, miniopticon real-time pcr detection system using sybr green supermix (takara, japan). the reactions were performed using the following program: 95 °c for 5 min and 40 cycles (95 °c for 30 s, 57 up to 61°c for 30 s and 72 °c for 30 s). wheat actin gene was used as the internal control. for each data point, the ct value was the average of ct values derived from three biological and three technical replicates, were normalized based on the ct of the control products (ta actin). the relative quantitative analysis preformed using the 2-δδct method (schmittgen and livak 2008) then subjected to a complete random design (crd) and least significant difference (lsd) test using sas software package (sas institute inc.). the correlations were visualized as a colored heat map. the heat map and the bi-plots were created by metaboanalyst (xia and wishart 2016). also, the graphs were performed using graphpad prism software. in addition, the gene expression of powdery mildew-sensitive genotype under normal condition was used as calibrator for calculating the relative gene expression. results and discussion microarray analysis in order to identify the differentially expressed genes in susceptible and resistant wheat genotypes responding to b. graminis, the publicly  available microarray data set published by xin et al. (2011) was used. the reproducibility of the microarray data was confirmed by the authors. based on the blast of the differentially expressed sequences against wheat transcription factor (tf) database, 5760 and 5315 probe sets were detected which 5427 and 4630 were differentially expressed by adjusted p-value <0.05, and 168 and 144 genes were differentially expressed based on e-value <1 × 10–5 cut-off in susceptible and resistant wheat genotypes, respectively (fig. 1). among 168 and 144 degs, 70 and 56 degs were upregulated and 98 and 88 degs were downregulated, respectively (fig. 2). in previous study, of the total of 61,127 probe sets, 44.57 and 42.43% were detected as expressed genes in susceptible and resistant wheat genotypes at 12 hpi by b. graminis, respectively (xin et al. 2011). according to results of present and previous studies, many genes are involved in wheat resistance to this disease. in another study, bruggmann et al. (2005) studied the epidermis-and mesophyll-specific transcript accumulation in powdery mildew-inoculated wheat leaves. the authors reported that out of 17000, 141 transcripts, were found to accumulate after b. graminis f. sp. hordei inoculation using microarray hybridization table 1. list of primer pairs using for validation of gene expression using qrt-pcr. pid gene symbol forward primer (5’-3’) reverse primer (5’-3’) taaffx.51406.1.s1_at gata gaagtcaaaccctccctcaag gcaaacaaacatctcacatttcc ta.9390.1.a1_at arf gagatcgcccgtctttagc accaacactacattcaaacaaag taaffx.78909.1.s1_at g2 ggtctctcgctcggtctc ctcatccacttgttcttcatcg taaffx.85891.1.s1_at bhlh aaggtgctggagaatcaagg ctcattgttcgctgggttc ta.25219.1.a1_at arf actactacaacatttcctcgtatc gacaactgacactgtattctgg ta.4054.2.s1_at arr gctgttactgtttgtccttctg tcttgtctcattccaccatcc taaffx.36896.1.a1_at myb caatgtcgtcaagaaggaaga ccgtcgtgctgagaaacc taaffx.37068.1.s1_at grf cggaacctactacgaccatc gattcagattgcctcaacatag taaffx.120915.1.s1_at far1 tttaccagtgatgttcttttct ctccagggtgtccaatgc taaffx.129201.1.s1_s_at c3h aaatgggaaattggacagatacc catagaaagagaccacataaagg ta.7033.1.s1_s_at hb aatgaagcacatgacgacaag accgacaatccaacactctg ta.21124.1.s1_x_at erf tccgccaaccaactgttag cagtcatcgtcgccaaagc ta.6443.2.s1_at bzip aaacggcgaacaacacagg accatcaaggagaacaacaac ta.4828.1.s1_a_at c3h actgctcgtcgtctcctac tgcgtaatgctactactgattc ta.2023.1.s1_at bhlh gccataacgcacatcactg attacacgaacaagaacctca reference gene actin gtgtaccctcagaggaataagg gtaccacacaatgtcgcttagg 66 panthea vosough-mohebbi et al. analysis. our results were consistent with the previous studies that pathogen infection activates a wide range of genes and pathways in the transcriptional networks in wheat plant (bolton et al. 2008; coram et al. 2008; bozkurt et al. 2010). among exclusively up regulated genes in the resistant genotype 12 hpi compared to its control, potential genes that may be responsible for b. graminis infection resistance were detected (table 2). for example, g2 (taaffx.78909.1.s1_at) was the top upregulated gene with a log2 fold change of 3.39, and its expression might has a function in stress and disease resistance (liu f et al. 2016; zeng et al. 2018). the bhlh1 (taaffx.85891.1.s1_at) was also in the top fifteen upregulated genes with a log2 fold change of 3.08, the plant bhlh transcription factors family have key function in regulation of developmental processes and environmental stresses (wang et al. 2015). however,  limited  information is available on their roles in wheat disease caused by pathogens infection. another bhlh family member, bhlh2, was also revealed to be upregulated in resistant genotype. gene expression analysis fifteen potential genes that may be responsible for b. graminis infection resistance were selected for gene expression analysis by real-time pcr. the genes including arf1, hb, c3h2, c3h1, bzip, myb, arr, bhlh1, g2, far1, bhlh2, gata, erf, arf2 and gr were selected among exclusively up regulated genes in the resistant genotype 12 hpi. the result of gene expression indicated that all the selected genes were upregulated in the resistant genotype, and hb, c3h2, c3h1, myb, arr, and bhlh1 genes were downregulated in the susceptible genotype at 12 hpi compared to their controls (fig 3). based on the results of cluster analysis, the top fifteen genes were classified in four main groups (fig. 4). the bzip and erf genes formed the first group, which their expressions were significant at 12 hpi in both genotypes compared to their control conditions (before inocr-up r-down s-up s-down 0 50 100 150 n um be r of g en es 5 6 8 8 7 0 9 8 figure 1. venn diagrams showing the common and unique differentially expressed genes in s (red) and r (blue) genotypes detected (p-value < 0.05) and s-evalue (yellow) and r-evalue (green) based on e-value <1 × 10–5 cut-off. figure 2. number of upand down-regulated genes responsive to powdery mildew in susceptible and resistant genotypes. table 2. top 15 exclusively upregulated genes in the resistant genotype 12 hpi. probe set id log2 (fold change) adj. p-value p-value taaffx.51406.1.s1_at 1.4868 0.0354 0.0023 ta.9390.1.a1_at 1.2165 0.0171 0.0006 taaffx.78909.1.s1_at 3.3896 0.0329 0.0021 taaffx.85891.1.s1_at 3.0839 0.0149 0.0004 ta.25219.1.a1_at 2.2206 0.0198 0.0008 ta.4054.2.s1_at 1.0219 0.0383 0.0027 taaffx.36896.1.a1_at 1.0314 0.0462 0.0037 taaffx.37068.1.s1_at 1.7241 0.0449 0.0036 taaffx.120915.1.s1_at 1.2725 0.0151 0.0004 taaffx.129201.1.s1_s_at 1.1133 0.0411 0.0031 ta.7033.1.s1_s_at 1.1329 0.0184 0.0006 ta.21124.1.s1_x_at 1.7122 0.0143 0.0004 ta.6443.2.s1_at 1.5355 0.0107 0.0002 ta.4828.1.s1_a_at 1.0126 0.0397 0.0029 ta.2023.1.s1_at 1.3044 0.0158 0.0005 67identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection s0h s12 h r0h r12 h 0 5 10 15 r el at iv e ge ne e xp re ss io n *** arf1 s0h s12 h r0h r12 h 0 2 4 6 r el at iv e ge ne e xp re ss io n * hb s0h s12 h r0h r12 h 0.0 0.5 1.0 1.5 2.0 r el at iv e ge ne e xp re ss io n ** c3h2 s0h s12 h r0h r12 h 0 1 2 3 4 r el at iv e ge ne e xp re ss io n * ** c3h1 s0h s12 h r0h r12 h 0 1 2 3 4 5 r el at iv e ge ne e xp re ss io n * ** bzip s0h s12 h r0h r12 h 0.0 0.5 1.0 1.5 r el at iv e ge ne e xp re ss io n ** myb s0h s12 h r0h r12 h 0 2 4 6 r el at iv e ge ne e xp re ss io n arr s0h s12 h r0h r12 h 0 1 2 3 4 r el at iv e ge ne e xp re ss io n ** bhlh1 s0h s12 h r0h r12 h 0 2 4 6 r el at iv e ge ne e xp re ss io n g 2 * s0h s12 h r0h r12 h 0.0 0.5 1.0 1.5 2.0 r el at iv e ge ne e xp re ss io n far1 s0h s12 h r0h r12 h 0 1 2 3 r el at iv e ge ne e xp re ss io n bhlh2 s0h s12 h r0h r12 h 0 1 2 3 r el at iv e ge ne e xp re ss io n gata s0h s12 h r0h r12 h 0 10 20 30 40 r el at iv e ge ne e xp re ss io n * **erf s0h s12 h r0h r12 h 0.0 0.5 1.0 1.5 2.0 2.5 r el at iv e ge ne e xp re ss io n arf2 s0h s12 h r0h r12 h 0 2 4 6 8 r el at iv e ge ne e xp re ss io n gr figure 3. expression patterns of the top 15 genes by qrt-pcr. student’s t-test was performed to analyze the changes in the gene expression 12 h after powdery mildew infection compared to 0 h in respective genotype. * and ** are statistically significant at 0.05 and 0.01 levels, respectively. 68 panthea vosough-mohebbi et al. ulation). there are different pathways including salicylic and jasmonic acids, and ethylene, which involved in the plant resistance against pathogens (yuan et al. 2019). the erf and bzip transcription factors are two main families responding to pathogen attack due to their importance, abundance, and availability of functionally well-characterized (amorim et al. 2017). the result of present study showed that the expression of bzip and erf genes were upregulated in the genotypes, however this upregulation in resistant genotype was higher than the susceptible genotype. many studies have shown that up-regulation and activation of bzip and erf transcription factor families are common as part of plant defense mechanism to response to pathogen attack (tateda et al. 2008; amorim et al. 2017; tezuka et al. 2019). for examples, the bzip60 gene was significantly up-regulated in nicotina benthamiana in response to pseudomonas cichorii inoculation, showing an involvement of the bzip in the plant innate immunity (tateda et al. 2008). an erf transcription factor in oriza sativa (oserf83) was expressed in leaves in response to blast fungus infection and led to blast resistance by regulating the expression of defense related genes (tezuka et al. 2019). the result of pca analysis, based on the first two main components showed that these two genes were far from the other genes (fig 5), indicating different expression patterns for the genes. this result was consistent with the cluster analysis. the high regulation and different expression patterns of these genes, indicating their important roles in responding to pathogen attack. also, the result of correlation analysis showed a low and positive correlation coefficient between these two genes. the second group consisted of three genes including arf1, arf2, and g2 genes. arf1 gene similar to the genes in the first group was significantly upregulated in the susceptible and resistant genotypes 12 hpi compared to their control, however the upregulation in resistant genotype was higher than the susceptible genotype. this result was confirmed by pca analysis, which had similar expression pattern with bzip and erf genes. in the present study the expression level of two auxin response factors were investigated. recently, the arfs were introduced as an active actor in plant resistance mechanism against dif ferent pat hogens attack (bouzroud et al. 2018). similar to our results, two arfs were detected in rice upon magnaporthe grisea and striga hermonthica infections (ghanashyam and jain 2009). differential expression of arf genes were observed in cotton in response to fusarium oxysporum f. sp vasinfectum infection (dowd et al. 2004). our results showed that the expression of both arf genes were upregulated in the genotypes 12 hpi compared to their controls, indicating the importance of auxin pathway in wheat resistance mechanism against the b. graminis. the result of gene expression revealed that g2 gene was significantly upregulated in the resistant genotype, while it was not significant for susceptible genotype 12 hpi compared to their controls. the result of real time pcr confirmed the result of microarray analysis, in both analyses g2 was highly upregulated in the resistant genotype. previous studies have shown that g2 plays an important role in disease defense mechanism figure 4. hierarchical clustering analysis of the top 15 differentially expressed genes among the susceptible and resistant samples. figure 5. bi-plot derived from pca analysis based on the top 15 genes. 69identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection in different plants such as arabidopsis (murmu et al. 2014) and rice (nakamura et al. 2009). over expression of a g2-like family (atglk1) in arabidopsis resulted in significant up-regulation of some genes involved in the defense mechanism and salicylic acid signaling pathway, which displayed stronger resistance to fusarium graminearum (savitch et al. 2007). the third group contained four genes, i.e. c3h1, c3h2, gr, and bhlh1. the results of pca analysis based on the two first main components confirmed the result of dendrogram, which these genes were close to each other than the other genes (fig. 5). the expression levels of c3h1, c3h2, and bhlh1 genes were significantly downregulated in the susceptible genotype, while the four genes were upregulated in the resistant genotype 12 hpi compared to their controls. also, the result of correlation analysis revealed that there were positive and significant correlation coefficients among these three genes (p < 0.01). osc3h12 and osdos genes (c3h family) positively and quantitatively regulates rice resistance to different diseases, which are likely associated with the jasmonic acid pathway (kong et al. 2006; deng et al. 2012). the bhlh transcription factors are important signaling components with dual roles in the regulation of defense responses thorough jasmonic acid pathway (wild et al. 2012; hu et al. 2013). the results of gene expression showed that the gr (grf) gene was upregulated in resistant genotype, however no change was observed in the susceptible genotype compared to their controls. grf-regulated genes are involved in some hormone biosynthesis pathways such as jasmonic acid, salicylic acid, ethylene, and auxin, which can activate the plant defense mechanisms and coordinate between developmental process and plant defense mechanisms (liu j et al. 2014). it seems that the genes in the third group caused to wheat resistance to the disease thorough jasmonic acid pathway. our result showed that the expression of c3h1, c3h2, and bhlh1 genes were downregulated in the susceptible genotype 12 hpi, which could be due to the process called stress-induced transcriptional attenuation (sita). in response to pathogens infections, the plant cells interrupt their daily routines to protect themselves from damage, cells start the production of new proteins to help damaged proteins and at the same time, many normally expressed genes rapidly downregulate in the sita process (aprile-garcia et al. 2019). the fourth group consisted of six genes, namely hb, bhlh2, myb, arr, far1, and gata. the expressions of hb and myb genes were significantly downregulated in the susceptible genotype 12 hpi compared to the control. also, there was a significantly positive correlation coefficient between them (p < 0.05). it seems that the downregulation of these two genes in the susceptible genotype were due to the sita process. cominelli et al. (2005) reported that an arabidopsis transcription factor figure 6. heat map of the correlations among the top 15 genes. figure 7. bi-plot derived from pca analysis based on susceptible and resistant samples. 70 panthea vosough-mohebbi et al. (atmyb60) involved in stomata movement, which rapidly downregulated by stress. one of the plants defense mechanism to pathogen attack, is closing their stomata to prevent pathogen entry (arnaud and hwang 2015). it seems that the susceptible genotype downregulated the myb gene to close the stomata and preventing pathogen entry, which can be a part of sita process. the results demonstrated that all genes in the fourth group were up regulated in the resistant genotype 12 hpi compared to the control, however none of them was statistically significant. the roles of these transcription factors in plant pathogen resistant were reported in different studies. for example, zhang et al. (2018) studied the expression pattern of tfs in resistant (vernicia montana) and susceptible (v. fordii) tung trees responding to fusarium wilt disease. the authors reported that the myb and bhlh families had the largest number of tfs among 59 different families in both v. fordii and v. montana species during the four infection stages. according to the result of correlation analysis all the genes in the fourth group had a significant correlation coefficient with each other (p < 0.05 or p < 0.01), although the highest correlation coefficient among the studied genes was between gata and far1 (r=0.94, p < 0.01). according to the pca analysis based on the genotypes, the two first main components confirmed more than 68% of total variance (fig. 7). it was obvious from the figure 7, the main variance of pc1 (50.4%) and pc2 (17.8%) were explained by infection treatment (0 and 12 hpi) and genotype factors, respectively. usually under similar conditions, genetic factors are the main source of variances among the samples (hassanabadi et al. 2019; farajpour et al., 2017). finally, the result of pca confirmed the results of microarray and real time pcr analyses. conclusions in the present study, the publicly available wheat microarray data set and the real time pcr analysis were used to study the transcriptomes and gene expression of susceptible (bolani) and resistant (kc2306) wheat genotypes in response to powdery mildew. a total of 5760 and 5315 probe sets were detected which 5427 and 4630 were differentially expressed by adjusted p-value < 0.05, in susceptible and resistant wheat genotypes, respectively. among exclusively up regulated genes in the resistant genotype 12 hpi compared to its control, fifteen potential genes that may be responsible for b. graminis inoculation resistance were detected. the results of real time pcr for the candidate genes showed that the genes were upregulated in the resistant genotype 12 hpi compared to its control, which validated the results 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to fusarium wilt disease. industrial crops and products. 122:716-725. caryologia international journal of cytology, cytosystematics and cytogenetics volume 73, issue 2 2020 firenze university press the first molecular identification of egyptian miocene petrified dicot woods (egyptians’ dream becomes a reality) shaimaa s. sobieh*, mona h. darwish gene flow patterns reinforce the ecological plasticity of tropidurus hispidus (squamata: tropiduridae) fernanda ito, danielle j. gama-maia, diego m. a. brito, rodrigo a. torres* the technique of plant dna barcoding: potential application in floriculture antonio giovino1,*, federico martinelli2,*, anna perrone3 cytogenetic of brachyura (decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species alessio iannucci1, stefano cannicci1,2,*, zhongyang lin3, karen wy yuen3, claudio ciofi1, roscoe stanyon1, sara fratini1 comparison of the evolution of orchids with that of bats antonio lima-de-faria identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection mehdi zahravi1,*, panthea vosough-mohebbi2, mehdi changizi3, shahab khaghani1, zahra-sadat shobbar4 populations genetic study of the medicinal species plantago afra l. (plantaginaceae) saeed mohsenzadeh*, masoud sheidai, fahimeh koohdar a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding timir baran jha1,*, partha sarathi saha2, sumita jha2 chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india harminder singh2, jaswant singh1,*, puneet kumar2, vijay kumar singhal1, bhupendra singh kholia2, lalit mohan tewari3 population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies raheleh tabaripour1,*, masoud sheidai1, seyed mehdi talebi2, zahra noormohammadi3 induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions girjesh kumar, shefali singh* karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 caryologia. international journal of cytology, cytosystematics and cytogenetics 72(3): 41-51, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-760 citation: s. wang, y. luo, t. yang, y. zhang, z. li, w. jin, y. fang (2019) genetic diversity of rhododendron simsii planch. natural populations at different altitudes in wujiashan mountain. caryologia 72(3): 41-51. doi: 10.13128/caryologia-760 published: december 13, 2019 copyright: © 2019 s. wang, y. luo, t. yang, y. zhang, z. li, w. jin, y. fang. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. genetic diversity of rhododendron simsii planch. natural populations at different altitudes in wujiashan mountain (central china) shuzhen wang, yanyan luo, tao yang, yujia zhang, zhiliang li, weibin jin, yuanping fang* hubei key laboratory of economic forest germplasm improvement and resources comprehensive utilization; hubei collaborative innovation center for the characteristic resources exploitation of dabie mountains; college of life science, huanggang normal university, huanggang, 438000, hubei province, p.r. china *correspondence author 3559541179@qq.com or wangshuzhen710@whu.edu.cn abstract. altitude could greatly influence species distribution and even their genetic diversity. however, it is unclear how altitude has affected the genetic diversity and population structure of rhododendron simsii planch., an dominant forestry species in north temperate forest. in this research, 22 polymorphic est-ssr markers were utilized to assess the genetic diversity of r. simsii population distributed at different altitudes of wujiashan mountain, a major peak of dabie mountains (central china). totally, 203 alleles were obtained, and each locus gave out 5 to 19 alleles. high genetic diversity existed, as nei’s gene diversity (h) and shannon’s information index (i) ranged from 0.728 to 0.920 and 1.430 to 2.690, with the mean value of 0.821 and 1.916, respectively. in particular, 11.1% of genetic differentiation was maintained between populations, while 88.9% occurred within populations. moreover, moderate gene flow (2.001) among populations was observed, which could effectively resist genetic drift. the genetic diversity of all these five r. simsii populations varied significantly with elevation, basically showing high-low-high pattern with elevation increase. without human intervention, genetic diversity of r. simsii populations might increase with the  altitude. at the significance level (p < 0.05), negative correlation was found between genetic diversity and attenuation rate of light intensity (r=-0.873). soil of wujiashan mountain was acid (the ph value ranged from 4.33 to 4.70), which was rich in organic matter, available phosphorus, available potassium, and alkali hydrolysable nitrogen, as these soil factors interacted with each other to affect the growth of r. simsii population. this research would contribute a lot to the knowledge of evolutionary history of r. simsii species and benefit subsequent management and conservation actions. key words. rhododendron simsii planch.; est-ssr; genetic diversity; altitude; germplasm protection. 42 shuzhen wang et al. introduction genetic studies are important for understanding the genetic structure of populations and their ability to respond to natural selection (lee 2002; allendorf and lundquist 2003). genetic diversity reflects the ability of plant species to adapt environment changes during evolution. moreover, understanding of genetic diversity in plants, including origin, maintenance and distribution, could give great insight into the modes of speciation, adaptation, as well as population dynamics (bussell 1999). genetic composition of a certain species is often influenced by various factors, including the history of introduction, founder effects, life-history characteristics, reproductive method, and even the effect of gene drift (liu et al. 1998; ye et al. 2003; dewalt and hamrick 2004; liang et al. 2008). in particular, life-history characteristics, including reproductive method, could affect genetic diversity withinand amongpopulations (dewalt and hamrick 2004). founder effects and genetic drift could reduce the heterozygosity and increase interpopulation differentiation (liang et al. 2008). moreover, spatial distribution of genetic structure, reflecting adaptation evolution, environmental changes and natural selection effect, is often closely related to breeding mechanisms of the species (ishihama et al. 2005). genetic and geographical structure in natural populations along elevational gradients are often influenced by life history, ecological traits, and biogeographic history (quiroga and premoli 2007; truong et al. 2007). elevation, or altitudinal gradient, is an assemblage of environmental variables, which could markedly influence the distribution of population genetic variation (hahn et al. 2012). therefore, understanding of current distribution pattern of population genetic diversity and differentiation along altitudinal gradients is vital for conservation and reasonable utilization (mcmahon et al. 2007). the rhododendron genus, belonging to ericaceae family, is widely distributed around the northern hemisphere and presents as different ecological types (popescu and kopp 2013). besides high horticultural and medicinal properties, rhododendron plants play important roles in the stability of ecological system. in particular, r. simsii is the dominant species in the community of “dabie mountains woods” (central china), (wang et al. 2017). however, rhododendron-based tourism, habitat fragmentation caused by human activities, as well as changes in ecological environment, all have exerted great influence towards natural rhododendron population (wang et al. 2017). therefore, research on genetic diversity and ecological conservation of wild r. simsii is essential. however, analysis of genetic diversity and population structure of wild r. simsii population is limited, especially the populations located on dabie mountains. microsatellite, or simple sequence repeats (ssr), is abundant, co-dominant, widely distributed in genomes, highly polymorphic, and easily detectable, which has been widely used in genotype mapping, population structure and genetic diversity analysis (ambreen et al. 2018; ukoskit et al. 2018). in particular, ssr marker developed from expressed sequence tags (est), the est-ssr, showed more convenience in genetic studies, which has a high transferability to related species (xu et al. 2018; zhang et al. 2018). in this research, est-ssr markers were used to investigate the genetic diversity of r. simsii populations at different altitudes in wujiashan mountain. materials and methods description of wujiashan mountain and materials wujiashan mountain (115°46’31.37”-115°50’39.20”e, 31°04’43.20”31°07’31.60”n, 3.02×104 hm2), is one of the beautiful spot in dabie mountains. according to our field investigation, the constructive species making up the brush and forest were mainly species belonging to the families of lauraceae, cornaceae, leguminosae, anacardiaceae, fagaceae, and caprifoliaceae. fresh leaves of r. simsii were collected at different altitudes on wujiashan mountain in august 2017 (table 1). particularly, the minimum interval between individuals was set as 100m. development of est-ssr markers transcriptome data (srp099282) of r. simsii flower tissue was used for the development of est-ssr markers table 1 the location of r. simsii populations studied sampled from wujiashan mountain. population code sampling altitudes number of individuals longitude (e) latitude (n) percentage of polymorphic loci 1 972m 15 115°47’18’’ 31°06’53’’ 100% 2 1,071m 15 115°47’06’’ 31°06’04’’ 100% 3 1,167m 15 115°47’01’’ 31°06’07’’ 100% 4 1,270m 15 115°46’49’’ 31°06’08’’ 100% 5 1,370m 15 115°46’40’’ 31°06’08’’ 100% 43genetic diversity of rhododendron simsii planch. natural populations at different altitudes in wujiashan mountain with microsatellite (misa, http://pgrc.ipkgatersleben. de/misa). these ssr-containing unigenes (di-nucleotide units) with sufficient flanking regions (more than 100bp) were chosen for prime pair design with online software primer 3 (wang et al. 2010). dna extraction and genetic diversity analysis based on est-ssr markers the modified ctab (cetyltrimethyl ammonium bromide) method was adopted to extract genomic dna, which was further diluted to 50ng/μl (wang et al. 2017). the 10μl pcr amplification system was set, including 5μl 2×taq plus pcr mastermix (tiangen, beijing, china), 0.2 μm for each primer, as well as 50 ng genomic dna. the pcr amplification conditions included initial denaturation at 95°c for 10 min, followed by 35 amplification cycles (94°c for 30 s, annealing at optimal temperature for 40 s, and 72°c for 50 s), as well as a 7 min elongation step at 72°c. then, the pcr amplification products were separated on 6% (w/v) denaturing polyacrylamide gels, which were further visualized by silver staining. analysis of soil nutrients and attenuation rate of light intensity in sample plots five randomized soil cores (3cm in diameter) were taken up from each sampling spot (0-15cm depth), which were dried off in air and sieved through the 1mm sieve according to wiśniowskakielian and klima (2010). available phosphorus and potassium forms were extracted from the soil with lactate reagent according to the egner-riehm’s method (sienkiewicz et al. 2011). in particular, the content of available phosphorus (mg/kg of the soil dry matter) were determined through spectrophotometric method using beckman du 640 apparatus, while content of available potassium were obtained with atomic absorption spectrometry (aas) using pu 9100x philips. furthermore, contents of alkali hydrolysable nitrogen were determined with alkaline persulfate digestion (ding et al. 2013). contents of the soil organic matter (som) were calculated with potassium  dichromate  oxidation  method. soil ph values were measured in 0.01mol/l cacl2 slurry (1:2.5 soil/solution) using a reference glass electrode (ding et al. 2013). the light intensity upon the upper leaf surface and below the bottom leaf of each r. simsii plant was measured with luminometer (as 810), and 50 plant were randomly selected in each sampling pot. the attenuation rate of light intensity was equal to the upper light intensity divided by lower light intensity. statistica (version 6.1, statsoft) was employed to determine the statistical parameters and correlation coefficients. data analysis dna bands were scored for each sample. population genetic parameters were calculated with popgene version 1.31 software, including number of alleles (na) per locus, effective number of alleles (ne) per locus, expected heterozygosity (he), observed heterozygosity (ho), tests for linkage disequilibrium (ld), nei’s (1973) gene diversity (h), shannon’s information index (i), totalpopulation inbreeding coefficient (fit), intra-population inbreeding coefficient (fis), inter-population genetic differentiation coefficient (fst), gene flow, genetic identify (gi), and genetic distance (d) between populations (wu et al. 2011). moreover, genetic distance matrix among pairs of populations resulting from popgene analysis was utilized to create a dendrogram by mega software version 4.0. in addition, the correlations between genetic diversity and altitudinal distances, as well as soil factors were tested using dmrt with the software spss 17.0. the statistical significance between populations was estimated by two-tailed student’s t test (p < 0.05). results genetic diversity of rhododendron populations among 57 est-ssr markers, 22 were polymorphic (table 2), which gave out 203 bands. r. simsii had high genetic diversity at species level, and the polymorphic percentage in five populations were all 100%. na and ne ranged from 5 to 19 and 3.674 to 12.437, with the mean value of 9.227 and 6.083, respectively (table 3). the length of amplified bands ranged from 161 to 268 bp. the average shannon’s information index (i) and nei’s gene diversity (h) were 1.916 and 0.821, respectively (table 3). in particular, the highest i and h was observed at est-ssr117 locus, while the lowest existed at ssr019 locus (table 3). moreover, ho and he ranged from 0.208 to 1.000 and 0.744 to 0.926, with the mean value of 0.862 and 0.828, respectively (table 3). the genetic diversity of population was lower than that of the species. at population level, the average na and ne were 5.56 and 4.23, and the mean i and h were 1.498 and 0.734, respectively (table 4). the level of genetic variation of these five populations from the highest to lowest revealed by i was pop 5> pop 1> pop 3> pop 2> pop 4. in particular, pop 5 gave out 44 shuzhen wang et al. the most alleles (128), while pop 4 produced the least alleles (119), which were all polymorphic (table 4). the i ranged from 1.423 (pop 4) to 1.565 (pop 5), while h ranged from 0.705 (pop 4) to 0.762 (pop 1). the mean ho and he ranged from 0.836 (pop 2) to 0.885 (pop 5) and 0.730 (pop 4) to 0.807 (pop 1), respectively. basically, the genetic diversity of five populations showed a high-low-high variation pattern, as genetic diversity of r. simsii populations sampled at high and low altitude was higher than populations collected at middle altitude. using an unpaired  two-tailed  student  t-test, the difference between populations was not statistically significant (p<0.05) genetic differentiation among populations at different altitudes significant genetic differentiation presented among these five r. simsii populaitons (p<0.001). an amova of the distance matrix for all individuals partitioned overall variation into two levels, including ‘among species’ and ‘among populations’. the fis and fit values ranged from -0.508 to 0.447 and -0.215 to 0.715, with the mean value of -0.178 and -0.047, respectively (table 3). the fis value was negative for all five populations, ranging from -0.223 (pop 4) to -0.136 (pop 3), inferring that relatively high level of outcross occurred within populations (table 4). fst value was calculated to be 0.111, suggesting that only 11.1 percent of overall genetic variation occurred between populations, while 88.9 percent took place within populations (table 3). furthermore, genetic variation mainly occurred at the ssr019 locus, followed by ssr105, ssr117, and ssr123 loci. in particular, gene flow was 2.001, which occurred frequently at ssr114, ssr097, ssr129, ssr082, and ssr090 loci (table 3). however, the gene flow was a low-frequency event at ssr019 locus with the nm value of 0.265, inferring that this locus might undergo genetic drift during population evolution (whitlock and mccauley 1999). cluster analysis of different r. simsii populations genetic distance between pop 1 and pop 3 was the biggest (d = 0.8169), while their genetic identify was the lowest (gi = 0.4418). however, genetic distance between pop 3 and pop 4 was the smallest (d = 0.3979), while their genetic identify was the highest (gi = 0.6717) (table 5). based on the matrix of genetic distance, upgtable 2 characteristics of ssr primers used in this research. shown for each primer pair are forward and reverse primer sequences, repeat motif, annealing temperature (ta), and the size range of alleles fragment (bp). locus forward primer sequence (5’-3’) reverse primer sequence (5’-3’) repeat motif ta (°c) size range (bp) ssr019 atcccatcccatctctctc cacagatgagagaagagagc (ct)25 55 202-212 ssr025 tcgtgttgggtttctattgt tccatcaaactaccaacacc (ct)25 55 236-256 ssr031 gcaatctttcctcccatctt cttctgaatgggtgctactt (ag)26 56 233-245 ssr032 gaaacgtgtctgttttctcc ctaccccaatttccactacc (ct)28 56 207-231 ssr070 tcttccgattccatcattcc tgggcgtgatttggttataa (ct)22 54 179-203 ssr078 ttccagttccaattcatcgg cccaacaacaattccatcac (ct)22 56 161-179 ssr081 gccctatccctcaactttac gaggagcgtggttagtaatt (tc)21 55 230-252 ssr082 gtatgggacctgtgatttcc ctccaactagctactccaac (ag)24 57 229-243 ssr090 ttgaagaacactcaagttgc acgtagaacattgctttcct (ga)21 56 187-201 ssr093 ggtatccggttttcatcact atacccactagcaacagaga (ga)23 55 234-248 ssr097 agaaaactgggagatgtgtc aggtgatcatctttgcatgt (ct)21 55 247-267 ssr105 cccctctttctctctaggat gagagagaagccgattacag (tc)22 56 186-200 ssr110 taacctgccagtggaattac tctacgtacgccattgaaat (ct)22 55 224-234 ssr111 ctgcagacatgacatgaaac tttgcttaccactcccattt (ag)21 55 244-260 ssr113 tattgtacagctcccctttg cctcaatgttctatcgacgt (ct)23 56 186-200 ssr114 tattgtacagctcccctttg gaacatgttaaagcgcttga (tc)21 54 171-183 ssr116 attgcttctgataccatccg tatcagctttcgagttgtcc (tc)21 55 211-223 ssr117 gctattcactcgtcaaatgc attgtgggaatgaaggtctc (ga)22 55 229-268 ssr123 cccttcctcttctcaaatcc cgtcattttcacacacagag (ct)23 54 174-189 ssr125 ctctcccaaaattagccgat gaattggctgttggatgatg (ct)21 55 234-246 ssr129 tgaagctgttttagactccc catgatgggaaagcaaagtg (tc)22 55 161-175 ssr130 ccatgacgaaccctattgat tcctgatattcctttgcaca (ag)21 56 235-245 45genetic diversity of rhododendron simsii planch. natural populations at different altitudes in wujiashan mountain ma cluster analysis assigned these five populations into two groups (figure 1). group i possessed pop 3, pop 4, and pop 5, while pop 1 and pop 2 were clustered into group ii. group i could be further divided into two subgroups, ia and ib. particularly, ib consisted of pop 3 and pop 4. population 3 appeared to be more closer with population 4 than other populations. the dendrogram indicated that r. simsii population clustering had obvious region specificity, as the first group included populations sampled at higher altitudes, while the second possessed populations collected at the lower altitudes of wujiashan mountain (figure 1). soil nutrients and attenuation rate of light intensity in five sample plots contents of available phosphorus and available potassium ranged from 13.696 mg/kg (pop 2) to 20.850 table 3 genetic diversity of r. simsii populations based on ssr markers, including number of alleles (na), effective number of alleles (ne), shannon’s information index (i), nei’s gene diversity (h), observed heterozygosity (ho), expected heterozygosity (he), intra-population inbreeding coefficient (fis), total-population inbreeding coefficient (fit), inter-population genetic differentiation coefficient (fst), and gene flow (nm). locus na ne i h ho he fis fit fst nm ssr019 5 3.674 1.430 0.728 0.208 0.744 0.447 0.715 0.486 0.265 ssr025 11 6.751 2.090 0.852 0.964 0.860 -0.286 -0.154 0.103 2.184 ssr031 7 4.018 1.598 0.751 0.900 0.757 -0.278 -0.182 0.075 3.105 ssr032 13 7.904 2.245 0.874 1.000 0.880 -0.243 -0.146 0.078 2.937 ssr070 13 9.047 2.361 0.890 1.000 0.896 -0.225 -0.130 0.078 2.960 ssr078 10 6.468 2.036 0.845 1.000 0.852 -0.338 -0.169 0.126 1.738 ssr081 12 5.787 2.005 0.827 1.000 0.833 -0.356 -0.189 0.123 1.785 ssr082 9 5.842 1.923 0.829 1.000 0.835 -0.269 -0.204 0.052 4.606 ssr090 8 6.630 1.961 0.849 1.000 0.857 -0.247 -0.175 0.058 4.048 ssr093 8 6.512 1.944 0.846 1.000 0.853 -0.276 -0.172 0.082 2.817 ssr097 11 6.054 2.015 0.835 1.000 0.841 -0.254 -0.193 0.049 4.874 ssr105 8 5.678 1.870 0.824 1.000 0.831 -0.508 -0.215 0.195 1.036 ssr110 6 3.947 1.527 0.747 0.561 0.752 0.201 0.251 0.062 3.754 ssr111 11 5.161 1.915 0.806 1.000 0.813 -0.363 -0.205 0.116 1.904 ssr113 8 6.383 1.949 0.843 0.754 0.850 0.019 -0.078 0.060 3.889 ssr114 7 5.070 1.758 0.803 0.732 0.810 0.053 0.097 0.047 5.116 ssr116 7 4.398 1.660 0.773 0.797 0.778 -0.117 -0.045 0.063 3.716 ssr117 19 12.437 2.690 0.920 0.843 0.926 -0.114 0.097 0.189 1.071 ssr123 9 7.861 2.126 0.873 0.968 0.880 -0.332 -0.109 0.168 1.241 ssr125 7 4.350 1.624 0.770 0.712 0.776 -0.016 0.059 0.074 3.133 ssr129 8 5.630 1.886 0.822 0.908 0.829 -0.161 -0.102 0.05 4.708 ssr130 6 4.221 1.538 0.763 0.623 0.769 0.080 0.208 0.139 1.547 mean 9.227 6.083 1.916 0.821 0.862 0.828 -0.178 -0.047 0.111 2.001 st. dev 3.146 1.990 0.295 0.050 0.201 0.050 table 4 genetic diversity of r. simsii populations at different altitudes. population codes na mean na mean ne i h ho he fis pop 1 120 5.46±1.57 4.52±1.30 1.551±0.271 0.762±0.065 0.884±0.222 0.807±0.069 -0.153±0.262 pop 2 121 5.50±1.50 3.97±1.27 1.457±0.321 0.717±0.110 0.836±0.239 0.750±0.114 -0.151±0.293 pop 3 124 5.64±1.68 4.27±1.35 1.495±0.396 0.727±0.148 0.849±0.249 0.757±0.154 -0.136±0.358 pop 4 119 5.41±1.71 3.92±1.21 1.423±0.387 0.705±0.154 0.866±0.257 0.730±0.159 -0.223±0.294 pop 5 128 5.82±1.62 4.50±1.30 1.565±0.291 0.757±0.077 0.885±0.200 0.784±0.079 -0.167±0.260 mean 122.4 5.56±0.17 4.23±0.28 1.498±0.060 0.734±0.025 46 shuzhen wang et al. mg/kg (pop 5) and 144.378 mg/kg (pop 1) to 306.197 mg/kg (pop 5), with the mean values of 17.329 mg/ kg and 204.198 mg/kg, respectively (figure 2a, 2b and supplementary file 1). the content of available phosphorus differed slightly between various populations, with a decreasing order of pop 5, pop 3, pop 4, pop 1, and pop 2 (figure 2a). moreover, content of soil organic matter in wujiashan mountain was 720.953 mg/kg (figure 2c and supplementary file 1). basically, the content of soil organic matter increased with altitude rising, which was highest in pop 5 (838.565mg/kg). along the elevation, contents of alkali hydrolysable nitrogen were also increased: the lowest value existed in pop 1, while the highest value was observed in pop 5 (figure 2d). overall, soil of wujiashan mountain was estimated to be rich in nutrients necessary for the growth of r. simsii. the ph value of soil ranged from 4.33 (pop 4) to 4.70 (pop 3), and the mean value was calculated to be 4.494 (figure 2e). moreover, the attenuation rate of light intensity in r. simsii populations differed significantly, varying from 0.438 to 0.594 (figure 2f). in particular, pop 5 had the lowest attenuation rate of light intensity (0.438), followed by pop 1 (0.455). however, the highest attenuation rate of light intensity was observed in pop 4 (0.594), followed by pop 3 (0.529). correlation between population genetic differentiation and environmental factors the correlation analysis showed that genetic diversity between populations was not significantly related to altitude (r(i, altitude)=-0.014, p>0.05; r (h, altitude) = -0.136, p>0.05). moreover, na, ho, he, and fis also showed no relationship with altitude: r(na, altitude)=0.599, p>0.05; r(ho, altitude)=0.599, p>0.05; r(he, altitude)=-0.343, p>0.05; r(fis, altitude)=-0.474, p>0.05. at the significance level (p < 0.05), negative correlation was observed between genetic diversity and attenuation rate of light intensity with r value of -0.873 (figure 3a). however, no significant correlation were observed between genetic diversity and available phosphorus, available potassium, alkali hydrolysable nitrogen, soil organic matter, as well as ph value of soil at the significance level (p < 0.05) by mantel’s test, with the r value ranging from 0.236 to 0.526. in particular, contents of available phosphorus were positively correlated with content of alkali hydrolysable nitrogen (r=0.953, p=0.012) and the content of soil organic matter (r=0.879, p=0.05) (figure 3b and c). furthermore, similar correlation also existed between alkali hydrolysable nitrogen content and soil organic matter content (r = 0.935, p = 0.020) (figure 3d). table 5 nei’s genetic identity (above diagonal) and genetic distance (below diagonal) between different populations. pop id pop 1 pop 2 pop 3 pop 4 pop 5 pop 1 0.5720 0.4418 0.4718 0.4865 pop 2 0.5587 0.6309 0.5660 0.5332 pop 3 0.8169 0.4605 0.6717 0.6194 pop 4 0.7512 0.5692 0.3979 0.6712 pop 5 0.7206 0.6288 0.4790 0.3987 table 6 contents of available phosphorus, available potassium, alkali hydrolysable nitrogen, soil organic matter, and the ph values of five sampling spots. populations available phosphorus (mg/ kg) available potassium (mg/kg) soil organic matter (g/kg) alkali hydrolysable nitrogen (mg/kg) ph value attenuation rate of light intensity (%) pop 1 14.578±6.429 144.378±21.666 670.808±39.513 192.356±38.317 4.67 0.455±0.165 pop 2 13.696±3.975 186.313±43.959 658.812±31.972 221.104±43.587 4.43 0.489±0.246 pop 3 20.302±8.97 167.584±10.924 734.673±40.214 327.148±38.510 4.70 0.529±0.229 pop 4 17.220±5.475 216.518±62.862 701.905±37.975 258.014±37.964 4.33 0.594±0.242 pop 5 20.850±2.125 306.197±32.939 838.565±35.178 375.484±42.802 4.34 0.438±0.294 mean 17.329 204.198 720.953 274.821 4.494 0.501 standard deviation 3.241 62.838 72.041 75.564 0.179 0.063 fig. 1. dendrogram of five r. simsii populations generated with meag4 cluster analysis. 47genetic diversity of rhododendron simsii planch. natural populations at different altitudes in wujiashan mountain fig. 2. contents of aavailable phosphorus content (a), available potassium content (b), soil organic matter content (c), alkali hydrolysable nitrogen content (d), soil acidity (e), and attenuation rate of light intensity (f) of five r. simsii populations. values were represented as mean value ± standard deviation. fig. 3. correlation between genetic diversity and attenuation rate of light intensity (a), content of alkali hydrolysable nitrogen and available phosphorus (b), soil organic matter and available phosphorus content (c), as well as soil organic matter and alkali hydrolysable nitrogen content (d). 48 shuzhen wang et al. discussion genetic diversity is the result of long-term evolution of a species, which represents the evolutionary potential (cheng et al. 2017). moreover, population evolution and the ability to adapt to environment may largely depend on genetic diversity. according to bussell (1999), deep research on origin, maintenance, as well as distribution of genetic diversity in a species could enhance the understanding of modes of speciation, adaptation, and even population dynamics in the future. r. simsii, one of the most valuable woody plants, is dominant shrub with narrow distribution in dabie mountains (li et al. 2015). global environmental change and travel increase have threatened native biodiversity of wild r. simsii, especially the populations located on dabie mountain, whose current status need for significant attention. in this study, high level of genetic diversity was observed in r. simsii populations, with i and he ranging from 1.423 to 1.565 and 0.730 to 0.807, respectively, as he ranging from 0.3 to 0.8 means that the tested population possessed high genetic diversity (frankham et al. 2002; edwards et al. 2014). the genetic diversity was significantly higher than corylus heterophylla populations in xingtangsi forest park (i=0.4790), acer ginnala sampled at different altitudes in qiliyu (i=0.5070), firmiana danxiaensis located in danxia landform of china (he: 0.364±0.019), r. decorum in southwest china (he: 0.758±0.048), erigeron arisolius (he: 0.748±0.069), and r. jinggangshanicum population (he: 0.642±0.200) sampled from mount jinggangshan of china (yan et al. 2010; di et al. 2014; chen et al. 2014; wang et al. 2013a; edwards et al. 2014; li et al. 2015). in our opinion, the ancestor of r. simsii located on wujiashan mountaian might have a rich genetic basis, which is well preserved during evolution. r. simsii, as perennial shrub with overlapping generations, is both wind-pollinated and insect-pollinated plant. sexual reproduction could increase genetic variation within population, which correspondingly allow natural selection to proceed effectively (ayres and ryan 1999; burt 2000). therefore, the high genetic diversity existed in r. simsii natural populations might be related to the biological characteristics and living conditions. furthermore, sexual reproduction might be another critical reason for high genetic diversity. heterozygote excess was found in this wide r. simsii populations (fis = -0.178), inferring that outcross might occurred, especially in pop 4 (fis = -0.223) (nagylaki 1998). relatively low levels of inbreeding coefficient and outcross also existed in r. jinggangshanicum (fis = 0.023), r. championiae (fis = 0.012), and r. moulmainense populations (fis = 0.045) (ng and corlett 2000; li et al. 2015).furthermore, 88.9 percent of genetic variation occurred within populations, while only 11.1 percent was maintained between populations (fst = 0.111, p < 0.001). in particular, genetic variation of r. simsii populationswas slightly lower than r. jinggangshanicum distributed on jinggangshan mountain (93.13%, p < 0.001), but higher than r. decorum sampled from southwest china (85.11%, p < 0.001) and r. concinnum collected in qinling mountains (85.3%, p < 0.001) (zhao et al. 2012; wang et al. 2013b; li et al. 2015). gene flow was2.001, higher than r. arboreum population (nm = 1.13). therefore, these r. simsii populations might effectively counteract the effect of genetic drift and resist the population differentiation (kuttapetty et al. 2014). dendrogram showed typical region specificity, so gene flow might easily occur between neighboring populations. genetic diversity of these five r. simsii populations varied significantly with elevation (pop 5> pop1>pop 3>pop 2>pop 4), and basically showed high-low-high pattern. in relation to pop 5 with the highest genetic diversity, the contents of available phosphorus, potassium, soil organic matter, and the alkali hydrolysable nitrogen were all the most, while the attenuation rate of light intensity was lowest. during field observation, we found that r. simsii population increased basically with altitudes, which reached the maximum at 1,280 meters. according to leimu et al. (2006), genetic diversity and population size was positively correlated, as well as fitness and population size. therefore, high genetic diversity at high altitude might be due to the large population size, as effective population size is sufficient to prevent the genetic drift caused by loss of genetic diversity during long-term evolution. moreover, populations located at 1,280 meters might also possess high level of ecological adapt-ability. furthermore, the community structure in wujiashan mountain had almost no artificial destruction, especially at the high altitude. local famers plant r. simsii as ornamental plant, therefore different genotypes might have been brought to the population at low altitudes. gene mutation and recombination further enhance the genetic diversity of r. simsii populations at low altitudes (liang et al. 2008). soil of wujiashan mountain was acid with the ph value ranging from 4.33 to 4.70, and was rich in organic matter, available phosphorus, available potassium, and alkali hydrolysable nitrogen. the typical acid soil is very suitable for the growth of r. simsii. substrate availability could influence microbial metabolic pathways to regulate carbon and even nutrient demand (mondini et al. 2006). the soil conditions might also exert influence towards the metabolic pathways of microbes associated with r. simsii, which further affect the growth of r. 49genetic diversity of rhododendron simsii planch. natural populations at different altitudes in wujiashan mountain simsii population. however, no obvious correlation was observed between these soil factors with genetic diversity of r. simsii populations, except the attenuation rate of light intensity. relatively high genetic diversity maintained within r. simsii populations located on wujiashan mountain was observed. no obvious correlation was observed between genetic diversity and altitude. however, genetic diversity was in negative correlation with attenuation rate of light intensity. in particular, the available phosphorus, potassium, soil organic matter, and the alkali hydrolysable nitrogen in soil might interact with each other to affect the growth of r. simsii population. this research will be beneficial for the understanding of evolutionary history and population dynamics of r. simsii population located on wujiashan mountain. in addition, the study is also important for preserving r. simsii genetic resources, as well as broadening genetic basis of rhododendron cultivars. acknowledgments this work was supported by research grants from national natural science foundation of china (nsfc 31500995), open fund of hubei key laboratory of economic forest germplasm improvement and resources comprehensive utilization (2017bx06), and fund of college students’  innovative  and entrepreneurial  activities of huanggang normal university. references ambreen h, kumar s, kumar a, agarwal m, jagannath a, goel s. 2018. association mapping for important agronomic traits in safflower 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markers. sci. silv. sin. 46(10): 50-56. ye wh, li j, cao hl, ge xj. 2003. genetic uniformity of alternanthera philoxeroides in south china. weed res. 43: 297-302. zhang z, xie w, zhang j, zhao x, zhao y, wang y. 2018. phenotypeand ssr-based estimates of genetic variation between and within two important elymus species in western and northern china. genes.  9(3): 147. zhao b, yin zf, xu m, wang qc. 2012. aflp analysis of genetic variation in wild populations of five rhododendron species in qinling mountain in china. biochem. syst. ecol. 45: 198-205. 51genetic diversity of rhododendron simsii planch. natural populations at different altitudes in wujiashan mountain supplementary file 1 the correlation coefficient (below) and significant level (above) among genetic diversity, contents of available phosphorus, available potassium, alkali hydrolysable nitrogen, soil organic matter, soil ph value, and the attenuation rate of light intensity. item genetic diversity available phosphorus available potassium alkali hydrolysable nitrogen soil organic matter ph value attenuation rate of light intensity genetic diversity —— 0.619 0.703 0.611 0.363 0.614 0.050 available phosphorus 0.304 —— 0.298 0.012 0.05 0.91 0.965 available potassium 0.236 0.587 —— 0.154 0.069 0.127 0.735 alkali hydrolysable nitrogen 0.311 0.953 0.739 —— 0.02 0.696 0.867 soil organic matter 0.526 0.879 0.849 0.935 —— 0.588 0.604 ph value 0.308 -0.071 -0.77 -0.0241 -0.33 —— 0.796 attenuation rate of light intensity -0.873 0.027 -0.021 -0.105 -0.317 -0.161 —— substantia an international journal of the history of chemistry vol. 2, n. 1 march 2018 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 72(3): 75-86, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-769 citation: n.k. bhagyanathan, j.e. thoppil (2019) active chemical constituents of cynanchum viminale and its cytotoxic effects via apoptotic signs on allium cepa root meristematic cells. caryologia 72(3): 75-86. doi: 10.13128/ caryologia-769 published: december 13, 2019 copyright: © 2019 n.k. bhagyanathan, j.e. thoppil. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. active chemical constituents of cynanchum viminale and its cytotoxic effects via apoptotic signs on allium cepa root meristematic cells neethu kannan bhagyanathan*, john ernest thoppil cell and molecular biology division, department of botany, university of calicut, kerala, india *corresponding author: neethu_dob@uoc.ac.in abstract. the present study evaluates the cytotoxic efficacy of methanolic extract of c. viminale on a. cepa root meristematic cells. dapi staining was used to study the chromosomal aberrations indued by extract of c. viminale. evans blue staining method was employed to estimate the cell death of root cells of a. cepa. the plant extract was found to impart severe cytological damges, specifically chromosomal aberrations at interphase and prophase stage of cell division. various apoptotic signs such as apoptotic body formation, nuclear budding, micronucleus, nuclear disintegration, nuclear breakage etc. were observed in meristematic cells of a. cepa. the results suggest the cytotoxic, preferably genotoxic effect of methanolic extract of c. viminale as evidenced by various apoptotic symptoms on a. cepa root cells. keywords. cynanchum viminale, aberrations, allium cepa, cytotoxicity, apoptosis introduction plants have long been used for millennia in traditional medicine against various ailments. instead of a conventional single compound-single target approach, a consortium of bioactive molecules against multiple targets is gaining more attention nowadays. the synergistic action of various phytochemical compounds acts on various target domains, thus increasing therapeutic efficacy and eliminating the side effects (cilla et al. 2015). the sarcostemma genus (preferably sarcostemma acidum) is considered as somalata or somavalli, also known as moon plant. it is a xerophytic, perennial leafless, jointed trailing shrub with green, cylindrical, fleshy glabrous, twining branches having milky white latex, leaves reduced to scales, opposite, flowers white or pale greenish white. the decoction of the plant is useful to gargle for throat and mouth infection, gonorrhoea, muscle pain etc. recent molecular studies resulted in the taxonomic dissolution of sarcostemma into cynanchum (meve & liedeschumann, 2012). allium cepa bioassay is an efficient procedure for assessing chromosome damages induced by plant extracts. it is considered as a preliminary cyto76 neethu kannan bhagyanathan, john ernest thoppil toxic screening test which shows high sensitivity and good correlation with mammalian test systems. it is also an important tool for environmental monitoring studies, employed to assess the impacts caused by xenobiotics (leme & marin-morales, 2009; khanna & sharma, 2013). the present study is an attempt to evaluate the phytochemical constituents of methanolic extract of c. viminale by gc/ms analysis and its cytotoxic screening with special emphasis on apoptotic signs. materials and methods plant material cynanchum viminale (l.) bassi (1768: 17) subsp. viminale was collected from karnataka, india (coordinates: 11.8083° n, 76.6927° e). the specimen was authenticated and a voucher specimen (cali no. 123742) was deposited at the herbarium of department of botany, university of calicut, malappuram, kerala, india. plant extract preparation 10 g of the ground plant materials were subjected to sequential extraction in n-hexane to remove non-polar components followed by 100 ml methanol. the extract thus obtained is then completely evaporated to remove the trace amount of methanol so as to avoid toxicity. stock solution was prepared in water and different concentrations of plant extracts (200, 400, 600, 800 and 1000 µg/ml) were then made from it. gc/ms analysis chemical composition was determined by gc– ms (shimadzu qp-2010 plus with thermal desorption system td 20, fitted with a 60 m × 0.25 mm × 0.25 m wcot column coated with diethylene glycol (abinnowax 7031428, japan). helium was used as a carrier gas at a flow rate of 1.21 ml/min at a column pressure of 77.6 kpa. both injector and detector temperatures were maintained at 260 °c. samples (6 μl) were injected into the column with a split ratio of 10:0. component separation was achieved following a linear temperature program of 70-260 °c at 3 ◦c/min and then held at 260 °c for 6 min, with a total run time of 44.98 min. the ms parameters used were: electron ionization (ei) voltage 70 ev, peak width 2 s, mass range 40-850 m/z and detector voltage 1.5 v. the constituents were identified by comparison of their linear retention indices. the ms fragmentation pattern was checked with those of other compounds of known composition, with pure compounds and by matching the ms fragmentation patterns with national institute of standards and technolog y (nist) mass spectra libraries and with those in the literature (adams, 2001). finally, their quantification was performed on the basis of their gc peak areas. cytotoxic screening on a. cepa prior to initiating the test, the outer dry scales of onion bulbs were removed without destroying the root primordia. they were allowed for rooting by placing in distilled water for 1-2 days. germinated bulbs with healthy roots (1-2 cm) were collected at a period of maximum mitotic activity (between 9 am and 10 am on sunny days) and washed with distilled water. the bases of bulbs were kept in vials containing different concentrations of plant extracts (200, 400, 600, 800 and 1000 μg/ml) in such a way that only roots were suspended in extracts. positive and negative controls were also kept viz., hydrogen peroxide (2%) and distilled water. root tips were collected from the different vials at 12 h, 24 h, and 48 h intervals. the collected samples were washed in distilled water and immediately fixed in modified carnoy’s fluid for 1 h. then the root tips were subjected to hydrolysis with 1n hcl for 5-10 min and washed in distilled water followed by incubation in pbs for 15 minutes. staining was done in dapi staining solution for 30 minutes in dark condition and washed in pbs by a modified method (begum & alam, 2016). root tips were squashed and mounted in 50% glycerol. slides were then prepared and the number of damaged cells and total cells were scored in 6 different fields of view using 40x of the fluorescent microscope (leica dfc 450c, germany) for cy togenetic effects. mitotic index (%) and aberration percentage (%) were calculated using the following formulae and values were expressed as mean±se from at least three independent experiments: mitotic index (%) = number of dividing cells total number of cells ×100 aberration percentage (%) = number of aberrated cells total number of cells ×100 77active chemical constituents of cynanchum viminale in situ visualization of cell death for the assessment of cell death, control and treated bulbs with intact roots were placed in evans blue staining solution for 15 min, followed by washing of the roots in running tap water for 30 min (baker & mock, 1994). subsequently, 10 root tips measuring equal length (10 mm) from control and the treated groups were excised and soaked in 3 ml of n, n-dimethylformamide for 1 h at room temperature. the absorbance of evans blue released was measured spectrophotometrically at 600 nm (elico sl 218, india). results gc/ms analysis the volatile composition of methanolic extract of c. viminale was determined by gc/ms. a total of 26 compounds were detected in the methanolic extracts of c. viminale by gc/ms. these compounds belonged to various classes viz., terpenoids, aldehydes, fatty acids, phenolics, fatty acid esters etc. the compounds identified in the methanolic extract of c. viminale by gc-ms analysis are enlisted in table 1 and gas chromatogram is given as fig. 1. the major compounds detected were carvone (31.57%), hexadecanoic acid (29.56%) and 9-cisoctadecenoic acid (10.57%). terpenes were the predominant class of compounds present in the extract; also aldehydes and alcohols in significant quantities. 2-hexyl-2-decenal, pentadecanal, and myristaldehyde were the aldehydes present in the extract. coniferyl alcohol, 2,4,4-trimethyl-2-penten-1-ol, (e)-2-nonenol and 1-heptanol were the alcohols present in the extract. nonanoic acid methyl ester, heptadecanoic acid methyl ester, isopropyl pentadecanoate and methyl docosanoate were the fatty acid esters present in the extract. phenolic compounds like p-vinylguaiacol, 3-tert-butyl-4-methoxyphenol and allylsyringol were detected in negligible amounts. an alkaloid, 6-bromo-5-methoxy-nb methoxycarbonyltryptamine was also detected in the analysis. the extract contained fatty acids such as myristic acid, table 1. chemical composition of c. viminale as analysed by gc/ms. sl no. rt compounds class content (%) 1 6.58 limonene terpene 8.06 2 9.68 carvone terpene 31.57 3 10.98 p-vinylguaiacol phenol 0.37 4 14.25 3-tert-butyl-4-methoxyphenol phenol 0.43 5 15.85 allylsyringol phenol 0.96 6 16.33 coniferyl alcohol alcohol 5.34 7 16.35 2-nitropropane alkane 0.12 8 17.24 myristaldehyde aldehyde 1.64 9 17.69 nitrous acid, butyl ester carboxylic acid ester 0.13 10 18.12 nonanoic acid, methyl ester fatty acid ester 0.47 11 18.45 myristic acid fatty acid 0.56 12 18.55 hexadecanoic acid fatty acid 29.56 13 18.82 2-methyl 1-butanol nitrite organic compound 0.1 14 18.91 acetic acid, methyl ester carboxylic acid ester 0.11 15 18.95 6-bromo-5-methoxy-nb methoxycarbonyltryptamine alkaloid 0.81 16 19.97 phytol diterpene alcohol 0.95 17 20.28 9-cis-octadecenoic acid fatty acid 10.57 18 22.33 pentadecanal aldehyde 0.37 19 22.63 isopropyl pentadecanoate fatty acid ester 0.9 20 24.11 2,4,4-trimethyl-2-penten-1-ol alcohol 0.33 21 24.23 methyl docosanoate fatty acid ester 0.12 22 24.27 (e)-2-nonenol alcohol 0.25 23 24.58 4-methyl pentanoic acid carboxylic acid 0.66 24 36.20 1,5-diazabicyclo[5.4.0]undec-5-ene amide 0.73 25 37.33 2-hexyl-2-decenal aldehyde 0.34 26 39.38 1-heptanol alcohol 4.55 78 neethu kannan bhagyanathan, john ernest thoppil fig. 1. gc chromatogram of methanolic extract of c. viminale [total ion current (tic) chromatogram]. 79active chemical constituents of cynanchum viminale 9-cis-octadecenoic acid, tridecanoic acid and hexadecanoic acid. among these, hexadecanoic acid was the predominant one. negligible quantity of carboxylic acid esters viz., nitrous acid butyl ester and acetic acid methyl ester were also present in the extract. other compounds belonging to amides, alkanes, alkenes, alkynes and heterocyclic organic compounds etc. were also detected in trace quantity. cytotoxic evaluation on a. cepa root meristem cytotoxic potential of c. viminale in terms of mitotic index and chromosomal aberrations were tested on a. cepa root tips. the concentrations of methanolic plant extracts of 200, 400, 600, 800 and 1000 μg/ml as well as incubation period of 12 h, 24 h, and 48 h were taken as the experimental conditions. time-and dose-dependent increase in chromosome aberrations were observed in a. cepa as visualized by dapi staining. effect on mitotic index reduction in mitotic index is an important factor concerning the cytotoxicity of plant extracts on a. cepa. at 12 h period of incubation, the percentage of dividing cells was 86.66 ± 1.07 in the 200 μg/ml concentration of c. viminale (fig. 2b). on increasing concentration, mitotic index is found to be gradually declined with respect to concentration and exposure time. at the highest concentration, 1000 μg/ml of c. viminale, mitotic index was observed as 31.11 ± 2.24%. in c. viminale extract treatment, mitotic index was found to be even lower than the positive control. the decrease in mitotic index was positively correlated with an increasing concentration of plant extracts. in addition to concentration, the time period is an important factor in genotoxicity and reduction of mitotic index. at the final time period of 48 h, mitotic index (5.47 ± 0.62%) was declined to much lower percentage in all concentrations tested than other two time periods considered. in the case of positive control, mitotic index was found to sharply decreased to 2.55 ± 0.56% at 48 h where was in negative control group, no reduction in mitotic index was observed. the progressive reduction in the number of dividing cells at increasing concentrations of plant extracts suggests that the plant extract has a mitodepressive effect on the cell division of a. cepa. effect on chromosomal aberrations chromosomal aberration percentage is also an endpoint parameter considered for cytotoxicity assays. time-and dose-dependent increase in chromosome aberrations was observed in a. cepa exposed to plant extracts (fig. 2a). at the lowest concentration 200 μg/ ml, chromosome aberrations were 13.98 ± 1.74%. as observed in the case of mitotic index of a. cepa root cells treated with plant extract, doseand time-dependent variation of chromosome aberrations were also observed. during 12 h treatment period and at the highfig. 2. a: determination of chromosomal aberrations on a. cepa by c. viminale; b: mitotic index; c: spectrophotometric determination of cell death by evans blue staining. 80 neethu kannan bhagyanathan, john ernest thoppil est concentration of methanolic extracts of c. viminale, chromosome aberrations observed were 46.88 ± 0.68%. hydrogen peroxide was used as the positive control, which induced serious clastogenic aberrations in a. cepa root cells in the form of prominent nuclear lesions. however, the plant extracts at 600, 800 and 1000 μg/ml concentrations induced more aberrations than the positive control. in the case of chromosomal aberrations, it was increased up to 86.24 ± 0.68 % for c. viminale at 1000 μg/ml concentration for 48 h. wide spectra of chromosomal aberrations were induced by the plant extract, more specifically numerous apoptotic symptoms were found to be prominent. the major chromosomal aberrations observed in the study was lesions, nuclear budding, nuclear peak, nuclear extrusion, nuclear fragmentation, bridged binucleate cell, giant cells, nuclear disintegration, nuclear erosion, hyperchromasia, fragmentation, cytoplasmic vacuolation etc. (fig. 3-4). nuclear buds were observed as frequent chromosomal aberration observed in higher concentration of plant extract and its various stages of development were also observed (fig. 5). it is noteworthy to observe the apoptotic symptoms such as apoptotic bodies, nuclear disintegration, micronucleus etc. in a. cepa cells treated with different concentrations of c. viminale plant extract. most of the damages were multiple aberrations such as bridged binucleate cell, giant cell with cytoplasmic shrinkage, shrunken and twisted cell with nuclear diminution, double budding and lesion, chromosome fragmentation in the hypoploid cell etc. which indicated the acute cytotoxic potential of the species of cynanchum. these results suggested the significant cytotoxic potential or more specifically, genotoxic potential of methanolic extracts of c. viminale on a. cepa meristematic cells mediated by apoptotic signs. in-situ visualization of cell death visualization of cell death of a. cepa root cells was performed by evans blue staining and their corresponding estimation of cell death was carried out spectrophotometrically at 12, 24 and 48 h of treatment periods. n, n-dimethylformamide was the solvent used to release evans blue from root cells and the solvent containing evans blue was then quantified by noting their absorbance. spectrophotometric determination of cell death suggested that severe cytotoxicity was observed in the higher concentration of plant extracts at 48 h of the incubation period. at 12 h of incubation of a. cepa root cells with methanolic extracts of c. viminale, absorbance was found to be gradually increasing with respect to the concentration. furthermore, cell death was highest in positive control and minimum for negative control. dose and time served as an important factor concerning the cell death of a. cepa by methanolic extracts of c. viminale. dosage and exposure time was found to be directly proportional to cell death. incubation of a. cepa root cells with methanolic extracts of c. viminale for 24 h resulted in the cell death of maximum absorbance 0.48 ± 0.02 (fig. 2c). finally, incubation of a. cepa with methanolic extracts of c. viminale for 48 h caused a profound cell death. the absorbance read was 0.51 ± 0.01, which corresponds to the cell death. negative control exerted negligible cell death and positive control treated a. cepa showed extremely severe cell death in terms of 0.73 ± 0.03 absorbance. discussion methanol has a higher dielectric constant than ethanol; which enables to extract more polar compounds in comparison with ethanol. as a safety concern, methanol content is completely removed by evaporating the extract and thus the further studies were carried out with various concentration of extracts prepared in water. the gc/ms analysis revealed the presence of 26 constituents in the methanolic extract of c. viminale (table 1). the peak with a maximum area of intensity of 31.57% corresponds to carvone followed by hexadecanoic acid (29.56%) and 9-cis-octadecenoic acid (10.57%). carvone is a monoterpene found as an important constituent of essential oil of spearmint, clove, syzygium etc. (kokkini et al. 1995; chaieb et al. 2007) and found to have insecticidal and genotoxic activity. apart from these, limonene is another monoterpene that occupied 8.06% of the total area. the cytotoxic activity of limonene was evaluated in amelanotic melanoma c32, renal cell adenocarcinoma achn, hormone-dependent prostate carcinoma lncap, and mcf-7 breast cancer cell lines by the sulfo rhodamine b assay (loizzo et al. 2007). p-vinyl guaiacol is a phenolic component present in 0.37% peak area. also, the compound is a major constituent of almost all essential oils from plants (bituminaria, ferula, torreya etc.) as reported before. 3-tert-butyl-4-methoxyphenol is a phenolic constituent and a potential antioxidant compound which was recognized from the essential oil of dictamnus dasycarpus which showed significant antimicrobial activity and cytotoxicity towards achn, mcf-7, zr-7530, mda-mb-435s, hep-g2 and bel-7402 cell lines (lei et al. 2008). coniferyl alcohol is present as 5.34% of the peak area of the total area of intensity. it is synthesized via 81active chemical constituents of cynanchum viminale fig. 3. chromosomal aberrations induced by extract of c. viminale on a. cepa. a: apoptotic breakage of nucleus at interphase; b: cytoplasmic vacuolation; c: apoptotic fragmentation of nucleus; d: binucleate cell showing micronuclei; e: binucleate cell with lesions; f: nuclear disintegration; g: apoptotic nuclear disintegration; h: nuclear peak; i: shrunken and twisted cell with nuclear diminution; j: nuclear disintegration; k: trinucleate cell; l: trinucleate cell showing different stages of nuclear budding; m: nuclear budding and micronucleus; bar: 10 μm. 82 neethu kannan bhagyanathan, john ernest thoppil fig. 4. chromosomal aberrations induced by extract of c. viminale on a. cepa. a: giant cell; b: giant cell with cytoplasmic shrinkage; c: formation of apoptotic bodies in the nucleus; d: bridged binucleate cell; e: nuclear extrusion; f: apoptotic body formation; g: nuclear and cytoplasmic lesions; h: cytomictic transfer of nuclear material; i: nuclear disintegration; j: binucleate cell; k: nuclear lesion; l: double budding and lesion; m: giant cell showing nuclear disintegration and lesion; n: double nuclear lesions; bar: 10 μm. 83active chemical constituents of cynanchum viminale the  phenylpropanoid biochemical pathway and it is an intermediate in the biosynthesis of eugenol, stilbenoids, and coumarin (kadir et al. 2015). myristic acid is another fatty acid component present in 0.56% in the whole content of plant extract. earlier the antioxidant and larvicidal activity against malaria and filariasis vectors were studied using the bioactive fraction of myristic acid from ammannia baccifera aerial extract (suman et al. 2013). phytol is another compound detected (0.95%) and it is a diterpene alcohol found ubiquitously in many plant species. it was well documented that phytol is having cancer preventive properties irrespective of their concentration in the plant (hema et al. 2011). in this study, c. viminale contained a bioactive fatty acid hexadecanoic acid which was found to be predominant (29.56%). hexadecanoic acid, a palmitic acid type compound was detected in extracts of various plants and have shown to possess hemolytic, antioxidant, anticancer, nematicide, 5-alpha reductase inhibition properties etc. (jananie et al. 2011; kalaivani et al. 2012). a systematic study with respect to the fatty acid composition of sarcostemma viminale has been carried out earlier, where hexadecanoic acid and octadecanoic acid were the major components (girme et al. 2014). 9-cis-octadecenoic acid, another fatty acid present as 10.57% of the total content of volatile compounds present in c. viminale. the derivatives or their esters have the potential to act against cancer or prevent cancer at the very initial stage itself (farina & chodahry, 2011). the present study evaluates cytotoxic efficacy of c. viminale mediated by acute apoptotic symptoms in a. cepa root cells. several researchers had demonstrated the efficacy of a. cepa bioassay for validating the cytotoxic potential of plants. the present observations showed a sharp decline in the mitotic index of a. cepa root cells as a result of treatment with different concentrations of the fig. 5. various stages of nuclear budding induced by extract of c. viminale. a: initiation of bud; b: growth of bud; c: protruding as fully formed bud; d: bud with a basal notch; e: detachment of bud from the nucleus; f: micronucleus. 84 neethu kannan bhagyanathan, john ernest thoppil plant extract, which is a clear indication of the mitotic depressive effect of the crude plant extracts. the positive control used for the study was h2o2, a serious clastogen which directly interacts with genetic material and result in prominent nuclear lesions in a. cepa which suggest that it interfere with cell cycle mechanism at the initial stage itself; so cells couldn’t be passed onto the next stages of cell cycle. the aberrations induced by plant extract had the potential to affect all phases of cell cycle. henceforth, these results suggest that the tested concentrations of c. viminale extract is inhibitory, turbagenic and mitodepressive on cell division of a. cepa, which is in agreement with akintonwa et al. (2009). the genotoxic effect of c. viminale was evidenced by a remarkable lowering of mitotic division in vegetative cells of a. cepa. in the experiments, mitotic activity showed a tendency to decrease to 5.47 ± 0.62% respectively for c. viminale at the highest concentration (1000 µg/ml) of plant extract at 48 h treatment. this reduction in the mitotic activity could be attributed to inhibition of dna synthesis or blockage of the cell cycle in g2 phase, thus preventing the cells from entering into mitosis (sudhakar et al. 2001). many serious chromosoma l aberrations were observed as a result of treatment with various concentrations of plant extract. of these, 90% of the damages were contributed to the genotoxic aberrations. treatment of a. cepa with c. viminale extract resulted in various apoptotic symptoms like nuclear buds, micronuclei, nuclear fragmentation, nuclear blebbing etc. most of the damages were nucleotoxic, whereas other aberrations were caused by the disturbance on the formation of spindle fibers during cell division. nuclear buds are one of the prominent aberrations observed in the bioassay experiment. four models have been proposed for the generation of nuclear buds. nuclear buds are formed in the s-phase, representing the expulsion of excess genetic material derived from the polyploidization process, which may subsequently lead to micronucleation (fernandes et al. 2007; lindberg et al. 2007)micronucleus-like bodies attached to the nucleus by a thin nucleoplasmic connection, have been proposed to be generated similarly to micronuclei during nuclear division or in s-phase as a stage in the extrusion of extra dna, possibly giving rise to micronuclei. to better understand these phenomena, we have characterized the contents of 894 nuclear buds and 1392 micronuclei in normal and folate-deprived 9-day cultures of human lymphocytes using fluorescence in situ hybridization with pancentromeric and pantelomeric dna probes. such information has not earlier been available for human primary cells. surprisingly, there appears to be no previous data on the occurrence of telomeres in micronuclei (or buds, whose chromatin replication has failed. nuclear bud formation from broken anaphase bridges (gisselsson and pettersson 2000) would appear to be an clear explanation, assuming that the typically stalked structure of a bud results from the collapse of the bridge when it is resolved. the mechanisms responsible for micronucleus have not been yet fully understood. it may have originated during anaphase from lagging acentric chromosomes or chromatid fragments caused by misrepair of dna breaks or unrepaired dna breaks (fenech et al. 2011; bonciu et al. 2018). nuclear blebs were also observed in a. cepa cells, consisting of nuclear material, with bud-shaped excrescences on the main nucleus, protruded from the nucleus, but without an obvious constriction or bridge between the protruding nuclear material and nucleus (wang et al. 2014). nuclear lesions and erosions are a type of nuclear disintegration, observed frequently in a. cepa cells as a result of treatment with c. viminale extract. these may suggest the direct action of phytochemical components on dna synthesis and it is a cytological evidence for the inhibitory action on dna biosynthesis and nuclear poisoning (saghirzadeh et al. 2008; ngozi, 2011). nuclear erosion, which may result from the disintegration of chromatid proteins, represents irreversible toxicity (karaismailoglu et al. 2013). nuclear extrusion or sometimes nucleolar extrusion was another type of clastogenic event frequently observed in a. cepa cells. it is known that the nuclear pore complex (npc) was the most important channel for nuclear material transport. the phenomenon that the nucleolar material was extruded from the nucleus into the cytoplasm could be explained by the fact that the proteins were affected after plant extract treatment, causing the npc to lose selectivity (qin et al. 2010). the fragmentation of nuclei may indicate cell death process and this may ultimately result in aneuploidy and then to cell death. this pattern of nuclear degeneration of nucleus were also observed in programmed cell death in the nucellus of tillandsia presenting various signals of degeneration like deformed shape, chromatin condensation, plasmalemma detachment etc. (brighigna et al. 2006). binucleate and trinucleate cells were the frequent aberrations observed in the study, due to the inhibition of cytokinesis in any of the control points of the cell cycle (özkara et al. 2015). moreover, shrunken root cells, nuclear blebs, marked nuclear chromatin condensation, fragmentation etc. clearly indicate the possibilities to tend towards apoptosis. these clastogenic, as well as apoptotic signs of aberrations, provide a clue that the plant c. viminale can be effectively utilized for anticancer studies. in addition, it is interesting to highlight the high frequency of multiple chromosomal aberrations [bridged 85active chemical constituents of cynanchum viminale binucleate cell, giant cell with cytoplasmic shrinkage, chromosome fragmentation in a hypoploid cell, giant cell showing nuclear disintegration and lesion, double nuclear lesions etc.] in cells of a. cepa rather than single aberration by treatment with c. viminale extracts. the above results point to the phytochemicals present in the extracts which might have disrupted the cell cycle mechanism since various cytotoxic compounds such as carvone, limonene etc. were detected in gc/ms analysis. they might have possibly interfered with the normal cell cycle process and led to cell death. the present results thus support the notion that the cytotoxic effect of plant extracts is due to the synergistic action of a broad array of phytochemicals, the total activity of which may result in health benefits. moreover, multiple aberrations of chromosomes might have attributed by the multiple compound-multiple target mechanism of interaction between phytochemical constituents of c. viminale and a. cepa cells. cytotoxic efficacy of c. viminale was then confirmed by estimating the cell death of a. cepa root cells. evans blue staining method works on the basis of its penetration to non-viable cells (panda et al. 2011). evans blue staining of treated and control roots of a. cepa points is considered as an indirect evidence of cell death by visualising the intensity of evans blue taken up by roots, suggesting the loss of viability of cells. the intensity of dye absorbed by root cells was directly proportional to the cell death; this could be seen within few minutes after the treatment, in corroboration with the result reported earlier (achary et al. 2008). cell death can be positively correlated with an increase in the concentration of plant extract and increase in duration of treatment. conclusion the cytotoxic effects were found to increase proportionately with the concentration of plant extract. the chromosomal aberrations 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history of chemistry vol. 2, n. 1 march 2018 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 73(2): 27-37, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-730 citation: a. giovino, f. martinelli, a. perrone (2020) the technique of plant dna barcoding: potential application in floriculture. caryologia 73(2): 27-37. doi: 10.13128/caryologia-730 received: december 12, 2019 accepted: april 1, 2020 published: july 31, 2020 copyright: © 2020 a. giovino, f. martinelli, a. perrone. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. the technique of plant dna barcoding: potential application in floriculture antonio giovino1,*, federico martinelli2,*, anna perrone3 1 council for agricultural research and economics (crea), research centre for plant protection and certification (crea-dc), bagheria, italy 2 department of biology, university of florence, sesto fiorentino, florence, 50019, italy 3 department of biological, chemical and pharmaceutical sciences and technologies university of palermo, viale delle scienze, palermo, 90128, italy *correspondonding authors. e-mail: antonio.giovino@crea.gov.it; federico.martinelli@ unifi.it abstract. the objective of this work was to assess the ability of the dna barcoding approach to identify different taxonomic groups from two flowering plant collections: 1) the most relevant commercial taxa (nursery production) and 2) mediterranean plants with ornamental attitude (new emerging species). “core markers”, rbcl and matk, were adoptedthe identification step of 100 taxa belonging to 20 families. a third marker, the intergenic spacer trnh-psba, was also tested, on 74 taxa, when the core markers were not able to discriminate well the analysed germplasm.dna barcode fragments were recovered for all the total taxa investigated (100%). the rbcl showed the best performances: the greatest amplification success, the best sequencing performance both in terms of the number of sequences obtained and in terms of quality of the sequences obtained. despite having recorded greater amplification difficulties, according to numerous other studies, matk has shown a good success in sequencing and quality of the obtained sequences (de vere et al. 2012), unlike what is indicated in some protocols that suggests for this region the need for further primers to be adopted for the sequencing phase (hollingsworth et. al 2011). results showed that sixty-one taxa overall (61%) were totally resolved at specific or subspecific level, by at least one of the three markers. the matk and rbcl locus respectively resolved 44% and 35% of the taxa. the core markers in multilocus approach led to the discrimination of a total of 49% taxa. the trnh-psba was able to discriminate 52% of taxa analysed and resulting determinant in the discrimination of 14 taxa. four families, including the major number of taxa (arecaeae, fabaceae, euphorbiaceae, asteraceae), were evaluated in terms of genetic distance (k2p% value). this work highlighted the potential of the barcoding approach for a rapid identification of plant species in order to solve taxonomic disputes and support commercial traceability of floreal products. keywords: dna barcoding, dna fingerprinting, floriculture,genetic identification. 1 introduction genetic certification of plant material is, today more than ever, a fundamental requirement to increase the competitiveness of plant nurseries, even 28 antonio giovino, federico martinelli, anna perrone in the ornamental sector. this represents a unique and effective tool for unambiguous determination of nature of plant species. these improvements will enhancethe floriculture sector through the successful obtainment of the following to objectives: 1) genetic identification (especially for native plants) and particularly to identify the link between genetic resources of ornamental interest and the relative territory of origin, thus promoting the products in harmony with the territory and with sustainability criteria, 2) traceability (native plants and imported plants) through the characterization of autochthonous products, plant material of supply chain and non-native incoming material. therefore, the genetic certification of plant material is an extremely important aspect for the resolution of related problems:1) taxonomic controversies (synonymy/homonymy) and species of difficult identification, 2) newly introduced programs, genetic improvement, 3) early identification of species with very long phenological cycles, 4)correspondence checks of vegetable species entering the markets, 5) protection of biodiversity, of native or endangered species. recently, dna barcoding has emergedas a new molecular tool for taxonomists (hebert, ratnasingham & dewaard, 2003). a dna barcodeis a universally accepted short dna sequence normally employed for the identification of species (savolainen et al., 2005), promoted for a variety of biological applications (hollingsworth, graham & little, 2011), including the identification of cryptic species, species discovery (bickford et al., 2007) and taxonomic revisions (simeone et al 2013). the genotype is nothing but the set of all the genes that make up the dna of an organism. thus dnabased taxonomy has proved to be a valuable support to the classical taxonomy allowing to face the growing need for accurate and accessible taxonomic information (tautz et al., 2003). in particular, the advent of molecular markers has marked a remarkable turning point in the world of plant genetics allowing the construction of association genetic maps and the identification of genes responsible for agronomic characters (giovino et al. 2015a). in taxonomic studies, markers are important for botanical classifications and the analysis of phylogenetic relationships (varshney et al., 2005). among the molecular techniques, a new approach to the study of biodiversity has become widespread, with all the problems related to this study: the dna barcoding, literally “dna barcode”. the name of this approach refers to the identification method by which a scanner distinguishes various commercial products using linear bar codes or “upc” (universal product code).this molecular investigation approach was first proposed to the scientific community in 2003 by the population geneticist paul hebert of the university of guelph (canada) (hebert et al 2003). in this work it was used for the identification of species, a gene sequence located in the region of the mitochondrial gene coi, coding for the subunit i of the cytochrome-c oxidase (also known as warburg’s respiratory fragment), therefore the variability of a molecular marker for the identification of biological identities is exploited.over the years, coi has been successfully used in various animal taxa, including birds (hebert et al., 2004b), arthropods (barrett and hebert, 2005), fish (ward et al., 2005) and lepidoptera (hebert et al., 2004a). in vegetables, coi has not proved to be an excellent marker for phylogenetic studies, due to the low evolutionary rate of the mitochondrial genome. in order to overcome this problem, other markers for dna barcoding of plants have been identified in recent years. these are dna sequences present in some sections of the chloroplast genome, such as the trnh-psba intergenic region, the matk gene or the rbcl gene, which have characteristics similar tocoxi useful for species identification. there are several requirements for a marker to be considered appropriate for dna barcoding. first of all it is advisable that the marker has a wide taxonomic coverage (also called universality), which would allow the applicability of the gene chosen as barcode markern to the largest possible number of taxa and have a high success rate of pcr and sequencing. a high resolution capacity of the gene is also important, i.e the ability of a given barcode to differentiate species. this is typically based on the amount of interspecific differences between dna sequences (polymorphism). another fundamental assumption is that the molecular marker chosen as a barcode should show a higher interspecific variability than intraspecific variability. inter and intra-specific variability are separated by a certain distance (discontinuity between intra and interspecific variability) called “barcoding gap” (meyer and paulay, 2005).the ideal marker therefore consists of a highly variable region, which provides for species discrimination, flanked by highly conserved regions for which adequate primers can be designed (saunders and kucera, 2010). therefore, for the plants, the barcoding protocols refer to the indications of the plant working group, which suggests the use of a multi-locus approach (hollingsworth et al., 2011; domina et al. 2017). the general objective of the research was to use the technique of dna barcoding to help nursery production thanks to the easy identification of new products, ornamental plants and ornamental-food valueto respond to new and growing market needs. 29the technique of plant dna barcoding: potential application in floriculture 2 materials and methods 2.1 plant collection native sicilian plant species of high ornamental value or dual aptitude for new introduction were selected, collected and morphologically analyzed. selection was also extended to autochthonous or exotic species already produced at faro srl (catania, italy) in order to gain more insights into: 1) taxonomic controversies (synonymy / homonymy); 2) early identification of species with very long phenological cycles; 3) correct identification of species with significant commercial impact. samples collection for dna analysis includes 100 plant species. (tab. 1). we have included 52 species commercialized by faro srl in addition to 36 native species that were present in the collection at the crea-dc (bagheria, italy). for all the selected species, a bank of freeze-dried plant material and the respective dna bank was set up at the crea-dc of bagheria for long-term conservation stock. before proceeding with the application of the molecular characterization protocols, it was necessary to carry out a preliminary characterization at a morphological level, the plant material under study is represented by 3 replicates for each species (or three distinct plants for each species), specifically from each of them tissue samples were taken, represented by young leaves, which constitute the plant material from which to proceed with dna extraction . every single sample was cataloged with an identification code (id) in order to set up a real germplasm collection, as well as for the establishment of a bank of germplasm dna (freeze-dried). for all the selected species, a bank of freeze-dried plant material and the respective dna bank was made at crea in bagheria, as an important stock for the conservation of the plant material in question. 2.2 molecular analysis for the molecular identification of the plants, young leaves, previously subjected to lyophilization, were used as starting material for dna extraction.dna was extracted from three biological replicates (lyophilized) for each taxonomic entity using ctab-related method (doyle & doyle, 1987). amplification and sequencing protocols of three regions of dna usingrbcl, matk and trnh-psba were performed,as defined by the consortium for the barcode of life (cbol). firstly, these plastid portions, named “core markers”, were used for genetic characterization. for those species in which the core markers were unsuccessful, a third marker was tested based on the trnh-psba intergenic region. this portion is in fact known to support a greater degree of discrimination between related species. a pipeline of the genetic characterization analysis is shown in figure 1. sequences of the rbcl, matk and trnh-psba primers used in the pcr amplification were the following: • rbcl-f: atgtcaccacaaacagagactaaagc • rbcl-r: gtaaaatcaagtccaccrcg • matk-3f kim: cgtacagtacttttgtgtttacgag • 4) m a t k-1r k i m : ac c c ag t c c at c t g gaaatcttggttc • 5) trnhf_05: cgcgcatggtggattcacaatcc • 6) psba3_f: gttatgcatgaacgtaatgctc in relation to the pcr conditions, the protocol suggested by the cbol plant working group (hollingsworth et al., 2009) was followed, and the amplifications were conducted with a gene®amp pcr system 9700 thermocycler (applied biosystems).the amplicons were run on 2% agarose gel, whose purpose is to ensure the successful amplification of the segments of dna involved, using the barcode primers used. the gels were analysed using the image acquisition “gel doc” of biorad, which allows to use a special “quantity one” software, to identify amplified dna bands. 2.3 data analysis the pcr products were purified and sequenced following the dyenamic™ et termination kit sequencing kit (amersham biosciences) using an automatic sequencer ab3730xl dna analyzer (applied biosystems). the fragments were sequenced both forward and in reverse, using the same primers adopted for pcr.through sequencer software 4.10 (gene codes corporation, usa) the electropherograms were carefully checked and eventually cleaned manually,and assembled in contigs. the obtained sequences were blasted and aligned using muscle software, implemented within mega 6 program (tamura et al. 2013) used for phylogenetic analysis. several parameters have been evaluated to be able to efficiently determine the real discriminating power of the barcoding markers used. two categories of parameters were taken into account: 1) thoserelated to technical performances and those useful for assessing the discriminated power. the number of pcr positive samples for each marker was calculated, both for the total number of biological replicates and number of taxa analyzed. dealing with sequencing success, the number of samples positive for the sequencing procedure was calculated, which concerned only the pcr-positive samples for each marker, both in relation to the total number of biological replicates and to the number of taxa. quality of the 30 antonio giovino, federico martinelli, anna perrone table 1. species selected for molecular investigations. famiglia specie acanthaceae acanthus mollis l. arecacea acoelorraphe wrightii h. wendl. ex becc. arengaengleri becc. caryota urens l. chamaerops humilis var. humilis/ chamaerops humilis l. chamaerops humilis var. argentea andré chamaeropshumilis l. “vulcano” chamaeropshumilis l. “etna star” howeaforsteriana (f. muell.) becc. livistonachinensis (jacq.) r.br. ex mart. phoenix canariensis chabaud phoenix dactylifera l. phoenix reclinata jacq. phoenix roebelenii o’brien sabal minor (jacq.) pers. sabal palmetto (walter) lodd. ex schult. & schult.f. trachycarpus fortune (hook. ) h. wendl. washingtonia robusta h. wendl. washingtonia filifera (linden ex andré) h. wendl. ex de bary butia capitata (mart.) beccari bismarckia nobilis hildebr. & h. wendl. brahea armata s. watson brahea edulis h.wendl. ex s.watson trithrinax campestris (burmeist.) drude&griseb. arecastrum romanzoffianum (cham.) becc. syagrus romanzoffiana (cham.) xanthorrhoeaceae aloe arborescens mill. aloe vera (l.) burm.f. aloe plicatilis (l.) mill.. aloe × spinosissima jahand. fabaceae spartium junceum l. ceratonia siliqua l genista madoniensis raimondo genista demarcoi brullo, scelsi & siracusa genista tyrrhenavals. genista cupanii guss. genista aetnensis (biv.) dc. genista aristata c.presl cistaceae cistus albidus l cistus salvifolius l. cistus x pulverulentus pourr. cistus × skanbergii lojac. cycadaceae cycascircinalis l. cycas revolutathunb. myrtaceae myrtus luma molina metrosideros excelsa sol. ex gaertn. myrtus communis l. famiglia specie lamiaceae rosmarinus officinalis l. salvia leucantha cav. lavandula angustifolia mill. lavandula stoechas l. sideritis italica (mill.) greuter&burdet salvia officinalis l. ericaceae arbutus unedo l. erica siculaguss. erica peduncularis c.presl erica multiflora l. asteraceae helichrysum italicum (roth) g. don helichrysum hyblaeum brullo helichrysum nebrodense heldr. helichrysum scandens guss. anthemis cupaniana tod. ex nyman centaurea sphaerocephala l. jacobaea gibbosa (guss.) b.nord. &greuter pallenis maritime (l.) greuter ptilostemon greuteri raimondo & domina senecio candidus (presl.) dc. /jacobaea candida (c.presl) b.nord. & greuter jacobaea ambigua(biv.) pelser&veldkamp anthemis maritima l. hieracium cophanense lojac. iridaceae iris pseudopumila tineo iris germanica l. strelitziaceae strelitzia augusta thunb strelitzia nicolai regel&k.koch strelitzia reginae banks tamaricaceae tamarix gallica l. convolvulaceae calystegia soldanella (l.) r. br. amaranthaceae diotis maritima (l.) desf. ex cass./achillea maritima (l.) ehrend. &ypguo liliaceae tulipa radii reboul brassicaceae brassica insularis moris brassica villosa subsp. tinei (lojac.) raimondo & mazzola brassica rupestris subsp. hispida raimondo & mazzola rosaceae rosa sicula tratt. rosa sempervirens l. rosa canina l. rosa corymbifera borkh. caryophyllaceae dianthus busambrae soldano & f. conti dianthus rupicola subsp. aeolicus (lojac.) brullo&miniss. dianthus rupicola biv. subsp. rupicola dianthus rupicola subsp. lopadusanum brullo & miniss. dianthus siculus c. presl 31the technique of plant dna barcoding: potential application in floriculture sequence was given by the quality of the peaks present on the electropherograms to indicate the precision and reliability of the sequences obtained. sequences with quality over 70% were considered suitable. the reported value indicates the average of biological replicates. fragment length was determined and referred to the average length of the fragments obtained for each marker, in relation to the total of biological replicates, following the analysis and cleaning of the electropherograms. the value of thepower of discrimination parameter was given by the number of taxa that have been univocally discriminated on the level of species (or subspecies).the discriminating power was assessed both for single locus and in multi-locus approach. the discrimination power of each locus was evaluated by phylogenetic analysis with mega6, conducted by comparing all the sequences generated in this study and using a subset of referring sequences related to each taxa found by bold database / genbank. the level of genetic divergence was determined and indicated the degree of variability between a group of sequences, obtained from the distance matrices calculated according to the parameter k2p% (kimura, 1980). it was calculated within some families considered most representative by number of species. number of variable sites was determined. it indicated the number of bases subject to variations within the gel phylogenetic group considered on the total length of the fragments obtained for each locus. like the previous one, it was calculated within some families considered most representative of the entire collection of analyzedplant species. 3 results and discussion results of discrimination outputs for each of the three markers are reported in tab. 2. using a total of 300 samples (including biological replicates), rbclobtained 93% pcr success, 95% sequencing success, with 90% sequence quality and an average fragment length of 569 bp. matk showed a success of pcr and sequencing, respectively of 70% and 93% and a quality of sequences of 80% with an average length of fragments of 766 bp. the use of trnh-psba marker showed pcr and sequencing success respectively of 80% and 91% and a sequence quality of 85% with an average fragment length of 518 bp. consideringa total number of 100 taxa tested, rbcl showed higher values than the other two markers, with pcr success of 97% and a success of sequencing of 99%, for matk the recorded values were of 81% for successful apmification and 96% for sequencing success, while trnh-psba marker showed respectively pcr and sequencing successof 89% and 94%. in relation to the above results, rbcl showed the best performances: the greatest amplification success, the best sequencing yield both in terms of the number of sequences obtained and in terms of the quality of the sequences obtained. the matk, despite having experienced greater amplification difficulties agreeing with numerous other studies (de vere et al. 2012), it showed a good success of sequencing and good quality of obatined sequences. this does not agree withprevious works that suggest the need to use matk with additional primers for sequencing purposes (hollingsworth et. to 2011). taxa identification was firstly carried out using “core markers” (rbcl and matk). the use of the third marker, the igs trnh-psba was reserved for those situations in which both core markfamiglia specie dianthus rupicola subsp. hermaensis (coss.) o. bolòs& vigo euphorbiaceae euphorbia ceratocarpa ten. euphorbia characias l. euphorbia dendroides l. euphorbia meuselii geltman euphorbia myrsinites l. euphorbia helioscopia l. euphorbia bivonae steud. euphorbia pithyusa subsp. cupanii (guss. ex bertol.) radcl.-sm. euphorbia amygdaloides l. table 2. technical performancesof markers used in dna barcoding techniques referred to the total of biological replicates (a) and tested taxa (b). (a)   rbcl matk trnh-psba number of tested samples* 300 300 222 successful amplification (93%) 279/300 (70%) 210/300 (80%) 177/222 successful sequencing (contigs) (95%) 265/279 (93%) 195/210 (91%) 161/177 high quality sequence (contigs) 90% 80% 85% fragment length (average in bp) 569 766 518 (b)   rbcl matk trnh-psba number of tested samples* 100 100 74 successful amplification (97%) 97/100 (81%) 81/100 (89%) 66/74 number of taxa successfully sequenced (99%) 96/97 (96%) 78/81 (94%) 62/66 32 antonio giovino, federico martinelli, anna perrone ers presented difficulties, due to lack of amplification, failure of sequencing reactions or insufficient discriminating power. the overall identification results at species level for each tested taxawere reported in tab. s1. out of a total of 100 taxa tested, 61% of taxa were successfully identified at the species level with at least one of the three locus, while 37% remained at the genus level. only the remaining 2% of the taxa remained undetermined due to the failure of all three markers employed.considering the individual markers, rbcl allowed a unique identification at the species level of 34 taxa (35%), matk of 34 taxa (44%) and trnh-psba of 32 taxa (52%) (tab. 3). matk showed greater percentage values of resolving power in terms of discrimination of taxa than rbcl, confirming the trends indicated by other studies (chen et al. 2010). when rbcl and matkwere not able to discriminate species (belonging to 14 taxa), trnh-psba was decisive in the identification of them, allowing to increase the total number of discriminated taxa from 47 to 61 taxa. the core markers, used in multi-locus, rbcl + matk, allowed the unambiguous identification at the species level of 38 taxa. further combinations of the two markers rbcl + trnh-psba and matk + trnhpsba allowed the discrimination of 32 taxa and 25 taxa respectively.the use of the multi-locus approach based on core markers appeared to be the most efficient, with a good compromise between the high technical performance of the rbcl and the best resolving power supported by the matk.the following families showed the highest success rate of species discrimination: asteraceae (9 uniquely discriminated taxa out of 13, caryophillaceae with 4 taxa of 6, fabacecae with 8 taxa out of 8, euphorbiaceae with 9 taxa out of 9, brassicaceae with 3 taxa out of 3, ericaceae with 4 taxa out of 4. minor successes in terms of unambiguous resolution at the species level, have been found for arecaceae, (7 taxa discriminated at the species level on a total of 24), and for the cistaceae (none). levels of genetic divergence for larger families were reported in tab. 4. the rbcl marker showed the lowest values of genetic divergence for arecaceae, with 0.7%, and the highest values for asteraceae, with 2.1%, while matk showed the lowest values for the arecaceae with 1.5% and the highest for fabaceae with 6.4%. trnh-psba showed the highest values for euphorbiaceae with 9.1% and the lowest for arecaceae with 2.9%. trnh-psba has confirmed high variability values and its ability to discriminate within very similar taxonomic groups (chase et al. 2007).in the arecaceae family, which in our study included 24 species from 15 different genera, the trnh-psba marker recorded the highest genetic divergence value with a percentage of 2.9%. the lowest values occurred with rbcl with a percentage of 0.7%, while matk showed intermediate values compared with the first two with a value of 1.5% (tab. 4). the rbcl was able to identify two species of arecaceae (acoelorraphe wrightii and caryota urens l.). when rbcl failed, matk was decisive for identification of 4 taxa (arenga engleri becc., phoenix roebelenii o’brien, sabal minor (jacq.) pers., bismarckia nobilis hildebrandt & h.wendl., 1881). other authors indicated rbcl and matk as highly decisive phylogenetic analysis of this family (asmussen et al. 2006).only in the case of washingtonia robusta h. wendl., the discrimination was possible through the use of both core markers. relating to fabaceae (8 species investigated from 3 different genera), the lowest values of genetic divergence were recorded with rbcl with 1.5% and the highest with trnh-psba with values of 7.4%. using matk a genetic divergence of 6.4% was obtained, discriminating 4 species out of 8. the matk was determinant for 1 taxa (genista aristata c.presl), while the trnh-psba was determinant for 2 taxa (genista tyrrhena vals., genista demarcoi brullo, scelsi & siracusa). considering that genista was the most represented genus (with 6 species), rbcl showed a better result than matk within this group, discriminating 5 species (spartium junceum l., ceratonia siliqua l., genista madoniensis raimondo, genista cupanii guss., genista aetnensis raf. ex biv.). this result appears to be in contrast with the potential expressed by matk within the fabaceae in other studies (gao et al 2011; gao and chen 2009). here, the genista group showed excellent levels of discrimination with this marker. relating to asteraceae (13 investigated species belonging to 8 different genera),matk showed values of genetic divergence of 4.4% and rbcl 2.1%. as for the trnh-psba, given the excessive variability shown by the analyzed sequences, a subdivision into genera. the lowest genetic divergence values were recorded for anthemis with 1% and higher for jacobaea with 3.3%. (tab. 4). relating to asteraceae, rbcl has allowed us to identify at the species level 4 taxa (centaurea sphaerocephala l., helichrysum nebrodense heldr., ptilostemon greuteri raimondo & domina, pallenis maritima (l.) greutable 3. discriminating power of barcoding markers. 33the technique of plant dna barcoding: potential application in floriculture ter), while the matk has discriminated 5 taxa resulting in particular in the discrimination of 2 species (helichrysum italicum (roth) g. don, hieracium cophanense lojac.). the trnh-psba was determinant in the resolution of a further 3 taxa (jacobaea gibbosa (guss.) peruzzi, jacobaea ambigua (biv.) pelser & veldk., senecio candidus (c. presl) dc. jacobaea gibbosa (guss.) peruzzi showed a wide variability compared to the other species of the genus jacobaea, departing from these in all three markers used. this highlighted the presence of different clusters within the species. for asteraceae, the discrimination was rather high in agreement with other studies that indicated high levels of discrimination success (gao et al 2010).within family euphorbiaceae (9 species investigated of a single genus) the lowest values of genetic divergence occurred with rbcl with 1.2%, the highest with trnh-psba with 9.1% and intermediate values with matk(4%). (tab. 4). figures s1-s2 showed the phylogenetic relationships using the three markers for euphorbiaceae. the rbcl identified 6 taxa (euphorbia bivonae steud., euphorbia ceratocarpa ten., euphorbia dendroides l., euphorbia helioscopia l., euphorbia myrsinites l., euphorbia pithyusa subsp. cupanii guss.). matk correctly identified 3 taxa and was decisive for 1 taxa (euphorbia amygdaloides l.), while the trnh-psba was determinant for 2 taxa (euphorbia characias l., euphorbia meuselii raimondo & mazzola). for the genera brassica, erica, cistus, chamaerops, dianthus, euphorbia and genista, the work of molecular identification was performed with the use of referring species found specifically for this study. this was due to the absence in the international databases of species similar to those selected in this study (aubriot et al. 2013; domina et al. 2017; giovino et al. 2015b). therefore, these species and their respective sequences are new will be added into international databases. for taxa discriminated on a species level with the dna barcoding methodology (green colour in figs 4 and 5), our data open the possibility of a real “identity certification” card for these plant species in order to trace their commercial products at marketing stage, in order to guarantee their unique identification and traceability, to protect both biodiversity and economic aspects of nursery productions as well as end-users.the certification and traceability system may follow a very precise path (fig. 2; fig. 3).this traceability can begin with the use of dna barcoding protocols for the identification of the species. consequently, the realization of a label where, in addition to the generic species, it will be possible to include molecular results, translated into a barcode swhich, by scanning with special barcode scanners will immediately make it possible to have all certain species’ indications. a big issue emerged from this study was the lack of reference sequences available for species and taxa comparison. this issue has determined the impossibility of discriminating some groups such as: livistona chinensis jacq, trachycarpus fortunei hook., phoenix dactylifera l.; phoenix reclinata jacq., trithrinax campestris burmeist., anthemis cupaniana tod. ex nyman, butia capitata (mart.) becc., senecio candidus (c. presl) dc, aloe arborescens mill., aloe plicatilis l., iris pseudopumila tineo, iris germanica l., salvia officinalis l., cistus salvifosius l., cistus x pulverulentus delilei, cistus albidus l., cistus skanbergii, dianthus rupicola subsp. aeolicus lojac., dianthus busambrae soldano & f. conti. this figure 1. flowchart summarizing steps for the genetic identification of samples using dna barcoding and selected markers. figure 2. workflow used for the molecular characterization of the plant species usable by companies using international cbol standards. figure 3. proposed type of genetic labels for traceability of plant species at commercial level. table 4. levels of genetic divergence for larger families. genetic divergences calculated with the parameter k2p% (kimura 1980). family rbcl matk   trnh-psba n. seq variable sites gd% n. seq variable sites gd% n. seq variable sites arecaceae 107 24/533 0,7 115 101/770 1,5   36 85/676 fabaceae 25 32/543 1,5 21 181/815 6,4   9 67/329 euphorbiaceae 27 39/540 1,2 14 88/769 4   18 164/736 asteraceae 67 54/563 2,1 83 157/797 4,4 jacobaea 14 22/416 helichrysum 14 44/533 anthemis 20 8/348 34 antonio giovino, federico martinelli, anna perrone evidence demonstrated the great importance of creating molecular databases that incorporate the widest possible biodiversity with universal markers. in addition, it highlighted the importance of creating a dedicated database of the main floricultural species of ornamental interest, which can support the practical application of the molecular protocol for the purposes of traceability and monitoring by control bodies (giovino et al. 2014). although dna barcoding can reach 80-90% of resolution levels, it can lack sufficient discrimination power in some families, including ericaceae, lamiaaceae, orchidaceae. this is due to the modest evolutionary distance between closely related species evolved from recent divergence, as suggested before (hollingsworth et al., 2009). although in some cases, the multi-locus approach can have a great success, the evaluation of additional barcoding regions in relation to the success of discrimination, requires the use of individual taxonomic groups with difficult discrimination (hollingsworth et al., 2011). in conclusions, this work confirmed the high performances of rbcl and matk markers usin a total of 100 plant taxa, belonging to 20 different families. the taxa successfully sequenced for at least one of the considered markers were 98 and 61% of the total evaluated ones at level of species or subspecies. considering that the failure of taxa is linked to particular genus, or species, with very low evolutionary divergence, this result confirms the potential of the barcoding approach for the rapid analysis of unknown samples. cryptic groups found in this study highlighted the already well-known technical problems due to the low level of matk amplification and sequencing success. anyway, this marker greater power of discrimination compared to rbcl.therefore, we can conclude that although the adoption of core markers appeared to be a good compromise, in some cases the multi-locus approach and the addition of the third trnh-psba marker can promote greater success, as demonstrated here. the evaluation of additional barcoding regions can be useful for increasing the success of discrimination, but thisdepends on the individual taxonomic groups showing problems of pcr amplification and sequencing with core markers. however, it is worthy to notice that a large sample of references related to eachtaxa is necessary to validate the accuracy of the method.this study highlighted the great importance of creating molecular databases incorporating the widest possible biodiversity with universal markers, developing a dedicated database, especiallyfor floricultural species with ornamental interest to enhance their traceability and monitoring of commercial exchanges by control national authorities. disclosure statement no fincial interests or benefits has been arisen from the application of our research. data availability statement we declare that all data presented here have been included in the present work and related supplemental material. data deposition most of the analysed species were submitted in the bold database within the project: fmed: manager giovino antonio (tab. 5). references asmussencb, dransfield j, deichmannv, barfodas, pintaudjc, baker wj. 2006. a new subfamily classification of the palm family (arecacee): evidence from plastid dna phylogeny. bot j linn soc. 151(1):15-38. aubriot x, lowry pp 2nd, cruaud c, couloux a, haevermans t. 2013. dna barcoding in a biodiversity 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rupicola subsp lopadusanum [matk:801,rbcla:562,trnh-psba:246] taxonomy: magnoliophyta, magnoliopsida, caryophyllales, caryophyllaceae, dianthus identifiers: d4.c[sampleid], pal96723[museumid] depository: palermo botanical garden, herbariummediterraneum collected in: italy, sicily, isole pelagie fmed013-12 genista madoniensis [rbcla:582] taxonomy: magnoliophyta, magnoliopsida, fabales, fabaceae, genista identifiers: g2u[sampleid], pal96710[museumid] depository: palermo botanical garden, herbariummediterraneum collected in: italy, sicily, palermo fmed014-12 genista demarcoi [matk:805,rbcla:577] taxonomy: magnoliophyta, magnoliopsida, fabales, fabaceae, genista identifiers: g4u[sampleid], pal96713[museumid] depository: palermo botanical garden, herbariummediterraneum collected in: italy, sicily, palermo fmed015-12 – hieracium cophanense [matk:817,rbcla:590] taxonomy: magnoliophyta, magnoliopsida, asterales, asteraceae, hieracium identifiers: h2.c[sampleid], pal96873[museumid] depository: palermo botanical garden, herbariummediterraneum collected in: italy, sicily, palermo fmed016-12 – helichrysum hyblaeum [matk:809,rbcla:596] taxonomy: magnoliophyta, magnoliopsida, asterales, asteraceae, helichrysum identifiers: h6.c[sampleid], pal96719[museumid] depository: palermo botanical garden, herbariummediterraneum collected in: italy, sicily, siracusa fmed023-12 – ptilostemon greuteri [matk:793,rbcla:577] taxonomy: magnoliophyta, magnoliopsida, asterales, asteraceae, ptilostemon identifiers: p1.c[sampleid], pal96705[museumid] depository: palermo botanical garden, herbariummediterraneum collected in: italy, sicily, trapani fmed027-13 centaurea [matk:805,rbcla:581] taxonomy: magnoliophyta, magnoliopsida, asterales, asteraceae, centaurea identifiers: c3.c[sampleid], pal96729[museumid] depository: palermo botanical garden, herbariummediterraneum collected in: italy, sicily, palermo fmed028-13 brassica villosa subsp. bivoniana [rbcla:568] taxonomy: magnoliophyta, magnoliopsida, 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plos biol. 3(12): 422. saunders gw, kucera h. 2010. an evaluation of rbcl, tufa, upa, lsu and its as dna barcode markers for the marine green macroalgae. cryptogamie algol. 31(4):487-528. savolainen vincent, cowan robyn s, vogler alfried p, roderick george k and lane richard towards writing the encyclopaedia of life: an introduction to dna barcoding360phil. trans. r. soc. bhttp://doi. org/10.1098/rstb.2005.1730. simeone mc, piredda r, papini a, vessella f, schirone b (2013) application of plastid and nuclear markers to dna barcoding of euro – mediterranean oaks fmed042-16 dianthus rupicola [matk:780,trnh-psba:247] taxonomy: magnoliophyta, magnoliopsida, caryophyllales, caryophyllaceae, dianthus identifiers: d9p[sampleid], pal108620[fieldid], pal108620[museumid] depository: palermo botanical garden, herbariummediterraneum collected in: spain, balearicislands, majorca fmed001-12 rosa sempervirens [matk:824,rbcla:590] taxonomy: magnoliophyta, magnoliopsida, rosales, rosaceae, rosa identifiers: r3-1[sampleid], sv term[fieldid] depository: research unit for mediterranean flower species collected in: italy, sicily, palermo fmed003-12 asteraceae [rbcla:577] taxonomy: magnoliophyta, magnoliopsida, asterales, asteraceae identifiers: a4u[sampleid], pal[museumid] depository: palermo botanical garden, herbariummediterraneum collected in: italy, sicily, palermo fmed004-12 anthemis [matk:806,rbcla:580] taxonomy: magnoliophyta, magnoliopsida, asterales, asteraceae, anthemis identifiers: a7.c[sampleid], pal[museumid] depository: palermo botanical garden, herbariummediterraneum collected in: italy, sicily, palermo fmed005-12 brassica insularis [rbcla:594] taxonomy: magnoliophyta, magnoliopsida, brassicales, brassicaceae, brassica identifiers: b2.23[sampleid], pal[museumid] depository: palermo botanical garden, herbariummediterraneum collected in: italy, sicily, isola di pantelleria fmed006-12 brassica [matk:779,rbcla:582] taxonomy: magnoliophyta, magnoliopsida, brassicales, brassicaceae, brassica identifiers: b5.c[sampleid], pal[museumid] depository: palermo botanical garden, herbariummediterraneum collected in: italy, sicily, palermo fmed009-12 dianthus rupicola subsp aeolicus [matk:623,rbcla:578,trnh-psba:256] taxonomy: magnoliophyta, magnoliopsida, caryophyllales, caryophyllaceae, dianthus identifiers: d1-2[sampleid], pal96703[museumid] depository: palermo botanical garden, herbariummediterraneum collected in: italy, sicily, messina 37the technique of plant dna barcoding: potential application in floriculture (quercus, fagaceae): problems, prospects and phylogenetic implications. botanical journal of the linnean society, 172(4): 478-499. tamura k, stecher g, peterson d, filipski a, kumar s. 2013. mega6: molecular evolutionary genetics analysis version 6.0. mol biol evol. 30(12): 27252729. tautz d, arctander p, minelli a, thomas rh, vogler ap. 2003. a plea for dna taxonomy. trends ecol. evol. 18(2): 70–74. varshney rk, graner a, sorrells me. 2005. genic microsatellite markers in plants: features and applications. trends biotechnol. 23(1):48-55. ward rd, zemlak ts, innes bh, last pr, hebert pdn. 2005. dna barcoding australia’s fish species. philos trans r soc lond b biol sci. 360(1462): 1847–1857. supplementary figures fig. 5. phylogenetic tree of euphorbiaceae family with neighbor joining for matk. fig. 4. phylogenetic tree of euphorbiaceae family with neighbor joining for rbcl marker. caryologia international journal of cytology, cytosystematics and cytogenetics volume 73, issue 2 2020 firenze university press the first molecular identification of egyptian miocene petrified dicot woods (egyptians’ dream becomes a reality) shaimaa s. sobieh*, mona h. darwish gene flow patterns reinforce the ecological plasticity of tropidurus hispidus (squamata: tropiduridae) fernanda ito, danielle j. gama-maia, diego m. a. brito, rodrigo a. torres* the technique of plant dna barcoding: potential application in floriculture antonio giovino1,*, federico martinelli2,*, anna perrone3 cytogenetic of brachyura (decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species alessio iannucci1, stefano cannicci1,2,*, zhongyang lin3, karen wy yuen3, claudio ciofi1, roscoe stanyon1, sara fratini1 comparison of the evolution of orchids with that of bats antonio lima-de-faria identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection mehdi zahravi1,*, panthea vosough-mohebbi2, mehdi changizi3, shahab khaghani1, zahra-sadat shobbar4 populations genetic study of the medicinal species plantago afra l. (plantaginaceae) saeed mohsenzadeh*, masoud sheidai, fahimeh koohdar a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding timir baran jha1,*, partha sarathi saha2, sumita jha2 chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india harminder singh2, jaswant singh1,*, puneet kumar2, vijay kumar singhal1, bhupendra singh kholia2, lalit mohan tewari3 population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies raheleh tabaripour1,*, masoud sheidai1, seyed mehdi talebi2, zahra noormohammadi3 induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions girjesh kumar, shefali singh* karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 caryologia. international journal of cytology, cytosystematics and cytogenetics 72(4): 41-49, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-298 citation: a.r. andrada, v. de los á. páez, m.s. caro, p. kumar (2019) meiotic irregularities associated to cytomixis in buddleja iresinoides (griseb.) hosseus. (buddlejaceae) and castilleja arvensis schltdl. & cham. (orobanchaceae). caryologia 72(4): 41-49. doi: 10.13128/caryologia-298 published: december 23, 2019 copyright: © 2019 a.r. andrada, v. de los á. páez, m.s. caro, p. kumar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. meiotic irregularities associated to cytomixis in buddleja iresinoides (griseb.) hosseus. (buddlejaceae) and castilleja arvensis schltdl. & cham. (orobanchaceae) aldo ruben andrada1,*, valeria de los ángeles páez1, m.s. caro1,2, p. kumar3 1 fundación miguel lillo. miguel lillo 251, san miguel de tucumán, tucumán, argentina 2 facultad de ciencias naturales e instituto miguel lillo. miguel lillo 205, san miguel de tucumán, tucumán, argentina 3 botanical survey of india, northern regional centre, dehradun 248 195, uttarakhand, india *corresponding author: arandrada@lillo.org.ar abstract. the current paper analyzes the male meiotic behavior in wild populations of buddleja iresinoides and castilleja arvensis from piedmont areas of the northwest region of argentina. castilleja arvensis showed tetraploid number of chromosome of 2n = 24. our results are not in agreement with the previously reported base number x = 19 for buddleja and the chromosome number n = 28 found for b. iresinoides is atypical in the genus. around 7 % pollen mother cells were aneuploid as they showed meiotic chromosome count of n = 20-21 bivalents. possible origin for such atypical chromosome number has been discussed in this paper. during the cytological studies we also came across pollen mother cells showing meiotic abnormalities such as cytomixis, chromatin stickiness and anaphase bridges with lagging chromatin. consequently microsporogenesis was also irregular showing dyads and triads. however, the percentage of these irregularities during meiosis and microsporogenesis was not higher, and pollen fertility was not affected to a great extent. cytomixis and other meiotic abnormalities in these species are reported here for the first time. keywords. buddleja iresinoides, castilleja arvensis, chromatin, pollen mother cells, cytomixis, chromosome numbers. introduction the migration of chromatin from the nucleus of one pollen mother cell (pmc) through specialized channels (named cytomictic channels) into an adjacent pmc was observed by gates (1911) who called it cytomixis. subsequently, risueno et al. (1969) during their investigations noticed that these intercellular channels were sufficiently large to permit the migration of chro42 a.r. andrada et al. matin/chromsomes and other cytoplasmic organelles. in addition, the studies of mursalimov et al. (2018) have established that plastids can pass into another cell through cytomictic channels. cytomictic connections were observed for the first time körnicke (1901) in pmcs of crocus sativus, but as the cytogenetic studies in plants advanced this phenomenon was also reported in meristematic, tapetal, integumental, nucellar and ovary cells in both angiosperms and gymnosperms (cooper, 1952; koul, 1990; guzicka & wozny, 2005; wang et al., 2004; oliveira-pierre & sousa, 2011; kumar et al., 2015; kumar & chaudhary, 2016; kumar & singhal, 2016; reis et al., 2016; mursalimov & deineko, 2017; mursalimov & deineko, 2018). as the cytogenetic analysis in higher plants expanded, cases of cytomixis were observed more frequently in accessions of cultured or natural plants populations. during our previous investigations, we observed the presence of cytomixis in different families of angiosperms such as pipperaceae, cuscutaceae, ranunculaceae and cactaceae from the northwest of argentina (noa) (andrada et al., 2009; lozzia et al., 2009; páez et al., 2013 a, b). we also investigated buddleja iresinoides (griseb.) hosseus. (buddlejaceae) and castilleja arvensis schltdl. & cham. (orobanchaceae) for male meiosis and pollen fertility and we observed cytomixis and meiotic irregularities. the genus buddleja consist of ca. 100 species and cultivars that occur in warm, tropical, and subtropical climates from the americas, africa and asia (tallenthalsell & watt, 2009). buddleja iresinoides is a shrubby plant, native to south america, distributed from bolivia to the northwest of argentina were it is found in catamarca, jujuy, salta and tucumán provinces. it is a dioecious plant with quadrangular stems and ovate-lanceolate leaves, tomentose flowers with a bell-shaped calyx and corolla, the latter of white or yellow color (fig.1a and b) (carrizo & isasmendi, 1994). castilleja mutis ex l. f. comprises approximately 200 species native from western north to south america (gonzález, 2013). castilleja arvensis is an annual hemiparasitic herb, growing on humid soils from mexico to the central region of argentina. this species is characterized by its erect, simple, hispid, leafy stems (fig. 1c). the leaves at the top of the stem are bract-like, gradually become smaller than the lower ones, and generally are red or purple colored (botta & cabrera, 1993). figure 1. morphological overview of the studied plants, a) general appearance of buddleja iresinoides, and b) inflorescence detail; c) general appearance of castilleja arvensis. 43meiotic irregularities associated to cytomixis in buddleja iresinoides and castilleja arvensis for buddleja chromosome numbers of 18 species are listed in ipcn (index to plant chromosome numbers) (goldblatt & johnson, 1979+). the basic chromosome number x = 19 is accepted and the majority of species present this number or higher ploidy levels as gametophytic number (norman, 2000; tallent-halsell & watt, 2009). chromosome numbers of 54 species are listed in ipcn for the genus castilleja (goldblatt & johnson, op. cit.). based on the published literature the basic chromosome number of x = 12 and one or more polyploid levels have been suggested (heckard, 1968; heckard & chuang, 1977; chuang & heckard, 1982; tank & olmstead, 2008; tank et al., 2009). the aim of the current paper is to analyze the male meiotic behavior in wild populations of b. iresinoides and c. arvensis in order to establish that meiotic irregularities are related to the phenomenon of cytomixis and, furthmore, to evaluate if they influence pollen fertility. materials and methods analyzed materials all the material studied in this investigation was collected from natural populations of buddleja iresinoides (figure 1a-b) and castilleja arvensis (figure 1c) in tucumán province. voucher samples were deposited at the phanerogamic herbarium of miguel lillo foundation (lil). buddleja iresinoides: argentina, prov. tucumán, dpto. concepción, loc. cochuna, 27°10’20” s, 65°55’39” w, alt. 1160 m, andrada r. s/n (lil 610862). castilleja arvensis: argentina, prov. tucumán, dpto. tafí viejo, loc. camino a la toma, 26°43’05,122” s, 65°17’45.53” w, alt. 878 m, 29-lx-2007, andrada r. s/n (lil610759). analysis of meiosis the material used consisted of flower buds from 5 randomly selected plants which were fixed in farmer solution (3 ethanol : 1 glacial acetic acid) for one day, immediately transferred to 70% ethanol and stored at 4 °c. anthers were first hydrolyzed in 1 n hcl at 60 °c for 20 minutes and then washed in distilled water. pollen mother cells were prepared by the squash technique and stained with a drop of hematoxylin propionic with ferric citrate (sáez, 1960; núnez, 1968). 100 pmcs at each stage of the meiosis were observed. size and fertility of pollen grains fixed flowers immediately after anthesis were selected in order to estimate pollen fertility rates. at least 100 pollen grains of each species were measured to determine the typical pollen size range. pollen grains were stained using müntzing solution (glycerin-acetic carmin 1:1) (sharma & sharma, 1965). well-filled pollen grains with uniformly stained cytoplasm were scored as apparently fertile/viable while the shrivelled/flaccid ones with unstained or poorly stained cytoplasm were counted as apparently sterile/unviable. at least 1000 pollen grains were analyzed for each taxon. photomicrographs were taken using a nikon eclipse e-200 microscope equipped with a moticam 1000 digital camera (1.3 mp). the graphics were designed with the software coreldraw x3. results analysis of meiosis: buddleja iresinoides: generally the meiosis at prometaphase i started totally normal (97%) with the presence of 28 bivalents at diakinesis (fig. 2a and b). cytomixis was a common phenomenon in different stages of meiosis. about 2% of the pmcs of telophase i (ti) showed simple cytomictic channels indicating transfer of chromatin and cytoplasmic material among proximate pmcs (fig. 2c), simple cytomictic channels connecting two or more cells were observed in 25% of mii (fig. 2d). furthermore, at tii cytomixis consisting of 1-2 channels between two cells were found in 45% of pmcs (fig. 2e). different kinds of irregularities were observed (table 1). at diakinesis, 7% of pmcs were aneuploid, and showed 20-21 bivalents. at metaphase i (mi), irregularities such as out of plate bivalents were observed in 9% of the pmcs (fig. 2f). in addition, 8% of the pmcs at ti stage were found to show anaphase bridges with lagging chromatin between two nuclei (fig. 2g). at mii and aii, respectively 6% and 4% of pmcs exhibited chromatin stickiness between contiguous nuclei. (figs. 2h-i). at the end of meiosis, abnormal sporads such as dyads and triads were present. (fig. 2j). the mean diameter for b. iresinoides pollen, as determined by light microscopy, was 13.3 μm (range of 12.9 to 13.6 μm) (table 1). pollen viability rates in b. iresinoides was 89 % (fig. 4a). castilleja arvensis: chromosome numbers in pmcs were not constant. the diakinesis showed 78% regular pmcs with a gametophytic number of n = 12 (fig. 3a). cytomixis was revealed to be a very frequent phenomenon during pachytene, and 92% of the pmcs were con44 a.r. andrada et al. nected by 1-5 cytomictic channels linking two or more adjacent meiocytes (fig. 3b). to a great extent, these channels were filled by chromatin strands indicaticating material transfer from one pmc to another. the donor cell sometimes transferred almost the whole of its chromosome complement to a recipient meiocyte leaving only a chromosome-like heteropycnotic body beside the nucleolus; the recipient meiocytes had bigger agglomerations of chromatin material (fig. 3c). at diakinesis, 22% meiocytes showed 1-5 cytomictic channels (fig. 3d). in 8.5% of pmcs, 1 or 2 cytomictic channels were found between the neighbour tetrads at the end of second division (fig. 3e). irregularities observed in this species occurred in different stages (table 1). during diakinesis there were present hyperploid pmcs with up to n = 20 (fig. 3d). during this stage, small-sized meiocytes with only a small nucleus were found. these small sized cells were covered by thick callose walls giving them an aspect of monads (fig. 3f). subsequent stages of first meiotic division (mi, ai and ti) were not observed in the preserved material. in mii, up to 7% of meiocytes were found to possess the hyperploid chromosome number of 14 at one pole (fig. 3g). in 5% pmcs at metaphase ii, it was found that chromosomes do not align on the metaphase plate and tend to lie towards the periphery of the cell wall (fig. 3h). dyads were also observed in 3% figure 2. meiosis in buddleja iresinoides. a) diakinesis with n = 28, b) graphic representation of figure a; c) cytomictic channel in ti connecting 2 cells; d) mii with cytomictic channels connecting 3 cells; e) cytomixis between tetrads; f) mi with two chromosomes away from the equatorial plate; g) ti showing anaphase bridges with lagging chromatin; h) mii with chromatin stickiness between two equatorial plates; i) aii with chromatin stickiness connecting 2 neighbour poles; j) dyad and triad. scale = 10 μm. figure 3. meiosis in castilleja arvensis. a) diakinesis with 12 bivalents; b) cells with multiple cytomictic channels in pachytene; c) donor pmc transferring almost all the chromatin to a neighbor pmc; d) two cells in diakinesis connected by 3 cytomictic channels; e) cytomictic channels between tetrads; f) small-sized meiocytes with a small nucleus during the division i; g) mii showing a plate with 14 chromosomes; h) mii showing chromosomes disconnected from equatorial plate; i) dyad; j) small-sized meiocytes with a small nucleus at the end of the division ii. scale = 10 μm. 45meiotic irregularities associated to cytomixis in buddleja iresinoides and castilleja arvensis cases (fig. 3i). interestingly, 2% of pmcs were of small sizes and small nuclei (fig. 3j). pollen size of castilleja arvensis was observed to range from 20.1 μm to 20.8 μm (the mean diameter was 20.5 μm) (table 1). pollen viability rates in castilleja arvensis was 95%, (fig. 4b). discussion our results are not in agreement with the base number x = 19 previously reported for buddleja (norman, 2000; tallent-halsell & watt, 2009) and the chromosome number n = 28 found for b. iresinoides is atypical in the genus. however, gadella (1980) suggested that x = 19 may have been derived from ancestral hybridization between two basic stocks with x = 12 and 7 (norman, 2000; oxelman et al., 2004). our results suggested that b. iresinoides could be an octoploid with a putative basic number x = 7 or its chromosome number, n = 28 may have derived through secondary aneuploidy from a diploid parent having n = 36. another hypothesis is that the unusual chromosome number n = 28 (2n = 56) in this population may have derived by fusion of an unreduced gamete n = 36 from a putative parent and another normal gamete n = 19 (total 2n = 55) followed by a chromosome gain (e.g. through cytomixis) to reach 2n = 56. similarly, the rest of irregular gametes found n = 20-21 could have increased their chromosome number by cytomixis; this is after normal gametes n = 19 “acted” like recipient cells increasing their chromosome complement in 1 or 2 additional chromosomes. the chromosome number of x = 12 had been suggested for the genus castilleja (heckard, 1968; heckard & chuang, 1977; chuang & heckard, 1982; tank & olmstead, 2008; tank et al., 2009) and c. arvensis showed tetraploid number of chromosome of 2n = 24. the phenomenon of cytomixis as well as dyads and chromosomes that didn’t attach to the equatorial plate were observed in both analyzed species. these latter kinds of meiotic irregularities could be caused by the cytomixis (kumar, 2010; kumar et al., 2010; kumar et al., 2013). the origin of cytomixis is still not clear and different opinions exist with respect to its causes and permanence during meiosis. oliveira-pierre & sousa (2011) concluded that the cytomixis could have multiple origins. however, these authors have cited relatively recent investigations which show that cytomictic channels always structurally occur in the same way: 1) in the beginning, plasmodesms loss their connections with smooth endoplasmic reticulum (desmotubules) and then starts the intrusion of cytoplasmic material into the plasmodesms that inncrease their size forming cytomictic channels (wei-cheng et al.,1988; oliveira-pierre & sousa, op. cit.); 2) during the cytomixis process both cellulase and pectinase enzymes are presented as playing a role in digesting the cell walls of pmcs involved in this phenomenon (wang et al.,1998); 3) in cells of germinal tissues of anthers during callose depositions that should block up the plamodesms occur disturbances, the connector channels increase their size and in this way facilitate the formation of cytomictic channels (falistocco et al., 1995; sheidai & fadaei, 2005; sheidai et al. 2006; sidorchuk et al., 2007). some authors argued that cytomixis is a process that occur in the early stages of meiotic division (at prophase i generally) supporting the idea that after passage of chromatin from one pmc to another, cells which acquired or donated chromatinic material tend to degenerate. this kind of result was obtained by koul (1990) through investigations carried out in alopecurus rundinaceus poir. on the other hand, there are authors that state that cytomixis could develop in all stages of meiosis (basavaiah & murthy, 1987 in urochloa panicoides p. beauv.; bellucci et al., 2003 in medicago sativa l.; malallah & attia, 2003 in diplotaxis harra boiss.; singhal & kumar, 2008 in meconopsis aculeata royle; singhal et al., 2009 in anemone rivularis buch.-ham. ex dc.). authors have different positions regarding the transfer of cell components through cytomixis. during cell division, heslop-harrison (1966) suggested that intercellular connections occur to foment the synchrony between meiocytes allowing homogeneity of organelles and cytoplasmic components among them. however, guanq-qin & gou-chang (2004) refused this hypothesis because they considered it inconsistent, being that in plants the tapetal cells are responsible for providing nutrients to the pmcs (not the passage from one pmc to another), figure 4. a-j: fertile/stained and sterile/unstained pollen grains in; a) buddleja iresinoides and, b) castilleja arvensis. scale = 10 μm. 46 a.r. andrada et al. between the last cells never has hitherto been observed cytoplasmic connections together the meiocytes. these authors attributed to cytomixis a more general function such as the mechanism that allows share regulatory and structural genetic products (e.g., mrnas, oragnelles, etc.) between connected cells, favoring thus a necessary homogenization of cytoplasmatic restructural events occurred during prophase i which could cause heterogeneity in the meiocytes and consequently could lead to loss of their quality (generating more abnormal but less normal gametes). during the pachytene stage, we have never observed cytomixis in pmcs of b. iresinoides but these started from telophase i. our observations are not in agreement with koul’s hypothesis (1990) according to which the cytomixis occurs in first stages of meiotic division. however, in c. arvensis cytomictic channels were present in prophase i in most of the meiocytes (92% of pmcs) suggesting that absence or presence of cytomixis does not essentially depend on the stage of cell division. participation of other factors that may play some role in cytomixis is still not clear. it is evident that the cytomixis can occur in different stages of meiosis from prophase i to tetrad formation as revealed in our results. our findings agree with guanq-qin & gou-chang (2004) who reported that cytomictic channels always are formed among cells at the same division stage. nevertheless, the above cited authors mentioned that cytomixis promote homogeneity between meiocytes, observations that contradict our results because we observed in c. arvensis pachytene the transfer of almost all the chromatin from donor cell to recipient cell and the presence of hypoploid and hyperploid pmcs. altogether, these irregularities (heterogeneity), probably produced by cytomixis, made up more than 30% of the observed cells. according to morphological characteristics, the small-sized meiocytes with a little nucleus observed in both the division i and the division ii correspond to apoptotic cells similar to the ones cited by different authors in both plants and animals (fuzinatto et al., 2007; kravets, 2013; andrada et al., 2016). the abnormal cells could be degraded by means of apoptosis, thus explaining the high percentage of pollen grains viability observed in c. arvensis. in this species this phenomenon occured two times: after pachytene at diakinesis and between the tetrads at the end of tii (both in division i and division ii after or during the two stages with major percentage of cytomixis). although this kind of abnormal cell was not observed in b. iresinoides it is possible that some similar mechanism could occur and the abnormal cells would be eliminated. by removing the abnormal pollen grains these plants ensure that gametes transferred being viable. in b. iresinoides transfer of chromatin from a donor cell to a recipient cell is not limited only to neighbouring meiocytes but also occurs between pmcs at same stage of division. according to ortíz et al. (2006) and andrada & páez (2014), this kind of connections could disturb the normal development of the phragmoplast during cytokinesis causing irregularities which could finish as unbalanced gametes and give rise to dyads as it was observed in b. iresinoides. although in c. arvensis connections among meiocytes from the same pmc was not observed, these were present in dyads once meiosis had been completed. in both species chromosomes which did not align to metaphase plate and stood near cell wall were found but in different stages in the two species examined. in b. iresinoides they occured during mi, whereas in c. arvensis this kind of irregularite was observed at mii even at the hyperploid cells. these chromosomes could occur due to transfer from a pmc to other neighbouring pmc through cytomixis channels during the early metaphase. in buddleja iresinoides, during the división ii the 57% of pmcs showed irregularities (stickness, cytomixis and bad debelop phragmoplast that finished in dyads and triads). this percentage is far to the 11% of irregular and inviable pollen grains revealed by münting’s stain, however, before start the division ii the regulatory mechanism that controls the normal course of the meiosis or the method to eliminate irregular pmcs still remains unknown. in addition, in b. iresinoides it is evident that the cytomixis (reaching the maximum value of 45% at tii) does not have a large impact on the development of non-viable pollen grains. gernand et al. analyzed the mechanisms underlying selective elimination of the paternal chromosomes during the development of wheat × pearl millet hybrid embryos and found that chromosome elimination frequently took place during meiosis. these cytological observations showed that parental genomes were spatially separated within the hybrid nucleus, and the pearl millet chromatin destined for elimination occupied peripheral positions. a similar phenomenon was found in this study; chromosomes were spatially separated within the pmcs where the chromatin occupied a predominantly peripheral position at metaphase i from b. iresinoides and at pachitene and mii from c. arvensis. in addition, given that the b. iresinoides and b. stachyoides cham. & schltdl. (chromosome number unknown) grow together, it is likely that the taxon studied contain chromosomes from b. stachyoides. this would have given rise to populations of hybrids with this atypical gametophytic number (n = 28). 47meiotic irregularities associated to cytomixis in buddleja iresinoides and castilleja arvensis in castilleja arvensis, among the frequent abnormalities (as stickness, cytomixis and bad debelop phragmoplast that finished in dyads and triads) the cytomictic channels at pachitene (table 1 and figure 3b) were salient. however, the cytomixis does not seem to be the main cause of pollen inviability. in this species, the apoptosis which was observed in both prophase and tii where occur the most of irregularities could regulate the number of abnormal pmcs during the meiosis and the non viable pollen grains would be obtained when simply some pmcs with different type of irregularities add together up to reach 5%. buddleja iresinoides and castilleja arvensis have high percentages of pollen grains stained (viables) close to 90% and small ranges of variation of size close to 0,7 µm. this fact suggest that polyploid cells (produced through of dyads and triads) which should develop giant pollen grains or “jumbo grains” were eliminated during the last steps of microsporogenesis. on the other hand, the limit size values of stained pollen grains may mask hyperploids and hypoploid cells as apparently normal and fertile pollen grains. future studies related to germinabaility of pollen grains could clarify the strange behavior of these species that show high percentages of irregularities during the meiosis and low production of sterile pollen grains. conclusions castilleja arvensis is a diploid taxon with n = 12 while the unusual number n = 28 from b. iresinoides suggest that the basic chromosome number for this genus could be less than x = 19. however, that this atypical number could have originated through passage of additional chromosomes from a donor cell to recipient cell by cytomixis or through hybridization process is possible too. in the present study, we have found that cytomixis is a process which is not stage specific and its frequency may vary from species to species which is evident from our results in b. iresinoides where maximum percentage of cytomixis occur during tii, whereas in c. arvensis it is more frequent in pachytene. in addition, this process could cause numerous irregularities that would result in (at the end of meiosis) genetically unbalanced gametes. furthermore depending upon the severity of meiotic irregularities it may hamper the reproductive success of species. cytomixis has been reported here for the first time from both buddlejaceae and orobanchaceae families. acknowledgements this work was made possible by the financial support of fundacaión miguel lillo (proyecto b-0013/1). we are grateful to the director of the genetic institute of miguel lillo foundation, g. e. ruiz de bigliardo for her critical reading of the manuscript. the authors are also very grateful to the director of the botanical survey of india, kolkata for necessary internet and library facilities. references andrada ar, lozzia me, cristóbal me (2009) cytological studies in piper tucumanum c. dc. and piper hieronymi c. dc. lilloa 46: 3–9. 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international journal of cytology, cytosystematics and cytogenetics 74(2): 111-119, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-775 caryologia international journal of cytology, cytosystematics and cytogenetics citation: patcharaporn chaiyasan, sumalee phimphan, teamjun sarasan, sippakorn juntaree, alongklod tanomtong, sitthisak pinmongkhonkul, weerayuth supiwong (2021) first report on nucleolar organizer regions (nors) polymorphism and constitutive heterochromatin of moonlight gourami, trichopodus microlepis (perciformes, osphronemidae). caryologia 74(2): 111-119. doi: 10.36253/caryologia-775 received: april 02, 2020 accepted: july 27, 2021 published: october 08, 2021 copyright: © 2021 patcharaporn chaiyasan, sumalee phimphan, teamjun sarasan, sippakorn juntaree, alongklod tanomtong, sitthisak pinmongkhonkul, weerayuth supiwong. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. first report on nucleolar organizer regions (nors) polymorphism and constitutive heterochromatin of moonlight gourami, trichopodus microlepis (perciformes, osphronemidae) patcharaporn chaiyasan1, sumalee phimphan2, teamjun sarasan1, sippakorn juntaree3, alongklod tanomtong1, sitthisak pinmongkhonkul4, weerayuth supiwong3,* 1 biology program, faculty of science, khon kaen university, muang, khon kaen 40002, thailand 2 biology program, faculty of science and technology, phetchabun rajabhat university, phetchabun 67000, thailand 3 faculty of interdisciplinary studies, khon kaen university, nong khai campus, muang, nong khai 43000, thailand 4 department of biology, school of science, university of phayao, muang, phayao 56000, thailand *corresponding author. e-mail: supiwong@hotmail.com abstract. nucleolar organizer regions (nors) polymorphism, constitutive heterochromatin and chromosomal analysis of moonlight gourami, trichopodus microlepis in thailand were firstly reported. specimens were collected from the chao phraya and mekong basins, thailand. the mitotic chromosomes were directly prepared from kidney tissues of ten males and ten females. conventional staining, ag-nor banding and cbanding techniques were applied to stain the chromosomes. the results shown that the diploid chromosome number of t. microlepis was 2n=46 and the fundamental number (nf) was 46 in both males and females. the karyotype consisted of 46 telocentric chromosomes classifying as 14 large and 32 medium chromosomes. no heteromorphic sex chromosome was observed in t. microlepis. the results also showed that the interstitial nucleolar organizer regions (nors) were clearly observed at the long arm of the chromosome pair 7. this is the first report on nors polymorphism in t. microlepis that a heteromorphic nor type in one female had a single nor-bearing chromosome of the chromosome pair 7, whereas 10 males and nine females had two nor-bearing chromosomes of the chromosome pair 7 with a homomorphic nor type. constitutive heterochromatin was located at all centromeres of all chromosome pairs. the karyotype formula of t. microlepis is 2n (46) = lt14 + mt32. keywords: moonlight gourami, trichopodus microlepis, karyotype, nucleolar organizer region, constitutive heterochromatin, chromosome. 112 patcharaporn chaiyasan et al. introduction trichopodu s which was formerly included in trichogaster (peapke, 2009; töpfer and schlindler, 2009) is a genus of tropical freshwater labyrinth fish of the gourami or family osphronemidae and subfamily trichogastrinae found in southeast asia. gouramis of the trichopodus genus are closely related to those of trichogaster (formerly colisa), species of both genera have long and thread-like pelvic fins (known as “feelers” in the aquarium trade) used to sense the environment. however, trichopodus species have shorter dorsal fin base and, when sexually mature, are much larger (peapke, 2009; töpfer and schlindler, 2009). there are currently six recognized species in this genus including trichopodus cantoris, pearl gourami (t. leerii), moonlight gourami (t. microlepis), snakeskin gourami (t. pectoralis), t. poptae and three spot gourami (t. trichopterus) (peapke, 2009). the moonlight gourami is a labyrinth fish native to the mekong river in cambodia, vietnam and the chao phraya basin, thailand (vidthayanon 2005). these fish are silvery coloured with a slightly greenish hue similar to the soft glow of moonlight (fig. 1). the moonlight gourami’s concavely sloped head distinguishes it from other gourami varieties. this peaceful, attractive species is a popular aquarium fish. although the gourami fishes are importance for national economy of thailand, there were quite scarce of cytogenetics in these fishes especially banding analysis in fish chromosomes. the study on fish chromosomes is the basic knowledge which can be applied for the several fields such as classification, evolution, heredity, systematic (gold et al. 1990, ueda et al. 2001, barat et al. 2002, barat and sahoo 2007, supiwong et al. 20019), breeding, rapid production of inbred lines and cytotaxonomy (kirpichnikov 1981). furthermore, cytogenetic studies on fish have also been used as biological indicator to determine the ecological toxicology (klinkhardt 1993, promsid et al. 2015) and cytogenetic techniques have been widely applied to improve farmed stocks in many aquaculture species in the world (beardmore et al. 2001, desprez et al. 2003, pradeep et al. 2012). an important characteristic of nucleolar organizer regions (nors) in fish is related to that it has interand intra-species polymorphism. nors characters can be a cytogenetic marker for cytotaxonomic studies and also have been used for studying of phylogenetic relationships among the cyprinid fishes (amemyia and gold 1988, galetti jr 1998, almeidatoledo et al. 2000). constitutive heterochromatin distributions on the chromosomes were widely studied in some fish groups (brinn et al. 2004, vicari et al. 2006, mesquita et al. 2008, takai 2012). generally, most constitutive heterochromatins locate at centromeric/pericentromeric regions of the chromosomes. some cases, these heterochromatins can be revealed at interstitial regions in some pomacentrid fishes to support that the chromosomal evolution in this family is related to the chromosome fusion (takai 2012). moreover, constitutive heterochromatin is also highly accumulated on the w chromosome in parodon hilarii (parodontidae) (moreira-filho et al. 1993), characidium fish (crenuchidae) (vicari et al. 2008) and lignobrycon myersi (triportheidae) (rodrigues et al. 2016). as mention before, chromosomal analysis is very important and clearly exhibits the benefits. moreover, the constitutive heterochromatin and polymorphism of nors characteristics in the t. microlepis were not studied. thus, the present study is the first report in t. microlepis from thailand using ag-nor banding and c-banding techniques. materials and methods sample collection, chromosome preparation and chromosome staining ten male and ten female specimens of t. microlepis (fig. 1) were obtained from the chao phraya river, sing buri province, the central part of thailand and the mekong basin, nong khai province, northeast of thailand. chromosomes were directly prepared in vivo as follows by supiwong et al. (2013, 2017). conventional staining was performed using 20% giemsa’s solution for 30 min (rooney 2001). ag-nor banding was carried out following by howell and black (1980) and c-banding was performed following from the method of sumner et al. (1972). figure 1. general characteristic of moonlight gourami, trichopodus microlepis (perciformes, osphronemidae). 113first report on nucleolar organizer regions polymorphism and constitutive heterochromatin of trichopodus microlepis chromosomal checks, karyotyping and idiograming chromosome counting was carried out on mitotic metaphase cells under light microscope for 30 cells per specimen to determine the diploid number (2n). twenty clearly observable and well-spread metaphase cells from each male and female were selected and photographed. the short arm length (ls) and the long arm length (ll) of each chromosome were measured to calculate the total length of the chromosome for 20 well-spread metaphase cells. the chromosome types were classified from method of turpin and lejeune (1965) as metacentric, submetacentric, acrocentric and telocentric chromosomes. the karyotyping and idiograming methods were according to turpin and lejeune (1965) and chaiyasut (1989). results and discussion diploid chromosome number, fundamental number and karyotype the diploid chromosome number (2n) of t. microlepis was found as 46 (figs. 2 and 3). this result is coincident with previous reports by koref-santibanez and paepke (1994) and seetapan and khamma-ai (2007). it is also the same 2n as in the other trichopodus spp. (abe 1975, koref-santibanez and paepke 1994, donsakul and magtoon 1988, seetapan and khamma-ai 2007, magtoon et al. 2007, supiwong et al. 2010), trichogaster chuna (koref-santibanez and paepke 1994) and trichogaster lalius (abe 1975, rishi 1976, koref-santibanez and paepke 1994). these species have the diploid chromosome number of 2n=46, which is an apparent modal diploid number of the trichopodus. accordingly, it can be concluded that chromosome number in this genus is conserved. however, it differs from the most species of the genus trichogaster (t. labiosa, t. fasciata, t. labiosus, t. sumatranus) which had 2n=48 (kaur and srivastava 1965, calton and denton 1974, abe 1975, rishi 1975, manna and prasad 1977, tripathy and das 1981, korefsantibanez and paepke 1994, rishi et al. 1994, sobita and bhagirath 2007, kushwaha et al. 2008) (table 1). the fundamental number (nf) of t. microlepis was 46 in both males and females. the karyotype consisted of 46 telocentric chromosomes (all as mono-arm chromosomes). these results are agreeable with the previous reports of both t. microlepis and all trichopodus species (abe 1975, donsakul and magtoon 1988, koref-santibanez and paepke 1994, magtoon et al. 2007, seetapan and khamma-ai 2007, supiwong et al. 2010). howevfigure 2. karyotypes of male (a) and female (b) of trichopodus microlepis, 2n=46 by conventional staining. scale bars = 5 µm. figure 3. karyotypes of trichopodus microlepis, 2n=46 by ag-nor banding (a) and c-banding techniques (b). the chromosome pair 7 show ag-nor and heteromorphic ag-nor (inserted box). scale bars = 5 µm. 114 patcharaporn chaiyasan et al. er, they are different from all of the genus trichogaster (kaur and srivastava 1965, calton and denton 1974, abe 1975, rishi 1975, manna and prasad 1977, tripathy and das 1981, koref-santibanez and paepke 1994, rishi et al. 1994, sobita and bhagirath 2007, kushwaha et al. 2008). the nfs of the genus trichogaster range from 48 to 86 and karyotypes composed of both monoand bi-arm chromosomes. nirchio et al. (2002) proposed that species with high nf is advanced state or apomorphic character whereas one with low nf is a primitive state or plesiomorphic character. t. microlepis including all species of the genus trichopodus have all monoarm chromosomes in karyotype whereas most species of the genus trichogaster display both monoarm and bi-arm chromosomes (table 1). thus, the trichoprodus seems to be more primitive karyotype than that in the trichogaster. the t. microlepis karyotype consisted of 14 large telocentric and 32 medium telocentric chromosomes (table 2). the karyotype formula for this species is 2n (46) = lt14 + mt32. there is no evidence of differentiated sex chromosomes in this species which accord to all species of this genus (abe 1975, donsakul and magtoon 1988, koref-santibanez and paepke 1994, magtoon et al. 2007, seetapan and khamma-ai 2007, supiwong et al. 2010). similar to several gourami fishes, no cytologically distinguishable sex chromosome was observed. chromosome markers from ag-nor banding and c-banding present study was accomplished by using ag-nor staining and c-banding in t. microlepis. the nors are used as makers to detect species specific character and indicate intraand inter species chromosomal polymorphism in many groups of fishes (ráb et al. 2008). the ag-nor positions were shown on the long arm near the centromere of the telocentric chromosome pair 7 (subcentromeric nor) in 10 male and nine female fish (fig. table 1. karyotype characteristics of some species in the subfamily trichogastrinae. species 2n nf karyotype nor reference trichogaster chuna 46 66 20m+26st/a – koref-santibanez and paepke (1994) t. labiosa 48 66 12m+6sm+12st+18a/t – manna and prasad (1977) 48 68 20m+10st+18a/t – koref-santibanez and paepke (1994) t. lalius 46 70 24m/sm+22a/t – abe (1975) 46 – 26m+1sm/st+19a/t – rishi (1976) 46 66 20m+8st+18a/t – koref-santibanez and paepke (1994) t. fasciata 48 48 48a/t – kaur and srivastava (1965) 48 74 14m+12sm+22a/t – rishi (1975) 48 78 8m+20sm+12st+8a/t – manna and prasad (1977) 48 78 18m+12sm+18a/t – tripathy and das (1981) 48 68 20m+12st+16a/t – koref-santibanez and paepke (1994) 48 80–81 16m+16sm+15a/t(16a/t) – rishi et al. (1994) 48 83 15m+16sm+4st+13a/t 6 sobita and bhagirath (2007) 48 86 16m+16sm+6st+10a/t 2 kushwaha et al. (2008) t. sumatranus 48 48 48st/a – calton and denton (1974) trichopodus leeri 46 46 46a/t – abe (1975) koref-santibanez and paepke (1994) 46 46 46a/t – seetapan and khamma-ai (2007) t. microlepis 46 46 46a/t – koref-santibanez and paepke (1994) 46 46 46a/t – seetapan and khamma-ai (2007) 46 46 46t 2 present study t. pectoralis 46 46 46a/t – koref-santibanez and paepke (1994) 46 46 46a/t – donsakul. and magtoon (1988) 46 46 46a/t – seetapan and khamma-ai (2007) t. trichopterus 46 46 46a/t – abe (1975), koref-santibanez and paepke (1994) 46 46 46a/t – magtoon et al. (2007) 46 46 46t/t 2 supiwong et al. (2010) remarks: 2n = diploid number, nf = the fundamental number, nor = nucleolar organizer region, m = metacentric, sm = submetacentric, st = subtelocentric, a = acrocentric, t = telocentric chromosomes and – = not available. 115first report on nucleolar organizer regions polymorphism and constitutive heterochromatin of trichopodus microlepis 3a). the single pair of nor is the same as in t. trichopterus (supiwong et al. 2010) and t. fasciata reported by kushwaha et al. (2008) but there is difference in t. fasciata which had three pairs of nors (sobita and bhagirath 2007) and betta splendens which had two pairs of nors (furgala-selezniow et al. 2008). gold and amemiya (1986) suggested that the occurrence of multiple nors in fishes was considered to be apomorphic or advance condition whereas single pair of nors was considered to be plesiomorphic or a primitive condition. considering for nor loci between t. microlepis and t. trichopterus, although both species had the single nor pair, the nor positions are difference. the present results revealed that t. microlepis had interstitial nors on the chromosome pair 7 whereas t. trichopterus had telomeric nors (region adjacent to the telomere) on the chromosome pair 2 (supiwong et al. 2010). therefore, the nor-bearing chromosome markers can be used as a tool for classification in this fish group. in addition, intraspecific nor heteromorphism between the homologous chromosomes of pair 7 was also displayed in one female specimen (fig. 3a, inserted box). this phenomenon is common event found previously in several fishes in thailand such as puntioplites proctozysron (supiwong et al. 2012), lutjanus johnii (phimphan et al. 2013), pterapogon kauderni (kasiroek et al. 2017) and hemibagrus wyckii (supiwong et al. 2017). constitutive heterochromatic blocks were observed at centromeric and pericentromeric regions of all chromosomes and with no clear interstitial and telomeric positive c-bands (fig. 3b). it indicates that the chromosomes of t. microlepis are conserved and non-related to chromosomal fusion or an increase in heterochromatin during evolution. present result is similar to some species in another family of the order perciformes such as geophagus brasiliensis and c. facetum in the cichlidae family (vicari et al. 2006), plectroglyphidodon lacrymatus, chrysiptera leucopoma, c. rex and neoglyphidodon melas in the pomacentridae family (takai 2012). however, there are several species which presented the complex types of positive c-bands. symphysodon haraldi, s. aequifasciatus and s. discus (cichlidae) had heterochromatic blocks on the pericentromeric regions of all chromosomes and the proximal regions of both arms of some table 2 mean length of short arm chromosome (ls), long arm chromosome (ll), total arm chromosome (lt), relative length (rl), centromeric index (ci) and standard deviation (sd) of rl, ci from 20 metaphases (both males and females) of the moonlight gourami (trichopodus microlepis) in thailand, 2n=46 chromosome pair ls ll lt rl±sd ci±sd type size 1 0.000 0.755 0.755 0.0306±0.0026 1.000±0.000 telocentric l 2 0.000 0.682 0.682 0.0276±0.0015 1.000±0.000 telocentric l 3 0.000 0.647 0.647 0.0261±0.0011 1.000±0.000 telocentric l 4 0.000 0.621 0.621 0.0251±0.0008 1.000±0.000 telocentric l 5 0.000 0.603 0.603 0.0243±0.0007 1.000±0.000 telocentric l 6 0.000 0.589 0.589 0.0237±0.0006 1.000±0.000 telocentric l 7* 0.000 0.578 0.578 0.0232±0.0005 1.000±0.000 telocentric l 8 0.000 0.567 0.567 0.0228±0.0004 1.000±0.000 telocentric m 9 0.000 0.559 0.559 0.0225±0.0004 1.000±0.000 telocentric m 10 0.000 0.549 0.549 0.0221±0.0004 1.000±0.000 telocentric m 11 0.000 0.538 0.538 0.0217±0.0004 1.000±0.000 telocentric m 12 0.000 0.529 0.529 0.0213±0.0004 1.000±0.000 telocentric m 13 0.000 0.521 0.521 0.0209±0.0004 1.000±0.000 telocentric m 14 0.000 0.513 0.513 0.0206±0.0004 1.000±0.000 telocentric m 15 0.000 0.506 0.506 0.0203±0.0004 1.000±0.000 telocentric m 16 0.000 0.498 0.498 0.0201±0.0004 1.000±0.000 telocentric m 17 0.000 0.491 0.491 0.0197±0.0005 1.000±0.000 telocentric m 18 0.000 0.479 0.479 0.0193±0.0006 1.000±0.000 telocentric m 19 0.000 0.470 0.470 0.0189±0.0006 1.000±0.000 telocentric m 20 0.000 0.457 0.457 0.0184±0.0005 1.000±0.000 telocentric m 21 0.000 0.442 0.442 0.0178±0.0007 1.000±0.000 telocentric m 22 0.000 0.425 0.425 0.0170±0.0009 1.000±0.000 telocentric m 23 0.000 0.397 0.397 0.0159±0.0015 1.000±0.000 telocentric m remarks: * = nor-bearing chromosome, l=large, and m=medium. 116 patcharaporn chaiyasan et al. chromosomes (mesquita et al. 2008), while n. nigroris (pomacentridae) exhibited the distribution of positive c-bands in most centromeric regions and including many terminal and interstitial regions (takai 2012). the idiogram shows a continuous length gradation of chromosomes. approximately two-fold of the size differences between the largest and smallest chromosomes were revealed. the marker chromosomes are the chromosome pair 1, which is the largest telocentric and the chromosome pair 23 is the smallest telocentric. the data of the chromosome measurement on mitotic metaphase cells (from all specimens) are shown in table 2. idiograms by conventional staining and cbanding are shown in fig. 4. in conclusion, nor phenotype and constitutive heterochromatin patterns on the chromosomes are specific to species in the genus trichopodus. for more information about the chromosomal diversity and chromosomal evolution 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(in thai) caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 1 2021 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 72(2): 29-35, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-257 citation: ö.s. aslantürk (2019) cytogenetic effects of fulvic acid on allium cepa l. root tip meristem cells. caryologia 72(2): 29-35. doi: 10.13128/ cayologia-257 published: december 5, 2019 copyright: © 2019 ö.s. aslantürk. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. cytogenetic effects of fulvic acid on allium cepa l. root tip meristem cells özlem sultan aslantürk adnan menderes university, faculty of art and science, department of biology, 09010 aydın, turkey e-mail: osaslanturk@adu.edu.tr abstract. fulvic acid is a class of compounds of humic substances and is found in a significant proportion of the substances in the environment. it has been used for many years in industry, agriculture, and complementary medicine. in this study, cytogenetic effects of fulvic acid purified from muğla milas hüsamlar leonardite (turkey) on allium cepa root tip meristem cells were investigated using the allium test. for this purpose, 10 mg/ml stock solution of fulvic acid was prepared by dissolving in citric acid and it was diluted with distilled water to 10, 20, 40, 80 and 100 µg/ml concentrations. onion bulbs were exposed to these concentrations of the fulvic acid for macroscopic and microscopic analysis. tap water was used as a negative control, 40 µg/ml citric acid was used as solvent control (fulvic acid solvent), and 0.02m ethyl methane sulfonate (ems) (a mutagenic, teratogenic, and possibly carcinogenic organic compound) was used as a positive control. there has been statistically significant stimulation of root growth depending on fulvic acid concentration in comparison with the control groups (p<0.05). furthermore, in fulvic acid treatment groups, breaks, stickiness and polar deviations appeared at very low rates, and total chromosome aberration ratios were insignificant compared to the control groups. these results suggest that fulvic acid does not have cytotoxic and genotoxic effects on a. cepa. keywords. allium test, chromosome aberrations, fulvic acid, mitotic index. 1. introduction fulvic acid is a class of compounds of humic substances, and it is a mixture of polyphenolic compounds formed through the degradation of organic substances such as plants, microbes and animals by chemical and biological processes (motojima et al. 2011). it is a type of humic acid. compared to other humic acid types, the fulvic acid is soluble in both acid and alkaline solutions, is lower in molecular weight, and has a greater biological activity (stevenson, 1994; bai et al. 2013; yong 2001; zhang et al. 2011). piccolo (2002) redefined fulvic acid as associations of small hydrophilic molecules in which there are enough acid functional groups to keep the fulvic clusters dispersed in solution at any ph. because, while humic acids precipitate when the ph is adjusted to 1-2, fulvic acids remain in solution after the alkaline extracts are acidified (canellas et al. 2015). 30 özlem sultan aslantürk recently, it has been reported that fulvic acid has nutraceutical, neuroprotective (cornejo et al. 2011; guzmán-martinez et al. 2013), antimicrobial, antioxidant, and anti-inflammatory properties (van rensburg et al. 2001; yamada et al. 2007; sherry et al. 2013). fulvic acid has also been used as a medicine by people in china, mexico, india, south america and russia for centuries. fulvic acid has a large capacity to retain transition metals, forming metalorganic complexes, which cause these metals to be more or less available for plants which include them into the food chain. the food industry also uses it as an ion exchanger, because it holds heavy metals very well (pena-mendez et al. 2005). over these last decades, more than 200 short-term bioassay utilizing plants, microorganisms, and insects have been developed and used to evaluate the environmental risks (marcato-romain et al. 2009). plant assays are highly sensitive, easy to use in an experiment, inexpensive, and good predictors of genotoxicity and carcinogenicity (ennever et al. 1988). the allium test has been used by many researchers as a bioindicator of environmental pollution (bagatini et al. 2009; leme and marin-morales 2009) and genotoxicity of various agents (aşkın çelik and aslantürk 2007, 2009, 2010) for a long time. with this test, mutagenic effects of substances may be analyzed by monitoring macroscopic parameters, like the appearance and growth of the roots or by genotoxic parameters, like type and frequency of chromosome aberrations, and abnormal cell division. another advantage of this test is the presence of an oxidase enzyme system, which is essential for promutagen evaluations (fiskesjö, 1985; nielsen and rank,1994 ). the allium test is important, since it is an excellent model in vivo, where roots grow in direct contact with the test substance enabling possible damage to dna of eukaryotes to be predicted. therefore, results from this test can be extrapolated for all animal and plant biodiversity (tedesco and laughinghouse iv 2012). although fulvic acid is found in a significant proportion of the substances in the environment, and has been used for many years in industry, agriculture and complementary medicine, there is still minimal scientific evidence of its biological properties. in this study, cytogenetic effects of fulvic acid on allium cepa root tip meristem cells were investigated using the allium test. 2. materials and methods 2.1. supply of fulvic acid fulvic acid purified from muğla milas hüsamlar leonardite (turkey) in chemical engineering laboratory of gazi university in ankara (turkey) was used in this research (sönmez 2011). this study was conducted between march and december 2017. 2.2. preparation of the fulvic acid solution the 10 mg/ml stock solution of fulvic acid was prepared by dissolving in citric acid, as it has a structure, which is soluble in weak acid. stock fulvic acid solution was diluted with distilled water to 10, 20, 40, 80 and 100 µg/ml concentrations. fresh solution was prepared just before the experiment. 2.3. allium test small bulbs (1.5–2.0 cm in diameter) of the common onion, a. cepa, (2n = 16) were purchased at a local supermarket in aydın, turkey. prior to initiating the test, the outer scales of the bulbs and the dry bottom plate were removed without damaging the root primordia. for each treatment, seven onion bulbs were placed on top of test tubes filled with tap water (ph 7.3) for 48 h. the test tubes were kept in an incubator at 22±1ºc. after 48 h, two unhealthy onions with the most poorly growing roots were removed and the other healthy onion bulbs in water were treated with 10, 20, 40, 80 and 100 µg/ml fulvic acid for 24 hours. 0,02m ethyl methane sulfonate was used as positive control for 3 h, 40 µg/ ml citric acid was used as solvent control, and tap water was used as negative control. citric acid is a  weak  organic acid  that has the chemical formula  c6h8o7. it occurs naturally in  citrus fruits. in  biochemistry, it is an intermediate product in the  citric acid cycle, which occurs in the  metabolism  of all  aerobic organisms (berovic and legisa, 2007). ethyl methanesulfonate  (ems) used as positive control in experiment is a mutagenic, teratogenic, and possibly carcinogenic  organic compound  and its chemical  formula c3h8so3. ems is often used in  genetics  as a mutagen. mutations induced by ems can then be studied in genetic screens or other assays (merck index, 1989). after the completion of treatment the roots were counted and their lengths were measured for each onion. to determine mean root length in a root bundle for each bulb, root lengths of experimental and control bulbs were measured with ruler at the end of treatment time. after then root tips were removed from the bulbs, fixed in 3:1 (v/v) ethanol:glacial acetic acid and stored overnight at 4ºc. the next day they were placed in 70% (v/v) aqueous alcohol and refrigerated until use. an average of five slides was made for each bulb using 31cytogenetic effects of fulvic acid on allium cepa l. root tip meristem cells five root tips which hydrolyzed in 1n hydrochloric acid (hcl) for 3 min, and microscope slides were prepared by squashing the stained root tips in 2% (w/v) acetic orcein. each slide was examined using olympus bx51 at a total magnification of 40×10. chromosomal aberrations were determined by scoring cells with bridges, fragments, sticky chromosomes, and polar deviations in 1000 cells per slide. also micronucleus formation was determined in 1000 cells per slide. 5000 cells scored in total for each bulb (fiskesjö 1993, 1997; pavlica et al. 2000). 2.4. statistical analysis statistical analyses were performed using the spss 20.0 software package program. data on physicochemical parameters, root length, root number, and mitotic index and chromosomal aberrations were compared using analysis of variance (one way anova) to confirm the variability of the data and validity of results. post-hoc test was used to describe the magnitude of variability. differences between corresponding controls and exposure treatments were considered statistically significant at p <0 .05. 3. results 3.1. morphological analysis the results of the morphological analysis (root number and root length) are presented in table 1. these results show that all tested concentrations of fulvic acid caused increase in the root growth, and average root number in comparison to negative control, positive control, and solvent control. the measured average root length is 2.26±1.07 cm in negative control, 1.40±0.40 cm in positive control, and 1.36 cm in solvent control. the average root length after 20 and 40 µg/ml fulvic acid treatment is found very high (4.10±0.41 and 4.22±0.69 cm, respectively) compared to controls (table 1). the number of roots also increased in fulvic acid treatment groups compared to control groups. the highest root number is found in group treated with 80 µg/ml fulvic acid (table 1). the root morphology in fulvic acid treated groups was thinner and more fragile compared to the negative control group. 3.2. cytogenetic analysis with the objective of investigating the possible mechanism involved in root growth stimulation, cytogenetic analysis was performed. fulvic acid was found to stimulate mitotic index. a statistically significant difference in the mitotic index of root meristems was found in negative, positive and solvent control. the increase in the mitotic index was found to be positively correlated with the increase in concentration of the fulvic acid (table 2). in the positive and solvent control groups, the mitotic index decreased significantly compared to the control group, and the mitotic index value approached zero in the solvent control group (table 2). cytogenetic alterations were investigated, and the results are described in table 2 and figure 1. table 2 presents the percentage of the aberrant cells in dividing cells. very few cells with polar deviation were observed in the negative control group. no chromosome aberration was observed except for polar deviation. in this group, total chromosome aberration was found very low (0.07%). the chromosome aberration rate in the positive control group was found to be significantly higher than the control group (36.09%). especially the anaphase bridge and stickiness have been observed to appear at a very high rate (p<0.05). in addition, breaks and polar deviations were observed in the positive control group. no chromosome aberration was observed in the solvent control group (citric acid), because there were only two divided cells in total and the mitotic index value was near zero in this group. the cell membrane and nucleus were deformed (fig. 1a). in the groups treated with fulvic acid, breaks, stickiness and polar deviations appeared at very low rates. total chromosome aberration percentages in these groups were insignificant compared to control and solvent control groups. the highest total chromosome aberration percentage in fulvic acid treated groups was 20 table 1. the average root numbers and root lengths in control and treatment groups after 24h treatment (analysis were carried by one way anova). concentrations average root number ± sd average root lengths (cm ± sd) negative control 19.2 ± 8.07 2.26 ± 1.07 solvent control 20.2 ± 8.87 1.36 ± 0.24 ems (positive control) 21.0 ± 7.81 1.40 ± 0.40 fa10 37.2 ± 4.43* 3.36 ± 0.54 fa20 31.8 ± 4.56 4.10 ± 0.41* fa40 37.6 ± 5.92* 4.22 ± 0.69* fa80 39.2 ± 5.17* 3.98 ± 1.29* fa100 34.8 ± 6.14* 3.68 ± 0.68 one way anova analysis *p<0.05 is significant (ems: 0.02m ethyl methane sulfonate; solvent control: 40 µg/ml citric acid; fa10: 10 µg/ml fulvic acid; fa20: 20 µg/ml fulvic acid; fa40: 40 µg/ml fulvic acid; fa80: 80 µg/ml fulvic acid; fa100: 100 µg/ml fulvic acid ). 32 özlem sultan aslantürk and 40 μg/ml, respectively (fig. 1d, e). this percentage is statistically insignificant when compared to the control and solvent control group. in addition, this percentage is very low compared to the chromosome aberration value obtained from the positive control group (ems), and the difference is statistically significant (p <0.05). in the positive control group (ems), chromosome aberration percentage has been found high, especially stickiness and anaphase bridge. micronucleus formation results are also present in table 2. micronucleus formation was found at a very low level of 0.04 ‰ in 10 µg/ml fulvic acid treated group only which was statistically not significant. no micronucleus formation was found in other experimental groups (including negative, positive and solvent controls). as a result of this study, root length and mitotic index results show that fulvic acid promotes root growth by inducing division in allium cepa root meristem cells. chromosome aberration and micronucleus results also indicate that fulvic acid does not induce cytotoxic and genotoxic effects in the root meristem cells. 4. discussion in this study, cytogenetic effects of fulvic acid were evaluated by analyzing root growth and root morphology. fulvic acid caused an increase in root growth and number, and there was a statistically significant difference between fulvic acid and control groups (negative, positive and solvent controls). cytoand genotoxicity were estimated by observing cytological parameters, such as the mitotic index and number of chromosome abnormalities, including chromosome breaks, stickiness, and polar deviations. the mitotic index (mi) of a. cepa meristem cells treated with the ems and citric acid (solvent control) was significantly decreased (0.83% and 0.01%, respectively) in comparison to negative control. although ems and citric acid significantly decreased the table 2. mitotic index values, percentage of chromosomal aberrations and thousandths of micronuclei in control and treatment groups after 24h treatment. concentrations total cells total dividing cells mitotic index (mi ± sd) breaks (%± sd) anaphase bridge (%± sd) stickiness (%± sd) polar deviation (%± sd) total aberrant cells (%± sd) micronuclei (%o ± sd) negative control 25000 1241 4.96 ± 0.31 0 ± 0 0 ± 0 0 ± 0 0.07 ± 0.03 0.07 ± 0.03 0 ± 0 solvent control 25000 2 0.01 ± 0.17* 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 ems (positive control) 25000 209 0.83 ± 0.11* 1.17± 0.26 10.47 ± 9.59*20.05 ± 6.90* 4.39 ± 6.03 36.09 ± 5.48* 0 ± 0 fa10 25000 2893 11.57 ± 1.93* 0.04 ± 0.01 0 ± 0 0± 0 0.51 ± 0.61 0.52 ± 0.60 0.04 ± 0.01 fa20 25000 3111 12.44 ±0.51* 0 ± 0 0 ± 0 0.03 ± 0.07 2.87 ± 0.53 2.90 ± 0.52 0 ± 0 fa40 25000 3559 14.23± 0.41* 0 ± 0 0 ± 0 0 ± 0 2.90 ± 0.42 2.90 ± 0.42 0 ± 0 fa80 25000 3566 14.26 ± 0.35* 0.03 ± 0.06 0 ± 0 0.03 ± 0.06 2.68 ± 0.51 2.73 ± 0.61 0 ± 0 fa100 25000 2988 11.95 ± 0.54* 0.03 ± 0.08 0 ± 0 0 ± 0 2.38 ± 0.17 2.41 ± 0.21 0 ± 0 one way anova analysis *p<0.05 is significant (ems: 0.02m ethyl methane sulfonate; solvent control: 40 µg/ml citric acid; fa10: 10 µg/ ml fulvic acid; fa20: 20 µg/ml fulvic acid; fa40: 40 µg/ml fulvic acid; fa80: 80 µg/ml fulvic acid; fa100: 100 µg/ml fulvic acid. 25000 cells/ group were evaluated for mi and ca) d a e b c fig. 1. a: membrane and nucleus deformation in solvent control (citric acid) group; b: stickiness; c: stickiness and polar deviation in positive control group; d: polar deviation in 20 µg/ml fulvic acid treatment group; e: polar deviation 80 µg/ml fulvic acid treatment group. 33cytogenetic effects of fulvic acid on allium cepa l. root tip meristem cells mitotic index, fulvic acid treatment increased the mitotic index in a. cepa meristem cells at all concentrations significantly (table 2). the mitotic index is measure of the mitotic activity of a cell population. it measures the proportion of cells in the m-phase of the cell cycle (rojas et al. 1993). therefore, the increase of mi in groups treated with fulvic acid, in comparison to negative control, suggests that fulvic acid could have proliferative effect on the meristem cells of a. cepa. the increased mitotic index in a. cepa root tip cells treated with fulvic acid is probably due to induction of dna synthesis and promotion of cell cycle. the mi results of fulvic acid treatment groups are consistent with literature data. previous studies suggest that humic substances including fulvic acid enhanced stimulation of seedling germination and growth of plants (kulikova et al. 2002; pena-méndez et al. 2005; van rensburg 2015). humic substances affect the development of organisms. being utilized as a substrate (a source of organic carbon) or nutrient source (n, p, trace elements and vitamins), humic substances can serve as a moiety of the biosynthesis chains. on the other hand, beneficial effects of humic substances on the plants are often attributed to hormone-like activity of these substances (nardi, 1994; nardi et al. 2002; piccolo et al. 1992; kulikova et al. 2002). since humic substances originate from the chemical and biological decomposition of plant and animal residues, and from metabolic activities of microorganisms, they might have characteristics of hormones. it was shown that humic substances enhanced plant growth by exhibiting auxin-like activity (kulikova et al. 2002). some researchers reported that humic and, in particular, fulvic acids showed some auxin, gibberellin or cytokinin-like activity (phuong and tichy 1973; nardi, 1994; kulikova et al. 2002). furthermore, fulvic acid, as a plant growth regulator, is involved in plant response to several environmental stress factors, and is reported to affect growth and development of plants (heil 2005; shahid et al. 2012). there are also studies of growth promoting effects of fulvic acid on plants as well as growth enhancing effects on animals (nardi et al. 2002; heil 2005; bai et al. 2013), because of its antioxidant, antimicrobial and anti-inflammatory properties (yamada et al. 2007; van rensburg et al. 2001; sherry et al. 2013). gao et al. (2017) have shown that when fulvic acid is used as food supplements for 60 days, it increases growth performance of paramisgurnus dabryanus (sauvage) and improves its intestinal health conditions (gao et al., 2017). also, bai et al. (2013) reported that supplementation of diets with fulvic acid is an effective way to increase growth performance, reduce backfat thickness, and improve meat quality in growing-finishing pig (bai et al. 2013). chromosome aberration and micronucleus results of this study show that fulvic acid does not induce genotoxic effects in the root meristem cells in comparison with control groups. although in the positive control group (ems), chromosome aberration rate (especially stickiness and anaphase bridges) has been found high, but in the fulvic acid treatment groups, breaks, stickiness and polar deviations appeared at very low rates. the total chromosome aberration percentages in fulvic acid treatment groups were found insignificant compared to control and solvent control groups (table 3). as a result of literature screening, different data on cytotoxic, genotoxic and mutagenic effects of fulvic acid have been reached. qui et al. (2007) reported that fulvic acid has protective effect against copper toxicity to the polychaete hydroidas elegans larvae, and such an effect is caused by the reduction in labile copper due to cufa (copper-fulvic acid) complexation (qui et al. 2007). also, it has been suggested that humic and fulvic acids have desmutagenic effect (inactivation of mutagens outside the cell) on vicia faba root tip cells treated with maleic hydrazide, whereas they have no antimutagenic effect (ferrara et al. 2000). ferrara et al. (2004) reported anticlastogenic, antitoxic and sorption effects of humic substances (soil humic acid, peat humic acid and peat fulvic acid) on the maleic hydrazide tested in leguminous plants, vicia faba and pisum sativum l (ferrara et al. 2004). however, no data has been found on the direct cytogenetic effects of fulvic acid in the literature. therefore, the data obtained in this study is important in terms of its contribution to the scientific literature in this regard. furthermore, fulvic acid is currently being used in planting and growing plants, especially in agriculture, and as complementary in treatment of human and animal health. its use is becoming increasingly widespread. therefore, it is important to determine whether this substance is safe for the environment, and animal and human health. the results of this study suggest that fulvic acid stimulate the root growth in a. cepa, and it does not have cytotoxic and genotoxic effects on a. cepa root meristem cells. these results 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journal of cytology, cytosystematics and cytogenetics volume 72, issue 2 2019 firenze university press karyotype analysis of a natural lycoris double-flowered hybrid jin-xia wang1, yuan-jin cao1, yu-chun han1, shou-biao zhou1,2, kun liu1,* insights on cytogenetic of the only strict african representative of genus prunus (p. africana): first genome size assessment, heterochromatin and rdna chromosome pattern justine germo nzweundji1, marie florence sandrine ngo ngwe2, sonja siljak-yakovlev3,* assessment of cytotoxicity and mutagenicity of insecticide demond ec25 in allium cepa and ames test arzu özkara cytogenetic effects of fulvic acid on allium cepa l. root tip meristem cells özlem sultan aslantürk evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of allium test ioan sarac1, elena bonciu2,*, monica butnariu1, irina petrescu1, emilian madosa1 fluorescence in situ hybridisation study of micronuclei in c3a cells following exposure to elf-magnetic fields luc verschaeve1,2,*, roel antonissen1, ans baeyens3, anne vral3, annemarie maes1 phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities nicoleta anca şuţan1,*, irina fierăscu2, radu fierăscu2, deliu ionica1, liliana cristina soare1 telomeric heterochromatin and meiotic recombination in three species of coleoptera (dorcadion olympicum ganglebauer, stephanorrhina princeps oberthür and macraspis tristis laporte) anne-marie dutrillaux, bernard dutrillaux* a whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of populus trichocarpa l. subjected to different stresses alberto vangelisti#, gabriele usai#, flavia mascagni#, lucia natali, tommaso giordani*, andrea cavallini differences in c-band patterns between the japanese house mice (mus musculus) in hokkaido and eastern honshu hikari myoshu, masahiro a. iwasa* karyotypic description and repetitive dna chromosome mapping of melipona interrupta latreille, 1811 (hymenoptera: meliponini) natália martins travenzoli1, ingrid cândido de oliveira barbosa2, gislene almeida carvalho-zilse2, tânia maria fernandes salomão3, denilce meneses lopes1,* caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 11-26, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-510 citation: m. meloni, c.a. dettori, a. reid, g. bacchetta, l. hugot, e. conti (2020) high genetic diversity and presence of genetic structure characterise the endemics ruta corsica and ruta lamarmorae (rutaceae). caryologia 73(1): 11-26. doi: 10.13128/caryologia-510 received: july, 2019 accepted: february, 2020 published: may 8, 2020 copyright: © 2020 m. meloni, c.a. dettori, a. reid, g. bacchetta, l. hugot, e. conti. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. high genetic diversity and presence of genetic structure characterise the endemics ruta corsica and ruta lamarmorae (rutaceae) marilena meloni1, caterina angela dettori2, andrea reid3, gianluigi bacchetta2,4,*, laetitia hugot5, elena conti1 1 institute of systematic botany, university of zurich, zollikerstrase 107, 8008 zurich, switzerland 2 centro conservazione biodiversità (ccb), dipartimento di scienze della vita e dell’ambiente università degli studi di cagliari. viale s. ignazio da laconi, 13 it-09123 cagliari, italy 3 department of environmental systems science, eth zurich, universitätstrasse 6, 8006 zurich, switzerland 4 banca del germoplasma della sardegna (bg-sar), hortus botanicus karalitanus (hbk), università degli studi di cagliari. viale sant’ignazio da laconi, 9-11, it-09123, cagliari, italy 5 conservatoire botanique national de corse, office de l’environnement de la corse, avenue jean nicoli, 20250 corte, france * corresponding author. e-mail: m.marilena75@gmail.com, c.angeladettori@gmail. com, areid@student.ethz.ch, bacchet@unica.it, laetitia.hugot@oec.fr, elena.conti@ systbot.uzh.ch. abstract. corsica and sardinia form one of the areas with highest biodiversity in the mediterranean and are considered one of the priority regions for conservation in europe. in order to preserve the high levels of endemism and biological diversity at different hierarchical levels, knowledge of the evolutionary history and current genetic structure of corso-sardinian endemics is instrumental. microsatellite markers were newly developed and used to study the genetic structure and taxonomic status of ruta corsica and ruta lamarmorae, rare endemics of corsica and sardinia, respectively, and previously considered a single species. our analyses identified high levels of genetic variation within each species (p=0.883, he=0.543 for r. corsica; p=0.972, he=0.627 for r. lamarmorae). intrinsic traits of the species (hermaphroditism, proterandry and polyploidy) and island-dependent factors (i.e. age, origin and history of the islands) might explain the detected high levels of genetic variation. we discovered differentiation between r. corsica and r. lamarmorae, and genetic structure within each species, which are consistent with the observation of low dispersal ability for both species. our genetic results support the recent taxonomic classification of r. corsica and r. lamarmorae as separate species and suggest that they diverge at only few loci. one r. corsica population (sa) strongly differed from all other studied populations and appeared to be the product of hybridization between the two species in structure analyses. our results provide important insights for the conservation of the two rare endemics. further genetic analyses are recommended for r. lamarmorae and for population sa (r. corsica). keyword. genetic diversity, endangered species, ruta, corsica, sardinia, microsatellite. 12 marilena meloni et al. introduction the mediterranean basin is characterised by high species richness (10.8 species/1000 km2, médail and quézel 1999) and considered one of the main “hot spots” of biodiversity in the world (médail and myers 2004, thompson 2005). additionally, this area is particularly rich in endemic taxa (about half of the approximately 25000 plant species native to this region are endemics), which are mainly concentrated in mountain chains and islands (médail and quézel 1997, 1999, thompson 2005, cañadas et al. 2014). corsica and sardinia (figure 1), the two largest islands of the western mediterranean basin, form one of the areas with highest plant diversity in the mediterranean and are particularly rich in endemics (médail and quézel 1997, thompson 2005, blondel et al. 2010). the sardinian flora consists of 2498 taxa, with about 11.61% endemic (290 species) to the island (conti et al. 2005, 2007; bacchetta et al. 2012; fenu et al. 2014). corsica’s flora consists of 2325 taxa, of which ca. 10% are endemics (230 species; jeanmonod and gamisans 2013). both islands are considered a major glacial refugium (médail and diadema 2009) and together host nine of the 50 most threatened plant species occurring in mediterranean islands (de montmollin and strahm 2005). the high level of biodiversity and the number of endemics found in corsica and sardinia are often ascribed to their noteworthy ecosystem diversity (bacchetta and pontecorvo 2005) and to past geologic and paleoclimatic processes. indeed, tectonic movements during the tertiary (from 66 to 2.58 million years ago, mya), the messinian salinity crisis (msc, ca. 5 mya), the establishment of a mediterranean climate type in the pliocene (2-3 mya), and climate changes associated with glacial and interglacial phases (pleistocene: 0.01-2 mya) have shaped the history of mediterranean plant lineages (hewitt 2000, gentili et al. 2015, médail and quézel 1997, thompson 2005). corsica and sardinia are continental fragment islands belonging to a single microplate (the corsosardinian (c-s) microplate; alvarez et al. 1972) and are currently separated by a narrow (11 km) and shallow (less than 50 m deep) water channel through the bonifacio strait. the c-s microplate was attached to southern france and northeastern spain until the late oligocene (28-30 mya), when it broke off and rafted eastward, until it collided with the apulian microplate (i.e., the current italian peninsula) ca. 18-20 mya (rosenbaum and lister 2004, speranza et al. 2002). it reached its current position in the middle of the western mediterranean ca. 9 mya (rosenbaum and lister 2004). the separation between corsica and sardinia may have begun as early as 15 mya and was complete by 9 mya (alvarez 1972, 1976, cherchi and montadert 1982, orsini et al. 1980). species that now occur in corsica and sardinia could have originated in different ways: 1) they were present in the region before the split of the c-s microplate from the iberian peninsula; 2) they reached the c-s microplate when it was temporarily connected with the apulian microplate during the miocene (ca.10-20 mya, rosenbaum et al. 2002); 3) they reached the c-s microplate through the land bridges that formed among corsica, sardinia, the apulian plate and the african continent during the msc (5 mya; hsü et al. 1977, krijgsman et al. 1999, mckenzie 1999) or 4) during the glacial marine regressions concomitant with the glacial cycles of the pleistocene (thompson 2005); 5) they reached the islands via long distance dispersal at any point in time (ldd). if insular populations differentiated sufficiently from their closest relatives due to isolation and/or extinction, they gave origin to island endemics. although corsica and sardinia are one of the priority regions for conservation in europe (myers et al. 2000, mittermeier et al. 2005), knowledge of the evolufigure 1. localities of populations taxonomically assigned to r. corsica (green dots) and r. lamarmorae (red dot) sampled for this study. ruta lamarmorae population was divided in two subpopulations. detailed information on each population is provided in table 1. 13genetic diversity and structure of ruta corsica and ruta lamarmorae tion and genetic characteristics of their endemic flora, instrumental for the long-term conservation of these species, is still poor. some molecular phylogenetic analyses have been performed to infer when and how c-s endemics reached the two islands (yesson et al. 2009, mansion et al. 2008, salvo et al. 2008, 2010) and few studies focused on the more recent history of these species (bacchetta et al. 2008; coppi et al. 2008; mameli et al. 2008; bacchetta et al. 2011; garrido et al. 2012). nevertheless, several questions remain unanswered, including: how did c-s endemics evolve after island colonization? what is their current genetic structure? are corsican and sardinian populations of the same species genetically differentiated? ruta corsica dc. and r. lamarmorae bacch., brullo & giusso are endemics of corsica and sardinia, respectively. the two species belong to the small genus ruta, which also includes four species widely distributed in the mediterranean (r. angustifolia pers., r. chalepensis l., r. montana l., and r. graveolens l.) and three species endemic to the canary islands (r. oreojasme webb & berth, r. pinnata l.f. and r. microcarpa svent.). ruta corsica and r. lamarmorae exhibit some features not found in the other species of the same genus (i.e. pulvinate, subspinescent habit; green-glaucescent leaves; white to pale yellow petals) and have been interpreted by taxonomists as relictual paleo-endemics (bacchetta et et al. 2006, cardona and contandriopoulos 1979), in other words as ancient lineages that were more widespread in the past and are now restricted to a local region (nekola 1999, mishler et al. 2014), in this case to the c-s microplate (arrigoni 1983, thompson 2005). they were treated as one species (i.e., r. corsica) until 2006, when they were split in two different taxa based on morphological (i.e., leaf shape and size of flowers, stamens and ovaries; bacchetta et al. 2006) and karyological differences (r. corsica is diploid, r. lamarmorae is tetraploid; contandriopoulos 1957, honsell 1957). phylogenetic analyses of chloroplast dna sequences from only two individuals each from corsica and sardinia supported the separation of r. corsica and r. lamarmorae, with individuals from the two islands grouped in mutually exclusive sister clades (salvo et al. 2008). molecular dating analyses and inference of ancestral areas of distribution for ruta species suggested that the genus originated during the eocene in eurasia and subsequently expanded westward and southward, colonising several landmasses of the forming mediterranean basin (salvo et al. 2010). the ancestor of the two endemics likely colonised the c-s block from the apulian plate (i.e., the emerging italian peninsula) during the early miocene. the divergence between the c-s endemics and the remaining ruta species apparently occurred during the middle miocene (ca. 14 mya). finally, r. corsica diverged from r. lamarmorae most likely in the pliocene (ca. 3.7 mya), when corsica and sardinia had already attained their current position in the middle of the western mediterranean sea and were separated by the bonifacio strait. the two islands were occasionally connected by land corridors during the msc of the miocene and during the glacial maxima of pleistocene climatic oscillations (salvo et al. 2010). table 1. description of populations of r. corsica and sub-populations of r. lamarmorae surveyed in this study. species population abbreviation location population size sample number coordinates altitude r. lamarmorae bc broncu spina ~1000 30 40° 07’ 34’’ n 9° 31’ 26’’ e 1650m ss su susciu ~1000 30 40° 01’ 02’’ n 9° 19’’ 33’’ e 1620m r. corsica mu muvrella 20 8 42° 24’ 0” n 8° 54 0”’ e 1050m mc monte cinto 150 16 42° 23’ 0’’ n 8° 55’ 0’’ e 1750m sa saltare 20 8 42° 21’ 41” n 8° 52’ 53”e 1180m gh ghisoni 40 23 42° 6’ 37” n 9° 9’ 16”e 1650m al albertacce 120 20 42° 17’ 39” n 8° 52’ 44”e 1300-1500m ba bastelica 750 21 42° 0’ 26” n 9° 6’ 4”e 900m 14 marilena meloni et al. given the inferred biogeographic history of r. corsica and r. lamarmorae and their current distribution in corsica and sardinia, respectively, these species represent an ideal case study to gain new knowledge on the genetic characteristics of the corso-sardinian endemic flora. the aims of the present study are thus to: (1) assess the current amount and distribution of genetic diversity for the two species, testing whether the taxonomic status of r. corsica and r. lamarmorae as separate species is warranted; and (2) use the results of genetic analyses to recommend proper conservation strategies for these species, with a particular focus on r. lamarmorae, recently listed as endangered according to the iucn criteria and categories (dettori et al. 2014a). materials and methods study species ruta lamarmorae was described as a species separate from r. corsica in 2006 (bacchetta et al.). it is a small, erect, perennial shrub, 15-50 cm tall, with woody, subspinescent branches. it is characterised by bipinnate, obovate-rounded leaves, 1.5-8 cm long. the whitish, pale yellow flowers (12-13 mm in diameter) are hermaphroditic and proterandrous. the capsules, 6-7 mm long, are obtuse at the apex. as with most members of its genus, r. lamarmorae is tetraploid, with 2n=36 (honsell 1957). it blooms in june-july, fruiting in august-october. it occurs mainly on siliceous substrates, at an altitude of 1500-1750 m a.s.l. ruta lamarmorae is found in a single, fragmented population in the gennargentu massif (central-eastern sardinia). it is categorized as endangered (en) according to the iucn criteria (2013; dettori et al. 2014a). the main threats to this species are habitat fragmentation, overgrazing and fires (bacchetta et al. 2006; dettori et al. 2014a). ruta corsica, first described in 1824 by de candolle, shares the same habit with r. lamarmorae and has overall similar leaves, flowers and fruits. it differs from r. lamarmorae in leaf shape (obovate to cuneate-oblong), smaller flower size (8-10 mm in diameter) and fruit size and shape (7-8 mm long, with apiculate apex; bacchetta et al. 2006). ruta corsica is the only diploid species of the genus, with 2n=18, while the other karyotyped species are tetraploid (contandriopoulos 1957). it blooms and fruits slightly later than r. lamarmorae (september-november vs. july-august, respectively). the species occurs at an altitude of 1000-1900 m asl, mainly on siliceous substrates, and is widespread on the main corsican massifs with about 15 populations. it is listed as least concern (lc) under the french red list of threatened species (uicn, france 2013). to date, no information is available on the mating system, the pollination biology and the mode of dispersal of the diaspores of neither of the two species. sample collection and dna extraction plant material was collected during summer 2010 (see table 1). since the only existing population of r. lamarmorae has a scattered distribution on the gennargentu massif, we sampled plants from the two opposite sides of the massif, in other words from the northwestern (bc, 30 individuals) and southwestern slopes (ss, 30 individuals; figure 1). because sub-populations bc and ss are large (thousands of individuals; gianluigi bacchetta, pers. obs.), sampling was carried out in order to minimize collection of related individuals and cover the entire occupied area. samples of r. corsica were collected from a total of 96 individuals from six populations chosen in order to cover the entire distribution range of the species (see figure 1 and table 1 for details on each population). leaf-tissue samples were dried and preserved in silica gel. total genomic dna was isolated from dried leaves using the qiagen® dneasy plant mini kit, following the manufacturer’s guidelines, with minor modifications. in particular, an increased volume of buffer ap1 (from 400 μl to 600 μl), buffer ap2 (from 130 μl to 200 μl) and rnase a (from 4 μl to 6 μl), as well as a longer incubation time with buffer ap1 (30 minutes) for cell lysis were applied. microsatellite development and genotyping dna isolated from one specimen of r. lamarmorae sampled from population bc was used by genetic marker services (brighton, uk, www.geneticmarkerservices.com) to develop an enriched library, design and test microsatellite primer pairs. enrichment involved incubating adaptor-ligated restricted dna with filterbonded synthetic repeat motifs, (ag)17, (ac)17, (aac)10, (ccg)10, (ctg)10 and (aat)10. twenty-one positive library colonies were selected for sequencing, from which 15 microsatellites were designed and tested. the primer sets were designed using primer 3.0 (rozen and skaletsky, 2000). each primer pair was tested for amplification ability and polymorphism on eight individuals of r. lamarmorae (four from bc and four from ss). cross-species amplification was tested on four individuals of r. corsica and four individuals of r. chalepensis (another species of the same genus with wider distribution also occurring in sardinia; salvo et al. 2010). polymerase chain reactions (pcrs) for primer screening 15genetic diversity and structure of ruta corsica and ruta lamarmorae were performed in 25 μl and contained approximately 50 ng of dna, 5 pmol of each unlabelled primer, 1.5 mm of mgcl2, 0.2 mm of each dntp, 1x pcr buffer and 0.5 u of supratherm dna polymerase (genecraft, cologne, germany). in-vitro amplifications consisted of: 60 s denaturation at 95°c, followed by 25 cycles of 95°c for 60 s, annealing at 55°c for 60 s and 72°c for 60 s, ending with a final extension at 72°c for 5 min. this first screening of microsatellites was performed on highresolution agarose gels. the primer pairs able to amplify polymorphic products were then used to genotype all sampled individuals via amplification with fluorescently labelled primers and separation of pcr products by capillary electrophoresis. amplifications were performed following the twostep method described by schuelke (2000), using ca. 20 ng of genomic dna, 2.5 μl of 10x reaction buffer, 0.5 μl of each dntp (10 mm), 1 μl of mgcl2 (50 mm), 0.2μl of the forward primer with m13(−21) tail at the 5’ end (10 μm), 0.5 μl of the reverse primer (10 μm), 0.5 μl of the fluorescently labelled m13(−21) primer (fam, ned, vic, pet; 10 μm) and 0.1 μl of taq dna polymerase (5 u/μl; bioline gmbh, luckenwalde, germany) in a final volume of 25μl. amplification reactions started with 94°c for 3 min, followed by 30 cycles of 94°c for 30 s,s 55°c for 45 s (see table 2), and 72°c for 1 min. the fluorescently labelled m13(−21) primer was incorporated in the following eight cycles of 94°c for 30 s, 53°c for 45 s, and 72°c for 1 min, followed by a final extension step of 72°c for 5 min. up to four pcr products of different primer sets were pooled for each individual and separated by capillary electrophoresis on an ab3130xl genetic analyzer. alleles were sized against the internal size standard genescan™ liz500™ (applied biosystems) and scored using genemapper® software version 4.0 (applied biosystems). the observed allele size of all genotyped individuals was decreased by 18bp in order to account for the m13(−21) universal sequence tag. population genetic analyses even though r. lamarmorae is known to be tetraploid (honsell 1957), a maximum of two alleles per locus and per individual were detected in all populations, thus showing disomic inheritance. because genetic analyses can be performed with standard population genetic tools developed for diploid organisms in tetraploid species characterised by disomic inheritance (stift et al. 2008; meloni et al. 2013), our analyses were conducted assuming a diploid status for r. lamarmorae. we assessed genetic diversity by quantifying the number of alleles (na), proportion of polymorphic loci (p), number of private alleles (ap), observed (ho) and expected (he) heterozygosity for each population across loci. populations were tested for deviations from hardyweinberg (hw) equilibrium using fisher’s exact test with the markov chain algorithm (guo and thompson 1992). the fixation index, fis, was estimated in order to assess departure from hardy-weinberg expectations due to non-random mating. fisher’s exact test was performed within each population to check for linkage disequilibrium (ld) between all different pairs of loci. these analyses were performed with the web-based genepop (raymond and rousset 1995; rousset 2008) and genalex v. 6.5 (peakall and smouse 2012). the program bottleneck (piry et al. 1999) was used to detect recent genetic bottlenecks in the studied populations. based on the observed number of alleles and the sample size of a population, the program computes the gene diversity expected under the assumption of mutation-drift equilibrium and compares it to hardy–weinberg gene diversity (he; nei 1978) to establish whether there is a significant deficit of gene diversity resulting from a recent bottleneck. as recommended by the authors of the program, a wilcoxon sign-rank test (luikart et al. 1998) was performed using 1000 bottleneck simulation replicates under the stepwise mutation model (smm). fstat 2.9.3 (goudet 1995) was used to estimate genetic differentiation among populations by fst. we also measured rst, an analogue of fst specific for microsatellite data, employing a stepwise mutation model (smm, slatkin 1995); rst was measured using the software spagedi 1.4 (hardy and vekemans 2002). the same program was used to perform an allele-size permutation test (10000 permutations) to evaluate the contribution of stepwise mutations to the observed genetic differentiation, hence the relative suitability of fst vs. rst (hardy et al. 2003). in addition, since fst can considerably underestimate differentiation when loci are highly variable (as commonly found with microsatellite markers; hedrick 2005; jost 2008; meirmans and hedrick 2011), we also calculated jost’s dest (jost 2008) using 1,000 bootstrap replicates in smogd (crawford 2010). an analysis of molecular variance (amova) was performed to assess the hierarchical partitioning of genetic variation among populations and species. we followed the procedure of excoffier et al. (1992), huff et al. (1993), peakall et al. (1995), and michalakis and excoffier (1996) by estimating fst and using 999 random permutations of the data in genalex v.6.5 (peakall and smouse 2012). with the same program, a principal coordinate analysis (pcoa) based on a genetic distance matrix was performed to visualise genetic relatedness among individuals. to test for isolation by distance, a 16 marilena meloni et al. mantel test (mantel 1967) was applied to the matrix of pairwise nei’s genetic distances (nei 1972, 1978) and the matrix of geographical distances with 999 random permutations in genalex v. 6.5 (peakall and smouse 2012). population structure was inferred using the bayesian clustering method implemented in structure (v. 2.3; pritchard et al. 2000; hubisz et al. 2009). the program uses a markov chain monte carlo (mcmc) procedure to estimate p(x|k), the posterior probability that the data fit the hypothesis of k clusters, and assigns individual genotypes to clusters by estimating the membership coefficient q for each individual based on allele frequencies at unlinked loci (independent of locality information). we tested all possible values of k from 1 to 9; for each k we ran an admixture model (each individual draws some fraction of the genome from each of the k populations) with correlated allele frequencies 20 times with a length of burnin period of 100,000 followed by 100,000 mcmc repetitions. to identify the best k, we measured δk (the rate of change in the log probability of data between successive k values), as suggested by evanno et al. (2005) and implemented in structure harvester (earl and vonholdt 2012). this method provides the most accurate estimate of the number of clusters k (evanno et al. 2005), but does not allow for discrimination between k=1 and k=2. therefore we also calculated the average posterior probability of the data for each value of k, ln p(x|k), as proposed by pritchard et al. (2000). after determining the most effective number of genetic groups (k) for our data, we ran structure with the admixture model and default parameter settings; the inferred genetic composition of individuals was then determined using 100,000 iterations after a burnin period length of 100,000. results microsatellite development and cross-species amplification of the 15 microsatellites newly developed for r. lamarmorae, 13 were suitable for genetic analyses on the target species (table s1). tests on cross-species amplification showed that 13 markers could be used for r. corsica while 11 could be amplified in the more distantly related r. chalepensis (table s1). genetic diversity eleven microsatellites showing a clear amplification pattern after capillary electrophoresis on both r. corsica and r. lamarmorae were used for genotyping (table 2). because all studied individuals of r. corsica and r. lamarmorae were heterozygotes for the same two alleles in locus rl9, this marker was excluded from population genetic analyses. in r. lamarmorae, all amplified loci were polymorphic; in r. corsica, locus rl16 was fixed in populations sa, gh, al, ba; population sa showed fixed alleles also at loci rl15, rl17 and rl18. the number of alleles identified across all loci ranged from 22 to 61 in r. corsica populations, and from 55 to 68 in the two r. lamarmorae sub-populations (table 3). private alleles were found in all populations except sa (table 1). all populations, except for gh, were at hw equilibrium (p>0.05). fis values were positive in the two sub-populations of r. lamarmorae, as well as in four out of six populations of r. corsica (mu, mc, gh and ba; table 3), meaning that the departure of genotype frequencies from hardy-weinberg expectations was always associated with a deficit of heterozygotes. conversely, an excess of heterozygotes was found in populations sa and al (r. corsica; table 3). significant linkage-disequilibrium (ld) at the 5% level was detected at one pair of loci for populations gh (rl6-rl18), al (rl13-rl15) and mu (rl12-rl17), three loci for population bc (rl4-rl6, rl13-rl15, rl11-rl17), four loci for sa (rl4-rl12, rl4-rl13, rl11-rl13, rl12-rl13) and ba (rl6-rl11, rl12-rl15, rl13-rl15, rl15-rl18), and eight loci for ss (rl4-rl5, rl5-rl11, rl5-rl13, rl6-rl13, rl11-rl13, rl15rl17, rl12-rl18, rl15-rl18). it was impossible to perform most of the tests for populations mu (25 out of 45 pairs of loci) and sa (30 out of 45 pairs of loci). gene diversity (he) was high in all populations, with a mean value of 0.627 for r. lamarmorae and 0.543 for r. corsica; the only population showing a relatively low value was sa (r. corsica), with he= 0.323 (table 3). the only population showing significant signs of a recent bottleneck was ba (r. corsica, p = 0.032), while all other populations were at mutation-drift equilibrium. genetic structure genetic differentiation among populations measured with fst was always statistically significant (p < 0.05); it was 0.086 between the two sub-populations of r. lamarmorae and ranged between 0.012 (mu-mc) and 0.240 (al-sa) in r. corsica (table 4). genetic differentiation among populations across the two species ranged between 0.050 (mu-ss) and 0.212 (sa-bc). population sa was the most differentiated, with 0.173<fst<0.240 (table 4). the overall genetic differentiation among populations was significant (p=0.01), with fst=0.097 for r. corsica and fst=0.086 for r. lamarmorae. simi17genetic diversity and structure of ruta corsica and ruta lamarmorae table 2. characteristics of 15 microsatellite markers developed in ruta lamarmorae. shown for each marker are the genbank accession number, forward and reverse sequences of the primer pair, repeat motif, and size of the expected pcr product (bp). primers in bold were used for genotyping; locus rl9 was excluded from statistical analyses as all genotyped individuals of r. corsica and r. lamarmorae were heterozygotes for the same two alleles. locus genbank accession no. primer sequence (5’-3’) repeat expected size (bp) rl4 kp098574 f: acatcagcccttcaactacg r: cctaaagaatcaattataagcaacc (tc)13 182 rl5 kp098575 f: tcgtagagccaatcacagga r: gctgcttcatttgctttgaa (ag)14 219 rl6 kp098576 f: ctcgatcgggaagaacttaca r: atccaatcccaacccaactt (ga)14 131 rl7 kp098577 f: ttgagtgaggagggttaggg r: tgagacacgccaatttcaga (ga)7 140 rl9 kp098579 f: cttgctcatgccacctttta r: acccacaagctcctcctgta (gt)9 145 rl10 kp098580 f: acatgttgcagaatttgaaagg r: ttaaagaggcaacaacctgga (ag)13 160 rl11 kp098581 f: gaaacgggcttcttcaacag r: gtacaggacatgatgcaaa (ga)15 113 rl12 kp098582 f: ctccgttcgcagaaagacac r: ttacgacccacacgcaagta (gt)8(ga)8 204 rl13 kp098583 f: caaccccatctgtgaatcct r: gcttgcactttggagtttttg (tc)12 185 rl15 kp098585 f: cgcctttcttacttgaaaaggtt r: tcaacaagacactgcaacca (tc)13 194 rl16 kp098586 f: tttcgtcaaaattgacatcagc r: tcaacaagacactgcaacca (tg)7 170 rl17 kp098587 f: tggagtttcatgtcggattg r: tgagataaccgcctcctacc (tg)7 242 rl18 kp098588 f: tcccaacgcacagtgaaata r: gtgtttcctcgaagctgctc (tg)10 166 rl19 kp098589 f: cgtcttgaaatgatgttacctg r: cctaacaaacaacacaaataataaatg (tc)16 225 rl20 kp098590 f: tgttttcgtgcaaaaattcg r: ggacttacctatccgaaaatgg (gt)19 246 table 3. genetic diversity parameters inferred from microsatellite analysis. na total number of alleles across loci, p proportion of polymorphic loci, ap number of private alleles, ho observed heterozygosity, he expected heterozygosity, fis fixation index; sd, standard deviation. for abbreviations of populations see table 1. population na p ap ho ± sd he ± sd fis ± sd bc 55 1 4 0.554 ± 0.082 0.614 ± 0.078 0.097 ± 0.100 ss 68 1 8 0.587 ± 0.076 0.639 ± 0.083 0.082 ± 0.027 overall r. lamarmorae 123 0.967 12 0.571 ± 0.055 0.627 ± 0.056 0.109± 0.052 mu 51 1 2 0.623 ± 0.075 0.688 ± 0.072 0.094 ± 0.056 mc 55 1 3 0.578 ± 0.077 0.630 ± 0.073 0.081 ± 0.076 sa 22 0.6 0 0.350 ± 0.118 0.323 ± 0.105 -0.083 ± 0.073 gh 53 0.9 1 0.546 ± 0.082 0.593 ± 0.084 0.080 ± 0.041 al 61 0.9 1 0.624 ± 0.085 0.624 ± 0.086 -0.001± 0.031 ba 53 0.9 3 0.497 ± 0.080 0.533 ± 0.081 0.070 ± 0.061 overall r. corsica 295 0.883 10 0.536 ± 0.036 0.543 ± 0.035 0.160 ± 0.024 18 marilena meloni et al. larly, the amova (based on fst) displayed that most of genetic variation was partitioned within-population (88%), while only 9% was found among populations and 3% between islands (figure 2). values of rst (analogous to fst, but specific to microsatellite data; slatkin 1995) were slightly smaller than those of fst (0.003< rst <0.253) (table s2). however, observed rst values were not significantly higher than permuted rst, suggesting that stepwise mutations did not contribute to population differentiation and that fst (based on allele identity) explains genetic differentiation among populations better than rst (based on allele-size information; hardy et al. 2003). although fst is widely used as a measure of population structure, its dependency on expected heterozygosity might underestimate the genetic differentiation among populations. higher levels of genetic differentiation among populations were detected, in fact, when the partition of diversity was based on the effective number of alleles (i.e. dest, jost 2008) rather than on the expected heterozygosity. we found dest=0.179 between the two sub-populations of r. lamarmorae, and 0.003 <dest<0.238 for r. corsica (table 4). across populations, total dest for r. corsica was 0.162. the pcoa showed some genetic structure among r. corsica populations: while mu, mc and gh appeared strongly genetically interconnected, al and ba overlapped only slightly, and sa was well separated from all other populations (figure 3a). a clear genetic differentiation was also present between the two sub-populations of r. lamarmorae (figure 3b). when considering both species together, population structure was still clearly present, although less marked: overall, the individuals of the two species diverged, but showed a certain degree of overlapping, populations within each species overlapped slightly more, and population sa, taxonomically assigned to r. corsica, remained well separated from all remaining populations (figure 3c). the mantel test for correlation between genetic differentiation and geographic distances among populations was not significant (p=0.579, r2 =0.0076). for both r. corsica and r. lamarmorae the ln probability of the data (ln p(x|k)) did not allow for discrimination between different values of k (table s3) while, based on the δk statistic, the best-supported number of a posteriori genetic clusters was k=3 for r. corsica (figure s1 a) and k=2 for r. lamarmorae (figure s1 b). for k=3, populations of r. corsica mu, mc, gh and al were grouped together, while sa and ba appeared genetically distinct (figure 4a). very little admixture seemed to occur between the two r. lamarmorae subpopulations for k=2 (figure 4b). when r. corsica and table 4. pairwise population estimates of fst (diagonal below) and dest (above diagonal). all values are statistically significant (95% ci). for abbreviations of populations see table 1. r. lamarmorae r. corsica bc ss mu mc sa gh al ba bc 0.179 0.146 0.155 0.303 0.234 0.256 0.254 ss 0.086 0.278 0.121 0.208 0.156 0.099 0.193 mu 0.087 0.050 0.003 0.177 0.076 0.065 0.103 mc 0.102 0.065 0.012 0.179 0.048 0.060 0.077 sa 0.212 0.173 0.176 0.173 0.168 0.238 0.158 gh 0.130 0.083 0.052 0.038 0.184 0.131 0.124 al 0.129 0.060 0.046 0.044 0.240 0.085 0.156 ba 0.160 0.136 0.092 0.071 0.213 0.088 0.122 figure 2. analysis of molecular variance (amova) based on ten microsatellite markers used to genotype 96 individuals taxonomically assigned to r. corsica (six populations) and 60 individuals taxonomically assigned to r. lamarmorae (one population divided in two sub-populations). the two regions considered in this analysis represent the two islands. 19genetic diversity and structure of ruta corsica and ruta lamarmorae r. lamarmorae were pooled together, the most effective number of genetic groups was k=4 according to pritchard’s method (pritchard et al. 2000, table s3) and k=2 according to evanno’s method (evanno et al. 2005, figure s1 c). figure 5 shows subfigures for k=4 and k=2; subfigures were included also for k=3 and 5<k<7, because populations subdivision was informative also for these values of k. for k=2, populations of the two species were well separated in two distinct r. corsica and r. lamarmorae clusters, except for population sa, taxonomically classified as r. corsica, whose individuals were genetically assigned with higher probability to populations of r. lamarmorae (figure 5). values of k=3 showed the separation of the two r. lamarmorae subpopulations, while population sa (of r. corsica) clustered with sub-population ss (of r. lamarmorae; figure 5). for k=4, r. lamarmorae sub-populations were genetically distinct, population sa was separated from all populations, while the remaining populations of r. corsica formed a single cluster (figure 5). values of k=5 showed the separation of the r. corsica population ba (figure 5). values of k=6 and k=7 showed more admixture. it is notable that population sa, taxonomically assigned to r. corsica, remained separated from all other populations for all k values (figure 5) discussion microsatellite development and cross-species amplification most of the microsatellites developed for this study were suitable for genetic analyses on r. lamarmorae (13 out of 15), r. corsica (13 out of 15) and the more distantly related r. chalepensis (11 out of 15), being amplifiable and polymorphic (table s1). despite the difference in ploidy level between r. corsica (diploid) and r. lamarmorae (tetraploid), the high transferability of the molecular markers suggests a low divergence between the genomes of the two species. genetic diversity among the microsatellite markers developed for the present study, 11 were useful for population genetic analyses on r. corsica and r. lamarmorae. our study revealed high levels of genetic diversity in both r. corsica (p=0.883, he=0.543) and r. lamarmorae (p=0.972, he=0.627). even though each species is restricted to a single island (figfigure 3. principal coordinate analysis (pcoa) based on the multilocus genotypes of 96 individuals of r. corsica (a), 60 individuals of r. lamarmorae (b), and the two species together (c). the percentage of the total variability explained by the first two components is indicated on brackets. each symbol represents a single plant from one of the eight studied populations. information on each population is provided in table 1. figure 4. proportional membership (q) to clusters identified by structure for: (a) 96 individuals from the six studied populations of r. corsica; (b) 60 individuals from the two studied subpopulations of r. lamarmorae. colours distinguish between the different clusters identified for each of the above three scenarios. each individual is represented by a single vertical bar and grouped by locality, indicated on the x-axis. information on each population (locality) is provided in table 1. 20 marilena meloni et al. ure 1), most populations showed fewer genetic signatures of isolation and small population size than typically expected for insular endemics (i.e., low gene diversity, high homozygosity, high ld; ellstrand 1993, frankham 1998, bouzat 2010). unexpectedly high values of genetic diversity were also found in the congener r. oreojasme, an endangered species endemic to the island of gran canaria in the canarian archipelago (a=7.625, p=0.984, ho=0.558, he=0.687; meloni et al. 2015). the occurrence of another endangered, insular endemic with high genetic diversity within the same genus suggests that r. corsica and r. lamarmorae share some intrinsic traits maintaining high levels of genetic variation with r. oreojasme. hermaphroditism and proterandry, characterising all three endemics, favour outcrossing, thus increasing genetic diversity. furthermore, polyploidy, occurring in all ruta species except r. corsica, might also contribute to elevated genetic variation, for polyploids are usually characterised by higher genetic diversity than diploids (soltis and soltis 2000). hermaphroditism, proterandry and polyploidy were associated with high levels of genetic variation also in other insular endemics outside of ruta (pérez de paz and caujapé-castells 2013). conversely, genetic analyses of r. microcarpa, a critically endangered species endemic to the island of la gomera (also in the canarian archipelago), revealed levels of genetic diversity lower than those observed in r. corsica, r. lamarmorae and r. oreojasme (ho=0.651, he=0.410; meloni et al. 2014). the relatively low genetic diversity found in r. microcarpa, resulting mainly from the almost total absence of sexual reproduction in this species (meloni et al. 2015), highlights the role of outcrossing in maintaining variation within and among populations. despite the relatively low number of population genetic studies on c-s species, a general trend of high genetic diversity was detected in published analyses of corsican and/or sardinian endemics. high levels of genetic variability were found, for example, in the sardinian centaurea horrida (he=0.780, mameli et al. 2008) and centaurea filiformis (he=0.678, pisanu et al. 2011; he = 0.576; giorgio binelli, pers. comm.), in the corso-sardinian ferula arrigonii (p=0.92, hw = 0.317, dettori et al. 2014b), and in the corsican mercurialis corsica (migliore et al. 2011). the occurrence of high genetic diversity in several c-s species suggests that also some geographic factors (i.e., age, physical dimension, geologic origin and history of the islands) might have contributed to the genetic diversity of r. corsica and r. lamarmorae as well as of several other c-s endemics. the complex geologic history of corsica and sardinia most likely influenced the evolution and current genetic diversity of their endemics in different ways. corsica and sardinia are considered old islands (they broke off from the iberian peninsula as the c-s microplate ca. 30-28 mya) and have been proposed to harbour many relictual endemics (médail and quézel 1999, grill et al. 2007). because they are continental fragment figure 5. proportional membership (q) to clusters identified by structure for 2<k<7. colours distinguish between the different k clusters. all studied individuals of r. lamarmorae (sub-populations bc and ss: 60 individuals) and r. corsica (populations mu, mc, sa, gh, al and ba: 96 individuals) are represented by vertical bars and grouped by locality, indicated on the x-axis. information on each population (locality) is provided in table 1. 21genetic diversity and structure of ruta corsica and ruta lamarmorae islands, it has been suggested that they might have hosted the ancestors of many of their current endemic taxa before their separation from the iberian peninsula in the oligocene, although this hypothesis has been corroborated by molecular dating analyses only in a few cases (for example, in araceae; mansion et al. 2008). subsequent to its split from the iberian peninsula, the c-s microplate was temporarily connected with the apulian microplate during the miocene. the vicariant origin of some c-s species during the oligocene and miocene might have favoured relatively high levels of genetic diversity, because they did not experience the loss of genetic variation associated to ldd and founder events typical, for example, of oceanic island colonization. furthermore, the long time-spans since colonisations (from either the iberian peninsula in the oligocene or the apulian microplate in the miocene) might have increased opportunities to accumulate genetic variation through mutation, recombination, drift and selection in cs endemics. additionally, multiple overland migrations might have occurred while corsica and sardinia were connected to neighbouring continental masses via land bridges at different points in time during the miocene and pleistocene, thus favouring genetic exchanges also within species with limited dispersal ability, as in the case of r. corsica and r. lamarmorae (gianluigi bacchetta, pers. obs.). furthermore, the relatively stable climate characterising corsica and sardinia during quaternary glacial/ interglacial periods (taberlet 1998, grill et al. 2007) might also have contributed to the high genetic diversity detected in their endemics, for it allowed populations of many species to persist through several climatic cycles and accumulate genetic differences via mutation and recombination. finally, climatic changes during the miocene and pleistocene might have also contributed to the high genetic variation detected in c-s species. indeed, land bridges among corsica, sardinia, the apulian plate and the african continent during the msc (hsü et al. 1977, krijgsman et al. 1999, mckenzie 1999) and quaternary glacial marine regressions (thompson 2005) may have decreased the genetic effects of isolation by favouring genetic exchange between c-s populations and populations of the same species occurring on other land masses. among the above mentioned islanddependent factors, the vicariant origin of the ancestor of r. corsica and r. lamarmorae following the msc, supported by molecular dating and biogeographic analyses (salvo et al. 2010), and the climatic stability of corsica and sardinia during pleistocene glacial cycles might have contributed to the relatively high levels of genetic diversity detected in the two species. ruta lamarmorae showed slightly higher values for most diversity parameters than r. corsica (p=0.967, ho=0.571, he=0.627 vs. p=0.83, ho=0.536, he=0.543). as mentioned above, the higher ploidy of the former might explain this difference. in addition to intrinsic traits, the small size of most r. corsica populations (laetitia hugot, pers. obs.) and its scattered distribution could also explain the lower diversity found in r. corsica, because its populations might be more affected by inbreeding and isolation than the larger population of r. lamarmorae (hamrick and godt 1989; ouborg et al. 2006). an exception to the trend of relatively high genetic diversity found in r. corsica and r. lamarmorae is represented by population sa (taxonomically assigned to r. corsica), which showed some genetic erosion, being characterised by values of polymorphism, allelic diversity and heterozygosity much lower than the other populations analysed in this study (na =22, p=0.6, ho=0.350, he=0.323, table 3). the low genetic diversity and the presence of alleles fixed at four loci suggest that genetic drift and inbreeding strongly influenced the genetic structure of this small population (20 individuals in total; laetitia hugot, unpublished data; ellstarnd et al 1993). they also suggest that the rate of genetic exchange between sa and other r. corsica populations, even the closest ones from a geographical point of view, is extremely low, and it seems unable to counteract the genetic effects of small population size. population genetic structure genetic differentiation among populations measured with fst and amova (table 4 and figure 2 respectively) was lower than expected, when considering the low dispersal ability of r. corsica and r. lamarmorae (gianluigi bacchetta, unpublished data). because fst is strongly influenced by levels of heterozygosity (meirmans and hedrick 2011), we cannot rule out the possibility that fst values (and amova, accordingly) may simply reflect the high levels of heterozygosity detected in our study species (table 3), and that in this case dest (which is based on the effective number of alleles and thus independent of levels of heterozygosity; jost 2008; table 4) might better describe genetic differentiation among populations. indeed, dest values are approximately two times larger than fst values (fst=0.097, dest=0.162 for r. corsica; fst=0.086, dest=0.179 for r. lamarmorae), suggesting that the studied populations might be more differentiated than implied by fst. high values of heterozygosity and lower levels of fst than dest were also found in the congener r. oreojasme (he= 0.687, fst = 0.097, dest = 0.275; meloni et al. 2015). 22 marilena meloni et al. the differentiation among populations described by dest (high for r. lamarmorae, relatively lower for r. corsica; table 4) was supported by pcoa (figure 3) and bayesian analyses (figures 4 and 5). the population structure detected in the two species (figures 3, 4 and 5) and the absence of isolation by distance reiterates that limited gene flow probably due to low dispersal ability has played a significant role in shaping current patterns of genetic differentiation. low dispersal ability seems to characterise particularly r. lamarmorae, whose subpopulations, showing the highest value of within-species dest and appearing genetically well separated in pcoa and bayesian analyses (figures 3b and 4), are only ca. 3 km apart from each other. analyses that included both species indicated some genetic differentiation between r. corsica and r lamarmorae (table 4, figures 3c and 5), lending some support to their taxonomic classification as separated species. however, some degree of admixture between them was detected. the difference in ploidy level (r. corsica is diploid, r. lamarmorae is tetraploid; contandriopoulos 1957, honsell 1957) and their geographic separation through the bonifacio strait suggest that relatively low levels of admixture detected might not be the effect of gene flow between islands. instead, this result might be better explained by the possibility that r. corsica and r. lamarmorae diverged at only few loci, probably as a consequence of low mutation rate and/ or the long generation time that characterises the two species. further analyses would be required to test this hypothesis. the high transferability of the newly developed microsatellite markers between the two species (table s1) supports the hypothesis of low divergence. in all genetic analyses, population sa (of r. corsica) strongly differed from all other studied populations (table 4), forming a distinct group in both pcoa (figures 3a and 3c) and structure analyses (figures 4 and 5). this result reinforces the conclusion that sa is more genetically isolated than the other studied populations. because geographic distance does not explain this genetic isolation, other factors seem to affect gene flow in this population. interestingly, in structure analyses that included r. corsica and r. lamarmorae, population sa showed evidence of genetic admixture between the two species for k=2 (the most accurate estimate of the number of clusters obtained measuring δk; evanno et al. 2005), was assigned to the same genetic group of r. lamarmorae sub-population ss for k=3, and resulted genetically differentiated from all other studied populations of both species for 4<k<7 (figures 5). further genetic and karyotype studies are required to clarify the reasons of the genetic differentiation of sa. genetic analyses on the congener r. chalepensis, occurring in both islands, might also help to clarify the occurrence and amount of gene flow between corsica and sardinia. conservation implications this study provides important insights into the genetic structure of r. corsica and r. lamarmorae, with potential applications for their effective conservation. the medium to high genetic diversity characterising these endemics suggests that both species are not at high risk of extinction due to genetic factors. nevertheless, their isolation, their very restricted distribution, the small size of r. corsica populations and, in the case of r. lamarmorae, the continued anthropogenic and environmental threats to its population (i.e., overgrazing, fires, presence of skiing infrastructures; bacchetta et al. 2006) might jeopardize conservation. moreover, mountain habitats are considered particularly sensitive to climatic change and are likely to show the effects of temperature increase earlier and more markedly than other ecosystems (grabherr et al. 2000; thuiller et al. 2005; patsiou et al. 2014). germination tests in r. lamarmorae show that an increase in winter temperatures could lead to reduced reproductive capability, because seeds may not experience the vernalization period required for germination (bacchetta et al., unpublished data). we expect a similar negative effect on germination in r. corsica, given the close relatedness with r. lamarmorae, the phenotypic traits they share, and the shared ecological preference for mountain habitats. similarly, a reduction in seed germination rate with increasing temperatures has been observed in other sardinian taxa that occur at medium to high altitudes, including lamyropsis microcephala (mattana et al. 2009), rhamnus persicifolia (bacchetta et al. 2011), ribes multiflorum subsp. sandalioticum (mattana et al. 2012), and vitis vinifera subsp. sylvestris (orrù et al. 2012). in situ conservation is essential to the long-term persistence of these two island endemics and should be aimed at preserving all extant populations, for they all carry unique alleles (a particular case is represented by population sa, whose low genetic diversity and high differentiation require further genetic analyses; tables 3 and 4, figures 3 and 5). for r. lamarmorae, seeds from sub-population bc were already collected for longterm storage at the bg-sar (the sardinian germplasm bank, university of cagliari). given the high differentiation detected between sub-populations bc and ss of r. lamarmorae, germplasm collection should be planned also from ss. genetic studies on the eastern part of the gennargentu massif are also advisable, because sub-populations from this area might show genetic differentiation as high as that found in the two r. lamarmorae subpopulations analysed here. finally, more detailed studies 23genetic diversity and structure of ruta corsica and ruta lamarmorae on the reproductive biology and dispersal ability of these species are fundamental for planning specific, and thus potentially successful, conservation programs and guarantee their long-term survival. acknowledgements m. m. and the project were funded by the swiss national science foundation (snsf) pmpdp3_129170. participation to a conference was funded by the claraz schenkung. this work is part of the doctoral thesis of c.a.d., funded by the biodiversity conservation center (university of cagliari). declaration of interest the authors declare that they have no conflict of interest. references alvarez w. 1972. rotation of corsica-sardinia microplate. nature-phys sci. 235:103–105. alvarez w. 1976. a former continuation of alps. geol soc am bull. 87:891–896. alvarez w, cocozza t, wezel fc. 1974. fragmentation of alpine orogenic belt by microplate dispersal. nature 248:309–314. arrigoni pv. 1983. aspetti corologici della flora sarda. lav soc ital biogeogr. 8:81–109. bacchetta g, pontecorvo cr. 2005. contribution to the knowledge of the endemic vascular flora of iglesiente (sw sardinia-italy). candollea 60:481-501. bacchetta g, brullo s, giusso del galdo g. 2006. ruta lamarmorae (rutaceae), a new species from sardinia. edinb j bot. 63:153–160. bacchetta g, coppi a, pontecorvo c, selvi f. 2008. systematics, phylogenetic relationships and conservation of the taxa of anchusa (boraginaceae) endemic to sardinia (italy). syst biodivers.:161–174. bacchetta g, fenu g, mattana e. 2012. a checklist of the exclusive vascular flora of sardinia with priority rankings for conservation. an jardín bot madrid 69:61–69. bacchetta g, fenu g, mattana e, zecca g, grassi f, casazza g, minuto l. 2011. genetic variability of the narrow endemic rhamnus persicifolia moris (rhamnaceae) and its implications for conservation. biochem syst ecol. 39:477–484. blondel j, aronson j, bodiou jy, boeuf g. 2010. the mediterranean region: biological diversity in space and time. 2nd ed. new york (ny): oxford university press. bouzat jl. 2010. conservation genetics of population bottlenecks: the role of chance, selection, and history. conserv genet. 11:463–478. cañadas em, fenu g, penas j, lorite j, mattana e, bacchetta g. 2014. hotspots within hotspots: endemic plant richness, environmental drivers, and implications for conservation. biol conserv. 170:282–291. cardona ma, contandriopoulos j. 1979. endemism and evolution in the islands of the western mediterranean (p. 133–169). plants and islands. london: academic press. cherchi a, montadert l. 1982. oligo-miocene rift of sardinia and the early history of the western mediterranean basin. nature 298:736–739. contandriopoulos j. 1957. caryologie et localisation des espèces végétales endémiques de la corse. bull soc bot france 104:53–55. conti f, abbate g, alessandrini a, blasi c. 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programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 phagocytic events, associated lipid peroxidation 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annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 73(3): 3-12, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-275 citation: n. atazadeh, m. sheidai, f. attar, f. koohdar (2020) species delimitation in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f.: morphometry and molecular analysis. caryologia 73(3): 3-12. doi: 10.13128/ caryologia-275 received: may 30, 2019 accepted: april 05, 2020 published: december 31, 2020 copyright: © 2020 n. atazadeh, m. sheidai, f. attar, f. koohdar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. species delimitation in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f.: morphometry and molecular analysis neda atazadeh1,*, masoud sheidai1, farideh attar2, fahimeh koohdar1 1 faculty of life sciences & biotechnology, shahid beheshti university, evin, tehran, iran 2 central herbarium of tehran university, school of biology, college of science, university of tehran, p.o. box: 14155, tehran, iran * corresponding author: email: atazadeh_neda@yahoo.com abstract. the genus cousinia of the tribe cardueae with about 700 species is one of the most diverse genera in central and southwest asia. the section cynaroides with 89 species is the largest section of the genus. due to the controversy in the number of cousinia species and their delineation, the first step in studying the genus is to identify and delimit presumed species. species delimitation is usually difficult in the species with overlaps in their morphological features. therefore, we used a combination of morphological and molecular markers (issrs) to carry out delimitation in 204 taxa of 68 cousinia species whitin the cynaroideae and platyacanthae sections. the species delineation based on morphometry and issr data were done by upgma clustering. the samples of each species were placed close to each other and formed a single sub-cluster, separated from the other studied cousinia species. in the present study the studied cousinia species within cynaroideae and platyacanthae sections could be delimited from each other based on issr and morphological data. therefore, using issr and morphological data can be useful in identifying and delineating crucial species. the mantel test performed between morphological distance and nei genetic distance produced non-significant correlation. this result also supports distance analyses of the trees and reveals that the two dendrograms are not correlated. some possible reasons for this incongruence are proposed: the high number of taxa in the genus cousinia, morphological traits homoplasious, convergent evolution and incomplete lineage sorting. keywords: cousinia, cynaroideae, issr, morphometry. introduction the genus cousinia cass. of the tribe cardueae (family asteraceae) with about 700 species is one of the most diverse genera in central and southwest (sw) asia after senecio l. (c. 1500 species) and vernonia schreb. (c. 1000 species) (tscherneva 1962; rechinger 1972, 1979; frodin 2004; attar and 4 neda atazadeh, masoud sheidai, farideh attar, fahimeh koohdar ghahreman 2006; susanna and garcia-jacas 2006; attar and djavadi 2010; mehregan and assadi 2016; minaeifar et al. 2016; rastegar et al. 2017, 2018). due to the extensive morphological variability in the genus, cousinia taxonomy is complicated and controversial (mabberley 1990; haffner 2000; susanna et al. 2003). the genus cousinia contains more than 400 species in sw asia, with the highest number of species in the flora iranica area, out of which 379 are endemic. these species are distributed in mountainous regions of iran, afghanistan and turkmenistan (rechinger 1986; knapp 1987). although the exact number of cousinia species in iran is still unknown, about 270 species have been reported till now (assadi 2009; attar and djavadi 2010). out of these, nearly 200 endemic cousinia species occur in iran (djavadi et al. 2007; zare et al. 2013). the cousinia species are distributed in 70 sections (rechinger 1986). the section cynaroideae bunge with 89 species is the largest section of the genus and contains irano-turkestanian elements (tscherneva 1962; rechinger 1972, 1979; hubermorath 1975; attar and djavadi 2010; rastegar et al. 2017, 2018). this sect. includes those species consisting of decurrent leaves and appendiculate bracts (tscherneva 1962; rechinger 1972, 1979; hubermorath 1975). iran with 77 taxa, of which 66 are endemic, seems to be the centre of diversity of the section cynaroideae (attar and ghahreman 2006). the section platyacanthae rech. f. has 6 species in flora iranica of which 5 species are endemic in iran (rechinger 1972). due to the controversy in the number of cousinia species and their delineation, the first step in studying the genus is to identify and delimit presumed species. species delimitation is usually difficult in the species with overlaps in their morphological features (wiens 2007). in such cases, combined morphological and molecular data have been used to delimit these taxonomic identities (duminil and di michele 2009; minaeifar et al. 2016; hassanpour et al. 2018; eftekharian et al. 2018). different molecular markers have been used in plant taxonomy and phylogeny, but some of them such as intersimple sequence repeats (issrs) seems to be very efficient in delineating species, varieties, ecotype and even genotypes of a single species (see for example, sheidai et al. 2012, 2013; safaei et al. 2016; eftekharian et al. 2018). therefore, we used a combination of morphological and molecular markers (issrs) to carry out cousinia species delimitation in sections cynaroideae and platyacanthae. materials and methods plant material the data investigated and discussed in the present study are based on 204 samples of 68 species in of the sections cynaroideae and platyacanthae. sixty-three species (189 specimens) of cynaroideae and five species (15 specimens) of platyacanthae were selected. the plant samples were collected from iran (table 1). the vouchtable 1. investigated cousinia species and their voucher information. no taxa section locality voucher no. 1 c. keredjensis bornm. & gauba cynaroides bunge tehran 21807(tuh) 2 c. elwendensis bornm. cynaroides bunge hamadan-alvand mountains 20566(tuh) 3 c. grandis c. a. mey. cynaroides bunge azarbaijan 21343(tuh) 4 c. disfulensis bornm. cynaroides bunge lorestankhorram abad 27589(tuh) 5 c. bornmulleri c. winkl. cynaroides bunge esfahan 22532(tuh) 6 c. behboudiana rech. f. & esfand. cynaroides bunge ghazvin 27629(tuh) 7 c. inflata boiss. & hausskn. cynaroides bunge kurdestan 39552(tuh) 8 c. eriocephala boiss. & hausskn. cynaroides bunge azarbaijan 22442(tuh) 9 c. calocephala jaub. & spach cynaroides bunge azarbaijan-mianeh 46276(tuh) 10 c. farsistanica bornm. cynaroides bunge kerman 28636(tuh) 11 c. jaccobsii rech. f. cynaroides bunge ilam 22370(tuh) 12 c. denaensis attar & djavadi cynaroides bunge boyer-ahmad 22495(tuh) 13 c. concinna boiss. & hausskn. cynaroides bunge kurdestan 20562(tuh) 14 c. grantii rech. f. cynaroides bunge azarbaijan 22490(tuh) 15 c. bobeckii rech. f. cynaroides bunge ardabil 46221(tuh) 16 c. barbeyi c. winkl. cynaroides bunge boyer-ahmad 22494(tuh) 17 c. kirrindica bornm. & rech. f. cynaroides bunge ilam 19711(tuh) 5species delimitation in the genus cousinia cass. no taxa section locality voucher no. 18 c. khorramabadensis bornm. cynaroides bunge lorestan 21851(tuh) 19 c. lactiflora rech. f. cynaroides bunge lorestan 46299(tuh) 20 c. phyllocephala bornm. & gauba cynaroides bunge lorestankhorram abad 46292(tuh) 21 c. lurorum bornm. cynaroides bunge kermanshahmahidasht 20568(tuh) 22 c. verbascifolia bunge cynaroides bunge khorasan-mashhad 43013(tuh) 23 c. monocephala bunge cynaroides bunge khorasanghouchan 21931(tuh) 24 c. shebliensis ghahreman cynaroides bunge azarbaijantabriz 20580(tuh) 25 c. millefontana rech. f. cynaroides bunge kurdestan-marivan 20227(tuh) 26 c. sanandajensis rech. f. cynaroides bunge hamadan 46287(tuh) 27 c. zardkuhensis attar & ghahreman cynaroides bunge chahar mahal& bakhtiari 21887(tuh) 28 c. pergamacea boiss. & hausskn. cynaroides bunge kurdestan 22571(tuh) 29 c. macrocephala c. a. mey. cynaroides bunge ardebilmeshkin shahr 42925(tuh) 30 c. onopordioides ledeb. cynaroides bunge khorasan: kashmar 28685(tuh) 31 c. aligudarzensis attar & ghahreman cynaroides bunge lorestan-aligudarz 27613(tuh) 32 c. dalahuensis attar & ghahreman cynaroides bunge kermanshahmahidasht 19929(tuh) 33 c. carolihenrici attar & ghahreman cynaroides bunge kurdestan 22455 (tuh) 34 c. khansarica attar & ghahreman cynaroides bunge esfahan: khansar 20037(tuh) 35 c. lurestanica attar & djavadi cynaroides bunge lorestan 21824(tuh) 36 c. parsana ghahreman cynaroides bunge hamadan 20553(tuh) 37 c. pasargadensis attar cynaroides bunge fars: dashte arjan 36294(tuh) 38 c. perspolitana attar & ghahreman cynaroides bunge fars: abadeh 22509(tuh) 39 c. silvanica attar cynaroides bunge w azarbaijan: urmie 24064(tuh) 40 c. shulabadensis attar & ghahreman cynaroides bunge lorestanshul abad 21874(tuh) 41 c. algurdina rech. f. cynaroides bunge azarbaijantabriz 30533(tuh) 42 c. mobayenii ghahreman & attar cynaroides bunge kermanshaheslamabad 20569(tuh) 43 c. sabalanica attar cynaroides bunge ardebil 22570(tuh) 44 c. kurdistanica attar cynaroides bunge kurdestanmaryvan 3232(tuh) 45 c. gaharensis attar & djavadi cynaroides bunge lorestanshulabad 38259(tuh) 46 c. kermanshahensis attar cynaroides bunge kermanshah: eslam-abad 19810(tuh) 47 c. fursei rech. f. cynaroides bunge kurdestan-marivan 18314(tuh) 48 c. chlorosphaera bornm. cynaroides bunge chahar mahal& bakhtiari: soreshjan 26244(tuh) 49 c. cynaroides c. a. mey cynaroides bunge ardebil 22581(tuh) 50 c. gilliatii rech. f. cynaroides bunge azarbaijan 21967(tuh) 51 c. iranica c. winkl. & strauss. cynaroides bunge arak 21881(tuh) 52 c. kotschyi boiss. cynaroides bunge azarbaijan 46244(tuh) 53 c. kopikaradaghensis rech. f. cynaroides bunge kurdestan: saqqez (tuh) 54 c. sagittata c. winkl. & strauss. cynaroides bunge arak 21822(tuh) 55 c. nana attar cynaroides bunge arak 14347(tuh) 56 c. sahandica attar & djavadi cynaroides bunge azarbaijan 46272(tuh) 57 c. lordeganensis mehregan cynaroides bunge chahar mahal& bakhtiari 46301(tuh) 58 c. hamadanensis rech. f. cynaroides bunge hamadanmalayer 46290(tuh) 59 c. subinflata bornm. cynaroides bunge kermanshah (tuh) 60 c. kornhuberi heimerl cynaroides bunge hamadan 22372(tuh) 61 c. sardashtensis rech. f. cynaroides bunge chahar mahal& bakhtiari 20073(tuh) 62 c. sefidiana rech. f. cynaroides bunge lorestan 21861(tuh) 63 c. platyacantha bunge platyacanthae rech. f. khorasan 43212(tuh) 64 c. freynii bornm. platyacanthae rech. f. semnanshahrud 27675(tuh) 65 c. reshingerorum bornm. platyacanthae rech. f. khorasan-torbate jam 39729(tuh) 66 c. bienerti bunge platyacanthae rech. f. khorasan-neyshabur 28682(tuh) 67 c. trachyphyllaria bornm. & rech. f. platyacanthae rech. f. khorasanghouchan 21932(tuh) 68 c. ecbatanensis bornm. cynaroides bunge hamadan 22371(tuh) 6 neda atazadeh, masoud sheidai, farideh attar, fahimeh koohdar er specimens have been deposited in the herbarium of tehran university (tuh) (table 1). dna extraction and pcr amplification total genomic dna was extracted from leaf tissue using protocol of the ctab-activated charcoal and poly venyl pyrrolidone (pvp) method (murray and thompson 1980). quality of extracted dna was examined by running on 0.8% agarose gels. each 20 ml pcr mixture contained 10 ml of 2_ pcr buffer, 0.5 mm of each primer, 200 mm of each dntp, 1 unit of taq dna polymerase (bioron, ludwigschafen, germany), and 1 ml of template genomic dna at 20 ng mle1. the pcr amplification program was performed in a techne thermocycler (germany) with the following program: 5 min at 94 °c, followed by 45 cycles of 30 s at 94 °c, 30 s at 54.6 °c, and 2 min at 72 °c, with a final extension step of 10 min at 72 c. the amplification products were visualized by running on 2% agarose gel, followed by ethidium bromide staining. the fragments size was estimated by using a 100-bp molecular size ladder (fermentas, germany). the experiment was replicated 3 times and constant issr bands were used for further analyses. ten issr primers, ubc 807, ubc 810, ubc 811, ubc 834, cag(ga)7, (ca)7ac, (ca)7at, (ca)7gt (ga)9a, and (ga)9t, commercialized by the university of british columbia, were used (godwin et al. 1997). morphological analysis in total, 19 morphological characters (quantitative and qualitative) were studied (table 2). morphological characters were coded accordingly. data were standardized (mean = 0, variance = 1) and used for multivariate analyses. upgma (unweighted paired group using average), and ward (minimum spherical variance) clustering based on euclidean distance and gower distances as well as principal coordinate analysis (pcoa) and multidimensional scaling (mds) methods were used for grouping of the species. principal components analysis (pca) was used to identify the most variable morphological characters. (podani 2000; safaei et al. 2016). data analyses were performed by past ver. 2.17 (hammer et al. 2012). molecular analysis the obtained issr bands were treated as binary characters (presence = 1, absence = 0). the number of table 2. morphological characters and their code. character code head diameter x<3 3≤x≤6 x>6 flower number x<80 80≤x≤150 x>150 bracts number x<80 80≤x≤120 x>120 appendages length of median bracts x<9 9≤x≤15 x>15 appendages width of median bracts x< 5 5≤x≤15 x>15 crolla length x< 20 20≤x≤25 x>25 habitate woodland alpine stepp leaves indumentum present absent stem leaves interruptedly decurrent countinuously decurrent undecurrent uppermost leaves distant from the head close to the head surrounding the head appendages present absent inner bracts indumentum smooth scabrous position of median bracts imbricated spreading recurved spreading-recurved imbricatedspreading appendages shape of median bracts sagitate triangular rhombic ovate lanceolate appendages margin of median bracts smooth 1-2 spins spinose receptacle bristles smooth scabrous corolla color yellow pink purple white ratio limb to anther tube longer shorter as long as anther tube color yellow pink purple white 7species delimitation in the genus cousinia cass. private bands versus common bands was determined. the genetic diversity parameters like nei’s gene diversity (h), shannon information index (i), number of ef fective a lleles, and percentage of poly morphism (freeland et al. 2011) were determined for each population. nei’s genetic distance was used for clustering (weising et al. 2005). neighbor joining (nj) and upgma (unweighted paired group using average) clustering were used for the species grouping after 100 times bootstrapping/permutations (freeland et al. 2011). the consensus tree was constructed from the obtained morphological and issr trees. similarly, tree distance was estimated accordingly. the mantel test between dendrograms was performed to check their agreement. past ver. 2.17 (hammer et al. 2012) and darwin ver. 5 (perrier & jacquemoud-collet 2006) programs were used for these analyses. amova (analysis of molecular variance) (with 1000 permutations) as implemented in genalex 6.4 (peakall and smouse 2006) was used to determine species genetic differentiation. gene flow was determined by: (1) calculating nm an estimate of gene flow from gst by popgene ver. 1.32 (1997) as: nm 1 ⁄4 0.5(1 e gst)/gst, (2) reticulation analysis that is based on the least square method as performed in t-rex (boc et al. 2012). results morphometry upgma dendrogram of the studied cousinia species based on morphological characters (figure 1) placed the studied samples of most of the species together and in a separate sub-cluster. this indicates that cousinia species can be differentiated by the used morphological features. upgma dendrogram also separated cousinia species of the two sections cynaroideae and platyacanthae.therefore, the morphological characters studied can delimit these sections too. pca analysis of morphological characters revealed that the first two pca components comprised about 79% of total variation. morphological characters like shape and length of the appendages of the median bracts, diameter of the heads, the no. of flowers and length of the corolla had the highest value of correlation with these components and are the most variable morphological features among the studied plants. in fact, these morphological features are of ta xonomic value in the two sections cynaroideae and platyacanthae. issr assay the used issr primers produced 36 reproducible bands/loci, out of which only 1 band was monomorphic, while the others were polymorphic bands. the highest number of issr bands occurred in c. keredjensis bornm. & gauba (20), while c. cynaroides c. a. mey had the lowest number of bands (5). a single private issr band occurred in c. keredjensis, while the other bands were common among the cousinia species. discriminating power of issr loci as determined by gst against nm (migration) analysis (table 3), revealed that almost all issr loci have excellent discriminating power (>0.95). therefore, issr markers are efficient in differentiating cousinia species studied. the highest value for nei genetic distance (0.87) occurred between c. bienerti bunge and c. elwendensis bornm., followed by c. freynii bornm. and c. elwendensis (0.81). similarly, the lowest value for the same (0.02) was observed between c. reshingerorum bornm. and c. bienerti. upgma dendrogram of the studied cousinia species based on issr data (figure 2) separated these species in distinct sub-clusters. therefore, issr molecular markers can be used in taxonomy of the genus. these molecular markers can also differentiate two sections of cynaroideae and platyacanthae. amova produced significant genetic difference among the studied cousinia species (p = 0.001), which indicates that the studied species are genetically differentiated. amova revealed that 99% of total genetic difference was due to among species genetic differentiation, while 1% was due to within species genetic variability. the species relationship illustrated by upgma dendrograms based on morphological features and molecular data were not congruent. it was also illustrated in the consensus tree of these dendrograms (figure 3). this tree revealed that only in some cases the studied cousinia species show the same relationship in both morphological and molecular trees. for instance, c. zardkuhensis attar & ghahreman (no. 27 in figure 3) and c. chlorosphaera bornm. (no. 48 in figure 3) were placed close to each other. the same applied for for c. platyacantha bunge (no. 63 in figure 3) and c. freynii (no. 64 in figure 3). similarly, three species of c. reshingerorum, c. bienerti and c. trachyphyllaria bornm. & rech. f. (no. 65-67 in figure 3) formed a distinct cluster in the obtained consensus tree. the rest of cousinia species studied were placed together in an unresolved cluster. this means that, their relationship is differently pictured in the obtained morphological and molecular dendrograms. tree distance between the obtained morpho8 neda atazadeh, masoud sheidai, farideh attar, fahimeh koohdar logical and issr dendrograms after adjusting the edges in each dendrogram was 0.36. similarly, comparison of these two dendrograms based on quartet tree distance method, produced 0.64 difference. both these results indicate that morphological relationship of the studied cousinia species, differed in great extent with issr based species relationship. the performed mantel test between morphological distance and nei genetic distance produced non-significant correlation (correlation r = 0.06, p = 0.113). this indicates that morphological divergence in the studied species is not correlated with genetic distance. discussion as mentioned by the authors, taxonomy and molecular phylogeny of the genus cousinia is complicated and unresolved mainly due to disagreement between the morphological and molecular phylogenetic studies (see figure 1. upgma dendrogram of the studied cousinia species based on morphological data. (the specie 1-68 are according to table 1). 9species delimitation in the genus cousinia cass. for example, sausana et al. 2003; lopez-vinyallonga et al. 2009). moreover, several overlapping morphological characteristics at the species level makes the species identification and delineation difficult (attar and djavadi 2010). in the present study, we could delimit the studied cousinia species based on both the used morphological and molecular data. we suggest that certain morphological characters like shape and the length of the appendages of the median bracts, diameter of the heads, the no. of flowers and the length of the corolla are taxonomically useful at the species level. interesting enough, the both sections cynaroideae and platyacanthae are separated from each other due to the difference in traits such as stem leaves and appendages of median bracts. therefore, these characters are of more practical utility, particularly in sectional level classification in the genus cousinia. the obtained species relationship based on morphological features are in agreement with previous studies. for example, within the section platyacanthae; c. platyacanthae and c. freynii were placed close each other due to the similarity in all features except color of corolla. their close affinity to each other was also noticed by asaadi and mehregan (flora of iran 2017). similarly, c. reshingerorum, c. bienerti and c. trachyphyllaria showed morphological resemblance due to the traits such as no. of the flowers, the length of the corolla, the color of the anther tube, inner bracts, receptacle bristles, diameter of head, ratio of limb/tube. close morphological affinity among these species was also illustrated by asaadi and mehregan (flora of iran 2017). in the section cynaroideae, issr data showed genetic affinity between c. grantii rech. f. and c.grandis c. a. mey., and also between c. verbascifolia bunge and c. monocephala bunge. these species also showed morphological similarities. the same holded true for c. pergamaceae boiss. & hausskn., c. millefontana rech. f., and c. carolihenrici attar & ghahreman; as well as for c. zardkuhensis and c. chlorosphaera. issr data revealed close affinity between c. disfulensis bornm., c. jaccobsii rech. f. and c.kermanshahensis attar; which is almost in agreement with the morphological data. the same applied for c. nana attar and c. kotschyi boiss. these results are almost in agreement with the taxonomic treatment of the section cynaroideae (attar and djavadi 2010). the other studied cousinia species in the section cynaroideae differed in their affinity in genetic tree versus morphological tree. this is in agreement with results of lopez-vinyallonga et al. (2009), as they also indicated that morphological traits are highly incongruent with molecular data in arctium‐cousinia complex and considered morphological characters homoplasious. in general, various reasons were suggested for this incongruence between molecular and morphological analyses: the high number of taxa in the genus cousinia, homoplasious of the morphological traits, convergent evolution (susanna et al. 2003; lopez-vinyallonga et al. 2009), incomplete lineage sorting (zhang et al. 2015); as well as the occurrence of intermediate forms and table 3. discrimination power of issr loci in studied cousinia species. locus sample size ht hs gst nm* locus1 204 0.1107 0.0000 1.0000 0.0000 locus2 204 0.4027 0.0000 1.0000 0.0000 locus3 204 0.4931 0.0000 1.0000 0.0000 locus4 204 0.2712 0.0000 1.0000 0.0000 locus5 204 0.3893 0.0000 1.0000 0.0000 locus6 204 0.0843 0.0000 1.0000 0.0000 locus7 204 0.0571 0.0000 1.0000 0.0000 locus8 204 0.3599 0.0000 1.0000 0.0000 locus9 204 0.3270 0.0000 1.0000 0.0000 locus10 204 0.4961 0.0000 1.0000 0.0000 locus11 204 0.3655 0.0088 0.9759 0.0124 locus12 204 0.2297 0.0000 1.0000 0.0000 locus13 204 0.4650 0.0000 1.0000 0.0000 locus14 204 0.2297 0.0000 1.0000 0.0000 locus15 204 0.3270 0.0000 1.0000 0.0000 locus16 204 0.1454 0.0088 0.9394 0.0323 locus17 204 0.2712 0.0000 1.0000 0.0000 locus18 204 0.1499 0.0132 0.9118 0.0484 locus19 204 0.0394 0.0088 0.7763 0.1441 locus20 204 0.0107 0.0088 0.1791 2.2922 locus21 204 0.0571 0.0000 1.0000 0.0000 locus22 204 0.1107 0.0000 1.0000 0.0000 locus23 204 0.2712 0.0000 1.0000 0.0000 locus24 204 0.4377 0.0000 1.0000 0.0000 locus25 204 0.4983 0.0000 1.0000 0.0000 locus26 204 0.1609 0.0000 1.0000 0.0000 locus27 204 0.2297 0.0000 1.0000 0.0000 locus28 204 0.2712 0.0000 1.0000 0.0000 locus29 204 0.0843 0.0000 1.0000 0.0000 locus30 204 0.2297 0.0000 1.0000 0.0000 locus31 204 0.2076 0.0000 1.0000 0.0000 locus32 204 0.0843 0.0000 1.0000 0.0000 locus33 204 0.0290 0.0000 1.0000 0.0000 locus34 204 0.0571 0.0000 1.0000 0.0000 locus35 204 0.1609 0.0000 1.0000 0.0000 locus36 204 0.0571 0.0000 1.0000 0.0000 mean 204 0.2270 0.0013 0.9941 0.0030 nm = estimate of gene flow from gst or gcs. e.g., nm = 0.5(1 gst)/gst. 10 neda atazadeh, masoud sheidai, farideh attar, fahimeh koohdar homoploid hybrid speciation, which there is little proof to prove them (mehregan & kadereit 2009; lopez-vinyallonga et al. 2009). furthermore, the genus cousinia with its relatively young geological age (ca. 8.7 mya) and high number of taxa is thoroughly unusually exposed to speciation (lopez-vinyallong et al. 2009). this is entirely consistent with the results reported by lopez-vinyallonga et al. (2009), as they also revealed that the dominant factor in speciation of the genus cousinia is allopatric geographic speciation. these may partly justify the complexity and incongruence of the relationships in the studied species of the genus cousinia. in conclusion, along with confirmation by the published literature, the current study proved that the morphological characters and issr molecular data are useful for the correct identification and species delimitation within the cynaroideae and platyacanthae sections. both quantitative and qualitative morphological characteristics are important and suitable for the identification of species of the genus cousinia. figure 2. upgma dendrogram of the studied cousinia species based on issr data. 11species delimitation in the genus cousinia cass. references assadi m. 2009. four new species of the genus cousinia cass. 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(apiaceae). caryologia 72(3): 23-34. doi: 10.13128/caryologia-755 published: december 13, 2019 copyright: © 2019 k. gautam, r. raina. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. floral architecture, breeding system, seed biology and chromosomal studies in endangered himalayan angelica glauca edgew. (apiaceae) kamini gautam1,2,*, ravinder raina1,3 1 dr. ysp university of horticulture and forestry, solan, himachal pradesh, india 2 grassland and silvipasture management division, icar-indian grassland and fodder research institute, jhansi, uttar pradesh, india 3 amity food and agriculture foundation, amity university, noida, uttar pradesh, india *correspondence author: kaminigautam1989@gmail.com abstract. endangered angelica glauca an important medicinal plant of temperate himalaya is valued for its roots which are used to treat several diseases besides food flavouring. reproductive biology studies conducted in this species for the first time have revealed i). presence of umbels of different orders with only bisexual flowers ii). occurrence of sterile seeds (without embryo) apart from fertile ones iii). seed set in only early blooming umbels (primary and lateral-i) iv). 2n=22 chromosomes besides presence of chromosomes in a group at metaphase and anaphase-i and cytomixis in some pollen mother cells and v). extreme protoandry and cross pollination behavior (upto 95%) of the species. these observations have implications for developing any conservation plan for the species. keywords. endangered, umbel order, apiaceae, cross pollination, low seed set, embryo less seeds. introduction apiaceae an angiosperm family consists of 300-455 genera and 30003750 species worldwide (pimenov and leonov 2004) and many of these species are highly valued for being economically and medicinally important (butola and badola 2006; sher et al. 2011). the genus angelica is one of the very important genera belonging to family apiaceae and is represented by about 110-115 species worldwide and almost 87 species in asia (pimenov and leonov 2004). this genus is represented by mainly three species viz. a. glauca edgew., a. archangelica l. and a. nubigena clarke in himalayan region. a. nubigena is poorly known species found in sikkim (pimenov and kljuykov 2003) and other species i.e. a. cyclocarpa (c.norman) m. hiroe and a. oreadum diels have also been reported from indian himalaya, pakistan and afghanistan (pimenov and kljuykov 2003). 24 kamini gautam, ravinder raina angelica glauca edgew. (family: apiaceae; english name: himalayan angelica; local name: chora, chokhara & gandrayan) is an endangered perennial temperate medicinal and aromatic herb distributed in moist and shady regions of himalaya at an altitude of 20003800m amsl (iucn 1993; samant et al. 1998; chauhan 1999; butola and budola 2004; samant et al.2009) in afganistan, pakistan and india (jammu and kashmir, himachal pradesh and uttrakhand) (bisht et al. 2003; butola and budola 2004; saeed and sabir 2008; butola and vashistha 2013).valued for roots which are used to treat dismenhorrea, metorrhagia, amenhorrea, polycystic ovary syndrome, rheumatism, infantile atrophe (bisht et al. 2003; butola and samant 2006; butola and budola 2008; butola and vashistha 2013; goswami et al. 2012), also acts as stimulant, cholagogue, cardio-active, carminative, sudorfic and expectorant (csir 1985; singh and rawat 2000; bisht et al. 2003; butola and samant 2006). besides this, roots also yield essential oil used to flavour liquor and food items (nautiyal and nautiyal 2004; butola and samant 2006). due to remote distribution of a. glauca in inaccessible areas of himalaya coupled with small size of flower because of being an apiaceae member, very less work has been carried out on its reproductive biology. however, breeding behavior and reproductive biology studies are crucial for understanding plant pollinator interaction, reproductive bottlenecks as well as for developing conservation plan. therefore reproductive biology has been studied in details for the first time in this species under the present investigation. material and methods studies were carried out at shillaru (2130m amsl, 30°45’00.48’’n, 76º59’12.22’’e; district shimla, himachal pradesh, india); at shilly (1550m amsl; 30°54’30’’n, 77° 07’30’’e; district solan, himachal pradesh, india) and medicinal plants laboratory of department of forest products, college of forestry, dr. y. s. parmar university of horticulture and forestry, nauni, solan-173230 (himachal pradesh, india) during years 2013-2016. population shillaru had almost 100 plants whereas in shilly population 50 plants were present. the vegetative and floral studies were conducted as per standard literature (lawrence 1951; weberling 1989; kaufman et al. 1989). pollen-ovule ratio was studied as per cruden (1977) and pollen viability was calculated on the basis of one percent acetocarmine staining test. young floral buds, fixed in absolute alcohol, glacial acetic acid and chloroform in the ratio of 1:1:1(v:v:v) for 24 hours, washed and then stored in 70% alcohol at low temperature, were used for meiotic studies. one percent acetocarmine stain was used for chromosomal staining by usual squash method. for open pollination plants with unopened healthy umbels were tagged and left as such, whereas for assessing autogamy umbels were enclosed (figure 2a) at pre flowering stage. increase in ovary size and its transformation into fruit was taken as the basis of fruit set. seed viability was tested by topographical tetrazolium test (ttz) test and germination by petri plate method. petri-plate germination test was performed at 23 ± 2°c in growth chamber and radicle protuberances were taken as sign of germination. topographical tetrazolium test (ttz) (0.1% ph 6.0) for 48 hours after extracting seeds was conducted by soaking seeds in water, then excised to expose embryo followed by immersing in ttz solution (0.1% ph 6.0) under dark conditions for 48 hours. darkly red stained embryos were taken as viable. statistical analysis was made as per crd factorial (seed germination and viability testing under laboratory conditions), rbd factorial (seed set % in field) as well as t-test (pollination studies). statistical analysis was conducted as per gomez and gomez (1984). ocular and stage micrometer (erma, tokyo, japan) were used for micro-measurements and microscopic examination was made using olympus trinocular research microscope (model ch20ibimf, new delhi, india). results morphology and floral architecture the qualitative and quantitative features are tabulated (table 1). plants of this species are erect perennial herbs, inflorescence is compound umbel with umbels of different order i.e. primary, lateral-i, lateral-ii and lateral -iii umbels based on whether they are borne on main stem or lateral branches (figure1a, b). percentage of plants with different umbel order varied in two studied populations (table 2) and quantitative features of umbels are presented in table 3. all the morphological features of stem, roots, leaves, inflorescence, flower, fruit and seeds were similar to earlier reports except for the presence of variation in seed size and presence of seeds without embryo which are is being reported for the first time in a. glauca (figure 2b, c). phenology sprouting starts in spring season (last week of april month onwards) and continues up to june month (first week). floral buds start appearing during july first week 25floral architecture, breeding system, seed biology and chromosomal studies in angelica glauca to mid of august month. primary umbel floral buds appear first followed by buds of lateral-i, lateral-ii and lateral-iii umbels. peak flowering occurs during august and is asynchronous even among plants occurring at same niche. within a plant also, phenological events are asynchronous among primary, lateral-i and lateral-ii umbels. fruit formation commence during the last week of august completing (full maturity) by september last week. fruit shedding occurs with beginning of october month onwards and physical as well as physiological changes leading to the prennation commence with the beginning of autumn season. this inactive phase lasts upto next spring season. breeding system studies floral biology anther dehiscence (figure 1c) asynchronously starts with the opening of floral buds through longitudinal slits and continues for 2-3 days. stigma become receptive (observed by in vivo artificial pollination and resultant pollen germination) when all the anthers of a flower are shed and style reached its maximum length after protruding out of stylopodium (figure 1d). at stigma receptive stage, stylopodium is with shiny surface. period of stigma receptivity depended upon umbel order. elongated, trinucleate (at shedding stage) and bicolpate pollen show 76% to 100% (average 92.64 ± 1.71%) stainability and their number/flower vary from 17400 to 40700 (average 26128.75± 1323.41). with two ovules/ flower, pollen-ovule ratio ranged from 8700 to 20350 (average 13064.37± 661.71). number of seeds produced ranged from 334 to 1024 (average 670.27 ± 65.24) in primary umbel and 112 to 344 (average 216.64 ± 18.88) in lateral-i umbel whereas, no seed set in lateral-ii and lateral-iii umbels was observed as these umbels dry before fruit formation. a d cprimary l i l ii l i l ii l ii l iii l iii b a hg fed cb kj i l fig. 2. a. glauca a. bagging for autogamy; b & c. seed without and with embryo respectively after ttz staining; d. pollen germination on receptive stigma; e. metaphase-i (n=11, 2n=22); f. ring formation at poles at anaphase-i; g. clumping of chromosomes at metaphase-i; h. pollen mother cells showing cytomixis i. decussate and tetrahedral tetrads; j. isobilateral tetrads; k. trinucleate pollen grains; l. seed viability through ttz test. fig. 1. a. glauca a. arrangement of umbel orders primary, lateral-i (l-i), lateral –ii (l-ii), lateral –iii (l-iii); b. schematic representation of a; c. anther dehiscence stage; d. protruding style at receptive stage. 26 kamini gautam, ravinder raina chromosomal studies in most of the pollen mother cells, the bivalent upto metaphase stage appeared to be clumped together without clear separation (figure 2g), however, some cells with separate 11 bivalents were also observed (figure 2e). anaphase-i was interesting as chromosomes at each pole were present in groups forming ring like structure and the number of chromosome in each group at each pole was 11 (figure 2f ). in 7-8% pollen mother cells, cytomixis (figure 2h) was observed, however other abnormalities like laggards, bridges, etc. were absent. pollen grains were trinucleate at pollen shedding stage (figure 2k). breeding system floral visitors like bees, flies, beetles, butterflies and ants were observed visiting its flowers. open pollination resulted in 670.27 ± 65.24 seeds/primary umbel and under autogamous conditions, only 21.82 ± 15.36 seeds/ primary umbel were set (table 4). based on the average number of flowers per primary umbels (591 ± 38.96) with two ovules per flower, 56.71 ± 5.52% seed set under open pollination conditions and 2.63 ± 1.82% under autogamous conditions was observed. out of is 56.71 ± 5.52% seed set under open conditions, after microscopic examinations, 27.49 ± 2.67% of such seeds was with embryo with the rest (29.22 ± 2.84%) being withtable 1. qualitative and quantitative morphological features of a. glauca. plant part qualitative quantitative habit and habitat erect perennial temperate and alpine herb. stem erect, cylindrical, hollow inside, smooth and swollen at nodes. covered with white powder and variously colored (entire shoot purple; purple upto middle and green at top or opposite; to green with purple patches). l: 172.67 ± 6.36 cm roots perennial consisting of tuberous roots, pale yellow to yellowish brown, surface smooth or wrinkled and occasionally tap root splits into two near collar region. tap l: 18.90 ± 1.66 cm b: 13.56 ± 0.97 mm secondary l:12.53 ± 1.23 cm b: 1.40 ± 0.36 mm leaves large, petiolated, tripinnate, alternate, with very long rachis. petiole base sheathing. leaflets: lance-ovate to ovate, tip narrowly-acute to acute, base cuneate, margin irregularly toothed and reticulate venation. adaxial surface of leaflets dark green and smooth. abaxial surface grayish white and smooth. cauline leaves/ plant: 5-12 inflorescence compound umbel with umbels of different orders. umbels/plant: 2 –9 involucre bracts 6-10 in number, linear and green colored. l: 2.73 ± 0.27 cm bracts 4-11 in number, linear and green colored. l: 1.72 ± 0.17 cm flower bisexual, pedicillate, epigynous, actinomorphic and pentamerous. spread: 3.39 ± 0.08 mm pedicel green colored, length decrease from peripheral towards the centre. calyx absent or obsolete. corolla petals five, free, valvate, obovate with inward curved tip, green in bud stage and whiter on maturity. l: 2.08 ± 0.03 mm b: 1.52 ± 0.07 mm androecium stamens five, green colored, bilobed, dorsifixed, exerted, alternate to petals, dehisce by longitudinal slits and filaments green colored. anthers remain bend inwards in bud stage and spread outwards at maturity. filament l: 3.15 ± 0.09 mm anther lobe l: 0.97 ± 0.02 mm anther lobe b: 0.77 ± 0.01 mm gynoecium ovary inferior, bicarpillary syncarpous, bilocular bearing single solitary ovule in each locule and placentation apical. style bifid, erect, white coloured and attain full development after anther dehiscence. stylar base swollen to form stylopodium. ovary l: 1.30 ± 0.07 mm b: 1.72 ± 0.06 mm style l:1.67 ± 0.06 mm ovule size: 0.65 ± 0.04 × 0.29 ± 0.01 mm fruit fruit mericarp, green colored, oblong, smooth, flat, pale white to brown, on maturity divides longitudinally into two halves joined with the help of carpophores bearing a single seed in each half. l: 1.66 ± 0.05 cm seed flat, pale whitish to brown, with five ridges, two lateral ridges form oblong membranous wings that surrounds the seed, wing color pale white or brown. small seed l: 0.59 ± 0.03 cm medium seed l: 0.94 ± 0.02 cm large seed l: 1.34 ± 0.04 cm floral formula ⚥ , ⨁, k 0 or obsolete, c5, a5, g (2) l: length; b: breadth. 27floral architecture, breeding system, seed biology and chromosomal studies in angelica glauca table 2. percentage of plants with different umbel order in a. glauca: population primary primary + laterali primary + lateral -i + lateral -ii primary + lateral -i + lateral -ii+ lateraliii shillaru 100% 24% 72% 4% shilly 100% 60% 40% 0 % table 3. quantitative features of umbels of different order in a. glauca: umbel order characters primary lateral-i lateral-ii lateral-iii number per plant 1 2-4 0-5 0-1 diameter 15.56 ± 0.52 cm × 15.6 ± 0.53 cm 11.59 ± 0.80 × 11.53 ± 0.81 cm 3.93 ± 0.21 cm × 3.93 ± 0.21 cm it was observed to be simple umbel, with upto 10 flowers, very weak and dried later on before blooming. umbelet number 18.50 ± 0.86 18.05 ± 0.85 15.08 ± 1.22 number of flowers 591 ± 38.96 539.35 ± 16.92 247.47 ± 31.09 diameter of peripheral umbelet 3.05 ± 0.07 cm × 3.05 ± 0.07 cm 2.32 cm ± 0.13 × 2.28 ± 0.14 cm 0.75 ± 0.08 cm × 0.75 ± 0.08 cm diameter of central umbelet 2.43 ± 0.06 cm × 2.42 ± 0.06 cm 1.61 ± 0.16 cm × 1.61 ± 0.16 cm 0.43 ± 0.03 cm × 0.43 ± 0.03 cm number of flowers in peripheral umbelets 33.65 ± 0.91 30.95 ± 1.01 16.05 ± 1.15 number of flowers in central umbelets 24.90 ± 0.91 20.8 ±0.89 10.9 ± 1.03 cm length of peripheral rays 8.2 ± 0.45 cm 5 ± 0.29 cm 1.17 ± 0.80 cm length of central rays 5.2 ± 0.35 cm 3.01 ± 0.26 cm 0.64 ± 0.05 cm length of flower stalk in peripheral flowers of peripheral umbelets 1.09 ± 0.07 cm 0.85 ± 0.04 cm 0.25 ± 0.02 cm length of flower stalk in central flowers of peripheral umbelets 0.48 ± 0.04 cm 0.32 ± 0.03 cm 0.16 ± 0.02 cm length of flower stalk in peripheral flowers of central umbelets 0.73 ± 0.04 cm 0.54 ± 0.03 cm 0.1 cm length of flower stalk in central flowers of central umbelets 0.36± 0.03 cm 0.19± 0.02 cm central flowers were underdeveloped table 4. impact of different pollination methods on seed set and viability in primary umbel of a. glauca: pollination conditions observations – primary umbel average number of seeds** per umbel* total seed** set* % seed set % (with embryo)* seed set % (without embryo)* seed viability %*** 100 seed# weight grams*with embryo without embryo open pollination 670.27 ± 65.24 56.71 ± 5.52 27.49 ± 2.67 29.22 ± 2.84 100 0.00 1.20 ± 0.44 g self pollination 21.82 ± 15.36 2.63 ± 1.82 1.27 ± 0.88 1.36 ± 0.93 100 0.00 0.85 ± 0.06 g t calculated value 9.22 8.87 8.87 8.87 4.19 * statistically significant. **on the basis of number of ovules involved in study. refers to all these structures that appeared to be like seed (with or without embryo). *** refers to seeds with embryo only. seeds without embryo did not show any positive viability due to absence of embryo. # refers to all these structures that appeared to be like seed (with or without embryo). 28 kamini gautam, ravinder raina out embryo. similarly out of the 2.63 ± 1.82% seed set under autogamous conditions, 1.27 ± 0.88% seed was with embryo with the rest 1.36 ± 0.93% without embryo respectively. 100 seed test weight under open pollination (1.20 ± 0.44 g) was statistically higher to 0.85 ± 0.06 g under autogamous pollination. ttz test revealed 100% seed viability (figure 2l) in seeds with embryo in both open as well as autogamous conditions and on the contrary none of the seed without embryo was found to be viable. seed biology seed size differences in seed size were noticed and were categorized into i). small, ii). medium and iii). large seeds (table 5). seed set percentage in different umbel orders of a. glauca 670.27 ± 65.24 seeds were obtained in primary umbel which was statistically higher than 216.64 ± 18.88 obtained in lateral-i umbels thus, seed set percentage was 56.71 ± 5.52% in primary and 20.08 ± 1.75% in lateral-i umbel. out of these only 27.49 ± 2.67% and 6.53 ± 0.57% with seeds with embryo were present in primary and lateral-i umbel respectively which was statistically significant. 100% viability in seed with embryo was observed irrespective of umbel order by ttz test. seed set by primary umbel had statistically significant 100 test seed weight (1.20± 0.44 g) as compared to 0.94 ± 0.28 g in lateral-i umbel (table 6). seeds (with embryo) set percentage as influenced by location, umbel order and position within umbel in shillaru population (2130 m amsl, district shimla, hp, india), statistically non-significant difference in percentage of seed with embryo among primary and lateral-i umbels as well as among peripheral and central regions of these umbel orders was observed (table 7). 53.80% (maximum) seeds with embryo were observed in peripheral regions of primary umbels and 32.38% (minimum) in central region of lateral-i umbels which was however statistically non significant. on overall basis, 48.47% seeds with embryo were obtained in primary and 32.52% in lateral-i umbel (table 7). in shilly population (1550 m amsl, district solan, hp, india), maximum (66.18%) seeds with embryo were obtained in central region of primary umbel and minimum (36.68%) in central region of lateral-i umbel which was however statistically non-significant (table 7). on overall basis maximum (56.56%) seeds with embryo were obtained in primary umbel which was statistically higher to minimum (40.11%) obtained in lateral-i umbels. maximum (51.43%) seeds with embryo were obtained in central region of umbels and minimum (45.24%) in peripheral regions of umbels which was, however statistically non-significant (table 7).amongst the two populations, on overall basis 48.34% (shilly) and 40.49% (shillaru) seeds with embryo were obtained which was statistically non-significant (table 8). table 5. different seed size classes in a. glauca. size class size of seeds (cm) 100 seed weight in grams (g) small 0.59 ± 0.03 (0.4-0.7) 0.72 ± 0.03 g medium 0.94 ± 0.02 (0.8-1.0) 1.02 ± 0.04 g large 1.34 ± 0.04 (1.1-1.6) 1.18 ± 0.07 g table 6. seed set percentage in different umbels of a. glauca: umbels observations average number of seed per umbel*# total seed set*# % seed set % (with embryo)* seed set % (without embryo)* seed viability %** 100 seed weight*** grams*with embryo without embryo primary umbel 670.27 ± 65.24 56.71 ± 5.52 27.49 ± 2.67 29.22 ± 2.84 100 0.00 1.20 ± 0.44 g lateral-i umbel 216.64 ± 18.88 20.08 ± 1.75 6.53 ± 0.57 13.55 ± 1.18 100 0.00 0.94 ± 0.28 g t calculated value 6.37 6.03 7.30 4.85 4.37 * statistically significant # on the basis of number of ovules involved in study. refers to all these structures that appeared to be like seed (with or without embryo). **refers to seeds with embryo only. seeds without embryo did not show any positive viability due to absence of embryo. *** refers to all these structures that appeared to be like seed (with or without embryo). 29floral architecture, breeding system, seed biology and chromosomal studies in angelica glauca seed size vis-à-vis percentage of seeds with embryo the large sized seed consisted of 52.12% seeds with embryo at shillaru as against 58.12% obtained at shilly, (statistically non significant). amongst the small seeds, only 37.67% (shillaru) and 39.15% (shilly) seeds were with embryo (table 9). amongst medium sized seeds 28.66% (shillaru) and 41.66% (shilly) were with embryo (table 9). there was observed no statistically significant difference amongst the two locations i.e. shilly and shillaru but significant difference in percentage of seed with embryo amongst seeds of different size class was observed at both locations (table 9) with large seeds having higher proportion of seeds with embryo (55.12%). seed germination seed size class wise, inter and intra population seed germination was conducted and shillaru (2130 m amsl, district shimla, hp, india) population gave maximum (31.00%) germination which was statistically higher (table 10). seeds of kilba (3200 m amsl, 31°31’ 18.17’’ n; 78°11’ 49.30’’e district kinnaur, hp, india) population did not germinate at all and in case of khan jungle (2300 m amsl, 30°49’ 13.40’’ n; 77°27’ 47.82’’ e, district sirmour, hp, india) population, large sized seeds gave maximum germination (24.00%) and medium seeds gave minimum germination (6.00%) (table 10). in case of jagatsukh (1982 m amsl, 32° 11’ 43.20’’ n; 77° 12’ 31.82’’ e, district kullu, hp, india) population large seeds gave maximum germination (8.00%) and small seeds did not germinate at all (table 10). in case of thandi dhar (2240 m amsl, 30° 54’ 51.42’’n; 77° 24’ 44.45’’e, district sirmour, hp, india) population, medium seeds gave maximum germination (26.67%) and large seeds gave minimum germination (8.33%) (table 10). in case of seeds from rohru forest division (2700 m amsl, 31°07’09.49’’n; 77°37’35.45’’e, district shimla, hp, india), small seeds gave maximum germination (34.00%) and large seeds minimum (13.33%) (table 10). in case of shillaru population, medium seeds gave maximum germination (48.00%) and large seeds gave minimum germination (10.00%) (table 10). on overall basis, non significant impact of seed size on seed germination was observed (table 11). table 7. percentage of seeds with embryo among different umbel order vis-à-vis umbel part in population in a. glauca. umbel order umbel part shillaru population seed set % shilly population seed set % peripheral central mean peripheral central mean primary 53.80 (47.13) 43.13 (39.15) 48.47 (43.14) 46.94 (46.21) 66.18 (58.12) 56.56 (52.16) lateral-i 32.65 (34.36) 32.38 (32.37) 32.52 (33.36) 43.55 (41.17) 36.68 (37.12) 40.11 (39.15) mean 42.23 (40.74) 37.76 (35.76) 45.24 (43.69) 51.43 (47.62) cd0.05 umbel order within umbel umbel order x within umbel ns* ns ns 12.13 ns ns values in parentheses are arc sine transformed values. * non significant. table 8. overall percentage of seeds (with embryo) comparison between two population of a. glauca. populations seed with embryo (%) shillaru 40.49% shilly 48.34% t calculated value 1.37* * non significant. table 9. percentage of seed with embryo amongst different seed size classes in a. glauca. population seed size small medium large mean shillaru 37.67 (34.96) 28.66(31.64) 52.12(46.34) 39.49(37.65) shilly 39.15(38.42) 41.66(39.90) 58.12(54.95) 46.31(44.42) mean 38.41 (36.69) 35.16(35.77) 55.12 (50.65) cd0.05 sites seed size site x size ns* 9.69 ns * non significant. values in parentheses are arc sine transformed values. 30 kamini gautam, ravinder raina discussion morphology and floral architecture the traded roots of a. glauca are sometimes adulterated by roots of pleurospermum angelicoides (wall. ex dc) benth. ex c. b. clarke and angelica archangelica l., thereby making morphological studies crucial to check the genuiness of the species. although the studied populations were of genuine a. glauca being similar in morphological features reported earlier (clarke 1885; kirtikar and basu 1984; bisht et al. 2003; nautiyal and nautiyal 2004; vashistha et al. 2006), yet with regard to the sex type present observations have established beyond doubt presence of only bisexual flowers that has been reported earlier by butola et al. (2010) also however, bisht et al. (2008) have reported a. glauca as andromonoecious (both bisexual and staminate flowers on same individual). apiaceae members exhibit diverse sexual expression with most of the species being andromonoecious, few bisexual (wild foeniculum vulgare mill.) and rest either dioecious (aciphylla or anisotome) or gynodioecious (gingidia, scandia and lignocarpa etc.) (koul et al. 1993; reuther 2013). seed size variation corresponding to test weight is being reported for the first time in this species and such variation was observed irrespective of the umbel order. however, the primary umbel followed by lateral-i umbel only set seed with almost nil seed set by lateral-ii and lateral-iii umbels. this suggests that only two former types of umbels should be targeted for seed harvest. seed size variation is also known in the apiaceae species like anethum graveolens l. and pastinaca sativa l. wherein such variations is correlated with umbel order as well as the portion of flowers within an umbel (hendrix 1984; hołubowicz and morozowska 2011). another important seed feature having implications for its reproductive fitness that has been observed is presence of seeds without embryo (thereby sterile) in the species. low seed germination in a. glauca is already known and seeds without embryo probably are the reason. low seed fertility due to the embryo less seeds may be the reason of its sporadic populations thereby leading to its rarity in nature. this is an important finding and any strategy towards sustainable utilization shall have to factor in this feature. this feature was irrespective of seed produced by different umbel order as well as pollination systems indicating physiological causes. reduced fertile seed output may have some advantages like allowing enough space for progeny to grow but limit their number. in self incompatible stevia rebaudiana bertoni, two types of sterile and fertile seeds are produced, however that is due to genetic reasons (raina et al. 2013). table 10. site wise open pollination set seed germination response vis-à-vis seed size in a. glauca. sites category small medium large mean khan jungle 20.00 (26.54) 6.00 (14.12) 24.00 (29.30) 16.67 (23.32) kilba 0.00 (0.00) 0.00 (0.00) jagatsukh 0.00 8.00 (16.37) 4.00 (8.18) thandidhar 23.33 (28.83) 26.67 (31.06) 8.33(16.72) 19.45 (25.54) rohru forest division 34.00(35.64) 13.33 (21.37) 23.67 (28.50) shillaru 35.00 (36.25) 48.00 (43.83) 10.00(18.27) 31.00 (32.79) cd0.05 1. size categories with in populations 2. between population with number of size categories 1 and 2 1 and 3 2 and 3 2 and 2 3 and 3 2.12 1.84 1.73 1.37 1.50 1.22 values in parentheses are arc sine transformed values. table 11. impact of seed size on germination percentage. seed size germination% small 22.47 (25.48) medium 20.17 (23.16) large 12.73 (20.48) cd0.05 ns* * non significant. 31floral architecture, breeding system, seed biology and chromosomal studies in angelica glauca phenology phenology of a. glauca follows the general pattern of temperate perennial herbs that undergo perennation during winter period only to sprout back after snow melting. flowering commences with the summer season with primary umbels blooming first followed by lateral-i, lateral-ii and lateral-iii umbels with peak flowering during august month. seed maturation and shedding commences from last week of august month till october. flowering is asynchronous among plants occurring in same niche and within plant too, different phenological events were asynchronous even among primary, lateral-i and lateral-ii umbels which appears to be an adaptation to limited pollinator services especially insects. vashistha et al. (2010) have also reported similar phenological events. floral biology flower of a. glauca have been observed to be protandrous with anther dehiscence beginning with the opening of floral buds that continues for 2-3 days. stigma become receptive after complete anther dehiscence that is also characterized by style extending full beyond stylopodium indicating complete intra floral dichogamy. shiny stylopodium is also indicator of stigma receptivity. late maturation of stigma coupled with elongation of style after anther dehiscence facilitate dichogamy in a. glauca and appears as an adaptation to avoid autogamy as well as inbreeding depression. protandry in a. glauca has also been reported by bisht et al. (2008). in chaerophyllum bulbosum l.(apiaceae), also styles elongates only after pollen is shed and sexual phases are clearly distinguishable indicating extreme ‘protandry’ (reuther and claßen-bockhoff 2013). foeniculum vulgare mill. other member of apiaceae, has also been found to be strongly ‘protandrous’ as pollen are released much before stigma receptivity (koul et al. 1996). as a. glauca thrives in hostile climatic condition, production of trinuclear pollen grains seems to be an adaptive feature for faster germination on stigma leading to reproductive assurance. pollen ovule ratio of 13064.37± 661.71, studied for the first time in present studies indicates the species to be an obligate outcrosser as per cruden (1977). foeniculum vulgare, another member of apiaceae, is also characterized by high pollen ovule ratio of 12005-14635 (koul et al. 1996). chromosomal studies the present gametic chromosome count of n=11 is in conformity with the previous diploid count of 2n=22 (kumar and singhal 2011) from northwest himalaya. however, grouping of bivalents at diakinensis and metaphase-i stage into a ring structure has been observed for the first time in this species. in, only few cells could clear 11 bivalents be observed at these stages. anaphase-i too was characterized by the presence of two rings of 11 chromosomes at each poles. presence of ring of 11 chromosomes at anaphase-i in a. glauca poles appears to be similar to the ‘renner’ complexes (entire haploid genomes which are inherited as single units) present in genus oenothera, wherein due to reciprocal translocations of chromosome arms, all the 14 chromosomes form two rings of seven chromosome each (greiner 2008). as has been discussed earlier, significant proportion of seeds of a. glauca were without embryo leading to reduced germination, which may be due to this meiotic anomaly. further studies on female meiosis, embryo development as well as more extensive studies on male meiosis are required to establish the consequences of meiotic anomaly in a. glauca. although meiotic abnormalities like laggards, bridges, etc. were not observed but in some pollen mother cells, cytomixis was observed in a. glauca. cytomixis often leads to abnormal meiotic behavior, variation in pollen grains size and low pollen viability or sterility e.g. in alopecurus arundinaceus poir. (koul 1990), polygonum tomentosum willd. (haroun 1995), hordeum vulgare l.(haroun 1996), brassica napus var. oleifera delile. and b. campestris var. oleifera dc (souza and pagliarini 1997), vicia faba l. (haroun et al. 2004), and meconopsis aculeate royle (singhal et al. 2008). despite chromosome arranged in rings as well as cytomixis, pollen stainability did not seem to be impacted much as it ranged from 76% to 100% in different plants of a. glauca studied. breeding system studies significant seed set under open pollination conditions in comparison to autogamous conditions established the species strongly favouring (about 95%) cross pollination. this aspect is being reported for the first time in this species. interestingly open pollination also resulted in much higher seed (with embryo) set (27.49 ± 2.67%) as compared to autogamous seed (1.27 ± 0.88%), again indicating strong presence of cross pollination. as it is generally presumed that selfing rates increase with increasing altitudes (schroter 1926; bliss 1962; garcia-camacho and totland 2009; korner and paulsen 2009) as pollinator abundance and activity become limiting factors due to hostile climatic conditions at higher altitudes (arroyo et al.1982, 2006; bingham and orthner 1998; medan et al. 2002). contrary to this view, the species under investigation (a. glauca) favours cross pollination 32 kamini gautam, ravinder raina and dichogamy seems to play a key role in its cross fertilization. eritricium nanum (vill.) schrad.ex gaudin (boraginaceae), chaetanthera renifolia  (j. remy) cabrera (asteraceae) and nardostachys grandiflora dc (valerianaceae) other temperate plant species, are also known for higher cross pollination rates at high altitudes (writh 2010; diaz et al. 2011; gautam and raina 2016). inflorescence attributes, high pollen ovule ratio and asynchronous opening of flowers in a. glauca are also evidence for its cross pollinating nature. however, low seed set in a. glauca limits, natural variation essential for genetic improvement. seed biology as has been already discussed only primary and lateral-i umbels set seeds. among these too primary umbel set significantly more (56.71 ± 5.52%) seed than lateral-i umbel (20.08 ± 1.75%). higher seed set in primary umbel in eryngium alpinum l. and carum carvi l. (family apiaceae) is already known (bouwmeester and smid 1995; gaudeul and bottraud 2004). interestingly, despite blooming, lateral-ii as well as lateral-iii umbels do not set any seed which seems due to their late development or restricted resource allocation and they seem to only for enhancing floral visibility of its plants for pollinator attraction. of the 56.71± 5.52% and 20.08 ± 1.75% seed set by primary and lateral-i umbels only27.49 ± 2.67% and 6.53 ± 0.57% seed is with embryo respectively indicating higher fertile seed production by primary umbels. this low fertile seed production in a. glauca may be the reason for generally low germination rates in this species. fruit and seed set percentage is generally low in late blooming flowers than early blooming ones (zimmerman and aide 1989), and several reasons like resource competition among the ovaries of an inflorescence (lee 1988); reduced pollen receipt by later blooming inflorescence (lee 1988); intrinsic features (berry and calvo 1991; diggle 1995) may be the reasons. non significant variations in fertile seed production between peripheral and central flowers of a umbel and also among seed from two ecologically different populations (shilly and shillaru) indicates that a. glauca as a strategy, limits fertile seed production either for nutrient resource conservation or to ensure better quality fertile seed that can produce a healthy progeny. seed germination seed germination behavior of any species impacts the genetic variability in any species and lower germination rates deprive such variation. inter population germination variation was observed amongst the six population viz. khan jungle, kilba, jagatsukh, thandidhar, rohru forest division and shillaru with shillaru population excelling others (31.00% seed germination) with no germination in seeds of kilba. apiaceae family members are known for generally low germination rates (koul et al. 1993) and in a. glauca poor and erratic germination with maximum of 8% germination is already reported (nautiyal et al. 2002; butola and budola, 2004; butola and samant 2006). present studies have revealed that low germination in a. glauca is not due to dormancy but production of seeds without embryo. conclusion only primary and lateral-i umbels set seeds as other lateral-ii and lateral-iii only attract pollinators without setting any seed. production of embryo less seeds (sterile seed) is a major reproductive bottleneck in this species. seed size variation occurs within same plant as well as within same umbel with large seeds having higher proportion of fertile seeds (with embryo). the species is strongly cross pollinating. acknowledgement this study was funded by department of biotechnology (dbt), government of india. the first author would like to acknowledge department of science and technology (dst), government of india for inspire (innovation in science pursuit for inspired research) fellowship.authors would also like to acknowledge director, hfri (himalayan forest research institute, conifer campus, panthaghati, shimla−171009, himachal pradesh, india) and dr. sandeep sharma (scientist−e, hfri) for providing necessary facilities for conducting research experiments. references arroyo mtk, munoz ms, henriquez c, bottraud ti, perez f. 2006.erratic pollination, high selfing levels and their correlates and consequences in an altitudinally widespread above tree-line species in the high andes of chile. acta oeco. 30(2): 248–257. arroyo mtk, primack r, armesto j. 1982.communitystudies in pollination ecology in the high temperate andes of central chile: pollination mechanisms and altitudinal variation. am j bot. 69(1): 82–97. 33floral architecture, breeding system, seed biology and chromosomal studies in angelica glauca berry pe, calvo rn. 1991. pollinator limitation and position dependent fruit set in the high andean orchid myrosmodes cochleare (orchidaceae). plant syst evol. 174(1): 93–101. bingham ra, orthner ar. 1998.efficient pollination of alpine plants. nature. 391(6664): 238–239. bisht ak, bhatt a, rawal rs, dhar u. 2008.assessment of reproductive potential of different populations of angelica glauca edgew: a critically endangered himalayan medicinal herb. j mt sci. 5(1): 84–90. bisht ak, manjkhola s, joshi m. 2003. comparative account of two high value species of himalayas: angelica glauca edgew. and angelica archangelica l. indian for.129 (10): 1241–1248. bliss lc.1962.adaptations of arctic and alpine plants to environmental conditions. arctic. 15(2): 117–144. bouwmeester hj,  smid hg. 1995. seed yield in caraway (carum carvi): role of pollination. j of agric sci. 124(2): 235–244. butola js and badola hk. 2006. chemical treatments to improve seedling emergence, vigour and survival in heracleum candicans wall. 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open access j med arom plants. 1(1): 7–12. weberling f.1989. morphology of flower and inflorescence. new york (usa) cambridge university press. wirth lr, graf r, gugerli f, landergott u, holderegger r. 2010. lower selfing rate at higher altitudes in the alpine plant eritrichium nanum (boraginaceae). am j bot. 97: 899–901. zimmerman j k and aide t m.1989. patterns of fruit production in a neotropical orchid: pollinator vs. resource limitation. am. j bot. 76(1):67–73. substantia an international journal of the history of chemistry vol. 2, n. 1 march 2018 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 72(1): 3-14, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-246 citation: m. khajavi, m. rahaie, a.ebrahimi (2019) the effect of tio2 and sio2 nanoparticles and salinity stress on expression of genes involved in parthenolide biosynthesis in feverfew (tanacetum parthenium l.). caryologia 72(1): 3-14. doi: 10.13128/cayologia-246 received: 30th july 2018 accepted: 24th december 2018 published: 10th may 2019 copyright: © 2019 m. khajavi, m. rahaie, a. ebrahimi. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. the effect of tio2 and sio2 nanoparticles and salinity stress on expression of genes involved in parthenolide biosynthesis in feverfew (tanacetum parthenium l.) mahshid khajavi1, mahdi rahaie2,*, asa ebrahimi1 1 department of biotechnology and plant breeding, faculty of agriculture, science and research branch of islamic azad university, tehran, iran 2 department of life science engineering, faculty of new sciences and technologies, university of tehran, tehran, iran * corresponding author: tel: +98-21-86093408; fax: +98-21-88497324; email: mrahaie@ut.ac.ir abstract. medicinal plants can produce various chemical compounds as secondary metabolites that have benefit to human. feverfew (tanacetum parthenium l.) is a medicinal plant belongs to the asteraceae family. this plant due to have parthenolide compounds has attracted much attention for medicinal value and pharmacological activity. due to the economic importance of the plant metabolite in cancer and migraine treatment, application of approaches for increasing the metabolite was the objective of this study. for this purpose, after cultivation in greenhouse, plants were treated with tio2 and sio2 nanoparticles and salinity stress at different times and concentrations. real time pcr used to evaluate the expression of tpgas, cost and tpcars genes which involved in secondary metabolites biosynthesis pathway (parthenolide and β-caryophyllen). it was found, sio2 nps can increase the expression of tpcars, cost and tpgas in the concentration of 25mm with increasing time from 6 to 24h. in this concentration (25mm), tio2 treatment, up-regulated the cost and tpgas in contrast, down-regulated the tpcars with increasing time from 6 to 24h. salinity treatment affected the expression of all three genes, so that with increasing time, the expression of all three genes was elevated. in conclusion, according to above and hplc results, it was shown the nanoparticles and salinity treatments can increase parthenolide synthesis in whole plant of feverfew and then they can be used as elicitor for more production of the metabolite. keywords. tanacetum parthenium l., nanoparticle, sio2, tio2, salinity stress, gene expression analysis. abbreviations: gas, germacrene a synthase; cost, costunolide synthase; cars, cariophyllene synthase; ptl, parthenolide; np, nanoparticle. introduction medicinal plants have a specific role in treatment and prevention of many human diseases. these plants are attracting more attention for produc4 mahshid khajavi, mahdi rahaie, asa ebrahimi ing safer medicine because it is believed that they rarely have side effects compared to chemical drugs. feverfew (tanacetum parthenium l.) (asteraceae), is a diploid (2n=2x=18) and perennial medicinal plant. this plant has medical applications on a wide range of disease such as migraine headaches, stomach aches, toothaches, insect bites, rheumatoid arthritis and infertility (pareek et al. 2011). these medicinal properties are due to the existence of chemical compounds which called secondary metabolites. terpenoids are the largest class of plant secondary metabolites (croteau et al. 2000). sesquiterpene lactones are the main group of terpenoids and frequently are derived from mevalonic acid (mva) pathway (van klink et al. 2003). t. parthenium (l.), contains many sesquiterpene lactones and parthenolide has the most concentration (comprises up to 85%) among the total sesquiterpenes (pareek et al. 2011). parthenolide is used for the treatment of migraine and shows specifically anticancer and anti-inflammatory activity, as well (tassorelli et al. 2005; walsh et al. 2011; mathema et al. 2012; al-fatlawi et al. 2015; wang and li, 2015). β-caryophyllene is another sesquiterpene lactone distributed in the essential oil of various plants. this compound has represented several biological activities, such as anti-inf lammator y, antibiotic, antioxidant (legault and pichette, 2007), anticancer (tundis et al. 2009) and antiproliferative activity (amiel et al. 2012). sesquiterpenes, like parthenolide and β-caryophyllene are synthesized via farnesyl diphosphate (fpp) in mevalonate pathway. tpgas and cost are two genes involved in parthenolide production. at the first step, tpgas converts farnesyl diphosphate to germacrene a, cost converts germacrene a acid to costunolide, and parthenolide is one of the deratives of costunolide. it has been revealed that costunolide synthase is a cytochrom p450 enzyme. tpcars is another sesquiterpene synthase in feverfew which is responsible for the production of β-caryophyllene. this enzyme converts farnesyl diphosphate to β-caryophyllene directly. (majdi et al. 2011; liu et al. 2011; menin et al. 2012; basha et al. 2016). several factors affect the production of secondary metabolites, and elicitors are one of the most efficient ones (zhao et al. 2005). plants and plant cells (in vitro culture) show the morphological, biochemical and physiological reactions to biological, chemical or physical factors, which is considered as “elicitors.” in fact, elicitation is an induced or enhanced synthesis process of secondary metabolites by the plants and it is a way to ensure their survival, persistence, and competitiveness (karuppusamy, 2009; kiong et al. 2005). elicitors are biological or nonbiological agents that cause biosynthesis and accumulation of secondary metabolites in plants through induction of defense responses (ramirez-estrada et al. 2016). the components of microbial cells and poly and oligosaccharides, chemicals such pesticides, heavy metals, and the signaling compounds in plant defense responses (growth factors, e.g. jasmonate) and physical factors such as hyperosmotic stress, uv, cold shock, ultrasound, and pulsed electric field can act as stimulators for hyperproduction of secondary metabolites (zhao et al. 2010; gueven and knorr, 2011; lin and wu, 2002). nanoparticles (nps) are new materials which show unique properties related to their physical size. the nanoparticles used in present work are sio2 and tio2 which are among the most used nanomaterials (servin et al. 2012; siddiqui and al-whaibi 2014). titania (tio2) has a wide range of applications such as cosmetics (anselmann, 2001), cancer treatment (kalbacova et al. 2008), sunscreens or food (lan et al. 2013). silica (sio2) is another popular metal oxide nps used in multiple varieties of applications such as disease labeling, drug delivery, photodynamic therapy (ohulchanskyy et al. 2007), cancer therapy (cheng et al. 2010; rosenholm et al. 2010 ), fertilizer and pest control (sakr, 2017; tripathi et al. 2014; laing et al. 2006). the effects of tio2 nps on different biological characters of the plant have been done in several studies. among them, it could be pointed out to the effect of tio2 on the growth and microrna expression profile of tobacco (frazier et al. 2014), effects of nano-tio2 on seed germination (castiglione et al. 2011), development and mitosis of root tip cells of vicia narbonensis l. and zea mays l. (castiglione et al. 2011), the effect of sio2 and tio2 nanoparticles on the expression of gpps gene (involved in thymoquinone biosynthetic pathway) in nigella sativa l. (kahila et al. 2017) and finally, mandeh et al. (2012) which studied in vitro influences of tio2 nanoparticles on barley tissue culture and quantitative and qualitative characteristics of calli were analyzed after each subculture. in the field of agriculture, sio2 has an inhibition effect on the pest (hongshu et al. 2009), carriers in drug delivery and absorption and transport of nutrients in plants (liu et al. 2006). the role of nano-sio2 in the characteristics of seed germination of tomato has also investigated (siddiqui and al-whaibi, 2014). salinity stress is a high concentration of soluble salts in soil and water. most common soil salinity is caused by high sodium (na+) and chloride (cl-) (tavakkoli et al. 2010). salt stress as an environmental factor is another factor that influences on gene expression (siddiqui and al-whaibi 2014). 5effect of tio2 and sio2 nanoparticles in tanacetum the effect of salinity on secondary metabolites in plants is various. there are different examples in this case. for example, during nacl stress in swertia chirata, significant increases ( p≤0.05) was occurred in secondary metabolites at 50mm and initial increase in 100mm nacl, which falls back to normal levels at the seventh day (abrol et al. 2012). in the present work, to evaluate the elicitation role of nanoparticles and abiotic stress on hyperproduction of parthenolide, it was investigated the effect of two nps (tio2 and sio2) and salinity stress on the expression of three related genes, tpgas, cost and tpcars that are involved in the biosynthetic pathway of ptl and β-caryophyllene in feverfew. materials and methods plant material and growth conditions the seeds of feverfew were provided by the medicinal plant institute of shahid beheshti university, tehran, iran. germinated seeds in pots were grown under controlled circumstances with a 16:8h photoperiod light/ dark, 25/18 °c for day/night and supplied with a photosynthetic photon flux density of 3000 lux (fig. 1). nanoparticles characterization the sio2 nanoparticles were purchased from tecnan inc. (tecnología navarra de nanoproductos s.l., spain). a size of 10–15 nm for nps was estimated (fig. 2). the xrd measurement clearly showed that the sio2 nps were amorphous. the elemental analysis of the nano-powder by icp-ms technique (thermo elemental vg pq-excell) showed a purity of 99.999%. the tio2 nanoparticles were provided from degussa inc., germany. the analyzed data from xrd showed ~25nm diameter and specific area equal to 55 m2g-1for the nanoparticles (ave. of 24.5 nm in diameter, a mixture of anatase and rutile with more proportion of anatase (89.2 %),). plant treatment with nps and salt the sio2 and tio2 nps, were separately prepared in two concentrations (25 and 50 mg/l) and the solution was used for watering of each pot. for each pot, 80 ml of 0.3m nacl solution was applied for doing salinity stress treatment. all tissues were collected at the certain times (6, 24 and 48h after irrigation) for gene expression analysis and phytochemical studies and were instantly flash frozen in liquid nitrogen then stored at -80°c until rna extraction. hplc analysis the chromatography assay was performed on a 25 cm×4.6 mm with pre-column, eurospher 100-5 c18 anafig. 2. the tem micrograph of sio2 nanoparticles. fig. 1. the grown plants in pots in green house under controlled condition. 6 mahshid khajavi, mahdi rahaie, asa ebrahimi lytical column provided by knauer (berline, germany) reversed phase matrix (5μm) (waters) and elution was carried out in a gradient system with acetonitrile as the organic phase (solvent a) and distilled water (solvent b) with the flow-rate of 1 ml min-1. the peaks were monitored at 220 nm wavelength. injection volume was 20 µl and the temperature was maintained at 25°c. all injections were repeated three times (n=3). calibration graphs were plotted subsequently for linear regression analysis of the peak area with concentration 1, 10, 25, 50, 80, 120, 150 and 200 mg l–1. rna extraction total rna was isolated from the leaf tissue by using the isolation total rna kit (denazist asia inc., mashhad, iran), according to the manufacturer’s instructions. the quantity and quality of extracted rna were determined respectively by using a spectrophotometer instrument (nanodrop2000c, thermo scientific, usa) and agarose gel electrophoresis. rna samples were stored at −80 °c until further analysis. gene selection and primer designing in the present work, tpgas and cost genes that involved in biosynthesis of ptl and tpcars as mediator in biosynthesis of β-caryophyllen at mevalonate pathway in feverfew (majdi et al. 2011), plus β-actin as a house keeping gene were selected and their sequences retrieved from the gene bank, ncbi (http://www. ncbi.nih.gov). four pairs of primers including β-actin (-5’agcatggtattgtgagcaact-3’, r-5’tgggtcatcttctctctgttagc-3’), tpgas (f-5’-taccagtttgagcgtgaaaga3’, r5’-caatcatgatcttgagctcgt3’),tpcars (f-5’-gagcatgtccaca a agtatttcac-3’, r-5’g catcg gaatatctttacacacag-3’) and cost (f5’gagacacaagaagaagtgagatcag-3’, r5’aaaggtgtaggagcatgta acctc-3’) were designed using primer 3 free software (http://biotools.umassmed. edu/bioapps/primer3_www.cgi). primers confirmation was performed by three criteria, including blast search in gene bank, single peak in qpcr and single band on gel electrophoresis. cdna synthesis cdna synthesis was performed by revert aid first strand cdna synthesis kit (thermo scientific, usa) with 200 u of m-mlv rt enzyme, oligo-dt and random hexamer primers according to manufacturer’s instruction. quantitative pcr quantification of cost, tpgas and tpcars genes expression levels in the samples were measured by qpcr using hot firepol evagreen qpcr master mix (solis biodyne, estonia). qpcr was performed according to manufacturer’s instruction in qiagen real-time pcr system (rotor-gene q, germany) using above primers. it should be mentioned that before qpcr, the specificity of all primers and optimization of pcr reactions was done with conventional pcr. the relative expression levels were calculated according to pfaffl method (pfaffl 2001). data statistics expression levels were calculated from the ct values obtained from triplicate biological samples. statistical significance analysis of relative gene expression level compared with the reference gene (β-actin) was performed with completely randomized design (crd). mean values of relative expression levels were compared with lsd test (p=0.05) using spss ver. 21.0 software. results in this study, the effects of tio2 and sio2 nanoparticles and salinity stress on expression of tpgas, and cost genes that are involved in the biosynthetic pathway of ptl in feverfew (fig. 3) were investigated. also change of tpcars gene expression which produces β-caryophyllen was analyzed. the present study focuses on elicitation effects of nanoparticles and salinity on parthenolide and β-caryophyllen productions in feverfew. gene expression analysis cost gene two concentrations (25 and 50mm) of sio2 np, in two periods of time (6 and 24h) were used. the results showed that, in both 25 and 50mm concentrations, the expression of cost gene was enhanced with increasing of time. while in both times, the gene expression in 25mm, was more than 50mm treatment; furthermore, the 7effect of tio2 and sio2 nanoparticles in tanacetum expression of the gene in 25mm and after 24h was very high and more than 7-folds in comparison to 6h (fig. 4a). the result of tio2 np was similar to sio2 treatment at 25mm concentration and increased the expression of cost gene. in 25mm concentration of tio2 np with increasing time from 6h to 24h, elevation of the gene expression by more than 6-fold (fig.4b) was observed. in opposite, the gene expression in 50mm of tio2 np was decreased by rising time until close to zero. as fig. 4 (a and b) shows, although the ratio of expression level for sio2 and tio2 nanoparticles in 6h/24h is nearly the same, but the absolute expression of the cost gene in tio2 treated samples (133.8 fold) is significantly more than sio2 treated plants (20.2 fold). the effect of salinity treatment on expression of cost gene were also measured; after 6h and 48h at 0.3 m concentration a gradual increasing trend (2-folds) in gene expression was observed (fig. 4c). tpgas gene tpgas is a gene involved in the parthenolide biosynthesis pathway. the expression analysis of the gene transcript pointed that, it is affected by sio2 and tio2 nps and salt stress treatment. the results represented that sio2 nps in 25mm concentration at 24h, significantly increased the expression of gas compared to 6h treatment (24.5 fold). in opposite, the tio2 nps treatment didn’t have a significant effect on the expression of the gene in both time and concentrations (fig. 5a and b). the effect of salinity stress was investigated with the same concentration used above. as shown in the fig.5c, the expression of tpgas, was statistically enhanced in 48h in comparison to 6h treatment, (~3 fold). tpcars gene tpcars is another gene which was analyzed in our experiment. the results of expression analysis showed that the gene was affected by sio2 and tio2 nps and salinity treatment, as well. as it can be seen in the fig. 6a and b, an increase (1.4 fold) and decrease (6 fold) in expression of tpcars in 25mm of sio2 and tio2 nps treatment were observed respectively, from 6h to 24h. in contrast, in the 50mm concentration of nps, no significant changes were detected during the time period. fig. 3. the suggested biosynthetic pathway for parthenolide and β-caryophyllen synthesis in feverfew (majdi et al., 2011). cars: caryophyllen sybthase; gas: germacrene a synthase; gao: germacrene a oxidase; cost: costunolide synthase; ps: parthenolide synthase. * 0 5 10 15 20 25 6h 24h r el at iv e e xp re ss io n l ev el np treatment time sio2 25mm sio2 50mm a b c * 0 20 40 60 80 100 120 140 160 6h 24h r el at iv e e xp re ss io n l ev el np treatment time tio2 25mm tio2 50mm * 0 20 40 60 80 100 120 140 160 6h 48h r el at iv e e xp re ss io n l ev el salinity treatment time * 0 5 10 15 20 25 30 35 40 6h 24h r el at iv e e pr es si on l ev el np treatmenttime sio2 25mm sio2 50mm a b c 0 5 10 15 20 25 30 35 40 45 6h 24h r el at iv e e xp re ss io n l ev el np treatment time tio2 25mm tio2 50mm * 0 20 40 60 80 100 120 140 160 180 6h 48h r el at iv e e xp re ss io n l ev el salinity treatmenttime fig. 5. the effect of elicitors on expression of tpgas gene in feverfew. a: sio2 np treatment; b: tio2 np treatment; c: salinity stress. fig. 4. the effect of elicitors on expression of cost gene in feverfew: a) sio2 np treatment; b) tio2 np treatment; c) salinity stress. 8 mahshid khajavi, mahdi rahaie, asa ebrahimi the effect of salinity stress as an environmental elicitor with 300mm of nacl in two times, including 6h and 48h was investigated. as shown in fig. 6c, the 48h salt stress elevated the expression of tpcars gene significantly (~12 fold) compared to 6h and 24h treatments. parthenolide concentrations in feverfew under different treatments to assay the parthenolide concentration in different treatments, leaves tissue were analyzed by hplc (fig. 7). there were significant differences between parthenolide concentrations (p< 0.05) in different treatments (fig. 8). the highest and least amount of parthenolide was observed in 25mm of tio2 after 24h and control plants with 378.61µg/mg and 136.02µg/mg, respectively. all treatments increased the concentration of parthenolide in feverfew leaves compared to control plants (p<0.05). the sio2 (25mm) after 6 and 24h treatments increased the leaves parthenolide content by 1.23 and 1.37 fold compared to control. the tio2 (25mm, 24h) raised the parthenolide amount in leaves far more than sio2 nps with 2.78 fold. discussion nanoparticles are a new class of men made materials which are emerging in these years. in hence, a new trend in biological sciences is toward investigation of interaction of the synthetic agents to organisms including plants. there are many reports which study the effect of them (tio2 and sio2) on plants in different kinds of morphological and molecular levels (siddiqui and al-whaibi 2014; castiglione et al. 2011; frazier et al. 2014; kahila et al. 2017), as well in vivo culture (mandeh et al. 2012). a number of studies also prove the key role of different elicitors, including chemical compounds (van fürden et al. 2005; esmaeilzadeh bahabadi et al. 2011; esmaeilzadeh bahabadi et al. 2014; * 0 0,5 1 1,5 2 2,5 3 6h 24h r el at iv e e xp re ss io n l ev el np treatment time sio2 25mm sio2 50mm a b c * 0 2 4 6 8 10 12 14 16 6h 24h r el at iv e e xp re ss io n l ev el np treatment time tio2 25 mg/l tio2 50 mg/l * 0 2 4 6 8 10 12 6h 48h r el at iv e e xp re ss io n l ev el salinity treatment time * * * 0 50 100 150 200 250 300 350 400 control sio 2 6h sio 2 24h tio2 24h pa rt he no lid e c on . ( µg /m l) treatment fig. 8. parthenolide concentration in tanacetum parthenium leaves at different treatments.fig. 6. the effect of elicitors on expression of tpcars gene in feverfew. a: sio2 np treatment; b: tio2 np treatment; c: salinity stress. fig. 7. the hplc analysis of extract from t. parthenium and 110 ppm of standard partenolide from sigma-aldrich company (usa). minutes: run time, au: absobtion intensity. 9effect of tio2 and sio2 nanoparticles in tanacetum wang et al. 2007; marsh et al. 2014), biotic (kim et al. 2001; jeong et al. 2005; savitha et al. 2006; wu et al. 2007; kang et al. 2009; gao et al. 2011; swaroopa et al. 2013; ahmed and baig. 2014) and abiotic (akula and ravishankar 2011; chan et al. 2010; szakiel et al. 2011) on metabolite production in different plants. however, nearly most of studies have been conducted on cell or hairy root culture and investigation on in vivo whole plant elicitation is limit. the aim of the present work was to determine the elicitation role of nanoparticles and salt stress on metabolite hyperproduction through gene expression in whole plant. nps and salinity treatments affect the parthenolide biosynthesis genes the effect of different kind of elicitors was investigated to find a clear view about the impact of elicitors on the expression of genes involved in the secondary metabolites biosynthesis pathway in t. parthenium. it seems that sio2 nps in low concentration can have a positive effect on parthenolide production in feverfew. in opposite to sio2, tio2 nps treatment created a different expression pattern of the tpgas, cost and tpcars genes. dimkpa et al. (2012) demonstrated the beneficial effects of cuo and zno nps on iaa production in vitro in the soil isolate p. chlororaphis o6. an increased iaa production with exposure to sublethal levels cuo nps was observed in their study. they explained an ion release and nonspecific mechanism for increased level of iaa due to cuo and zno nps treatment, respectively. the salt stress up-regulated all the mentioned genes in our study. it seems, this is an evolutionary mechanism in medicinal plant to tolerance abiotic stress with producing of metabolites including ptl compound in feverfew. a comparison between the salts stress and the nanoparticles effect on the genes and hyperproduction of parthenolide, mentioned that, the nps are more effective than salt stress, because they affected two key genes, including tpgas and cost which catalyzes two steps of parthenolide biosynthesis pathway compared to salt stress which affect only on tpcars gene. the drought and salt stresses cause common reactions in plants. cellular dehydration then osmotic stress and transfer of water from the cytoplasm to vacuoles are a result of both stresses (akula and ravishankar, 2011). salinity stress often causes both ionic and osmotic stress in plants, which increases or decreases specific secondary metabolites in plants (mahajan and tuteja, 2005). for example, parida and das (2005) found that anthocyanins are increased in response to salt stress. oppositely, daneshmand et al. (2010) demonstrated that salinity stress decreases the anthocyanin level in the salt-sensitive species. as the plant polyamines are involved in plant response to salinity, changes of free and bound polyamines levels has been reported in sunflower (helianthus annuus l.) roots under salinity stress (mutlu and bozcuk, 2007). the endogenous ja (jasmonic acid) under salt stress in tomato cultivars has been found (pedranzani et al. 2003). polyphenols are another group of metabolites which their synthesis and accumulation is usually is stimulated as a reaction to biotic or abiotic stresses (dixon and paiva, 1995; muthukumarasamy et al. 2000). in some of plants such red peppers (navarro et al. 2006); a raise in polyphenol content with increasing level of salt in different tissues has also been reported (parida and das, 2005). positive correlation between tpgas and cost expression and parthenolide concentration under nps treatments a positive correlation between tpgas and cost expression and parthenolide concentration was observed in sio2 and tio2 treatments (25mm and 24h) compared to control plants, in contrast to tpcars of which its expression had a negative correlation with increasing treatment time. although few studies have revealed the genotoxic potential of tio2 nanoparticles in the plant systems (a. cepa and n. tabacum) with dna damaging effect (ghosh et al. 2010), in this work, tio2 nanoparticles had a positive effect on parthenolide production. tpgas encodes the enzyme that highly likely catalyzes the first step in parthenolide biosynthesis, germacrene a synthase (fig.3) (majdi et al. 2011). then, it seems that the feverfew plant responds to the nps by up-regulation of the responsive genes and then metabolite production. tpcars (β-caryophyllene synthase) encodes β -caryophyllene which is an anti-inflammatory (martin et al. 1993; tambe et al. 1996) and anti-carcinogenic (kubo et al. 1996; zheng et al. 1992) compound and a common and quite widely distributed sesquiterpene in plants (knudsen et al. 1993; kubo et al. 1996). similar to artemisia annua plant (chen et al. 2011), in tanacetum parthenium (bouwmeester et al. 2002) farnesyl diphosphate (fpp) (fig.3) is an initial point and serves as a basic precursor to synthesize the various classes of sesquiterpenes with divergent structures and functions by different synthases such tpcars through competitive pathways. therefore, depending on their competition with the available fpp pool, the critical step catalyzed by different sesquiterpene synthases shows a metabolic regulating to direct the cellular carbon flux 10 mahshid khajavi, mahdi rahaie, asa ebrahimi towards parthenolide or other sesquiterpenes. therefore, it seems, the nanoparticles directly and exclusively induce the genes toward more production of parthenolide compared to β-caryophyllene. different studies have proved hyperproduction of secondary metabolite related to use of elicitors, including jasmonate (walker et al. 2002; zhao et al. 2010; tocci et al. 2012; tocci et al. 2011; gadzovska et al. 2013; cui et al. 2014). it has been found that jasmonic acid (ja) and its methyl esters, methyl jasmonate (meja), are important signaling compounds in the process of elicitation leading to the hyper production of various secondary metabolites (walker et al. 2002). this compound plays a key role in signal transduction processes involved in defense responses in plant and has shown that is effective to induce the production of secondary metabolites in cell cultures (walker et al. 2002; zhao et al. 2010; tocci et al. 2012; tocci et al. 2011; gadzovska et al. 2013; cui et al. 2014). the effect of the elicitors on flavonoid production was reported by wang et al. (2015). they showed that the flavonoid content is promoted by meja and sa induction, which were 2.1 and 1.5 times higher in comparison to control cultures, respectively. a list of reports on different plant species have demonstrated a positive and strong correlation between terpene content and the level of the related mrna transcripts, which proves the terpenoid biosynthesis is majorly regulated at the transcript level (nagegowda, 2010). conclusion the results of our work, showed a significant effect of nanoparticles in low concentration and salinity stress on the expression of two genes involved in partheonlide biosynthesis pathway, also tpcars as a caryophyllen synthase in feverfew. it seems due to the positive effect of sio2 np compared to tio2, on the expression of all key genes for parthenolide and β-caryophyllen productions in feverfew, these nanoparticles can be used as efficient elicitors and useful additives to soil for increasing of secondary metabolite production in the whole plant, practically. as it was mentioned above, the metabolites are used as medicinal compounds for patients with migraine disease and different type of cancers; in hence, our results can suggest a simple and cost effective technique for mass production of them. however, due to a dominant view among researchers about the destructive role of nanoparticles in the environment, it is necessary for more research for investigation about side effect of the nanoparticles on plant growth and development in future. in conclusion, in this work, it was tried to explain the role of nanoparticle by this point of view which the nanomaterial, can also be valuable for more providing of human medicinal necessities from herbs and then, it opened a new window toward nanoparticle application for medicinal plants studies. it should be mentioned that, in this project the short-term effects of elicitors on secondary metabolite production were investigated and further studies are needed to determine the long-term impact of these elicitors on plants gene expression. acknowledgements we would to thank the university of tehran for instrumental supports and all people who help us in this work. author contribution statement mr conceived and designed research. mk conducted experiments. mr contributed new reagents or analytical tools. mr analyzed data. mk, mr and ae wrote the manuscript. all authors read and approved the manuscript. availability of data and materials the datasets during and/or analyzed during the current study available from the corresponding author on reasonable request. references abrol e, vyas d, koul s. 2012. metabolic shift from secondary metabolite production to induction of anti-oxidative enzymes during nacl stress in swertia chirata buch.-ham. acta physiol plant. 34(2):541546. ahmed sa, baig mm.2014. biotic elicitor enhanced production of psoralen in suspension cultures of psoralea corylifolia l. saudi j biol sci. 21(5):499-504. akula r, ravishankar ga.2011. 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cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-255 citation: n. anca şuţan, i. fierăscu, r. fierăscu, d. ionica, l.c. soare (2019) phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities. caryologia 72(2): 53-61. doi: 10.13128/cayologia-255 published: december 5, 2019 copyright: © 2019 n. anca şuţan, i. fierăscu, r. fierăscu, d. ionica, l.c. soare. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities nicoleta anca şuţan1,*, irina fierăscu2, radu fierăscu2, deliu ionica1, liliana cristina soare1 1 university of piteşti, faculty of sciences, physical education and informatics, department of natural sciences, 1 targu din vale str., 110040 pitesti, romania 2 the national institute for chemistry and petrochemistry – icechim, 202 spl. independentei, 060021, bucharest, romania *corresponding author: ancasutan@yahoo.com abstract. ferns are traditionally used by some nations to treat rheumatism, lungs, gynecology, blood and digestion dysfunctions, and several others illnesses. the present study evaluates the bioactivity of methanol and ethanol extracts from polystichum setiferum (forssk.) moore ex woyn. in an allium cepa test and disk diffusion test. in the allium cepa test the methanol and ethanol extracts induced a significant time-related increase in the mitotic index. the tested extracts were non-mutagenic by used assay, with no occurrence either the structural or numerical aberrations detected. the extracts were also evaluated in terms of trace elements (by edxrf) and qualitative composition (by uv-vis, ftir and total phenolic content). in the disk diffusion test, methanol extracts from leaves determined a small inhibition of bacterial growth for enterobacter cloacae and citrobacter freundii strains relatively to control sample (methanol). the ethanol extracts were more efficient, the diameter of inhibition growth zones measured from 7 to 10 mm, the most affected strain was chryseobacterium meningosepticum. keywords. polystichum, extracts, ftir, edxrf, uv-vis, cytogenotoxicity, antimicrobial activity. introduction appearance of antibiotic microbial resistance is present all over the world and is an increasingly serious threat to global public health. from the developing countries to the developed ones, bacterial antibiotic resistance problem it is very serious because cases of infection are treated by the lack of medicines or using them in excess (mundy et al. 2016). nosocomial infections usually represent an infrequent phenomenon. if it most developed countries this phenomenon is controlled, in many developing countries it is not reported (serban et al. 2012). in order to obtain alternative ways to combat bacteria that cause infections, herbal medicine represents one of the most important fields of tradi54 nicoleta anca şuţan et al. tional medicine all over the world, special attention has being paid to natural extracts with potential antimicrobial biological active compounds (chanda et al. 2011; jeyaseelan et al. 2012; chanda et al. 2013; fierascu et al. 2015). the natural products have been considered as a source of potential medicines, specific for each region or country (dubey and padhy 2013; panahi et al. 2014; gyawali and ibrahim 2014; georgiev 2014; schnekenburger et al. 2014; atanasov et al. 2015). in this context the study of ferns can be beneficial for this domain. studies focused on some ferns used as crude extracts, standardized extracts, purified substances or in the form of nanoparticles showed pharmacological activity (santos et al. 2010; soare and sutan 2018). the total flavonoid contents, antioxidant and anticancer activities, and acetylcholinesterase (ache) inhibition potential of fern extracts were investigated in detail (xia et al. 2014), but not the antimicrobial effect and cytotoxicity. some ferns species were traditionally used for scabies, eczema, jaundice including wounds and skin disease (kirtikar and basu 1999). polystichum is one of the most diverse genera of ferns with 360–400 species distributed worldwide (morero et al. 2015). polystichum setiferum (forssk.) moore ex woyn. is an evergreen or semi-evergreen fern native to southern and western europe. of the four species of polystichum present in romania, polystichum setiferum (forssk.) moore ex woyn. is the most common species (sârbu et al. 2013). totally, one hundred fern species have been described for their ethnomedicinal applications and chemical constituents in a fern ethnomedicinal plant database (thakar et al. 2015). rhizomes of polystichum setiferum (forssk.) moore ex woyn. tied around the neck of child are used to cure dysentery during the primary teeth development (kumar et al. 2013), parts of polystichum pungens (kaulf.) c. presl are used for the treatment of wounds (grierson and afolayan 1999), aerial parts of polystichum munitum (kaulf.) c. presl are used to stimulate digestion (lans et al. 2007), decoctions obtained from the rhizomes of polystichum pungens (kaulf.) c. presl roth are used to treat intestinal worms and as a general anthelminthic, powdered dried fronds to heal wounds, and fresh fronds as poultice (lall and kishore 2014), sporophyll extracts of polystichum squarrosum (d. don) fee and polystichum moluscens (bl.) t. moore are used as antibacterial agent (singh 1999), decoction obtained from fronds of polystichum woronowii fomin is used as antiinflamatory and anti-hepatitis agent and decoction obtained from leaf of polystichum aculeatum (l.) schott as anthelminthic (bahadori et al. 2015). the present study aims to comparative analyse the chemical composition of the methanol and ethanol extracts of polystichum setiferum (forssk.) moore ex woyn., their antimicrobial effects on five bacterial strains: escherichia coli (atcc 25922), staphylococcus aureus (atcc 25923), enterobacter cloacae and citrobacter freundii (both clinical strains) and elizabethkingia meningoseptica (formerly chryseobacterium meningosepticum, strain isolated from soil) and their cy totoxic effects on meritematic root cells of allium cepa l. assay. materials and methods preparation of plant extracts leaves and rhizomes of polystichum setiferum (forssk.) moore ex woyn. were collected from vîlsan valley, argeş county, romania from site n 45º25’40.2”, e 024º42’33.1”, altitude 1062 m. part of the plant material is preserved (-18 ºc) in botanic laboratory of the department of natural sciences, university of pitesti for future references. the voucher specimen is register at the botanical collection of county museum argeş with number 11326/25.05.2016. the leaves and rhizomes with scales were washed thoroughly and rinsed with bidistilled water and preserved at -18 ºc. frozen leaves and rhizomes were chopped at room temperature. leaves and rhizomes ethanol and methanol extracts (pel leaves ethanol extract, per rhizomes ethanol extract, pml leaves methanol extract, pmr rhizomes methanol extract) were prepared by mixing 100 g of each type of vegetable material in 1000 ml alcohol for 48 h at room temperature (22°c). the obtained extracts were filtered using whatman filter paper no. 1. the dried weight was obtained by shade drying to constant mass the plant materials, in order to remove excess moisture. the average dried masses for 15 samples were 29.01%±2.80 for rhizomes and, respectively, 32.25%±2.66 for leaves. evaluation of chemical composition of leaf and rhizome extracts for fourier tra nsform infrared spectroscopy (ftir), was used a varian 3100 excalibur spectrometer equipped with a harrick praying mantis diffuse reflectance (drift) accessory. the ir spectrum was collected in the region 4000–500 cm-1 at a resolution of 2 cm-1. the spectrum was analysed with the program analyzer ir – knowitall (bio-rad 2005). 55phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities for uv-vis evaluation was used an uv vis unicam helios α thermo orion spectrometer from 200 to 900 nm, at the resolution of 1 nm, with 1 nm slit width and automatic scan rate. the obtained results were processed using specific data analysis software (origin pro 8.0). for energy dispersive x-ray fluorescence determinations was used a pw4025 – minipal – panalytical energy dispersive xrf spectrometer with rhodium anode. the xrf determinations have been carried out in helium atmosphere, for a period of 300 seconds, without any filter, at proper voltage and current intensity. determination of total phenolic contents the concentration of total phenolic was measured by colorimetric method with folinciocalteu reagent (merck kgaa germany), according with a method previously presented (fierascu et al. 2015). the method involves the reduction of folin-ciocalteu reagent by phenolic compounds, with formation of a blue complex; the absorbance was read at 765 nm on the uv-vis spectrophotometer. the measurements were compared to a standard curve prepared with gallic acid (99%, merck kgaa germany) solutions at different concentrations (10-55 µg/ml). the total phenolic content was expressed as milligrams of gallic acid equivalents per 100 gram of dried weight. the dried weight was obtained by shade drying to constant mass the plant materials, in order to remove excess moisture. antioxidant activity antioxidant activity of the extracts was determined following the dpph assay, following a protocol previously exhaustively described (fierascu et al. 2015, fierascu et al. 2014). the absorbance was read at 517 nm after an incubation period of 30 minutes. the antioxidant activity (aa %) percentage was calculated using the formula: aa (%) = [(acontrol-asample)⁄a_control]×100, where: acontrol is the absorbance of the dpph solution without sample, asample is the absorbance of the extract mixed with 0.02 mg/ml dpph solution. evaluation of antimicrobial properties antibacterial effects of fern methanolic and ethanolic extracts were tested by disk diffusion method on five bacterial strains: escherichia coli (atcc 25922), staphylococcus aureus (atcc 25923), enterobacter cloacae and citrobacter freundii (both clinical strains) and elizabethkingia meningoseptica (formerly chryseobacterium meningosepticum, strain isolated from soil). frequency and rank of isolated bacteria, identified and tested by antibiogram in microbiology laboratories within major hospitals in bucharest present these strains in the top of the isolated germs that produce nosocomial infections (serban et al. 2012). as positive control was used antibiotic (ampicillin 10µg per disc from bioanalyse), while the negative control was the solvent specific for each extract (ethanol 96% and methanol 99.8%). an overnight (16 to 24 h) culture at 37°c of each bacterial strain was used (bacterial suspension was prepared by suspending one wire loop from the stock into 2 ml nutrient broth). in disk diffusion method sterile filter paper discs with 6 mm diameter were used. for diffusion method was used nutrient agar in petri dishes. each bacterial culture was homogeneous inoculated (in three directions) on the entire surface of nutrient agar in petri dishes. the disc with ampicillin (pre-warmed to room temperature) was placed on the surface of inoculated solid medium. four discs of sterilized filter paper were put on the nutrient agar; 5µl of each fern extract were used to impregnate the discs using micropipette. for negative control, two sterile discs of paper filter were used (5µl ethanol, respectively 5µl methanol). the petri dishes were incubated inverted for 24 h at 37°c. the antibacterial effects of fern extracts were estimated by measuring the diameter of inhibition growth zone (in millimeters), as a clear zones surrounding paper filter discs. the experiment was repeated three times and the results were in terms of average of measured values. evaluation of cytotoxic activity of leaf and rhizome extracts cytotoxic potential of leaf and rhizome extracts was evaluated by changes in mitotic index (mi) and phase indexes (prophase, metaphase, anaphase, telophase), induced in root tips cells of allium cepa l. (sutan et al. 2016). equal-sized onion bulbs, from a local variety, were purchased from local market. the outer scales were carefully removed and the bottoms were scraped to expose root primordia. rhizogenesis and root growth were induced on 30 ml jars filled with distilled water until to 0.5-1cm average length of the roots. after 48 hours, freshly emerged roots were treated with leaf and rhizome extracts for 6, 12 and 24 hours. distilled water, ethanol and metha56 nicoleta anca şuţan et al. nol were used as controls. cytological analysis were performed on squash slides prepared as follow: the root were fixed in a mixture of ethanol + glacial acetic acid (3:1) for 12 hours at 4°c, than were transferred to a watch glass in preheated 1n hcl at 60°c for 14 minutes and subsequently were immersed in preheated aceto-orcein solution at 60°c for 14 minutes. the tips of the roots were cut on a glass slide in a drop of 45% acetic acid, covered with coverglass, and squashed by tapping with matchstick. about 3000 cells from 9 root tips were scored for each treatment. the cells at different stages of mitosis were noticed. mitotic index (mi) was computed by determining the mitotic cell frequency (prophase, metaphase, anaphase and telophase) by the total number of cells observed and multiplying the result by 100 (tedesco and laughinghouse iv 2012). the number of cells at various mitosis stages (prophase, metaphase, anaphase, telophase) was calculated as percentage to number of dividing cells. results are presented as the mean ± standard error of more independent experiments. the data was analysed for statistical significance using analysis of variance (one way anova) and tukey test was used to determine significant differences among means. significant differences were set at p≤0.05. results phytochemical analysis the mineral content of the extracts was evaluated using a non-destructive technique, x-ray fluorescence. the extracts contain traces (in the ppm concentration range) of mg, p, ca, cr, mn, fe, ni and cu. also, pml contains minor traces of k and the ethanol extracts contains traces of co, not observed for the methanol extracts (fig. 1a). the ftir spectra presented in fig. 1b showed that the plant have compounds such as aldehyde, ketone, alcohol, carboxylic acid, amides, ethers and phenolic compounds. the peaks in ftir spectra in figure 1b are attributed to the following type of compounds, as previously reported (sutan et al., 2016): hydroxy compounds (oh stretching) – 3649 cm-1, carboxylic acid (oh stretching – 2982 cm-1, 1406 cm-1), carbonyl compounds (c-h stretching – 2901 cm-1, c=o stretching – 1668 cm-1), water (2133 cm-1), allenes (c=c=c bond -1923 cm-1), aromatic ring (1454 cm-1), alcohol (1323 cm-1, 1260 cm-1), aromatic hydrocarbons (880 cm-1) and mineral components (539 cm-1). the peaks at 1067 cm-1 and 1040 cm1 are attributed to characteristic functional groups of polyflavonoids and, respectively, –c-ogroups of the polyols such as flavones and terpenoids. uv-vis spectrum (fig. 2) of the leaves extract (pml and pel) seems to be a complex mixture of pigments: chlorophyll a – around 415 nm and 660-670 nm; chlorophyll b – 450 and 640 nm, and in less extent, carotenoids, while the rhizomes extracts (pmr and per) contains traces of chlorophyll a and minor traces of chlorophyll b. the total phenolic content and antioxidant activity of the extracts (table 1) shows a direct correlation between the phenolic content and the antioxidant potential. fig. 1. edxrf spectra (a) and ftir spectra (b) of the ethanol and methanol extracts of polystichum setiferum (forssk.) moore ex woyn. 57phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities antimicrobial activity antimicrobial activity of fern extracts is illustrated in table 2. statistical analysis of experimental data shows that the highest value for inhibition zone (9.5±0.28 mm) was determined for pel against elizabethkingia meningoseptica. except citrobacter freundii, the microorganisms being tested displayed significant differences of ≤2 mm between inhibition zone induced by pel and corresponding concentration of ethanol. in comparison, the smallest inhibition zone was noted for the methanol extracts, irrespective of the part of the plant material used in extraction or the type of microorganisms tested. mitotic index variation the effect of polystichum setiferum (forssk.) moore ex woyn. extracts is relevant, but there is not data about their effect on mi, so the mi of root tips cells to leaves and rhizome extracts was evaluated (fig. 3). the highest frequency of cells undergoing mitosis was noted in control samples. the mi in meristematic root cells of allium cepa l. treated with polystichum setiferum (forssk.) moore ex woyn. methanol and ethanol extracts of leaves follow similar trends: decreased at the minimum of 6h treatment and rise progressively with increasing of time exposure to 12 and 24h. the mi values ascertained for pel were slightly lower, when comparing with pml, that may be the consequence of higher concentration of metals in ethanol extracts, as edxrf analysis demonstrates. fig. 2. uv-vis spectra of the ethanol and methanol extracts of polystichum setiferum (forssk.) moore ex woyn. table 1. total phenolic content and antioxidant activity of the ethanol and methanol extracts of polystichum setiferum (forssk.) moore ex woyn. extract total phenolic content (mg gae/100 g dry mass) antioxidant activity (%) pmr 187.25±0.78 95.58±0.95 pml 44.64±0.16 74.32±0.62 per 161.24±0.65 92.55±0.68 pel 23.57±0.12 90.38±0.55 table 2. antimicrobial activity of the extracts of leaves and rhizomes of polystichum setiferum (forssk.) moore ex woyn. (inhibition zone – in mm). extracts/ control tested microorganisms escherichia coli atcc 25922 staphylococcus aureus atcc 25923 enterobacter cloacae citrobacter freundii elizabethkingia meningoseptica iz iz iz iz iz pmr 7.00±0.28 fghi r 6.16±0.16 hi 6.16±0.16 hi r pml 7.00±0.00 fghi r 6.5±0.18 hi 6.83±0.16 ghi r per 8.00±0.00 f r 7.33±0.33 fgh 9.16±0.16 e 6.66±0.33 ghi pel 9.33±0.33 e 8.00±0.00 f 7.66±0.33 fg 7.33±0.33 fgh 9.5±0.28 e ampicillin 14±0.00 d 25.66±0.33 c 30.33±0.33 a 9.33±0.33 e 29±1.00 b ethanol 7.33±0.33 fgh 6.16±0.16 hi 6.66±0.33 hi 7.66±0.33 fg 7.33±0.88 fgh methanol 7.00±0.57 fghi 6±0.00 i 6.66±0.57 hi 6.33±0.57 hi 6.33±0.57 hi *means with the same letter are not significantly different from each other (tukey test, p>0.05) * r – resistant 58 nicoleta anca şuţan et al. distribution of mitotic phases ethanol and methanol extracts affected the percentage of mitotic phases for all tested times (fig. 4). we showed here that treatments with methanol extracts of leaves and rizomes of polystichum setiferum (forssk.) moore ex woyn., except pml-24h, had caused mitotic arrest in meristematic root cells of allium cepa l., accumulating telophase cells. prophase cells frequency decreased significantly in these experimental conditions. in contrast, the frequency of telophase cells decreased in root tips treated with ethanol extracts, except pel-12h and pel-24h. discussion as a general remark, the ethanol seems to extract with higher efficiency the metals from the samples, as all the metals are in higher concentration in the ethanol extracts, while p has the same concentration in all the samples. it can be observed that the methanol extracts have higher intensity of the absorbance bands, which can be correlated with the results obtained for the total phenolic content and with the uv-vis analysis. it can be noticed that all extracts presents strong absorption bands corresponding to phenolic acids (more intense for the rhizomes extracts). also, even if the methanol seems to be a more efficient solvent for the extraction of the phenolic compounds, the ethanol seems to be more efficient for the extraction of pigments. antioxidant activity has been noticed for other species of polystichum genera, such as polystichum lepidocaulon (hooker) j. smith, polystichum polyblepharum (roem ex. kunze) c. presl (shin 2010) and polystichum semifertile (c.b. clarke) ching (ding et al. 2008). according to shin (2010) the crude extracts obtained from some ferns, such as those of the genera davallia, hypolepis, pteridium, cyrtomium, dryopteris, polystichum, dicranopteris, lycopodium, osmunda, adiantum, coniogramme, polypodium, pyrrosia, pteris, lygodium, selaginella, thelypteris, athyrium, matteuccia, onoclea și woodsia have strong antioxidant properties, sometimes substantially more effective than other natural or synthetic antioxidants. statistical analysis of experimental data indicates that pel had a significant antimicrobial activity against gram negative bacteria, escherichia coli and elizabethkingia meningoseptica; furthermore, pel was solely responsible for any antimicrobial activity noticed against staphylococcus aureus, as comparing with per, pml and pmr. aerobic gram-negative bacilli citrobacter freundii showed statistic significant sensitivity to per. the antimicrobial assay shows no correlation between the phenolic content and the antimicrobial potential. thus, the antimicrobial effect observed might be assigned to the other type of compounds identified by ftir, as well as to the metals identified by x-ray fluorescence in higher concentration in the ethanol extracts. recent studies indicate that different metals cause various types of injuries to microbial cells as a result of generation of reactive oxygen species (ros), membrane damage, interruption of electron transport, protein dysfunction or dna damage and inhibition of dna replication (lemire et al. 2013; dizaj et al. 2014). antibacterial properties of extracts obtained from leaves of a various fern species, such as asplenium nidus, fig. 3. the influence of the extracts of leaves and rhizomes of polystichum setiferum (forssk.) moore ex woyn. on the mitotic index in meristematic root cells of allium cepa l. (*the interpretation of the significance of the differences by means of the tukey test, p<0.05). fig. 4. the influence of the alcoholic extract of the rhizome and leaves of polystichum setiferum (forssk.) moore ex woyn. on the distribution of the mitotic phases in the meristematic root cells of allium cepa l. (*interpretation of the significance of the differences, by means of the tukey test, p<0.05). 59phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities blechnum orientale, cibotium barometz, dicranopteris linearis var. linearis, against gram positive bacteria (e.g. bacillus cereus, micrococcus luteus, staphylococcus aureus, as well as gram negative bacteria (e.g. escherichia coli, pseudomonas aeruginosa, salmonella enterica (formely salmonella choleraesuis), enterobacter aerogenes, klebsiella pneumoniae) have been noticed by lai et al. (2009). these results justify and certify the use of these species in traditional medicine. the allium root tip biossay was widely applied as bioindicator of pesticides, fertilizers and heavy metals cytogenotoxic effects, but is especially relevant for evaluation of the bioactivity of plant extracts (bonciu et al., 2018). taking a comparative approach to the cytogenetic effects of extracts of leaves and rhizome, respectivelly, methanol and ethanol extracts of rhizome have had a stronger mitodepressive effect over the meristematic root cells. the decrease of mi in the root tips of a. cepa l. has already been highlighted as indicator of the antiproliferative activity of different extracts, such as frescura et al. (2012) evaluating the luehea divaricata extracts, kuhn et al. (2015) who studied the leaves and fruits of eugenia uniflora infusions, sutan et al (2018) assessing the aconitum toxicum reichenb. rhizome extracts. the mi of samples that has been treated with per for 12h was significantly lower than in the control. the higher values of mi determined in the root meristems treated with methanol extracts may be directed related to the higher content of phenolic acids (fig. 1b). due to their redox properties, which allow them to act as reducing agents or hydrogen-atom donors (amarowicz et al. 2010), phenolic compounds exert protective effect against damaged mediated by ros. as the oxidative stress exerts its effect on the cell cycle shortly after the stress was imposed (reichheld et al. 1999, cited by west et al. 2014), this variation may be the consequence of stress adaptation that prevents cell death or constitute a radical route to cell death. results presented by west et al. (2014) regarding the response of primary root of arabidopsis to salt stress suggested that plants have a general mechanism that rapidly blocks cell cycle progression under stress conditions, presumably to impede transition to a stage where the cells are susceptible to damage (e.g. m-phase), simultaneously the cellular defence system being activated. stress-adapted cells undergo cell cycle stages at default rates. the root tips incubated in alcoholic extracts of leaves and rhizome have revealed these variations of mi values, without showing any chromosome breaking action. comparing to ethanol and methanol effects on mitosis in onion root tips, mitotic inhibition induced by extracts of leaves and rhizomes of polystichum setiferum (forssk.) moore ex woyn. was more intense. decreasing and significant decreasing of mitotic index in root meristems of allium cepa l. suggest the mitodepressive potential of alcoholic extracts of polystichum setiferum (forssk.) moore ex woyn., although mitotic delay induced by low concentration of ethylic alcohol were previously noticed by ancara and nuti ronchi (1967). increased frequency of telophase cells may be the consequence of high activity of cdk/mitotic cyclin complex which inhibits the pathway that promotes exit from mitosis. mitosis progress requires the ubiquitination of proteins whose proteolysis is necessary for chromatid separation and pre-replication complexes assembles, so that the cell is ready to begin dna replication at the next s phase. when ubiquitination of proteins is inhibited, telophase arrest is induced (searle and sanchez 2007). ros interference with nuclear envelope dynamics was evidenced by the delayed breakdown of the nuclear envelope at late prophase and its delayed reconstitution at telophase (livanos et al. 2012), which lead to delayed cell exit from telophase. this delay may be due to experimental disturbance of ros homeostasis, thus affecting microtubule dynamics and organization (livanos et al. 2012). it has also been suggested that arrested telophase cells perish by apoptosis (swe and sit 1997). mitotic and chromosomal abnormalities were detected at insignificant levels comparing with controls. conclusion the chemical analyses conducted showed a direct correlation between the solvent used for extraction and the total phenolic content. the rhizomes extracts showed a good antioxidant potential, also in good correlation with the total phenolic content. the antibacterial effect of ethanol extract was stronger against bacteria from soil than clinical bacterial strains. methanol extracts of fern demonstrated some effects on tested bacterial strains, just clinical ones were slight inhibited by these extracts. mitoinhibitory effect of leaves and rhizome extracts of polystichum setiferum (forssk.) moore ex woyn. without cytotoxic and clastogenic effects suggest its antimitotic drugs potential. although this is an important advance in our understanding of extracts effects further researches 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cytotoxic and genotoxic potential of some heavy metals by use of allium test ioan sarac1, elena bonciu2,*, monica butnariu1, irina petrescu1, emilian madosa1 fluorescence in situ hybridisation study of micronuclei in c3a cells following exposure to elf-magnetic fields luc verschaeve1,2,*, roel antonissen1, ans baeyens3, anne vral3, annemarie maes1 phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities nicoleta anca şuţan1,*, irina fierăscu2, radu fierăscu2, deliu ionica1, liliana cristina soare1 telomeric heterochromatin and meiotic recombination in three species of coleoptera (dorcadion olympicum ganglebauer, stephanorrhina princeps oberthür and macraspis tristis laporte) anne-marie dutrillaux, bernard dutrillaux* a whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of populus trichocarpa l. subjected to different stresses alberto vangelisti#, gabriele usai#, flavia mascagni#, lucia natali, tommaso giordani*, andrea cavallini differences in c-band patterns between the japanese house mice (mus musculus) in hokkaido and eastern honshu hikari myoshu, masahiro a. iwasa* karyotypic description and repetitive dna chromosome mapping of melipona interrupta latreille, 1811 (hymenoptera: meliponini) natália martins travenzoli1, ingrid cândido de oliveira barbosa2, gislene almeida carvalho-zilse2, tânia maria fernandes salomão3, denilce meneses lopes1,* caryologia. international journal of cytology, cytosystematics and cytogenetics 74(2): 57-63, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-613 caryologia international journal of cytology, cytosystematics and cytogenetics citation: adriano silva dos santos, karina de cassia faria (2021) identification of regions of constitutive heterochromatin and sites of ribosomal dna (rdna) in rhogeessa hussoni (genoways & baker, 1996) (chiroptera; mammalia; vespertilionidae). caryologia 74(2): 57-63. doi: 10.36253/caryologia-613 received: march 09, 2019 accepted: april 26, 2021 published: october 08, 2021 copyright: © 2021 adriano silva dos santos, karina de cassia faria. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid ass: 0000-0002-0492-197x identification of regions of constitutive heterochromatin and sites of ribosomal dna (rdna) in rhogeessa hussoni (genoways & baker, 1996) (chiroptera; mammalia; vespertilionidae) adriano silva dos santos1, karina de cassia faria2,* 1 graduate program in genetics, medical school of ribeirão preto, university of são paulo usp, ribeirão preto, sp, brazil 2 faculty of agricultural, biological and applied social sciences, mato grosso state university – unemat, nova xavantina, mt, brazil *corresponding author. e-mail: karinafaria@unemat.br abstract. there i s s carce i nformation a bout t he g eographical d istribution, b iological and cytogenetic data from the rhogeessa hussoni. this s tudy a ims t o c haracterize i ts chromosome composition through chromosome bandings to visualize regions of constitutive heterochromatin (cgb bands) and sites of ribosomal dna (rdna) in r. hussoni’s karyotype. a female specimen of r. hussoni was collected in the “parque municipal mário viana” conservation unit, nova xavantina, mato grosso, central-west region of brazil. the k aryotype c onstitution w as 2 n=52 a nd n f=54. th e cb g ba nds evidenced a sex x chromosome nearly completely constituted by heterochromatin. the rhogeessa hussoni has two sites of rdna located in a single pair (pair 25) of autosomal chromosomes. we carried out the first c ytogenetic c haracterization o f r . h ussoni, s upplementing knowledge about regions of heterochromatin and ribosomal dna in this species, thus contributing to future elucidations about the genetic diversification in t he genus rogheessa. keywords: bats, cytogenetics, x chromosome, heterochromatin, ribosomal dna. introduction the family vespertilionidae is constituted by the genera eptesicus, histiotus, lasiurus, myotis, and rhogeessa. the genus rhogeessa is composed by eleven species: r. túmida, r. aeneus, r. parvula, r. mira, r. minutilla, r. genowaysi, r. bickhami, r. alleni, r. gracilis, r. io and r. hussoni. the species occur exclusively in neotropical regions (laval 1973; genoways and baker 1996; ramiréz et al. 2014), and among them, r. minutilla, r. hussoni, and r. io are restricted to regions of south america (gardner 2008). the rhogeessa hussoni occurs in suriname and brazil and, in the latter, covers the states of 58 adriano silva dos santos, karina de cassia faria maranhão, bahia (gardner 2008). recently had its distribution extended to the states of sergipe (mikalauskas et al. 2011), mato grosso, minas gerais, and pará (aires et al. 2011). despite this broad spatial distribution, accurate taxonomic resolution in the genus rhogeessa is still required (baird et al. 2009). identifications based only in external morphological features led for some time to incongruences in distinctions of some species grouped in the complex rhogeessa tumida-parvula (laval 1973; gardner 2008; baird et al. 2009). the use of molecular and cytogenetic markers provided more conclusive information to distinguish some species (baird et al. 2009), and cytogenetic markers showed singularities in the chromosomal constitution of the following species: r. genowaysi (2n=42) (roots and baker 1998), r. parvula (2n=44) (roots and baker 2007), r. túmida (2n=34), r. aenus (2n=32) (bickham and baker 1977), r. io (2n=30) (gardner 2008), and r. hussoni (2n=52) (genoways and baker 1996; gardner 2008). an explanation for this diverse karyotype within the genus rhogeessa is the occurrence of chromosomal rearrangements by means of centric fusions and fissions (baker et al. 1985; baird et al. 2009). despite the efforts to acknowledge the variety of species of the genus rhogeessa, the information about the r. hussoni and r. io occurring in the brazilian territory is still scarce (nogueira et al. 2014), with overlapping areas in the state of mato grosso (gardner 2008; aires et al. 2011). rhogeessa hussoni and r. io are considered cryptic species, because of the difficult taxonomic designation through external morphological data (gardner 2008; gurgel-filho et al. 2015). however, it is possible to distinguish the two species through their basic diploid number, being r. hussoni 2n=52 and r. io 2n=30 (bickham and baker 1977; genoways and baker 1996). the existing cytogenetic information about r. hussoni come from a specimen in the region of suriname (genoways and baker 1996; gardner 2008), and the authors presented only the basic karyotype of the species. however, there are no cytogenetic studies with mappings of specific chromosome regions for this species. in this study, we characterized the chromosome composition of a specimen of r. hussoni captured in brazil, by identifying the sites of ribosomal dna (rdna) and the regions of constitutive heterochromatin (cbg bands) in this specimen’s karyotype. material and methods the capture of a female specimen of rhogeessa hussoni was made using mist nets (7.0 x 3.0) in the “parque municipal mário viana” conservation unit, located in the municipality of nova xavantina, state of mato grosso, brazil. the area is characterized by the phytophysiognomy of cerradão (14°42’02.6” s e 052° 21’01.5” w), inside the brazilian cerrado biome, with plant formations of continuous canopy and tree coverage ranging from 50% to 90% (silva et al. 2008). the region’s climate is tropical rainy (aw) according to köppen’s classification system (vianello and alves 2012), with a dry season from april to september, and a rainy season from october to march (pirani et al. 2009). the female specimen of rhogeessa hussoni was captured under the license 18276–1 from ibama/sisbio/ mt, and its identification was made based on specialized literature (vizotto and taddei 1973; genoways and baker 1996; gardner 2008; díaz et al. 2011). after the capture, the bat was kept in a cage until the following morning, when cytogenetic procedures were made. after extracting the biological material, the animal was mounted and deposited in the scientific collection of chiroptera of mato grosso state university, campus of nova xavantina (unemat/nx), registered under the number rm 276. the chromosome preparations were obtained directly from bone marrow, following morielle-versute et al. (1996). the diploid number and the fundamental autosomal number were determined using the giemsa staining method (2%). the procedure of howell and black (1980) was adapted to identif y regions of ribosomal dna (rdna). the blocks of constitutive heterochromatin were determined using the technique of summer (1973) with minor modifications: the chromosomal preparations were treated with 2n hcl solutions, barium hydroxide, and 2xssc at a temperature of 42 ° c, and then stained with giemsa solution. the karyotype was determined with based on the first cytogenetic description of the species in suriname. the slides with chromosome preparations for each procedure were photo-documented using an optical microscope with observation at 1000x (axio vison® -nikon digital sight ds-u3). next, the analysis and mounting of karyotypes were made using adobe photoshop, version 7.0.1. after the cytogenetic analyses, taxonomic characterizations were made to confirm species identification. external and cranial measurements were taken using a digital pachymeter (precision 0.01 mm) after the taxonomic characterizations. the measured features were: forearm length, total skull length, basal condyle length, canine condyle length, basal length, palatal length, length of upper teeth series, length of interior teeth series, mandible length, width of cingula (canine teeth), external width of molar teeth, interorbital width, pos59identification of regions of constitutive heterochromatin and sites of ribosomal dna (rdna) in rhogeessa hussoni torbital width, zygomatic width, skull width, mastoid width, palatal width, skull height, and occipital height (fig. 1 and supplementary material). results the morphometrical analyses confirmed that the exemplar was an r. hussoni. we analyzed more than thirty metaphases to determine the diploid number (2n) and the number of autosomal arms (nf). the specimen of r. hussoni presented 2n = 52 and nf = 54 (fig. 2). the set of autosomes is constituted by 23 pairs of acrocentric or subtelocentric chromosomes of large to small dimensions (pairs 1-12, 14-17, 19-25). the sexual set is composed of two x chromosomes, medium size, with submetacentric morphology. the nucleolar organizing regions were evidenced in the pair of autosomal chromosomes number 25 (fig. 2). with the c banding technique, we showed the formation of heterochromatin blocks in pericentromeric regions of all autosomal chromosomes. regarding the sexual pair, one of the x chrofigure 1. dorsal view of skull (a), ventral view (b), lateral view (c), and ventral view of mandible (d) (rm 276), specimen of r. hussoni (adult female), deposited in the scientific collection of chiroptera/unemat, campus nova xavantina, state of mato grosso, brazil (bar scale 6.4mm). 60 adriano silva dos santos, karina de cassia faria mosomes has a structure composed almost entirely by heterochromatin regions, whereas a small portion of heterochromatin is located in the pericentromeric region in the other x chromosome (fig. 3a). in interphase nuclei, we observed the presence of regions of more condensed heterochromatin, which may suggest the inactivation of one of the x chromosomes (fig. 3b). discussion for species of the genus rhogeessa, the sole use of morphological features does not allow a clear taxonomic designation, since rhogessa hussoni and r. io show overlap in their forearm size (gardner 2008). however, cy togenetic information provided fundamental data to designate species of the genus (genoways and baker 1996, gardner 2008). the species rhogeessa io and r. hussoni have geographical distribution in brazilian territory with overlapping areas in the state of mato grosso (gardner 2008; gurgel-filho et al. 2015). the fist karyotype description of r. hussoni was of a specimen in suriname (genoways and baker 1996), with 2n=52, whose authors assumed a meta-submetacentric morphology in sex chromosome x, which was followed in the present study. the morphology of the y chromosome is not mentioned because there are still no cytogenetic descriptions of male specimens. figure 2. karyotype of rhogeessa hussoni (2n = 52 and nf = 54). the highlighted box shows respectively the chromosome pair 25, with rdna sites, and sex chromosomes. figure 3. (a) karyotype of rhogeessa hussoni with c banding, showing blocks of heterochromatin in pericentromeric regions of all chromosomes. the highlighted box shows the pair of sex chromosomes. (b) an interphase nucleus with evidence of barr corpuscle (indicated by the arrow). 61identification of regions of constitutive heterochromatin and sites of ribosomal dna (rdna) in rhogeessa hussoni the karyotype of myotis (2n=44) has been indicated as the similar ancestral karyotype for the family vespertilionidae. some species of the genus eptesicus (2n=50, nf=48) are proposed as the closest evolutionary kinship of rhogeessa (bickham 1979). comparisons of g-bands patterns between chromosomes of myotis velifer and representatives of the genus rhogeessa (r. parvula, r. túmida, r. aenus, and.r. io) showed homology between the autosomal chromosomes pairs 16/17 and 20/18, with rearrangements of chromosomal fusions shared by species of rhogeessa presenting meta-submetacentric morphology (bickham 1979; baker et al. 1985). in the present study, the pair of chromosomes with metasubmetacentric morphology 13 of r. hussoni seems to correspond to pair 16/17 of myotis and other species of rhogeessa. homology between g-bands patterns of pairs 20/18, which corresponds to the morphology of chromosome 18 found in r. hussoni, suggest a synapomorphy shared by species of the genus rhogeessa (bickham and baker 1977; baker et al. 1985). information about k inship relations has been obtained for some representatives of the family vespertilionidae through comparative genomic techniques and g-bands patterns (volleth et al. 2002, 2012; sotero-caio et al. 2017). however, there is not a clear knowledge about chromosomal rearrangements in the genus rhogeessa due to lacking data of cytogenetic bandings for some species. the presence of a pair of ribosomal dna sites was evidenced in r. hussoni. likewise, studies have shown a single pair of rdna in r. tumida (2n=34), eptesicus fuscus (2n=50) (baker et al. 1992), and lasiurus cinereus (2n=28) (marchesin and morielle-versute 2004), indicating a maintenance in the number of rdna sites among species of the genera rhogeessa, eptesicus, and lasiurus within the family vespertilionidae. nonetheless, the presence of four chromosome pairs with rdna sites is acknowledged in myotis keaysi (2n=44) (baker et al. 1992). the blocks of heterochromatin in r. hussoni are ordered in pericentromeric regions in most autosomal chromosomes and in almost all the structure of one of the sex chromosomes x. (fig. 3a). in lasiurus ega, the presence of constitutive heterochromatin has also been evidenced along all the short arm of the x chromosome (marchesin and morielle-versute 2004), in the same location of regions of heterochromatin in the sex chromosomes of r. hussoni. the organization of constitutive heterochromatin is dynamic among species. the existence of a copy of x chromosome, almost totally constituted by heterochromatin, with function in regulating the genic expression and gene dose compensation between complements xx and xy is acknowledged in several mammal species (avner and heard 2001). in r. hussoni, the presence of one x chromosome nearly wholly constituted by heterochromatin blocks, with regions of highly condensed interphase nuclei suggest a possible function in regulating gene expression of the x chromosome. a large amount of information about cytogenetic and molecular comparisons for representative species of myotis, eptesicus, and lasiurus is known (bickham 1979; varella-garcia et al. 1989; volleth et al. 2002, 2012; larsen et al. 2012; seim et al. 2013; supanuam et al. 2012; furman et al. 2014). still, little is known about structural chromosome relations among the species of the genus rhogeessa (bickham 1979; baker et al. 1985; baker et al. 1992), and there are no studies about phylogenetic relationships based on molecular and cytogenetic markers for r. hussoni (baird et al. 2009). in the present study, we carried out the first cytogenetic characterization with chromosome bandings for r. hussoni, broadening knowledge about this species’ chromosome composition. further studies about ecological aspects, geographical distribution, molecular biology, and phylogenetic inferences are necessary to better understand and preserve the species, considering that the kinship relationship within the genus rhogeessa is not clear. besides, the species is classified as deficient in data from the international union for conservation of nature (iucn), reinforcing the importance of more data in order to better understand the status of ecological threat (sampaio et al. 2016). acknowledgments to researchers ricardo f. sousa and júlio m. alvarenga, for their aid in collecting biological material. to mato grosso state university (unemat), for the scientific undergraduate grant (unemat-probic). to the university of são paulo (usp) and capes (coordination for the improvement of higher education personnel) for their post-graduate academic education and financial investment. references aires cc, nascimento fo, césari a. 2011. mammalia, chiroptera, vespertilionidae, rhogeessa hussoni genoways and baker 1996: distribution extension and taxonomic notes. check list. 7(2):117-119. avner p, heard e. 2001. x-chromosome inactivation: couting choice and initiation. nature reviews genetics. 2(1):59-67. 62 adriano silva dos santos, karina de cassia faria baird ab, hillis dm, patton jc, bickham jw. 2009. speciation by monobrachial centric fusions: a 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basal condyle length (11.97); canine condyle length (11.83); basal length (10.27); palatal length (6.02); upper teeth series length (4.50); inferior teeth series length (4.85); mandible length (8.59); external width of cingula canines (4.05); external width of molar teeth (6.04); interorbital width (3.89); postorbital width (3.45); zygomatic width (8.49); skull width (6.24); mastoid width (7.25); palatal width (3.43); skull height (4.20); occipital height (5.10) (fig. 1). the dental formula is constituted by: i: 1/3; c: 1/1; pm: 1/2; m: 3/3. caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 1 2021 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 72(1): 45-53, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-250 citation: i̇. genç, m. firat (2019) karyological study of the genus gundelia (compositae) in turkey. caryologia 72(1): 45-53. doi: 10.13128/cayologia-250 received: 13th june 2018 accepted: 4th december 2018 published: 10th may 2019 copyright: © 2019 i̇. genç, m. firat. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyological study of the genus gundelia (compositae) in turkey i̇lker genç1,*, mehmet firat2 1 department of pharmaceutical botany, faculty of pharmacy, i̇stanbul university, i̇stanbul, turkey 2 department of biology education, faculty of education, van yüzüncü yıl university, van, turkey * corresponding author: gencilker1@gmail.com abstract. karyotypes in 12 taxa of gundelia are compared, based on feulgen-stained somatic metaphase chromosomes. the karyotypes of g. anatolica, g. asperrima, g. cilicica, g. colemerikensis, g. dersim, g. glabra, g. komagenensis, g. mesopotamica, g. munzuriensis and g. vitekii are described for the first time. karyological analyses indicate relationships among the species with respect to their asymmetry indices. all gundelia species studied were diploid with 2n = 2x = 18 chromosomes. all karyotypes are symmetrical, consisting of metacentric and submetacentric chromosomes. the submetacentric chromosomes of all the investigated specimens contain a secondary constriction. three chromosome types were identified according to the position of the secondary constrictions. the chromosomes ranged in size from 2.00 µm to 7.02 µm. the total haploid chromosome length (thl) varied from 24.97 μm (g. asperrima) to 42.56 μm (g. rosea). to determine the karyological relationships among taxa, pcoa (principal coordinate analysis) with six uncorrelated parameters was performed. keywords. chromosome number, karyotype asymmetry, secondary constriction, cichorieae, scolyminae. introduction gundelia l. was first recorded in one of the earliest natural history collections made in the near east by the german physician, botanist and traveller, leonhard rauwolf (1535–1596) at 16. century. however, the plant was first evaluated in silybum then eryngium groups. about 125 years later, in the early years of the 18th century, joseph pitton de tournefort saw the plant in the natural habitat. moreover, he concluded that the plant should be called gundelia (hind 2013). finally, the plant was named gundelia tourneforti by linnaeus, in accordance with the binomial nomenclature (linnaeus 1753). the infrafamilial position of gundelia has also changed over time (hind 2013). the last tribal position of genus gundelia was cichorieae lam. & dc., subtribe scolyminae less., along with catananche l., hymenonema cass., and scolymus l. (kilian et al. 2009). the genus grows in the semi-desert areas, and it is distributed in the mediterranean to central/eastern asia (karis et al. 2001). 46 i̇lker genç, mehmet firat some different species and varieties have been described over the years, although many authors have treated gundelia as monospecific and the wide variation in corolla colour was considered unrelated to gross morphology (komarov 1961; kupicha 1975; feinbrun dothan 1978; meik le 1985; rechinger 1989). however, in the last decades, researches on gundelia increased. numerous new species have been published as a result of research on live and more abundant materials. the genus is currently represented by 16 species, of which 12 (10 endemic) in turkey (vitek et al. 2010, 2014, 2017; nersesyan 2014; armağan 2016; fırat 2016, 2017a, 2017b, 2017c; vitek and noroozii 2017; vitek 2018). these are gundelia anatolica fırat, g. asperrima (trautv.) fırat, g. cilicica fırat, g. colemerikensis fırat, g. dersim vitek, yüce & ergin, g. glabra mill., g. komagenensis fırat, g. mesopotamica fırat, g. munzuriensis vitek, yüce & ergin, g. rosea m.hossain & al-taey (non-endemic), g. tournefortii l. (non-endemic), and g. vitekii armağan. the chromosome numbers of cichorieae range between 2n = 14x = 126 chromosomes in sonchus (beuzenberg and hair 1984; dawson 2000) and 2n = 2x = 6 in some species of crepis (ikeda 1988; gupta and gill 1989; dimitrova and greilhuber 2001). the basic chromosome number in the majority of the subtribes is x = 9, or a descending series starting with x = 9 to x = 3. subtribe scolyminae has the basic chromosome numbers x = 9 and 10 (kilian et al. 2009). the only chromosome number gundelia is 2n =18 (waisel 1962; al-taey and hossain 1984; ghaffari and chariat-panahi 1985; nersesyan and nazarova. 1989; ghukasyan and janjughazyan 2015). fig. 1. synflorescences of the studied taxa. (a) g. anatolica; (b) g. asperrima; (c) g. cilicica; (d) g. colemerikensis; (e) g. dersim; (f ) g. glabra; (g) g. komagenensis; (h) g. mesopotamica; (i) g. munzuriensis; (j) g. rosea; (k) g. tournefortii; (l) g. vitekii. 47karyological study of the genus gundelia (compositae) in turkey this study aimed to determine the chromosome numbers and karyomorphology of al the 12 gundelia species occurring in turkey (figure 1). materials and methods twelve gundelia species were analysed in this study. a list of examined specimens is provided in table 1. all endemic taxa were collected from their type localities. voucher specimens were deposited in the herbarium of university of van yüzüncü yıl (vanf) and in a private herbarium (herb. m. fırat). for karyological observations, four to eight individuals were used for each species in this study. mitotic metaphase cells of root tips were obtained from germinated seeds which were collected in natural habitats from turkey. mitotic chromosomes were prepared from root tips and pre-treated with 0.002 m 8-hydroxyquinoline at +4 °c for 24 h. roots were fixed for a minimum of 2 h in absolute ethanol:glacial acetic acid, (3:1,v/v), hydrolysed at 60 °c in 1 n hcl for 12 min. and stained with the feulgen method. finally, root tips were squashed in 1% aceto-orcein. permanent slides were prepared with entellan mounting medium. microphotographs of good quality metaphase plates were taken using an olympus bx53 (tokyo, japan) microscope equipped with a high-resolution digital camera. metaphase observations and chromosome measures were made using the image analysis systems kameram (argeni̇t microsystems, i̇stanbul, turkey). the somatic chromosome number and karyotype details were studied in five to eighteen well-spread metaphase plates from different individuals; mean values were used for the analysis. chromosome pairs were identified and arranged on the basis of their length and any other evident karyomorphological features. the nomenclature used for describing karyotype composition followed levan et al. (1964). to determine the karyological relationships among taxa, we performed a pcoa (principal coordinate analysis) with six uncorrelated parameters as suggested by peruzzi and altinordu (2014). these parameters are chromosome number (2n), basic chromosome number (x), total haploid length (thl), mean centromeric asymmetry (mca), coefficient of variation of chromosome length (cvcl), and coefficient of variation of centromeric index (cvci) (paszko 2006; peruzzi et al. 2009; zuo and yuan 2011, peruzzi and eroğlu 2013). the software past 3.03 (hammer et al. 2001, hammer 2018) was used to perform this analysis. mitotic metaphase chromosomes are given in figure 3. idiograms of these taxa are arranged in order of centromere position and then decreasing the length of homologue chromosome pairs (figure 4). in this study, chromosome types were determined according to the position of the secondary constrictions of gundelia chromosomes for the purpose of chromosome comparison. general description of these chromotable 1. localities and voucher specimens of gundelia taxa examined in the present study. taxa locality collector (m. fırat) /voucher g. anatolica fırat kırıkkale: delice province, tuzkayası region, dry steppe, 700 m, 10 july 2017 33866 g. asperrima (trautv.) fırat erzurum: palandöken mountain, mountain slope, steppe, 2444 m, 30 july 2017 33845 g. cilicica fırat mersin: erdemli province, tozlu village, open forrest, 1460 m, 11 july 2017 33868 g. colemerikensis fırat hakkâri: hakkâri province (colemerîk) berçelan plateau, open erode region and steppe, 2284 m, 7 july 2017 33861 g. dersim vitek, yüce & ergin tunceli (dersim): ovacık, c. 11.7 km sw ovacık, 1.9 km ne ziyaret (fountains of river munzur), 1300 m, 28 july 2017 33872 g. glabra mill. bayburt: kop mountain arround, near kop village, 1897 m, 19 july 2017 33867 g. komagenensis fırat adıyaman: kahta province, nemrut mountain, rocky steppe, 1445 m, 30 july 2017 33783 g. mesopotamica fırat mardin: 2-3 km from mardin to nusaybin (nisêbîn), eroded slopes, aride steppe, 807 m, 11 july 2017 33865 g. munzurensis vitek, yüce & ergin tunceli (dersim): ovacık, c. 2 km sw ovacık, near road ovacık plain, 1275 m, 28 july 2017 33871 g. rosea m.hossain & al-taey hakkari: şemdinli province, sad mountain, derîyê kera region, meadows and stony slopes, 1662 m, 7 july 2017 33862 g. tournefortii l. hatay: reyhanli district, near syria (aleppe) border, 121 m, 9 july 2017 33864 g. vitekii armağan tunceli (dersim): c. 8-9 km n of tunceli, mountain slope nw of tüllük bucaği, rocky steppe, 1760 m, 28 july 2017 33870 48 i̇lker genç, mehmet firat some types is given below, followed by the karyotype description. type a: longest metacentric chromosomes with two constrictions, secondary constrictions in the distal position of the long arm. type b: submetacentric chromosomes with two constrictions, secondary constrictions nearly in the median position of the long arm. type c: submetacentric chromosomes with two constrictions and secondary constrictions located very close to the centromere on the short arm. results all species showed basic chromosome number x = 9 and diploids with 2n = 18 (figures 3 and 4). chromosome measurements and karyotype formula of the twelve analysed species are indicated in table 2. total haploid length, asymmetry indices, chromosome types and flower number within the partial synflorescences in the middle part are summarised in table 3. secondary constrictions were observed at the long or short arms of all submetacentric chromosomes, and in the distal regions of the long arms of some of the longest table 2. karyotype formula according to levan et al. (1964) and measurements of the investigated taxa. (sc: the shortest chromosome length; lc: the longest chromosome length; p: mean long arm length; q: mean short arm length; sd: standard deviation; m: metacentric; sm: submetacentric). sc–lc q (μm) mean (±sd) p (μm) mean (±sd) p+q mean (±sd) karyotype formula g. anatolica 2.90 – 5.15 1.64(±0.30) 2.19(±0.44) 3.83(±0.65) 16 m + 2 sm g. asperrima 2.00 – 4.01 1.19(±0.23) 1.58(±0.42) 2.77(±0.60) 14 m + 4 sm g. cilicica 2.77 – 5.35 1.67(±0.35) 2.14(±0.46) 3.81(±0.76) 16 m + 2 sm g. colemerikensis 2.40 – 4.90 1.43(±0.33) 1.89(±0.50) 3.32(±0.74) 14 m + 4 sm g. dersim 2.78 – 5.78 1.66(±0.40) 2.18(±0.50) 3.84(±0.83) 16 m + 2 sm g. glabra 2.86 – 5.61 1.70(±0.35) 2.25(±0.56) 3.96(±0.82) 14 m + 4 sm g. komagenensis 3.10 – 6.08 1.79(±0.37) 2.42(±0.61) 4.21(±0.87) 14 m + 4 sm g. mesopotamica 2.73 – 5.27 1.55(±0.36) 2.08(±0.47) 3.63(±0.71) 14 m + 4 sm g. munzurensis 2.28 – 4.43 1.33(±0.26) 1.77(±0.43) 3.10(±0.63) 14 m + 4 sm g. rosea 3.30 – 7.02 2.03(±0.49) 2.70(±0.58) 4.73(±1.01) 16 m + 2 sm g. tournefortii 2.31 – 4.45 1.33(±0.30) 1.68(±0.37) 3.02(±0.61) 16 m + 2 sm g. vitekii 2.49 – 5.04 1.45(±0.31) 1.95(±0.51) 3.40(±0.74) 14 m + 4 sm table 3. karyo-morphometric parameters, symmetry indices, chromosome types and cephaloid flowers number for investigated taxa (thl: total haploid length; mca: mean centromeric asymmetry; cvcl: coefficient of variation of chromosome length; cvci: coefficient of variation of centromeric index). thl mca cvcl cvci chromosome types cephaloid. flowers numbers g. anatolica 34.48 14.13 16.87 9.73 _/b/_ 6 g. asperrima 24.97 13.08 21.58 9.83 a/b/c 3(–4) g. cilicica 34.26 12.06 20.01 7.44 _/_/c 6(–7) g. colemerikensis 29.84 13.03 22.42 10.75 _/b/c (3–)5(–6) g. dersim 34.52 13.27 21.57 9.56 a/_/c 6–7 g. glabra 35.60 13.27 20.84 9.94 _/b/c 3–5(–6) g. komagenensis 37.90 13.95 20.56 11.94 _/b/c 3(–4) g. mesopotamica 32.64 14.38 19.70 12.44 a/b/c 6–7 g. munzurensis 27.87 13.27 20.24 10.21 a/b/c 3–5 g. rosea 42.56 14.40 21.42 8.12 a/_/c (6–) 7–8 g. tournefortii 27.16 11.41 20.20 8.83 a/_/c (5–)6(–7) g. vitekii 30.57 13.76 21.64 11.82 a/b/c 3(–5) 49karyological study of the genus gundelia (compositae) in turkey metacentric chromosomes (figure 2). moreover, three chromosome types were determined according to the position of the secondary constrictions (figure 2). the chromosomes ranged in size from 2.00 µm to 7.02 µm. g. asperrima showed the smallest mean chromosome length (2.77 µm), while g. rosea the biggest (4.73 µm). similarly, the smallest mean short arm length (q) was observed in g. asperrima (1.19 μm) and the largest mean long arm length (p) was observed in g. rosea (2.70 μm). the idiograms of the analysed species are shown in figure 3. fig. 2. chromosome types according to the position of the secondary constrictions (indicated by arrows). fig. 3. somatic chromosomes (2n = 18) in the studied taxa. (a) g. anatolica; (b) g. asperrima; (c) g. cilicica; (d) g. colemerikensis; (e) g. dersim; (f ) g. glabra; (g) g. komagenensis; (h) g. mesopotamica; (i) g. munzuriensis; (j) g. rosea; (k) g. tournefortii; (l) g. vitekii. scale bars 3 μm. 50 i̇lker genç, mehmet firat fig. 4. haploid idiograms in the studied taxa. (a) g. anatolica; (b) g. asperrima; (c) g. cilicica; (d) g. colemerikensis; (e) g. dersim; (f ) g. glabra; (g) g. komagenensis; (h) g. mesopotamica; (i) g. munzuriensis; (j) g. rosea; (k) g. tournefortii; (l) g. vitekii. 51karyological study of the genus gundelia (compositae) in turkey the chromosomes with secondary constriction ranged from 2 to 6 in number. the total haploid chromosome length (thl) varied from 24.97 μm (g. asperrima) to 42.56 μm (g. rosea). karyotypes of the analysed species exhibit a predominance of metacentric chromosomes. however, one or two submetacentric chromosomes were detected in each taxon. due to the prevalence of metacentric pairs and to the absence of strong differences between smaller and larger chromosomes, asymmetry indices were in general low. however, some species show a tendency to have karyotypes distinct on asymmetry grounds: gundelia rosea, with relatively high intrachromosomal (mca) and g. tournefortii with low intrachromosomal asymmetry, also, g. vitekii with high interchromosomal (cvcl) and g. anatolica low interchromosomal asymmetry. discussion karyotype data for all taxa are reported for the first time in the present study with the exception g. tournefortii and g. rosea. the present investigation on gundelia supports earlier data about 2n = 2x = 18 (waisel 1962; al-taey and hossain 1984; ghaffari and chariat-panahi 1985; nersesyan and nazarova 1989; ghukasyan and janjughazyan 2015). the article by nersesyan and nazarova (1989) is the most detailed work published on the karyology of gundelia tournefortii. three chromosome types were also seen in that study. however, according to our results, type b chromosome was not observed in gundelia tournefortii chromosomes. however, earlier works accepted only one species in the genus, and this could be the cause of this difference. according to trautvetter (1876), f lower number within the partial synflorescences differs between different populations of gundelia. moreover, the partial synflorescences in the middle part of the synflorescence are formed by 3 to 8 flowers according to the articles published in recent years. these flowers have been defined as “cephaloid flowers” in the following sections of the article. the taxa examined in this article were evaluated according to cephaloid flowers, and two groups have been recognised. group i consists of mainly 3(-5) cephaloid flowers; group ii consists of mainly 6 or more cephaloid flowers (table 3). in addition to this, according to chromosome types (a-c), taxa are divided into two main groups too. in agreement with our results, these groups are quite overlapping. namely, type b chromosomes were present in all the taxa of group i and no type b chromosomes was observed in the taxa of group ii with the exception of g. mesopotamica and g. anatolica. these species differ from others by having 6-8 cephaloid flowered and type b chromosomes. also, g. anatolica is the only species in which there are no type c chromosomes. according to tarıkahya hacıoğlu and fırat (2017), g. fig. 5. pcoa analysis based on six quantitative karyological parameters of investigated taxa. 52 i̇lker genç, mehmet firat anatolica is a derived species. this difference in chromosomal morphology supports this argument. according to pcoa analysis, the species belonging to the same group tend to cluster together substantially (figure 5). the presence of type a and type b chromosomes does not show variation at intraspecific level, these chromosome types were observed in all metaphase stages. however, type a chromosomes showed some infraspecific variation. in conclusion, this study illustrated that two different groups can be distinguished, according to chromosome morphology. therefore, the chromosome types can be used, in addition to other characters, for species identification and classification in the genus gundelia. references al-taey ra, hossain m. 1984. studies in gundelia: 1 a new species from iraq. notes roy. bot. gard. edinburgh. 42: 39–44. armağan m. 2016. gundelia vitekii (compositae), a new species from turkey. ann. naturhist. mus. wien, b 118: 129–134. beuzenberg ej, hair jb. 1984. contributions to a chromosome atlas of the new zealand flora 27: compositae. new zealand j. bot. 22: 353–356 dawson mi. 2000. index of chromosome numbers of indigenous new zealand spermatophytes. new zealand j. bot. 38: 47–150. dimitrova d, greilhuber j. 2001. c-banding patterns and quantitative karyotype characteristics of bulgarian species of crepis (asteraceae). pl. biol. 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ros reactive oxygen species; o2– superoxide radicals; h2o2 hydrogen peroxide; oh• hydroxyl radicals; sod superoxide dismutases; cat catalases; apx ascorbate peroxidases; gst glutathione s-transferases; gpx glutathione peroxidases. introduction anise hyssop (agastache foeniculum) from the family lamiaceae is as an important medicinal plant. the essential oil (eo) of anise hyssop is mainly 34 seyyedeh farahnaz talebi, mohammad jamal saharkhiz, maryam jafarkhani kermani , yavar sharafi biosynthesized in its leaves and flowers which contain significant amounts of methyl chavicol. in medicinal plants, secondary metabolites are fundamentally produced by genetic pathways, although environmental factors also strongly influence their biosynthesis (zhang, 2015). biotic and abiotic environmental factors, specifically salinity and drought conditions, affect growth parameters, medicinal plants’ survival, and their essential oil yield (heidari et al. 2008, heydari et al. 2020, sharafi et al. 2017). podda et al. (2013) stated that salinity is one of the most important abiotic stresses in agriculture affecting the plant growth and agricultural productivity. high levels of soil salinity have toxic effects on the absorption of nutrients from the root system in the plant through osmotic processes which, in turn, reduces essential oil production and modifies their composition in medicinal and aromatic species (sarmoum et al. 2019). it is essential to determine the environmental factors under which medicinal and aromatic plants offer higher yields and improve quality. high salinity can disturb essential physiological processes due to factors such as water deficits, nutritional imbalance, hyper-osmotic stress, ion imbalance, metabolic disorders, and appearance or disappearance of some proteins which may eventually lead to death (meng et al. 2016). these culminate in reduction of growth, yield, and quality of plants. therefore, the over expression of genes encoding the biosynthetic enzymes may increase proline concentration in plant cells (apse and blumwald, 2002; rabiei et al. 2011). on the other hand, oxidation reactions from choline to glycine betaine enhance plant resistance to salinity (apse and blumwald, 2002). saline stress increases production of reactive oxygen species (ros) including superoxide radicals (o2 –), hydrogen peroxide (h2o2), and hydroxyl radicals (oh•) which cause oxidative damage to different cellular components including membrane lipids, proteins, and nucleic acids (hasanuzzaman et al., 2020). plants use low molecular mass antioxidants such as ascorbic acid, superoxide dismutase (sod), catalases (cat), ascorbate peroxidases (apx), glutathione s-transferases (gst) and glutathione peroxidases (gpx) to scavenge ros (apse and blumwald, 2002). several mechanisms have been developed in plants under salt stress, one of which is the control of ion movement across tonoplasts to maintain a low na+  concentration in the cytoplasm (brini and masmoudi, 2012). apse and blumwald (2002) showed that plants could use several strategies to keep a high k+/na+ ratio in the cytosol to control the entry of na+ ions into and out of cells. polyploidy has been used in horticulture as a breeding tool to improve morphological, physiological, and physio-biochemical characteristics (kermani et al. 2003, talebi et al. 2017). some polyploids are tolerant to environmental stresses such as drought (li et al. 2009), heat (zhang et al. 2010), nutrient-poor soils (kolar et al. 2014), and salinity (mouhaya et al. 2010, podda et al. 2013). this increased tolerance may be related to duplicate gene expression or simply associated with evolutionary processes. meanwhile, few studies have specifically reported the relationship between ploidy level and abiotic tolerance in plants (podda et al. 2013). polyploidy plants had enabled better adaptation to some detrimental environmental conditions (parisod et  al., 2010) and enhanced tolerance to a range of abiotic stresses and biotic, souch as soil salinity (chao et al., 2013). polyploidy improved resistance to salt stress in rice (tu et al., 2014), and citrus tetraploid genotypes (mouhaya et al., 2010). salt resistance in polyploidy plants was related to reduced sensitivity of plasma membrane k+permeable channels in the meristem root zone and increased sensitivity of ca2+-permeable channels in the elongation and mature root zones to h2o2 (liu et al., 2019). omami et al. (2006) reported that cat is one of the major antioxidant enzymes which breaks down h2o2 to oxygen and water. chao et al. (2013) reported that autopolyploidy induces resistance to salinity and may represent an adaptive outcome of the enhanced k+ accumulation of plants with higher ploidy. bagheri and mansouri (2014) found that polyploidy raised protein and sugar content under saline conditions. in another study, munns (2002) suggested that the soil salt reduced water absorption and growth rate which could be due to loss of cellular turgor pressure and hormonal signals produced by the roots. when the amounts of salt rise to toxic levels in the plant cell, it is transported to leaves, which results in reduction of the photosynthetic leaf area and premature leaf senescence (munns, 2002). in salttolerant plants, there is a low rate of na+ and cltransport to leaves where these ions are sorted in vacuoles in a way to prevent their build-up in cytoplasm, cell walls, and avoid salt toxicity (greenway and munns, 1980). aromatic plants that are salt stress tolerant should also maintain their growth and secondary metabolite production (aziz et al. 2008; ahmadi et al. 2013). tabatabaie et al. (2007) showed that abiotic stress changed the quantity and quality of essential oil and thus reduced the market value of the mentha piperita plants. aziz et al. (2008) reported that essential oil yields of peppermint (mentha piperita l.), pennyroyal (mentha pulegium l.), and apple mint (mentha suaveolens ehrh.) diminished under salt stress, compared with controls. currently, there is no information available regarding the effects of salt stress on induced polyploid anise hyssop plants compared with diploid parents. accord35polyploidy increases tolerance to salt stress in anise hyssop (agastache foeniculum [pursh.] kuntze) ingly, the purpose of this study was to compare the effect of salt stress on tetraploid and diploid plants by measuring growth rate, antioxidant enzyme activity, and essential oil content of this plant. materials and methods the tetraploid (2n=4x=36) and diploid (2n=2x=18) explants of anise hyssop (agastache foeniculum [pursh.] kuntze) that were used in this study were obtained from our previous study (talebi et al. 2017). these plants were grown under greenhouse conditions (16/8 h light/dark cycle, 21°c and 15°c day/night temperature and 60 % humidity). the tetraploid and diploid explants were cultured on an murashige and skoog medium medium containing 0.6 mg/l 6-benzylaminopurine (bap) and 0.2 mg/l 1-naphthaleneacetic acid (naa) and sub-cultured every four weeks (fig. 1). the cultures were incubated under controlled conditions of temperature (25±2°c), light (20002500 lux for 16 h/d provided by fluorescent tubes), and 60-70% humidity. adaptation of micropropagated plantlets was carried out in pots filled with sand and vermiculite (1:1, v:v) in a greenhouse. initially, all plants were irrigated with a nutrient solution with half strength hoagland’s for 4 weeks and then irrigated every 3 days with full-strength hoagland’s solution containing salt (nacl) at 0, 50, 100, and 150 mm (hoagland and arnon 1950). the cultures were then incubated under a photoperiod of 16 hr light and 8 hr dark, light intensity of 20002500 lux, and at a temperature of 21°c day and 15°c night and 60% humidity. morphological traits such as survival percentage and plant growth (leaf and shoot number, stem length) were measured. essential oil content was measured after three months. this content was determined using hydro-distillation by placing the aerial parts of dried plants (10 g) in a modified clevenger apparatus for 3 hours (ozturk et al. 2004) whereafter the essential oil content (w/w %) was calculated. the composition of essential oil was analyzed by gc-ms (agilent technologies 5977a gc/msd system, usa) analysis, using a fused silica capillary hp-5 column (30 m × 0.32 mm i.d.; film thickness 0.25 µm with an agilent gas chromatograph series 7890a equipped with a flame ionization detector (fid). the injector and detector temperatures were kept at 250°c and 280°c, respectively. nitrogen was used as carrier gas at a flow rate of 1 ml/min; oven temperature program was 60210°c at the rate of 4°c.min, which was then programmed to 240°c at the rate of 20°c.min, and finally, held isothermally for 8.5 min. the split ratio was 1:50 and the gc-ms analysis was carried out by agilent gas chromatograph equipped with fused silica capillary hp5ms column (30 m × 0.25 mm i.d.; film thickness 0.25 µm) coupled with 5975c mass spectrometer. helium was used as carrier gas with an ionization voltage of 70 ev. ion source and interface temperatures were 230°c and 280°c, respectively. finally, the mass ranged from 45 to 550 amu (atomic mass unit). the activity of antioxidant enzymes such as cat and pod was measured according to the method of chance and maehly (1955). experiments were analyzed in a factorial design based on a completely randomized design. analysis of variance was performed and comparisons of means were conducted using duncan’s multiple range test (dmrt) at the 0.01 or 0.05 levels of probability. all analyses were performed using sas and mstatc software. results it was observed that the survival percentage of diploid and tetraploid plants decreased with elevation of nacl concentrations. the diploids survived at 100mm nacl, while tetraploids were able to survive at a higher salt concentration of 150 mm (fig. 2, 3). diploid plants did not tolerate 150 mm nacl and died under these conditions, while 21% tetraploid plants survived at 150 mm nacl. the results revealed that stem length, leaf and shoot number significantly declined in tetraploid and diploid plantlets of anise hyssop under salt stress. in diploids and tetraploids, the highest stem length and number figure 1. micropropagation of tetraploid and diploid plants of anise hyssop. 36 seyyedeh farahnaz talebi, mohammad jamal saharkhiz, maryam jafarkhani kermani , yavar sharafi of leaves and shoots was observed in the control, while the lowest stem length and leaf and shoot number was detected at 150 mm nacl (figs. 4, 5, 6). th e results indicated that cat activity was enhanced at 50 and 100 mm nacl treatments in diploids and tetraploids of anise hyssop. although the cat activity decreased at 150 mm nacl in both diploids and tetraploids, it remained higher in 150 mm nacl treatment compared with the control (table 1). fig 8? illustrates that the plants treated with 100 mm nacl had the highest pod activity. however, the activity of antioxidant enzymes was higher in the tetraploid plants (table 1). th e essential oil content extracted from the diploid and tetraploid plants is displayed in fig. 7. th e results indicated that salinity reduced the essential oil content in diploid and tetraploid plants as compared with essential oil produced in control plants. th e maximum essential oil percentages in diploid (1.37%) and tetraploid (2.82%) plants were obtained from control plants. th e minimum essential oil content was observed in 150 mm nacl in diploid (0.71%) and tetraploid (1.97%) plants. th e reductions in essential oil content were greater in diploids than in tetraploids under salt conditions. th e results of components identifi ed through gas chromatography (gc/ms) in diploid and tetraploid plants are reported in table 2. in tetraploid plants, with an increase in salt stress, the percentage of methyl chavicol, anisaldehyde, and β-caryophyllene rose, while the percentage of α-th ujene, terpinene, and germacrene d did not change. however, several other constituents decreased at the maximum salt concentration tested. figure 2. eff ect of salt stress on survival percent in diploid and tetraploid plants. figure 3. eff ect of salt stress on survival of diploid and tetraploid plants. figure 4. eff ect of salt stress on leaf number in diploid and tetraploid plants. figure 5. eff ect of salt stress on shoot number in diploid and tetraploid plants. figure 6. eff ect of salt stress on stem length in diploid and tetraploid plants. 37polyploidy increases tolerance to salt stress in anise hyssop (agastache foeniculum [pursh.] kuntze) data revealed that the percentage of all chemical constituents of essential oil in the diploid plants decreased with elevated nacl concentration. in contrast, the changes in the essential oil constituent levels in the tetraploid plants were relatively lower than in diploid plants under the salt stress conditions. discussion according to the results of this study, salt stress reduced survival percentage and plant growth in tetraploid and diploid plants. th e main reason for this reduction may be attributed to suppression of growth due to changes in developmental pathways under saline conditions. salt stress reduced leaf growth and leaves exhibited wilting and chlorosis in diploid plants (meng et al. 2011, wang et al. 2013). studies of munns (2002) showed that plants treated under saline conditions had decreased water availability as well as sodium chloride toxicity. munns (2002) reported that salt-induced drought stress decreased the ability of the plant to absorb water and nutrients from the soil. th e ability of plant cells to prevent na+ transport into the growing tissues is critically important for maintaining metabolic processes during cell growth against the toxic eff ects of na+ (khorasaninejad et al. 2010). khorasaninejad et al. (2010) reported that reduction in dry weight under salinity stress may be related to inhibition of hydrolysis of reserved foods and their translocation to the growing shoots. similar decreases in growth parameters under salt stress were found in salvia offi cinalis (ben taarit et al., 2009), thyme (ezz el-din et al. 2009), and basil (said-al ahl and mahmoud, 2010). in this study, the highest activity of antioxidant enzymes was observed in the plants treated with 100 mm nacl. increasing salinity beyond 100 mm nacl signifi cantly decreased the activity of antioxidant enzymes. under salt stress conditions, reactive oxygen species (ros) increase in chloroplasts (meng et al. 2016). generally, salt stress results in an increased accumulation of ros, such as h2o2, which may act as a signal molecule during stress conditions, which in turn induces gene expression encoding antioxidant enzymes (breusegem et al. 2001). tseng (2007) showed that salt stress tolerance in cabbage was enhanced with the production of cuprozincsuperoxide dismutase (cu/zn sod) and catalase (cat) in chloroplasts. th e levels of plant hormones such as abscisic acid (aba) increase with high salt concentrations. aba plays an important role in the mechanism of salt tolerance (omami et al. 2006). chao et al. (2013) found that autopolyploid plants have greater tolerance to salinity compared with diploids, which could be related to the enhanced k+ in the tetraploid plants. meng et al. (2016) reported that salt stress facilitated increased h2o2 production, antioxidative enzymes, non-enzymatic antioxidants, and protein activity in tetraploid plants compared with diploid plants. on the other hand, gene expression and synthesis of plant hormones such as aba grow under salt conditions (riddle et al. 2010). tu et al. (2014) found that tetraploid rice showed less root growth inhibition, accumulated a higher proline content and lower malondialdehyde (mda) content, and exhibited a higher frequency of normal epidermal cells than diploid rice did under salt conditions. th e response of salt-tolerant organisms to salinity stress involves synthesis and accumulation of osmo-protective compounds, which are small, non-toxic compounds and can stabilize proteins, cellular structures and increase the osmotic pressure of the cell (yancey et al. 1982). th e high levels of proline and glycine betaine were correlated with improved tolerance to salinity (apse and blumwald, 2002). similar results were observed in melissa offi cinalis (ozturk et al. 2004), majorana hortensis (shalan et al. 2006), th ymus vulgaris (najafi an et al. 2009), and mentha pulegium (queslati et al. 2010). figure 7. eff ect of salt stress on essential oil content in diploid and tetraploid plants. table 1. infl uence of diff erent concentrations of nacl on selected antioxidant enzyme activity. means with the same letters in each column are not signifi cantly diff erent at p < 0.01%. pod activity (µmol min-1 mg-1 protein) cat activity (µmol min-1 mg-1 protein) tetraploid (%) diploid (%) tetraploid (%) diploid (%) treatment 1.17 d 0.67d 2.31 d 1.07 d control 1.33 b 0.84 b 2.43 b 1.24 b 50mm 1.62 a 1.16 a 2.50 a 1.34 a 100mm 1.21 c 0.71 c 2.37 c 1.14 c 150mm 38 seyyedeh farahnaz talebi, mohammad jamal saharkhiz, maryam jafarkhani kermani , yavar sharafi table 2. the effect of salt stress on essential oil composition in diploid and tetraploid plants. ** means followed by the same letter in each row are not significantly different by lsd test (p< 0.05). compounds diploid nacl (mm) tetraploid nacl (mm) control 50mm 100mm 150mm control 50mm 100mm 150mm α-thujene 0.52a 0.51b 0.46c 0.44d 0.64a 0.61c 0.63b 0.64a α-pinene 0.61a 0.59b 0.54c 0.52d 0.42a 0.41b 0.34c 0.27d camphene 0.52a 0.50b 0.42c 0.39d 0.41a 0.41a 0.32b 0.22c 1 -octen-3-ol 0.28a 0.17b 0.11c 0.09d 0.35a 0.31b 0.23c 0.22d 3-octanone 0.37a 0.31b 0.22c 0.16d 0.14a 0.09b 0.00c 0.00c sabinene 0.18a 0.14b 0.7c 0.00d 0.30a 0.23b 0.11c 0.02d β-pinene 0.52a 0.44b 0.39c 0.21d 0.34a 0.26b 0.16c 0.06d 3-octanol 0.04a 0.00b 0.00b 0.00b 0.10a 0.05b 0.00c 0.00c myrcene 0.04a 0.00b 0.00b 0.00b 0.06a 0.00b 0.00c 0.00c p-cymene 0.63a 0.54b 0.49c 0.36d 0.84a 0.84a 0.75b 0.73c 1 ,8cineole 3.24a 3.23b 3.18c 3.13d 3.05a 2.98b 2.87c 2.86d limonene 2.69a 2.61b 2.56c 2.53d 3.02a 2.92b 2.94c 2.93d γ-terpinene 0.37a 0.29b 0.15c 0.07d 0.32a 0.30b 0.29c 0.32d trans-sabinene hydrate 0.04a 0.00b 0.00b 0.00b 0.04a 0.00b 0.00b 0.00b cis-linalool oxide 0.08a 0.03b 0.00c 0.00c 0.05a 0.00b 0.00b 0.00b trans-linalool oxide 0.05a 0.00b 0.00b 0.00b 0.06a 0.00b 0.00b 0.00b linalool 0.55a 0.46b 0.43c 0.39d 0.61a 0.53b 0.47c 0.42d 1 -octen-3-yl acetate 0.28a 0.19b 0.12c 0.09d 0.37a 0.31b 0.27c 0.26d α-campholenal 0.02a 0.00b 0.00b 0.00b 0.02a 0.00b 0.00b 0.00b camphor 0.04a 0.00b 0.00b 0.00b 0.07a 0.03b 0.00c 0.00c trans-pinocarveol 0.27a 0.16b 0.08c 0.00d 0.30a 0.26b 0.18c 0.09d trans-verbenol 0.04a 0.00b 0.00b 0.00b 0.02a 0.00b 0.00b 0.00b pinocawone 0.03a 0.00b 0.00b 0.00b 0.03a 0.00b 0.00b 0.00b borneol 0.52a 0.47b 0.42c 0.39d 0.28a 0.22b 0.19c 0.11d terpinen-4-ol 0.02a 0.00b 0.00b 0.00b 0.05a 0.00b 0.00b 0.00b methyl chavicol 78.77a 78.73b 78.68c 78.61d 81.11d 81.13a 81.13b 81.15a piperitone 0.35a 0.26b 0.18c 0.03d 0.20a 0.14b 0.09c 0.00d anisaldehyde 0.68a 0.54b 0.43c 0.34d 0.81b 0.80c 0.82ba 0.82a bornylacetate 0.54a 0.47b 0.31c 0.28d 0.42a 0.37b 0.33c 0.26d β-bourbonene 0.58a 0.56b 0.51c 0.45d 0.44a 0.40b 0.39c 0.37d β-caryophyllene 0.72a 0.65b 0.41c 0.35d 0.61c 0.56d 0.63b 0.65a (e)-α-bergamotene 0.05a 0.00b 0.00b 0.00b 0.03a 0.00b 0.00b 0.00b α-humulene 0.04a 0.00b 0.00b 0.00b 0.02a 0.00b 0.00b 0.00b germacrene d 0.24a 0.17b 0.00c 0.00c 0.30a 0.30a 0.29b 0.30a β-selinene 0.02a 0.00b 0.00b 0.00b 0.03a 0.00b 0.00b 0.00b valencene 0.02a 0.00b 0.00b 0.00b 0.03a 0.00b 0.00b 0.00b bicyclogermacrene 0.20a 0.13b 0.00c 0.00c 0.21a 0.17b 0.08c 0.00d β-bisabolene 0.02a 0.00b 0.00b 0.00b 0.01a 0.00b 0.00b 0.00b γ-cadinene 0.04a 0.00b 0.00b 0.00b 0.02a 0.00b 0.00b 0.00b δ-cadinene 0.04a 0.00b 0.00b 0.00b 0.06a 0.00b 0.00b 0.00b spathulenol 0.33a 0.26b 0.23c 0.10d 0.45a 0.39b 0.36c 0.27d caryophyllene oxide 0.30a 0.33b 0.25c 0.07d 0.48a 0.42b 0.31c 0.27d globulol 1.45a 1.29b 1.13c 0.57d 1.72a 1.72a 1.67b 1.67b 39polyploidy increases tolerance to salt stress in anise hyssop (agastache foeniculum [pursh.] kuntze) in our study, salinity reduced the essential oil content in diploid and tetraploid plants compared with control plants. data showed that treatment of tetraploid plants with different concentrations of nacl had a different response in terms of essential oil composition and production. in the diploid plants, the percentage of all chemical constituents of essential oil decreased with elevation of nacl concentration. aziz et al. (2008) found that essential oil synthesis in peppermint was very sensitive to stress. further, olfa et al. (2009) reported that essential oil content in marjoram (origanum majorana) was reduced consistently with rising salt concentration. salinity stress requires additional energy for plant cells; therefore, the amount of carbon for growth and flower initiation and essential oil synthesis is reduced during stress (cheesman 1988). reductions in essential oil content could be due to decreases and changes in photosynthesis systems, essential oil biosynthesis and metabolic pathways (aziz et al. 2008). however, belaqziz et al. (2009) reported that oil content of thymus maroccanus did not change with elevation of salt concentration. the results of the present investigation demonstrated that anise hyssop is sensitive to salt stress. however, tetraploid plants were more resistant to salt stress than diploids. this was most probably due to the bigger cell size and fewer cells in the unit area in tetraploids compared with diploids (comai, 2005). thus, the responses of polyploid plants may differ in terms of morphological, physiological, cellular and biochemical aspects (shafieizargar et al. 2013). riddle et al. (2010) reported that polyploidy induction increased chromosome number, dna content, gene expression, and enzyme activity per cell. in addition, according to our previous study, the polyploid plants of anise hyssop had a larger stomata size and density, chloroplast number, morphological features (leaf length and width, distance between the nodes, leaf area, plant height, fresh and dry weight, and spikes length), and physio-biochemical characteristics (net photosynthesis, protein content, catalase and peroxidase activity) (talebi et al. 2017). thus, tetraploid plants could naturally tolerate salt stress better than diploid plants. according to zhang et al. (2015), the response of the autotetraploid apple seedlings to salt stress was better than that of the diploid. other reports have also suggested that polyploidy induction is an efficient way to increase abiotic stress tolerance in spathiphyllum wallisii (van laere et al. 2010), dendranthema nankingense (liu et al. 2011), brassica rapa l. (meng et al. 2011), and nicotiana benthamiana (deng et al. 2012). conclusion according to the results obtained in the present study, salt stress reduced survival percentage, stem length, leaf and shoot number in tetraploid and diploid plants. the minimum growth rates were detected at 150 mm nacl in both diploids and tetraploids. however, since tetraploid plants had higher rates of growth compared with diploids, they showed a higher percentage survival and growth compared with diploids under salt stress conditions. the highest activity of antioxidant enzymes for the two ploidy levels was observed in the plants treated with 100 mm nacl. tetraploid plants were more resistant to salt stress than diploids. increasing salt concentration caused a significant reduction in the essential oil content in both tetraploid and diploid plants. nonetheless, tetraploid plants showed different responses under different salinity stress conditions when the percentage of essential oil composition was measured. in the diploid plants, the percentage of all chemical constituents of essential oil decreased with increasing nacl concentration. the results of our work suggest that in anise hyssop, tetraploid plants have a better protective mechanism than diploid plants against saline conditions. acknowledgements the authors wish to extend their thanks and appreciation to shiraz and shahed universities research and technology councils for their financial and technical supports. references ahmadi t, jafarkhani kermani m, mashayekhi k, hasanloo t, shariatpanahi me. 2013. comparing plant morphology, fertility and secondary metabolites in rosa hybrida cv iceberg and its chromosome-doubled progenies. int res j of app and bas sci 4:3840-3849. apse mp, blumwald e. 2002. engineering salt tolerance in plants. plant biotech. 13:146–150. aziz ee, al-amier h, craker le. 2008. influence of salt stress on growth and essential oil production in peppermint, pennyroyal, and apple mint. j herbs spices and 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www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/cayologia-253 citation: j.g. nzweundji, m.f.s. ngo ngwe, s. siljak-yakovlev (2019) insights on cytogenetic of the only strict african representative of genus prunus (p. africana): first genome size assessment, heterochromatin and rdna chromosome pattern. caryologia 72(2): 9-19. doi: 10.13128/cayologia-253 published: december 5, 2019 copyright: © 2019 j.g. nzweundji, m.f.s. ngo ngwe, s. siljak-yakovlev. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. insights on cytogenetic of the only strict african representative of genus prunus (p. africana): first genome size assessment, heterochromatin and rdna chromosome pattern justine germo nzweundji1, marie florence sandrine ngo ngwe2, sonja siljak-yakovlev3,* 1 medicinal plants and traditional medicine research centre, institute of medical research and medicinal plants studies (impm), p. o. box 6163 yaoundé, cameroon 2 national herbarium of cameroon-institute of agricultural research for development, bp 1601 yaoundé-cameroon 3 ecologie, systématique et evolution, univ. paris-sud, cnrs, agroparistech, université paris-saclay, bât 360, 91405 orsay, france *corresponding author: sonia.yakovlev@u-psud.fr abstract. prunus africana is a multipurpose evergreen species endemic to africa and an endangered species because of overexploitation. the great importance of this species resides particularly in the use of its bark against benign prostatic hyperplasia. as for most tropical trees and generally woody species, cytogenetic studies are scarce. standard and molecular cytogenetic approaches have been implemented for the first time to study p. africana from cameroon. this is the tetraploid species with a chromosome number of 2n=4x=32. genome size estimated by flow cytometry was 2c=1.44 pg. five loci (ten signals) of 35s rrna genes were observed after fluorescence in situ hybridization. ten g-c rich dna regions were detected by chromomycin a3 fluorochrome banding. all chromomycin positive bands were co-localized with 35 s rdna signals. prunus africana, the only strict african representative of genus prunus, is in need of the conservation strategy and in situ management that we are also discussing in this work. keywords. chromomycin fluorochrome banding, chromosome number and 2c dna value, fluorescent in situ hybridization (fish), heterochromatin and rrna gene patterns. introduction the genus prunus s. l. comprise from 200 to 430 species according different authors: over 200 (rehder 1940), approximately 250 (wen et al. 2008), 400 (maghuly et al. 2010), and 430 (according wielgorskaya 1995 and niklas 1997). prunus africana (hook. f.) kalkman, (syn. pygeum africanum hook.f.) belonging to the rosaceae family (subfamily amygdaloideae), known as african cherry (kalkman 1965), is the only species of the genus prunus endemic 10 justine germo nzweundji, marie florence sandrine ngo ngwe, sonja siljak-yakovlev to africa (fig. 1). it belongs to subgenus laurocerasus which comprises all evergreen species of the genus prunus (kalkman 1965; bodeker et al. 2014). this species of the afromontane flora is present along the central and southern part of africa, as well as madagascar and on the islands of bioko, são-tomé, and grande comore (kalkman 1965; cunningham et al. 1997; white 1983; hall et al. 2000). the main habitats of this species are fallow land, primary and secondary forests. it grows in montane and sub montane forest at a relatively high altitude, from 600 m to 3000 m in the tropical africa according to kalkman (1965) and stewart (2003a, b). the eastern limit of the distribution of the species is in cameroon, where prunus africana is found on mountain ranges called “volcanic line of cameroon” which includes the regions of southwest, northwest, west and adamaoua, and in some sites of the central region (melle et al. 2016). this species has several vernacular names in cameroon (cunningham and mbenkum 1993) but it is commonly called african pygeum which is the name attributed to this plant during its first description by hooker (1864) as pygeum africanum hook.f. and later was moved to the genus prunus subg. laurocerasus by kalkman (1965). prunus africana attains a height of 30 to 40 m (stewart 2003; kadu et al. 2012). its mature bark resembles the skin of the crocodile and its wood is brown and red. however, the morphology of the species varies according to its habitat, especially concerning the diameter and the height of the trees. the flowers of prunus africana are hermaphrodite (hall et al. 2000) and the flowering period coincides with low seasonal rainfall and the lowest temperatures, including november to february. its fruiting is irregular, two to three months intervening between flowering and fruit maturity, and occurs every 2 to 3 years (geldenhuys 1981). the first flowering takes place when the tree is between 15 and 20 years old, when it is not exploited (simons et al. 1998). seeds are recalcitrant (schaefer, 1990) and lose their germinative power after three weeks (ondigui 2001). both self-pollination and insect cross-pollination (were et al. 2001) occur in prunus africana; however, cross-pollination is the preferential reproductive system of this species (tonye et al. 2000). prunus africana is used for multiple purposes, including artisanal and medicinal uses (cunningham et al. 2002). the great importance given to this tree resides particularly in the use of its bark against benign prostatic hyperplasia, first discovered and patented by debat (1966) and since studied by several authors (njamnshi and ekati 2008; kadu et al. 2012). the prunus leaves and roots are also used in the traditional pharmacopoeia as febrifuge for the treatment of upset stomach, pulmonary infection (chest pain), malaria, fever, sexually transmitted diseases and injuries (carter 1992; cunnigham and mbenkum 1993). apart from this medicinal use, its wood is used locally as firewood and for construction, especially the construction of wagons (stewart 2003a and b). this plant gives important economic opportunities to rural communities, and over the past decades interest for p. africana has changed from traditional to international commercial use. since 1972, cameroon has been the major source of bark trade (cunningham and mbenkum 1993). despite the multiplicity of its uses, the declining of p. africana populations has been observed in many areas due to the overexploitation of its bark, which threatens a gradual disappearance of the species mainly by commercial harvesting (cameroon, equatorial guinea, kenya, drc, uganda, madagascar) and by habitat degradation and fragmentation accompanied by invasive alien species which is the case in the southern africa (jimu et al. 2011). this overexploitation led the world alliance for nature to classify p. africana as a vulnerable species which was listed in appendix ii of the convention on international trade in endangered species of fauna and flora (cites) in 1994, becoming effective in 1995 (sunderland and tako 1999). to ensure sustainable utilization and management of p. africana in many african countries where it grows, exportation requires a permission. before other considerations, the conservation and in situ management of p. africana requires a good knowledge fig. 1. global distribution of p. africana according hall et al. (2000) with geographic position of collection sites in cameroon (box). 11insights on cytogenetic of the only strict african representative of genus prunus (p. africana) of the genetic structure of this species with low potential of colonization. for prunus africana, many studies have been carried out on genetic diversity (barker et al. 1994; dawson 1999; avana et al. 2004; muchugi et al. 2006; atnafu 2007; clair and howe 2011; kadu et al. 2013; mihretie et al. 2015, nantongo et al. 2016), biochemical property (tchouakionie 2014; nzweundji 2015) but cytogenetic studies are still lacking, hence the present study. the karyological studies in the genus prunus were principally limited on chromosome count. if we consider, as wen et al. (2008), that the genus prunus comprises about 250 species, the chromosome number is available for more than 76 percent of species (191/250 according to available chromosome databases). the genus presents the basic chromosome number of x=8 (darlington 1927, 1928) and at least three ploidy levels, diploid, tetraploid and hexaploid (oginuma 1987; bennett and leitch 1995; iwatsubo et al. 2002; maghuly et al. 2010). however, some species have been studied by molecular cytogenetics as p. amygdalus stokes (corredor et al. 2004), p. persica (l.) batsch peach (yamamoto et al. 1999; yamamoto 2012) and p. subhirtella hook. (maghuly et al. 2010). genome size is a fundamental parameter in many genetic and molecular biological studies. knowledge of the genome size or the 2c value is important for basic and applied studies involving genome organization, species relationships, phylogeny and even taxonomy and biodiversity (bennett 1984; bennett and leitch 2005). at present, flow cytometry is the main method for evaluation of nuclear dna content because it is both rapid and precise, and reveals even the small differences in dna content (marie and brown 1993; doležel and bartoš 2005). however, till today, and according to available databases the genome size of about only 25 species of prunus has been estimated (arumuganathan and earle 1991; dickson et al. 1992; baird et al. 1994; bennett and leitch 1995; loureiro et al. 2007; siljak-yakovlevet al. 2010; gainza-cortés 2014; žabka et al. 2018), which presents 10% for the genus. despite the importance of genome size, to the best of our knowledge there is no report on the dna amount of prunus africana, which is the only species of prunus originating from africa. it is the same case for cytogenetical characterization of this species, despite the fact that for other species of this genus the banding techniques and fluorescent in situ hybridization (fish) has been developed in order to detect respectively the difference between morphologically similar chromosomes and localization of useful genes (soodan et al. 1988; corredor et al. 2004; maghuly et al. 2010; yamamoto 2012). therefore, we consider that it was necessary to characterize the genome of p. africana: 1) by assessment of dna content using the flow cytometry technique; 2) chromosome counting for determination of ploidy level using the standard cytogenetic method; 3) distribution patterns of gc-rich heterochromatin using the fluorochrome banding; 4) chromosome localization of 18s-5.8s-26s (35s) rrna genes using a fluorescent in situ hybridization. the results will be compared and discussed with those available for other species of the genus prunus. materials and methods origin and conditioning of plant material the seed samples (50 seeds from two populations) of prunus africana were collected in mont oku at 2400 m and mont cameroon at 2500 m of altitude in cameroon (fig. 1, box). the hard endocarps of seeds were removed and seeds of uniform size (about 0.7 cm) were used for germination. they were germinated directly in glass dishes containing watered filter paper (fig. 2c). cultures were visually checked daily and irrigated with tap water when necessary under laboratory conditions at about 24°c. after 6 weeks, the root meristems of germinated seeds were used for cytogenetic studies. germinated seeds were transferred to pots and after 4 weeks the leaves were collected for flow cytometry (fig. 2d). genome size assessment by flow cytometry the total nuclear dna content was determined by flow cytometry according to marie and brown (1993). solanum lycopersicum, 2c= 1.99 pg (lepers-andrzewski et al. 2011) was used as internal standard. leaf tissue of prunus africana and solanum lycopersicum was placed in a plastic petri dish and chopped together with a razor blade in 600 µl of cold gif nuclear buffer which is slightly modified galbraith’s buffer (galbraith et al. 1983): 45 mm mgcl2, 30 mm sodium citrate, 60 mm 4-morpholinepropane sulfonate ph 7, 0.1 % (w/v) triton x-100, 1% polyvinylpyrrolidone (~10,000mr, sigma p6755), 5 mm sodium metabisulfite and 10 µg/ml rnase (sigma aldrich, saint quentin, france). nuclear suspensions were filtered through nylon mesh (pore size 50 mm, cell trics, partec) and stained with 50 mg ml-1propidiumiodide (pi: sigma-aldrich, france). after 5 min incubation on ice, nuclear suspensions were analyzed. five individuals per accessions were analyzed in order to obtain the mean dna content. at least 5000 to 10, 000 nuclei were analyzed for each sample using a cyflow sl3, partec, 532-nm laser cytometer (munster, ger12 justine germo nzweundji, marie florence sandrine ngo ngwe, sonja siljak-yakovlev many). nuclear dna content was estimated using the linear relationship between the fluorescent signals from stained nuclei of p. africana specimens and the internal standard (s. lycopersicum) according to the following equation: 2c dna content/nucleus = [sample 2c peak mean × standard 2c dna] (pg) standard 2c peak mean the symbol c corresponds to the holoploid nuclear genome size (the whole chromosome complement with chromosome number n), 1c and 2c being, respectively, the dna contents of the haploid (n) and diploid (2n) sets of chromosomes, irrespective of ploidy level (greilhuber et al. 2005). the conversion from picograms (pg) to base fig. 2. prunus africana: a) 6-years-old tree; b) fruit two-lobed drupe, with a seed in each lobe; c) germination of seeds in petri dishes; d) seedlings. 13insights on cytogenetic of the only strict african representative of genus prunus (p. africana) pairs (bp) was done as follows: 1 pg dna = 978 mbp (doležel et al. 2003). determination of chromosome number root tips, obtained from potted plants were pretreated with 0.002 m 8-hydroxyquinoline at 16°c for 3h. fixation was performed in freshly prepared 3:1(v/v) ethanol-acetic acid for at least 24h. fixed root tips were stored for a few days in the first fixative, or several months in 70% ethanol at 4°c. for chromosome counting the root tips were hydrolyzed in 1n hcl for 10 min at 60°c and stained in schiff reagent following the standard feulgen method, or in an enzymatic mixture for 45 min at 37°c. the squash was performed in a drop of acetic carmine. the chromosome number was also verified on slides prepared for fluorochrome banding by chromomycin a3 or for fish for which they were counterstained with dapi (4’, 6-diamidini-2-phenylindole). chromosome plates were observed under a zeiss axiophot microscope. the chromosome number was determined for at least 10 individuals, from several wellspread metaphases per root tip. preparation of protoplasts for fluorochrome banding and fluorescence in situ hybridization (fish) fixed root tips were washed in 0.01 m citrate buffer ph 4.6 for 15 min, then incubated in an enzymatic mixture for 45 min at 37°c. this enzymatic mixture was composed of 4% cellulase r10 (onozuka yakult honsha co.), 1% pectolyase y23 (seishin pharmaceutical, co., tokyo, japan), and 4% hemicellulase (sigma chemical co.) in 0.01m citrate buffer at ph 4.6. these digested meristems were squashed onto a drop of freshly prepared 45% acetic acid and the preparations were observed at a phase contrast microscope. the best slides were frozen at -80º c over night and then the cover slips were removed and the slides were rinsed with absolute ethanol and air-dried. fluorochrome banding for detection of gc-rich dna regions, the chromosomes were stained with chromomycin a3 (cma3) fluorochrome using schweizer’s (1976) method, with slight modifications, from siljak-yakovlev et al. (2002) concerning the concentration of chromomycin (0.2 mg/ml) and time of staining (60 min). after chromosome observation, the best slides were distained in 3  :1 ethanolacetic acid, dehydrated in a graded ethanol series (70%, 90%,100%), air-dried for at least 12h at room temperature, and then used for fish experiment. florescence in situ hybridization fish was performed for the detection of 35s rdna loci. the 35s rdna probe was a clone of 4-kb from ecori fragment, including 18s-5.8s-26s rdna sequences from arabidopsis thaliana (l.) heynh. labelled with the direct cy3 fluorochrome (amersham, courtaboeuf, france) by nick translation, according to the manufacturer’s protocol. in situ hybridization was carried out following heslop-harrison et al. (1991) with some minor modifications. slides were counter-stained and mounted in vectashied medium (vector laboratories, peterborough, uk) with dapi. chromosome plates were observed using an epifluorescence zeiss axiophot microscope with different combinations of excitation and emission filter sets (01, 07, 15 and triple filter set 25). hybridization signals were analyzed and the best metaphase plates were photographed using a highly sensitive ccd camera (retiga 200r, princeton instruments, every, france) and image analyzer (metavue, every, france). results nuclear dna content and chromosome number in this study both the genome size and chromosome number of prunus africana were determined for the first time. nuclear dna content of p. africana was 2c=1.44 pg or 1408 mbp (1pg dna = 978 mbp according to doležel et al. 2003), ranking the taxon in the category of very small genomes (2c ≤ 2.8 pg, according to leitch et al. 1998). the chromosome number showed that p. africana is a tetraploid species with 2n=4x=32 small (~ 1 to 2 µm) chromosomes (fig. 3) and basic chromosome number x=8. this number, which showed a high stability, has been verified with all used techniques: squash in acetic acid after staining in carmine acetic (fig. 3a); protoplast technique with chromosome speeded in acetic acid without staining (fig. 3b – phase contrast picture), after fluorochrome bandings with dapi staining (fig. 3c) and cma3 (fig. 3d), and after fish experiment (fig. 3f). rrna genes and heterochromatin pattern fluorochrome banding revealed ten g-c rich heterochromatin regions (cma+ bands), which were always 14 justine germo nzweundji, marie florence sandrine ngo ngwe, sonja siljak-yakovlev associated with rdna loci (fig. 3d). the karyotype of p. africana is characterized by five loci of 35s rdna (visible as ten red hybridization signals), which were located in terminal or subtelomeric positions. these strong red signals appeared as weakly stained regions after dapi counterstaining which indicated their richness in g-c bases (fig. 3e, arrows). the number and the position of 35s rdna loci are presented in fig. 3f. some centromeric 35s signals were of weaker fluorescent intensity than terminal signals and do not appear in all observed metaphase plates. dapi stained chromosomes did not display any visible bands (fig. 3c). discussion phylogenetic context the subfamily amygdaloideae comprises nine tribes and for several of them the relationships were uncertain in previous phylogenetic study (potter et al. 2007). in recent work of xiang et al. (2016) the tribe neillieae is supported as the basal lineage of amygdaloideae, followed by the tribe spiraeeae. the only species of lyonothamneae, previously placed as the basal lineage, becomes the sister to amygdaleae. this phylogenetic context is also very well supported by the basic chromosome number. the two basal clades have x = 9, which is also the ancestral basic number of rosaceae (vilanova et al. 2008), and two tribes lyonothamneae and amygdaleae, formed the only clade with x = 8 in subfamily amygdaloideae. it is, at the same time, the only events of decreasing dysploidy in this subfamily while in the subfamily rosoideae this phenomenon occurred several times because x = 7 is the most frequent base number. the closest genera of prunus, maddenia and pygeum also have x = 8. in the phylogenetic frame prunus africana is always in the same clade especially with the asian tropical species of the genus pygeum which shows that they are very close (wel et al. 2008). chromosome number and nuclear dna content to verify the genome size and chromosome number data for the genus prunus and to attribute the status of novelty for investigated species, we used updated databases: kew plant dna c-values database (http://data. kew.org/cvalues), flower, a plant dna flow cytometry database (http://botany.natur.cuni.cz/flower/index.php), index to plant chromosome numbers (ipcn) tropicos (http://www.tropicos.org/project/ipcn) and the chromosome counts database (ccdb), (http://ccdb.tau. ac.il/search/). in the genus prunus, which has been highly studied from many viewpoints, the number of species with available genome size information is very low, only about 25 (10 %) species have genome size records to date, whereas chromosome numbers have been reported for at least 76 % of the taxa. it is a very good report knowing that chromosome number has been determined to date for about only 25% of angiosperms taxa (stuessy 2009) or for only 20% according to rice (2015). the basic chromosome number of prunus genus is x=8 (darlington 1927, 1928) and their ploidy level ranges from diploid (2n=2x=16) to tetraploid (2n=4x=32) fig. 3. a) schiff stained metaphase plate showing 2n=32 chromosomes. b) non-colored metaphase chromosome plate obtained after enzymatic hydrolysis photographed under microscope with phasecontrast. c) dapi stained chromosomes without visible bands. d) the same metaphase plate (d, e and f) after the fluorochrome banding with chromomycin a3. the cma+ bands corresponded to dna regions rich in g-c bases. e) dapi stained chromosome after fish experiment showing dapi negative bands (arrows) corresponding to a-t low dna regions. f) fish experiment revealed ten 35 rdna (red) signals which corresponded to ten cma+ bands (see fig. c). bar scale = 10 µm. 15insights on cytogenetic of the only strict african representative of genus prunus (p. africana) and hexaploid (2n=6x=48) (bennett and leitch 1995; maghuly et al. 2010; siljak-yakovlev et al. 2010). prunus africana is a tetraploid species (2n=32) with genome size 2c=1.44 pg. based on available bibliographic data genome size for diploid prunus species ranges from 0.55 to 1.00 pg (dickson et al. 1992) and for tetraploids from 1.14 to 1.30 (arumuganathan and earle 1991; siljakyakovlev et al., 2010). the 2c values of p. africana (1.44 pg) was slightly bigger that in other tetraploids. the only hexaploid species measured until now is p. domestica l. (darlington and wylie 1955) with 2c=1.85 pg (arumuganathan and earle 1991; bennett and leitch 1995). horjales et al. (2003) reported 2c=2.35 pg for p. padus l. and zonneveld et al. (2005) 7.30 pg for p. laurocerasus l. which indicated the existence of higher ploidy levels that 6x in this genus. heterochromatin and 35s rdna pattern recorded in this study the data concerning heterochromatin and rrna genes patterns for prunus africana were reported for the first time. in table 1 we presente available molecular cytogenetics data for some species of the genus prunus and compare with the results obtained in this work. we observed 10 g-c rich dna regions in all the samples used for this experiment. previous studies performed for prunus persica (yamamoto et al. 1999; yamamoto 2012), p. salicina lindl. (yamamoto 2012) and p. armeniaca l. (yamamoto 2012; arumuganathan and earle 1991) revealed six and in p. mume five to eight g-c rich dna regions (yamamoto 2012). the gc-rich heterochromatin was always co-localized with 35s rdna loci. the same observation has been reported by yamamoto et al. (1999) and yamamoto (2012) for prunus persica. the co-localization of gc-rich heterochromatin and ribosomal genes has been frequently reported; e.g. in retama (benmiloud-mahieddine et al. 2011); fraxinus (siljak-yakovlev et al. 2014); tanacetum  l.  (olanj et al. 2015); eucalyptus (riberio et al. 2016); sclerocaria (bationo-kando et al. 2016). other rdna patterns have been described in several prunus species. this is the case of 5s and 18s-5.8s-25s ribosomal rna genes which have been located in prunus persica (yamamoto et al. 1999), in p. amygdalus batsch (corredor et al. 2004) and in two others species of cherry rootstock; p. subhirtella miq. (corredor et al. (2004); maghuly et al. 2010) and p. incisa x serrula (maghuly et al. 2010). in these last studies the diploid p. subhirtella presented six 35s rdna signals as in the majority of diploid prunus species (yamamoto 2012), while the recent tetraploid hybrid p. incisa x serrula presented namely the double (12 signals). the diploid ancestor of p. africana probably had three loci of 35s rdna whose numbers decrease from six to five loci during the chromosomal restructuring after polyploidisation. conclusion the present paper has focused on the cytogenetic characterization of prunus africana, contributing to the better knowledge of this useful african tree. howtable 1. prunus species for which some molecular cytogenetic data are available. comparison with our results of p. africana. species 2n (ploidy level) 2c dna in pg 35s rdna signals number (position) cma+/dapi bands number references p. africana (hook. f.) kalkman 32 (4x) 1.44 10 (terminal, satellite) 10 present work p. amygdalus stokes 16 (2x) 0.66 6 (terminal satellite) corredor et al. 2004 p. armeniaca l. 16 (2x) 0.60 6 yamamoto 2012; arumuganathan and earle 1991 p. incisa x serrula 32 (4x) 1.22 12 (terminal satellite) maghuly et al. 2010 p. mume (siebold) siebold & zucc 16 (2x), 32 (4x) 5 to 8 yamamoto 2012 p. persica (l.) batsch peach 16 (2x) 0.55 6 (terminal, satellite, proximal) 6 yamamoto et al. 1999, yamamoto 2012 p. salicina lindl. 16 (2x) 6 yamamoto 2012 p. subhirtella hook. probably 16 (2x) 0.61 6 (terminal) corredor et al. (2004); maghuly et al. 2010 16 justine germo nzweundji, marie florence sandrine ngo ngwe, sonja siljak-yakovlev ever, future studies from populations collected in other regions would provide more information for the sustainable management of this endangered species. currently, p. africana is on the iucn red list and has been classified as a priority for conservation by fao. in cameroon and at the international level, provisions have been made and laws have been drawn up to ensure the rational exploitation of this species. these investigations will make possible to identify the priority areas for the conservation of this species but also to establish a best management plans for the sustainability of genetic resources of prunus africana. acknowledgements we would like to thank michael bourge for his expertise of the flow cytometry of i2bc (www.i2bc. paris-saclay.fr), gif-sur-yvette, france. samuel pyke is acknowledged for critical reading of the manuscript and english text revision. this work was partially supported by grants from the  international union of biochemistry and molecular biology (grant mfs ngo ngwe). references arumuganathan k, earle e. 1991. nuclear dna content of some important plant species. plant molecular biology reporter 9: 208-218. atnafu h. 2007. genetic variation in some natural population of prunus africana from ethiopia as revealed by randomly amplified polymorphic dna (rapd). 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cells özlem sultan aslantürk evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of allium test ioan sarac1, elena bonciu2,*, monica butnariu1, irina petrescu1, emilian madosa1 fluorescence in situ hybridisation study of micronuclei in c3a cells following exposure to elf-magnetic fields luc verschaeve1,2,*, roel antonissen1, ans baeyens3, anne vral3, annemarie maes1 phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities nicoleta anca şuţan1,*, irina fierăscu2, radu fierăscu2, deliu ionica1, liliana cristina soare1 telomeric heterochromatin and meiotic recombination in three species of coleoptera (dorcadion olympicum ganglebauer, stephanorrhina princeps oberthür and macraspis tristis laporte) anne-marie dutrillaux, bernard dutrillaux* a whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of populus trichocarpa l. subjected to different stresses alberto vangelisti#, gabriele usai#, flavia mascagni#, lucia natali, tommaso giordani*, andrea cavallini differences in c-band patterns between the japanese house mice (mus musculus) in hokkaido and eastern honshu hikari myoshu, masahiro a. iwasa* karyotypic description and repetitive dna chromosome mapping of melipona interrupta latreille, 1811 (hymenoptera: meliponini) natália martins travenzoli1, ingrid cândido de oliveira barbosa2, gislene almeida carvalho-zilse2, tânia maria fernandes salomão3, denilce meneses lopes1,* caryologia. international journal of cytology, cytosystematics and cytogenetics 73(4): 11-16, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-811 caryologia international journal of cytology, cytosystematics and cytogenetics citation: a. turco, a. albano, p. medagli, s. d’emerico (2020) contribution to the study of wild orchidaceae, genus platanthera l.c.m. richard. karyotype and c-banding analysis of two species from italy. caryologia 73(4): 11-16. doi: 10.13128/caryologia-811 received: january 08, 2020 accepted: november 18, 2020 published: may 19, 2021 copyright: © 2020 a. turco, a. albano, p. medagli, s. d’emerico. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid at: 0000-0001-9071-344x aa: 0000-0002-0874-320x contribution to the study of wild orchidaceae, genus platanthera l.c.m. richard. karyotype and c-banding analysis of two species from italy alessio turco1,*, antonella albano1, pietro medagli1, saverio d’emerico2 1 dept. of biological and environmental sciences and technologies, university of salento, lecce, italy 2 “aldo moro” university of bari, bari, italy *corresponding author. e-mail: alessio.turco@unisalento.it abstract. this study examined the chromosome numbers and karyotypes of two taxa of the genus platanthera (orchidaceae) from italy. cytological analyses showed 2n = 2x = 42 in p. chlorantha and p. algeriensis. karyotype analysis revealed similarity between the species. the karyotypes are as follows: p. chlorantha consists of 34 metacentric + 8 submetacentric pairs and p. algeriensis consists of 36 metacentric + 6 submetacentric pairs. both species possess a rather symmetrical karyotype. p. chlorantha has a very similar c-banding pattern to p. algeriensis. dapi bright blocks were observed in p. chlorantha. these analyses also show the close relationship between the studied species. keywords: chromosome number, c-banding, heterochromatin content, karyotypes, orchidaceae, platanthera algeriensis, p. chlorantha. introduction the genus platanthera rich., also known as “butterfly orchids”, belongs to the subtribe orchidinae (subfamily orchidoideae) and consists of 100 to 200 species (wood 2001; delforge 2016; efimov 2016 and references therein). the geographical distribution of platanthera species covers most of the temperate areas of europe, north africa, asia, new guinea and north and central america (hulténand fries 1986; wood 2001; efimov 2016), with 12 species widespread in europe, six of which are found in italy (delforge 2016). this genus is divided into 5 sections (efimov, 2016), three of which are found in europe: p. hyperborea, the most ancient clade, which has a fareastern and north-american distribution, with only p. hyperborea (l.) lindl. present in iceland; the p. oligantha clade, with a circumpolar distribution, and finally the eurasian section platanthera (delforge, 2016). platanthera species are terrestrial, photosynthetic and – in very few cases – epiphytic or lithophytic. they are found in a variety of habitats, including meadows, temperate and boreal forests, bogs, fens, marshes and prairies. european pla12 alessio turco, antonella albano, pietro medagli, saverio d’emerico tanthera species are geophytes, characterised by a broad anther, 2 elongated root-tuberoids, a dorsal sepal and petals combining to form a helmet, a stigma without processes and an enlarged receptive surface with a nectariferous spur. in cytological analyses performed on taxa of the genus platanthera, the chromosome number was found to be 2n = 2x = 42 (cauwet-marc and balayer 1986; yokota 1987; yang and zhu 1988; dalgaard 1989; d’emerico 2001 and references therein). to date however, only four taxa exhibit polyploidy: platanthera hyperborea (dalgaard 1989) and p. huronensis (nutt.) lindl. (sheviak and bracht 1998), both with 2n = 4x = 84 chromosomes; p. obtusata (banks ex pursh) lindl., which may be triploid in some populations with 2n = 63 (tanaka and kamemoto 1984); and the nordic–siberian p. oligantha turcz. (= p. obtusata subsp. oligantha), which according to webb (1980) is hexaploid (2n = 126). as already mentioned above, six of these species are found in italy: platanthera algeriensis batt. & trab. 1892, p. bifolia subsp. bifolia (l.) rich. 1817, p. bifolia subsp. osca lorenz, romolini, romano & soca 2015, p. bifolia subsp. subalpine brügger, p. chlorantha (custer) rchb. and p. kuenkelei subsp. kuenkelei var. sardoa lorenz, akhalk, baumann, cortis, cogoni & scrugli 2012. in this study, karyotype morphology and the distribution of heterochromatin in italian specimens of p. chlorantha and p. algeriensis were studied for the first time. the aim of this study was to verify chromosome numbers and to compare the heterochromatin pattern of the above-mentioned species in order to verify similarities between them. materials and methods the material studied in this investigation was gathered from natural populations of platanthera chlorantha and p. algeriensis in apulia and sardinia (table 1). mitotic chromosomes were prepared from immature ovaries, pre-treated with 0.3% colchicine at room temperature for 2h. for feulgen staining they were fixed for 5 min in 5:1:1:1 (v/v) absolute ethanol, chloroform, glacial acetic acid and formalin, hydrolysed at 20 °c in 5.5 n hcl for 20 min (battaglia 1957) and stained in freshly prepared feulgen solution. for c-banding, ovaries were fixed in 3:1 (v/v) ethanol–glacial acetic acid and stored in a deep-freeze for up to several months. subsequently, they were squashed in 45% acetic acid; coverslips were removed by the dry ice method and the preparations were air-dried overnight. the slides were then immersed in 0.2n hcl at 60 °c for 3 min, thoroughly rinsed in distilled water and then treated with 4% ba(oh)2 at 20°c for 4 min. after thorough rinsing they were incubated in 2xssc at 60°c for 1h, and then stained in 3-4% giemsa (bdh) at ph 7 (d’emerico et al. 1996). for dapi (4–6-diamidino2-phenylindole) staining, ovaries were treated as for c-banding and stained using a buffered dapi solution (0.6 mg/ml) for 5 min, followed by rinsing and mounting in glycerol buffer (1:1 v/v). chromosome pairs were identified and arranged on the basis of their length and any other evident karyomorphological feature. heterochromatin content was assessed using micromeasure 3.3, a freeware program from colorado state university (reeves 2001). karyoty pe symmetry indices – mca (mean centromeric asymmetry) and cvcl (coefficient of variation of chromosome length) – were used for the evaluation of karyotype asymmetry (peruzzi et al. 2009). the nomenclature used for describing karyotype composition followed levan et al. (1964). a list of the examined specimens and their sampling locations is given in table 1. results and discussion analysis of the somatic metaphases showed that the diploid chromosome number is 2n = 2x = 42 in both platanthera chlorantha and p. algeriensis. p. chlorantha, known as the “greater butterf ly orchid” (lima-de-faria 2020) was found to be diploid with 2n = 2x = 42 chromosomes (fig. 1a), in agreement with previous reports (scrugli 1980; averyanov et al. 1985; cauwet-marc and balayer 1986), with chromotable 1. taxon, sites, chromosome number, formula and percent heterochromatin in set of the chromosomes of species platanthera chloranta and p. algeriensis. m, metacentric; sm, submetacentric. taxon site chromosome number (2n) formula % het in set p. chlorantha martina franca (ta) balvano (pz) 42 34m+8sm 25.50 p. algeriensis aritzo (nu) 42 34m+2m(sm)+6sm 22.43 13contribution to the study of wild orchidaceae, genus platanthera somes that range in size from 4.04 to 1.8 µm at metaphase, and the arm length ratio was from 1.03 to 3.00. six well spread-out metaphases were paired on the basis of chromosome size and centromere position and used for chromosome measurements. the karyotype consisted of 34 metacentric and 8 submetacentric chromosome pairs (fig. 2a). the complement showed two chromosome pairs with secondary constrictions on the long arm (pair 2) and the short arm (pair 8). this species possesses a fairly symmetrical karyotype (mca = 18.50±1.09 and cvcl = 21.53±0.29), with metacentric chromosomes being the most frequent. it is interesting to note that this species has similarities in terms of dimensions and structure (such as the visibility of centromeres) with karyotypes of the anacamptis group (2n = 2x = 36) (d’emerico et al. 1996). similarities with chamorchis alpina (l.) rich. (2n = 2x = 42) (d’emerico and grünanger 2001) and dactylorhiza romana (sebast.) soò (2n = 2x = 40) (d’emerico et al. 2002) can also be observed. the c-banding analysis shows that constitutive heterochromatin was located in the centromeric regions of numerous chromosomes (fig. 3a). one pair of chromosomes had the subtelomeric c-bands only on the short arm. interphase nuclei exhibited a number of chromocentres equal to that of the constant bands (fig. 3b). the centromeric regions of numerous chromosomes had bright fluorescence after staining with dapi (fig. 3c). in platanthera algeriensis, somatic cells showed 2n = 2x = 42 chromosomes (fig. 1b). this species is similar to p. chlorantha apart from its greener flowers and its very different preferred habitat. in europe this species is restricted to a few sites in corsica, sardinia, mainland italy and spain, but it also occurs in algeria. in this species, similarities to the karyotype structure and c-banding of p. chlorantha were observed. chromosome lengths were found to be between 3.90 and 2.18 µm. the karyotype consisted of 34 metacentric, 2 metacentric/submetacentric and 6 submetacentric chromosome pairs (fig. 2b). in addition, this species possesses a symmetrical karyotype (mca = 21.21±0.83 and cvcl = 18.19±0.60). constitutive heterochromatin was also detected in the centromere regions of numerous chromosomes (fig. 3d). the present analysis of chromosome evolution showed that the species p. chlorantha and p. algeriensis are very close. indeed, specimens of the two species in the present study exhibited practically the same karyotype and c-banding pattern. however, p. algeriensis seems to differ from p. chlorantha in that it has lower heterochromatin content (fig. 3-d vs. fig. 3-a). the small figure 1. mitotic metaphase with feulgen staining of platanthera chlorantha (a) and p. algeriensis (b); 2n = 2x = 42. bar = 5 µm. figure 2. diploid karyotypes of platanthera chlorantha (a) and p. algeriensis (b). bar = 5 µm. 14 alessio turco, antonella albano, pietro medagli, saverio d’emerico differences in the c-banding patterns found between the two species seem to indicate limited rearrangement of constitutive heterochromatin during their evolution. in a study based on plastid dna sequence variation, pavarese et al. (2011) found that platanthera algeriensis was characterized by haplotypes a and b, both of which are shared with p. chlorantha. in another study, italian platanthera chlorantha were found to form a single group with p. algeriensis from tunisia and sardinia (bateman et al. 2012). concluding remarks the chromosome number 2n = 2x = 42 has been reported in 13 of the 17 genera for which data are available, although the subtribe orchidinae includes about 50 genera (d’emerico 2001; felix and guerra 2005). in this subtribe, the internal transcribed spacer (its) phylogenies (bateman et al. 2001; bateman et al. 2003; jin et al. 2017) show that the group pseudorchis-amerorchisgalearis-platanthera s.l. includes genera with the chromosome number 2n = 2x = 42 (löve and simon 1968; löve 1981; cauwet-marc and balayer 1986). also it is suggested that dactylorhiza s.l. and gymnadenia s.l., which have 2n = 2x = 40, probably derive from 42 chromosomes (pridgeon et al. 1997; bateman et al. 2009). as pointed out in the discussion, it is interesting to note the remarkable karyomorphological similarity in the species platanthera chlorantha, p. algeriensis (this work), chamorchis alpina (d’emerico and grunanger 2001), galearis diantha (schltr.) p. f. hunt (luo 2004), which have 2n = 2x = 42 chromosomes and dactylorhiza romana (d’emerico et al. 2002), which has 2n = 2x = 40 chromosomes. last but not least, in spite of the extensive cytogenetic literature that has built up over the years, little is known about the karyotype structure in other species of the genus platanthera and related genera. furthermore, to our knowledge, there are few studies of constitutive heterochromatin content, despite the fact that c-banding patterns provide extra information useful in assigning genomes. we have a limited quantity of data on the platanthera genus but it is possible to make some considerations on heterochromatin content. indeed, platanthera chlorantha and p. algeriensis show centromeric and subtelomeric heterochromatin, with a higher percentage in the former. on this basis, an evolutionary comparison is possible with the genera dactylorhiza and gymnadenia. previous cy tological studies using the traditional giemsa c-banding technique have shown significant heterochromatin content in some species of these two genera (d’emerico et al. 2002; d’emerico and grunanger 2001; baumann et al. 2012). for example, in the genus gymnadenia, g. rhellicani (teppner and klein) teppner and klein, g. conopsea and g. odoratissima (l.) rich. have been found to possess numerous chromosomes with centromeric and telomeric heterochromatin. similar banding patterns were previously observed in three species of the genus dactylorhiza, including two diploids (d. romana, d. saccifera (brogn.) soò) and one polyploid (d. urvilleana subs. phoenissa b. baumann and h. baumann). moreover, the distribution of c-banding patterns in dactylorhiza karyotypes is of great cytological interest, although its nature can only be conjectured for the time being. specifically, d. romana specimens have shown the chromosome numbers 2n = 40+1b and 2n = 40+2b, with one or two heterochromatic supernumerary chromosomes, which seems to suggest a possible evolutionary trend from 2n = 42 to 2n = 40 (d’emerico et al. 2002). figure 3. giemsa c-banded mitotic metaphase of platanthera chlorantha (a) and p. algeriensis (d); 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(1990) karyomorphological studies on habenaria, orchidaceae and allied genera from japan j sci hiroshima univ. 23:53–161. caryologia. international journal of cytology, cytosystematics and cytogenetics 72(4): 85-92, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-405 citation: s.t. nabavi, f. farahan, m. sheidai, k. poursakhi, m.r. naeini (2019) population genetic study of ziziphus jujuba mill.: insight in to wild and cultivated plants genetic structure. caryologia 72(4): 85-92. doi: 10.13128/ caryologia-405 published: december 23, 2019 copyright: © 2019 s.t. nabavi, f. farahan, m. sheidai, k. poursakhi, m.r. naeini. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. population genetic study of ziziphus jujuba mill.: insight in to wild and cultivated plants genetic structure seyyedeh tahereh nabavi1, farah farahan2, masoud sheidai3,*, katayoun poursakhi1, mohammad reza naeini4 1 department of horticulture science, isfahan (khorasgan) branch, islamic azad university, isfahan, iran 2 department of microbiology, qom branch, islamic azad university, qom, iran 3 faculty of life sciences and biotechnology, shahid beheshti university, tehran, iran 4 department of horticulture crops research, qom agricultural and natural resources research and education center,areeo, qom, iran *corresponding author: msheidai@yahoo.com abstract. ziziphus jujuba (jujube) of buckthorn family (rhamnaceae) is an important medicinal crop plant cultivated in different provinces of iran. it has also wild populations in some geographical areas. we carried out population genetic study on 8 populations of cultivated versus wild jujuba by using issr molecular markers to produce data on population genetic structure, gene flow, and genetic variability in the studied populations. we also aimed to investigate genetic differentiation between wild and cultivated plants and identify the potential gene pools of this medicinal plant species. the studied populations had a moderate genetic variability and were grouped in two major groups by pcoa plot. amova revealed significant genetic difference among these cultivars. mantel test showed significant correlation between genetic distance and geographical distance in the studied populations. pcoa analysis showed genetic differentiation between wild and cultivated plants within each province. structure analysis identified two potential gene pools for jujube cultivars. data obtained may be used in genetic conservation and future breeding programs of this medicinal plant species in the country. keyword. ziziphus jujube, issr, structure. introduction the genus ziziphus mill. of the buckthorn family (rhamnaceae), contains about 40 species that are deciduous evergreen trees or shrubs and are distributed in the tropical and subtropical regions of the world (sing et al. 2007). south and southeast asia are considered to be the center of both evolution and distribution of ziziphus species (sing et al. 2007). these plant species are of medicinal value and are known to be self-incompatible and produce inter-specific hybrids (asatryan and tel-zur 2013, 2014). 86 seyyedeh tahereh nabavi et al. z. jujuba (jujube) is one of the well known species of the genus with great medicinal value. it is mainly distributed in southwestern asia. traditional use of the species dates back to 2,500 years ago, as revealed in the original chinese materia medica records. the fruit, seed, and bark are used to alleviate stress and insomnia and as appetite stimulants, digestive aids, antiarrhythmics, and contraceptives (vahedi et al. 2008). the fruit is eaten fresh or dried and made into candy; tea, or syrup (gupta et al. 2004; jiang et al. 2007). moreover, some specific saponins, as well as ethyl acetate and water extracts of the fruit and bark, have explored the potential cytotoxicity of jujube. these extracts bring about apoptosis and differential cell cycle arrest, moreover, activity against certain human cancer cell lines has been demonstrated in vitro (lee et al. 2004; huang et al. 2007;vahedi et al. 2008). ziziphus jujube is an important plant species to the mankind, due to which its cultivation and conservation gained high importance within recent years. moreover, as jujube has wide geographical distribution and forms many local populations, it is important to be studied from population genetic point of view. the species with extensive geographical distribution can be adapted to adverse environmental conditions and harbor different gene content that may be used in future breeding programs and establishing genetic-rich germ plasm collections (sheidai et al. 2013, 2014, 2016). different molecular markers were used to investigate the genetic diversity in z. jujuba cultivars or wild individuals. for instance, random amplified polymorphic dna (rapd), amplified fragment length polymorphisms (aflp), sequence-related amplified polymorphisms (sr ap), simple sequence repeats (ssr), inter-simple sequence repeats (issr), and chloroplast microsatellite (cp-ssr) markers were used to study cultivar relationships and genetic variability (see for example, zhao and liu 2003; peng et al. 2000; liu et al.2005; wang et al. 2007; singh et al. 2007; wang et al. 2014; zhang et al. 2014; huang et al. 2015). population genetic study is an important step for genetic evaluation of medicinally important species as it gives insight on the genetic structure, genetic diversity and gene flow versus genetic fragmentation of these plant species. it also produces data on the number of potential gene pools for conservation and breeding strategies (sheidai et al. 2013, 2014, 2016). therefore, the aim of present study was to produce data on genetic diversity, population genetic structure and to compare the cultivars and wild populations of ziziphus jujuba of iran. we investigated 150 plants of both cultivated as well as wild jujube growing in 23 localities within 8 provinces. for genetic study we used issr molecular markers, as these markers are very useful tool to detect genetic polymorphism, are inexpensive and readily adaptable technique for routine germplasm fingerprinting. they can be used to illustrate genetic relationship between accessions or genotypes and construction of genetic linkage maps (sheidai et al. 2013, 2014, 2016). the suitability of issrs was reported by alansi et al. (2016), who studied genetic diversity in populations of ziziphus spina-christi (l.) willd. material and methods plant materials in total 80 plants were studied in 8 provinces (fig. 1). ten plants were randomly selected in each population and used for molecular studied. issr assay for molecular studies, the fresh leaves were randomly collected from 53 randomly selected plants in the studied area and were dried in silica gel powder. the genomic dna was extracted using ctab-activated charcoal protocol (križ man et al., 2006). the extraction procedure was based on activated charcoal and polyvifigure 1. distribution map of zizphus jujube populations studied. 87population genetic study of ziziphus jujuba mill.: insight in to wild and cultivated plants genetic structure nylpyrrolidone (pvp) for binding of polyphenolics during extraction and under mild extraction and precipitation conditions. this promoted high-molecularweight dna isolation without interfering contaminants. quality of extracted dna was examined by running on 0.8% agarose gel. ten issr primers, ubc 807, ubc 810, ubc 811, ubc 834,cag(ga)7, (ca)7ac, (ca)7at, (ca)7gt (ga)9a, and (ga)9t, commercialized by the university of british columbia, were used. pcr reactions were performed in a 25-μl volume containing 10 mmtris-hcl buff er at ph 8, 50 mm kcl, 1.5 mm mgcl2 , 0.2 mm of each dntp (bioron, germany), 0.2 μm of a single primer, 20 ng of genomic dna, and 3 u of taq dna polymerase (bioron). amplification reactions were performed in a techne thermocycler (germany) with the following program: 5 min for initial denaturation step at 94 °c, 30 s at 94 °c, 1 min at 55 °c, and 1 min at 72 °c. th e reaction was completed by a fi nal extension step of 7 min at 72 °c. the amplification products were visualized by running on 2% agarose gel, followed by ethidium bromide staining. the fragment sizes were estimated using a 100-bp molecular size ladder (fermentas, germany). the experiment was replicated 3 times and constant issr bands were used for further analyses. data analyses the issr bands obtained were treated as binary characters and coded accordingly (presence = 1, absence = 0). the numbers of private versus common alleles were determined. the shared loci among populations were determined by popgene ver. 1.3 (2000). genetic diversity parameters like, new gene diversity (he), shannon information index (i), the number of effective alleles, and percentage of polymorphism (weising 2005), were determined by using genalex 6.4 (peakall and smouse, 2006). for genetic grouping of the studied cultivated and wild plants, nei genetic distance was determined (weising, 2005), and used in clustering as well as ordination methods (podani 2000). genetic differentiation of the studied populations was determined by amova after 1000 permutations as performed in genalex 6.4 (peakall and smouse, 2006). the mantel test (podani, 2000) after 5000 permutation was performed to study the association between genetic distance and geographical distance of the studied populations. genetic structure of the populations was studied by model-based clustering as performed by structure software ver. 2.3 (pritchard et al., 2000). we used the admixture ancestry model under the correlated allele frequency model. a markov chain monte carlo simulation was run 20 times for each value of k (1-8) after a burn-in period of 10 5. data were scored as dominant markers and analysis followed the method suggested by falush et al. (2007). for the optimal value of k in the population studied, we used the structure harvester website (earl and von holdt, 2012) was used to perform the evanno method to identify the proper value of k (evanno et al., 2005). to study genetic differentiation between wild and cultivated plants, we performed pcoa (principal coordinate analysis) analysis within each province. results we obtained 31 issr bands (loci) in total (table 1). the highest number of bands (17 bands) occurred in population 1 (soth khorasan), and 2 (fars) (16 bands), respectively. some of the populations had private bands with population 4 (sistan-o-baloochestan) having the highest number (4 private bands). few common bands occurred in the studied populations too. these are shared alleles among these populations. genetic diversity parameters determined in z. jujuba populations are presented in table 3. the percentage of genetic polymorphism obtained ranged from 3.25 in population 7 (golestan) to 51.61 in population 2 fars). a moderate level of genetic polymorphism (>30%) also occurred in populations 3, and 4 (dnorth-khorasan, andsistan-o-baloochestan, respectively). the highest mean value of new gene diversity (he) occurred in populations 1 to 4 (0.10-0.16, table 2). table 1. details of issr bands in z. jujube populations. population pop1 pop2 pop3 pop4 pop5 pop6 pop7 pop8 no. bands 16 17 13 15 12 10 8 13 no. bands freq. >= 5% 16 17 13 15 12 10 8 13 no. private bands 1 2 0 4 0 1 0 1 no. lcomm bands (<=50%) 6 7 6 5 4 3 3 5 88 seyyedeh tahereh nabavi et al. detailed analysis of issr loci revealed that 16 issr loci (50% of all issr loci), have high gst value i.e. >0.50 (equivalent of fst). this indicates that, these loci are different in the studied populations and lead to population genetic differentiation. this issr locus had a low value of nm and therefore, they are not shared by all the populations. on the contrary, 14 issr loci had nm value >1, and low get value. they are the common alleles shared by the studied populations. the mean nm value of the studied populations was 0.38, which is very low and indicates lack of extensive gene flow among the studied populations. the nei’s genetic identity and genetic distance of the studied populations are provided in table 3. genetic similarities between 0.70 to 0.96% were observed in the studied populations. the highest genetic identity occurred between populations 1 and 2 (0.96%). genetic differential of z. jujube populations based on nei genetic distance, pcoa plot was constructed for the studied cultivars and wild populations, separately (fig. 2). the plot constructed for the cultivars, placed z. jujube populations in two main groups. populations 2, 3 and 4 formed the first main group, while populations 1, 6, 7, and 8, comprised the second major group. some trees in population1 and 5 were intermixed in both groups. this is due to within population genetic variability and the common shared alleys in these two populations. similarly, pcoa analysis of the wild populations revealed that these populations differ genetically from each other as they are placed in separate groups (fig. 3). therefore, both cultivated and wild plants of the studied provinces are genetically differentiated from each other. moreover, amova produced significant genetic difference among z. jujube populations (phipt = 0.57, p = 0.001). amova revealed that 57% of total genetic variability occurred among populations while, 43% of genetic variability was due to within population difference. paired-sample amova also produced significant difference among the studied populations. these results indicate that although the studied z. jujube cultable 2. genetic variability parameters determined in ziziphus jujube populations based on issr markers (populations numbers are according to fig. 1). pop n na ne i he uhe p% pop1 10.000 0.968 1.240 0.223 0.146 0.154 45.16% pop2 10.000 1.065 1.262 0.252 0.164 0.172 51.61% pop3 10.000 0.742 1.180 0.161 0.107 0.112 32.26% pop4 10.000 0.871 1.193 0.177 0.115 0.121 38.71% pop5 10.000 0.613 1.141 0.125 0.084 0.088 22.58% pop6 10.000 0.484 1.105 0.091 0.061 0.065 16.13% pop7 10.000 0.290 1.028 0.021 0.015 0.016 3.23% pop8 10.000 0.710 1.167 0.145 0.097 0.102 29.03% n = no. of studied plants, na = no. of polymorphic alleles, ne = effective no. of alleles, he = new gene diversity, uhe = unbiassed gene diversity, and p% = percentage of polymorphism. table 3. nei genetic identity versus genetic distance in the z. jujube populations (populations numbers are according to fig1. nei’s genetic identity (above diagonal) and genetic distance (below diagonal). pop id 1 2 3 4 5 6 7 8 1 **** 0.9609 0.8920 0.8411 0.8574 0.8735 0.8445 0.9128 2 0.0399 **** 0.9189 0.8675 0.8526 0.8032 0.7677 0.8477 3 0.1143 0.0846 **** 0.9076 0.8505 0.7602 0.7187 0.7955 4 0.1731 0.1422 0.0969 **** 0.8741 0.7263 0.6824 0.7415 5 0.1538 0.1595 0.1619 0.1345 **** 0.8011 0.7621 0.7748 6 0.1353 0.2192 0.2742 0.3198 0.2218 **** 0.9434 0.9539 7 0.1691 0.2644 0.3303 0.3821 0.2717 0.0583 **** 0.9548 8 0.0913 0.1652 0.2288 0.2991 0.2552 0.0472 0.0462 **** figure 2. pcoa plot of issr data in z. jujube populations. figure 3. pcoa plot of z. jujube wild populations based on issr data. 89population genetic study of ziziphus jujuba mill.: insight in to wild and cultivated plants genetic structure tivars and wild populations differ genetically from each other, but also some degree of within population of genetic variability do occur in each population. wild versus cultivated z. jujuba plants in the other attempt, we investigated the genetic differentiation of wild versus cultivated plants within each locality. in three provinces namely, 1fare, 2golestan, and 3-kerman, both cultivated and wild plants were present. the comparison of issr bands in these plants revealed almost complete genetic differentiation of wild and cultivated plants in fars province, while in two other provinces, they were genetically differentiated to some degree (fig. 4). this indicates that these two types of z. jujube, are not genetically alike and we may have still novel genes in wild plants that can be introduced in to cultivated plants genome. these genetic variability are of high importance in medicinal plant conservation and breeding. assocition between genetic diversity and geographical features correlation analysis performed did not show significant association between gene diversity with either altitude or latitude in the studied populations (fig. 5). the same hold true for percentage of genetic polymorphism. this may happen due to cultivation practice and selection made by local gardeners which interfere with local natural adaptation. however, mantel test (fig. 6) between geographical distance (combined distance of longitude and altitude) and genetic distance produced significant correlation (p<0.01). therefore, with increase in geographical distance, an increase in genetic difference of the populations occurred. this is called isolation by distance (ibd). this indicates that the combined effect of geographical features as well as genetic background of the studied cultivars bring about significant genetic differentiation among z. jujube populations. genetic structure of z. jujube populations the genetic structure of the studied populations and degree of genetic admixture among populations were determined by structure analysis. the structure plot (fig. 7) revealed presence of different allele combinations (differently colored segments) in the z. jujube populations. however, some degree of shared common alleles (similarly colored segments) was observed in populations 1, 2 and 3, and also in populations 6, 7, and 8. populations 4 and 5 contained distinct allele combinations. figure 4. pcoa plot of wild versus cultivated z. jujube plants within fars province. figure 5. correlation analysis of genetic diversity and genetic polymorphism with geographical features in z. jujube populations. figuse 6. mantel test plot between genetic distance and geographical distance of z. jujube populations. 90 seyyedeh tahereh nabavi et al. evanno test produced optimal number of genetic group k = 2. therefore, 13 studied ziziphus jujube populations studied could be grouped in 2 genetic groups. structure plot based on k = 2 (fig. 8), revealed that populations 2-4 comprise the first genetic group, while populations 6-8 comprise the second genetic group. moreover, populations 1 and 5 stands somewhere in between these two groups. this is in complete agreement with pcoa plot results presented before. discussion in spite of medicinal importance (vahedi et al. 2008) and wide geographical distribution of ziziphus jububa in our country, we had no detailed information on its genetic variability and structure.the present study revealed the presence of a moderate genetic variability in the cultivated populations. it also showed genetic differentiation between wild versus cultivated plants within each province. therefore, we can use these plants in a core germ plasm collection of z. jujube for conservation and breeding purpose (sheidai et al. 2013, 2014, 2016). alansi et al. (2016), studied genetic diversity in populations of ziziphus spina-christi (l.) willd. by using issr markers and reported the genetic diversity value of 0.26, total genetic diversity ht = 0.266, and intra-population genetic diversity, hs = 0.2199. in present study, amova revealed significant genetic difference among z. jujube cultivars, and also identified a good level of genetic variability within studied population. moreover, gst and nm results revealed that about 50% of issr loci was either private on not shared by all populations, and 50% were exchange in populations via gene flow. this may be to some degree related to out-crossing nature of z. jujube. zhang et al. (2015) studied genetic variability and differentiation in cultivated jujube and wild jujube by using ssr molecular markers. they reported high levels of genetic diversity (he=0.659 and hs=0.674) within populations, and moderate differentiation among studied populations (fst=0.091, rst= 0.068, gst=0.271). they a lso reported a high degree of gene f low (nm=6.572) and weak correlation between genetic and geographical distances (r 2 =0.026, p>0.05), and suggested that gene flow occurred frequently among populations. amova showed that most of the existing genetic diversity was distributed within populations (88 %), and only 12 % occurred among populations, therefore, the studied populations were not differentiated. on the other hand, singh et al. (2017) investigated genetic variation and relationships among cultivars of ziziphus mauritiana (lamk.) native of india by using start codon targeted (scot), issr, and ribosomal dna (rdna) markers. they reported high level of polymorphism among scot (61.6%) and issr (61%) markers. scot and issr dendrograms delineated all the cultivars of z. mauritiana into well-supported distinct clusters. these populations were genetically differentiated as also was indicated with high get values. difference in the results of these studies is probably due to difference in geographical isolation of the studied populations. in present study, the distance between populations is great as they are located in different provinces ranging from south to north of the country with no intermediately plant populations among them (fig. 1). genetic differentiation of the studied populations may be attributed to a combination of adaptation to different environmental conditions and limited capacity for longdistance dispersal (zhang et al. 2015). however, we also noticed good genetic differentiation within each province between wild and cultivated z. jujube plants; this is probably due to effects of cultivation practice and artificial selection made by jujube growers in the gardens. such selection pressure is absent in wild plants. in conclusion, we have presented data on genetic variability and genetic structure of both z. jujube cultivars and wild plants in the country. two main gene pools were identified for jujube cultivars which may be used in future genetic conservation and hybridization programs of this important medicinal plant. figure 7. structure plot of z. jujube populations based on k = 8. figure 8. structure plot of z. jujube populations based on k = 2. 91population genetic study of ziziphus jujuba mill.: insight in to wild and cultivated plants genetic structure references asatryan a, tel-zur n. 2014. intraspecific and interspecific crossability in three ziziphus species (rhamnaceae). genetic resources and crop evolution. 208:390–399. asatryan a, tel-zur n. 2013. pollen tube growth and self-incompatibility in three ziziphus species (rhamnaceae). flora. 208:390–399. earl da, von holdt bm . 2012. structure harvester: a website and program for visualizing structure output and implementing the evanno method. conservation genetics resources 4: 359– 361. evanno g, regnaut s, goudet j. 2005. detecting the number of clusters of individuals using the software structure: a simulation study. molecular ecology.14: 2611–2620. gupta m, mazumder uk, vamsi ml, sivakumar t, kandar cc. 2004. anti-steroidogenic activity of the two indian medicinal plants in mice. journal of ethnopharmacology. 90(1):21–25. križman m, jakše j, baričevič d, javornik b , prošek m. 2006. robust ctab-activated charcoal protocol for plant dna extraction. acta agriculturae slovenica. 87:427–433. lee sm, park jg, lee yh. 2004. anti-complementary activity of triterpenoides from fruits of zizyphus jujuba . biological and pharmaceutical bulletin. 27:1883– 1886. peakall r, smouse pe. 2006. genalex 6: genetic analysis in excel. population genetic software for teaching and research. molecular ecology notes. 6: 288–295. sheidai m, zanganeh s, haji-ramezanali r, nouroozi m, noormohammadi z, ghsemzadeh-baraki s. 2013. genetic diversity and population structure in four cirsium (asteraceae) species. biologia. 68: 384–397. sheidai m, ziaee s, farahani f, talebi sm, noormohammadi z, hasheminejad ahangarani farahani y .2014. infra-specific genetic and morphological diversityin linum album (linaceae). biologia. 69: 32e39 sheidai m, taban f, talebi sm, noormohammadi z. 2016. genetic and morphological diversity in stachys lavandulifolia (lamiaceae) populations. biologija. 62(1): 9-24. singh a, sharma p, singh r. 2007. assessment of genetic diversity in ziziphus mauritiana using inter-simple sequence repeat markers. journal of plant biochemistry and biotechnology. 16:35–40. singh, s. k., chhajer, s., pathak, r., bhatt, r. k.,kalia, r. k. 2017. genetic diversity of indian jujube cultivars using scot, issr, and rdna markers. tree genetics & genomes. 13: 12 doi 10.1007/s11295–016-1092-x. vahedi f, fathi najafi m, bozari k. 2008. evaluation of inhibitory effect and apoptosis induction of zizyphus jujuba on tumor cell lines, an in vitro preliminary study. cytotechnology. 56:105–111. weising k, nybom h, wolff k, kahl g.2005. dna fingerprinting in plants. in: principles, methods, and applications. 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(lamiaceae). caryologia 74(1): 53-61. doi: 10.36253/caryologia-729 received: november 24, 2019 accepted: april 26, 2021 published: july 20, 2021 copyright: © 2021 m. hasaninejad, z. jamzad, s. afsharzadeh, h. saeidi. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. chromosome counts of eight iranian endemic species of nepeta l. (lamiaceae) maryam hasaninejad1, ziba jamzad2, saeid afsharzadeh1,*, hojjatollah saeidi1 1 department of biology, faculty of biological science and technology, university of isfahan, isfahan, iran. 2 research institute of forests and rangelands, agricultural research, education and extension organization (areeo), tehran, iran. *corresponding author. e-mail: s.afshar@sci.ui.ac.ir abstract. in this survey, the chromosome counts of eight nepeta l. species were investigated and the karyotypic diversity among these species was studied. the examined species belong to n. cephalotes boiss. species group, namely n. eremokosmos rech.f., n. gloeocephala rech. f., cephalotes boiss., n. pungens (bunge) benth., n. ispahanica boiss., n. mahanensis jamzad & simonds, n. hormozganica jamzad and n. denudata benth. collected from different habitats in iran. the ploidy levels, karyotype formula, chromosome length range, total karyotype length, several karyotype asymmetries values and stebbins classification were determined in this study. results showed the same chromosome number, 2n = 2x= 18 for all studied species. the basic chromosome number for the above mentioned species are x = 9. also, the smallest chromosome length is 1.02 μm in n. mahanensis. the largest chromosome length is 2.3 μm in n. ispahanica. the chromosomes of species were metacentric or submetacentric. according to the stebbins classification, these species were located into three classes 1a, 2a and 3a. the chromosome numbers for six of studied species are reported here for the first time. keywords: chromosome number, cytotaxonomy, endemics, lamiaceae, karyotype, iran, nepeta l. introduction the family lamiaceae consists of 7173 species in 236 genera worldwide. many of its species have a great importance due to their economic values (harley et al. 2004). nepeta l. (catmint) is a genus belonging to the subfamily nepetoideae (cantino et al. 1992). it is one of the largest genera within nepetoideae, growing as annual, herbaceous perennial and fruticose plants (rechinger 1982; jamzad 2003a, 2012; kaya and dirmenci 2008). endemic species, constituting valuable floristic elements are those which are confined to a particular geographic region. the narrow endemic species are not only scientifically interesting but also very important from conservation point of view. therefore, the identification of endemic plants, their 54 maryam hasaninejad, ziba jamzad, saeid afsharzadeh, hojjatollah saeidi conservation and genetic resources are interesting for the scientific community (ghaffari et al. 2005). in iran, there are 165 endemic taxa of lamiaceae including 42 endemic nepeta species. iran is one of the centers of diversity for the genus nepeta (jamzad et al. 2003b). most species of the lamiaceae have medicinal values. there are numerous known species in the family that are used as analgesic drugs in traditional medicine (uritu et al. 2018). medicinal properties of the lamiaceae species are often ascribed to their high content of volatile compounds (khoury et al. 2016) and glandular hairs represent important sites for the synthesis of natural bioactive compounds (giuliani et al. 2020). nepeta is an important genus in lamiaceae and is specified by terpenoid-type compounds and phenolic constituents, which exert several activities such as an antimicrobial, repellent against major pathogen vector mosquitoes, insecticide, larvicide against anopheles stephensi, cytotoxic anticarcinogen, antioxidant, anticonvulsant, analgesic, anti-inflammatory agent, and antidepressant, disclosing its importance in medicinal and agricultural fields (süntar et al. 2018). species of this genus have been studied in the fields of morphology-anatomy (kaya and dirmenci 2008; acar et al. 2011), palynology (jamzad et al. 2000; celenk et al. 2008; moon et al. 2008). chemical composition (baser et al. 2000; asgarpanah et al. 2014) and molecular phylogeny (jamzad et al. 2003b). the lack of sufficient data on the karyomorphology of the genus is probably due to the small size of its chromosomes (esra et al. 2020). many karyological data concerning chromosome numbers of the genus have already been reported as x = 6, 7, 8, 9, 11, 12, 15, 13, 17, 18. (ipcn, http://www.tropicos. org/project/ipcn, darlington and wylie, 1955; goldblatt and johnson 1979–2017; chen et al. 2018) and there are a few reports from iran (aryavand 1977; ghaffari and kelich 2006; kharazian et al. 2013; payandeh et al. 2015; akbarpur mamagani et al. 2016; hasaninejad et al. 2020). it should be admitted that the numerical variation in chromosome numbers within a genus is quite common. the chromosome numbers and karyotype studies are not only useful in predicting morphological similarities and diversity among species, but also, they are valuable sources of taxonomic and biosystematic information. regarding to the complexities in taxonomy of the genus nepeta, the phylogenetic relationships of species and the chromosomal evolutionary trend may elucidate the systematics, and lead to a comprehensive infrageneric classification of the genus. in this study, we aim to do a cytotaxonomic study of the genus, and follow up the process of chromosomal evolution and its use in the classification of this genus. here we report part of our results on the chromosome counts of a natural species group, recognized previously as section capituliferae benth. p.p. (bentham 1848) and group five (jamzad et al. 2003b), with mostly iranian endemic species. materials and methods seeds of 8 species were collected from different habitats of iran are, as listed in table 1. the voucher specimens of the examined species are preserved in the herbarium of the research institute of forests and rangelands of iran (tari). for mitotic studies, the seeds were germinated at 25 °c on wet filter paper in petri dishes. after germination, roots of 0.5-1cm were selected for pretreatment. root tips were pretreated for 1 h in α-monobromonaphthalene at 4 °c, washed and fixed in carnoy solution (3:1 absolute ethanol glacial acetic acid) overnight. the root tips were hydrolyzed for 5-8 minutes in 1n hcl at room temperature, washed and stained in 2% hematoxylin for 1 h. table 1. the voucher details of studied nepeta species. no species geographical location 1 n. cephalotes boiss. iran, tehran, jajroud highway towards jajrood 1544 m, golipour, 106883, tari. 2 n. denudata benth. iran, hamedan, near razan, 1889 m, golipour, 106879, tari. 3 n. eremokosmos rech.f. iran, semnan, sorkhe, 1355 m, golipour, 106880, tari. 4 n. gloeocephala rech. f. iran, yazd, taft, nasr abad, gilok village in the river, 2800m, mirhoseini, 95002, tari. 5 n. hormozganica jamzad iran, hormozgan, n. bandar abbas, n. slop of m. bokhon, 834 m, ajani,105647, tari. 6 n. ispahanica boiss. iran, kerman, rayen to the first garow, fazlabad village road, 2618 m, golipour, 106881, tari. 7 n. mahanensis jamzad & simonds iran, kerman east silo mahan-before to khaki-asphalt road, hossein abad 1980 m, golipour, 106882, tari. 8 n. pungens (bunge) benth. iran, chaharmahal va bakhtiari, shahrekord, babahidar, the first road to the village of sepidaneh, 2340 m, ajani & hasaninejad, 107079, tari. 55chromosome counts of eight iranian endemic species of nepeta l. (lamiaceae) olympus bh-2 photomicroscope provided the clearest mitotic metaphase among 5 cells and measured by micro measure software 3.3. karyotypes were prepared and chromosome pairs were classified according to levan et al. (1964) and the metacentric and sub-metacentric chromosomes were symbolized using the letters m and sm, respectively. the chromosomes were arranged according to their lengths. the long arm (q), short arm (p), mean length of the chromosome (cl), and total chromosome length (tcl) were measured. karyotype symmetry was determined according to stebbins (1971) and total form percentage (tf, 100 × σs/c) (huziwara 1962). results there was no difference between basic chromosome numbers of the eight studied species and they were x = 9. the details of each species are as follow: nepeta cephalotes is an irano-turanian endemic species and grows in central and northwest of iran. this species showed a diploid chromosome number 2n = 2x = 18 (figure 1a) and the basic chromosome number of x = 9. karyotype consisted of 9 pairs of submetacentric chromosomes (tables 2, 3; figure 2a). the chromosome length ranged from 1.14 to 2.07μm. the chromosome number of this species is reported here for the first time. nepeta denudata is an endemic perennial species, with a distribution range in central, northeast, and west of iran. the results showed that this species is also diploid with chromosome number of 2n = 18 (figure 1b). the karyotype was formed of eight pairs of submetacentric and one pair of metacentric chromosomes (tables 2, 3; figure 2b). the mean length of chromosome varied from 1.1 to 1.9μm. the chromosome number of this specie is reported here for the first time. nepeta eremokosmos is a narrow endemic species. it grows in a limited geographical area in central iran. the studied specimens showed a diploid chromosome number of 2n = 2x = 18 in this taxon (figure 1 c) and basic chromosome number of x = 9. karyotype in this taxon consisted of 9 pairs of submetacentric chromosomes table 2. karyotype formula according to levan et al. (1964) of the studied nepeta species: 2n– chromosome number; x– basic chromosome number; pl– ploidy level; kf– karyotype formula r– range; sc– the shortest chromosome length; lc– the longest chromosome length. no species 2n x pl kf r (sc–lc) (μm) 1 n. cephalotes 18 9 2x 9sm 1.14-2.07 2 n. denudata 18 9 2x 8sm+m 1.17-1.90 3 n. eremokosmos 18 9 2x 9sm 1.31-1.99 4 n. gloeocephala 18 9 2x 6m+3sm 1.05-1.98 5 n. hormozganica 18 9 2x 5m+4sm 1.15-1.73 6 n. ispahanica 18 9 2x 7sm+2m 1.47-2.30 7 n. mahanensis 18 9 2x 9m 1.02-1.74 8 n. pungens 18 9 2x 7sm+2m 1.11-2.12 table 3. karyomorphological parameters of studied nepeta species: ar– arm ratio; ci– mean centromeric index; p– mean length of the short arm; q– mean length of the long arm; tcl– the total chromosome length of the haploid complement; cl– mean length of the chromosome; tf– total form percentage and stebbins– classification of karyotypes in relation to their degree of asymmetry according to stebbins (1971). no species ar (l/s) (μm) ci (μm) p mean (μm) q mean (μm) tcl clmean (μm) tf(%) stebbins 1 n. cephalotes 2.18 0.32 0.47 1.10 14.47 1.61 31.41 3a 2 n. denudata 2.12 0.33 0.50 1.06 13.98 1.55 32.01 3a 3 n. eremokosmos 1.91 0.35 0.56 1.07 14.61 1.62 34.37 2a 4 n. gloeocephala 1.58 0.36 0.59 0.93 13.71 1.52 38.81 2a 5 n.hormozganica 1.67 0.33 0.52 0.87 12.58 1.40 37.47 1a 6 n. ispahanica 1.83 0.35 0.64 1.18 16.44 1.83 35.28 2a 7 n. mahanensis 1.34 0.43 0.58 0.78 12.29 1.37 42.79 1a 8 n. pungens 1.89 0.35 0.57 1.08 14.86 1.65 34.60 2a 56 maryam hasaninejad, ziba jamzad, saeid afsharzadeh, hojjatollah saeidi figure 1a-h. somatic chromosomes of nepeta (an. cephalotes; bn. denudata; cn. eremokosmos; dn. gloeocephala; en. hormozganica; fn. ispahanica; gn. mahanensis; hn. pungens). scale bars: 10 µm. 57chromosome counts of eight iranian endemic species of nepeta l. (lamiaceae) figure 2a-h. idiograms of the karyotypes of nepeta (an. cephalotes; bn. denudata; cn. eremokosmos; dn. gloeocephala; en. hormozganica; fn. ispahanica; gn. mahanensis; hn. pungens). scale bars: 10 µm. 58 maryam hasaninejad, ziba jamzad, saeid afsharzadeh, hojjatollah saeidi (tables 2, 3; figure 2c). the chromosome length is 1.3 to 1.9 μm. this is the first chromosome count for this species. nepeta gloeocephala is an endemic species found in few localities in central iran. chromosome number of 2n = 2x = 18 and somatic chromosome count in this species showed an x = 9 (figure 1d). karyotype was included 6 pairs of metacentric and 3 pairs of submetacentric chromosomes in this specie (tables 2, 3; figure 2d). the chromosome length varied from 1.05 to 1.98 μm. this is the first chromosome number reported for this taxon. nepeta hormozganica is an annual species from saharo-sindian region, growing in south iran. the diploid chromosome number of 2n = 18 was counted in this species (figure 1e). five chromosome pairs were metacentric and four pairs were submetacentric (tables 2, 3; figure 2e). the chromosome length was in the range of 1.1 to 1.7 μm. the chromosome number of this species is reported here for the first time. nepeta ispahanica is a regional endemic annual species growing in west, northeast, central, south, and southeast of iran. it is also distributed in afghanistan. the studied specimens showed a diploid chromosome number of 2n = 2x = 18 (figure 1f) and basic chromosome number of x = 9. n. ispahanica had 7 pairs of submetacentric and 2 pairs of metacentric chromosomes (tables 2, 3; figure 2f). the chromosome length ranged from 1.4 to 2.2 μm. this is the first chromosome count for this species. nepeta mahanensis is a narrow endemic annual species. this species grows in a limited geographical area in kerman province. chromosome number in this species was 2n = 18 (figure 1g). the karyotype was formed of 9 pairs of metacentric chromosomes (table 2, 3; figure 2g). the chromosome length varied from 1.02 to 1.74 μm. this is the second report on the chromosome numbers of this species. the result of this study is in agreement with the previous report conducted by payandeh et al. (2015) for n. mahanensis (x = 9; 2n = 18). nepeta pungens is a regional endemic species with wide distribution in central, northwest, west, northeast and southwest of iran, afghanistan, turkmenistan and central asia. nepeta pungens (2n = 2x = 18) had 7 pairs of submetacentric and 2 pair of metacentric chromosomes (figure 1 h). the chromosome length was in the range of 1.1 to 2.1μm. this is the second report on the chromosome numbers of this species (table 2, 3; figure 2 h). however, the result of this study was not in agreement with the previous report conducted by kharazian et al. (2013) for the n. pungens (x = 11; 2n = 22). discussion according to the index to plant chromosome numbers (ipcn, http://w w w.tropicos.org/project/ipcn) (goldblatt and johnson 1979-2017), in lamiaceae, the chromosome numbers vary from 2n = 10 to 2n = 240 in different genera and species. alloplipoid and autoploid changes can be an important reason for this diversity. extensive cytological studies of the different genera, including thymus l., ajuga l., lamium l., salvia l., scutellaria l. and elsholtzia willd. had revealed the presence of diploid, tetraploid, hexaploid and octaploid species in the family lamiaceae (rather et al. 2018). the chromosome numbers together with other factors can alter breeding strategy in plants (fehr 1991; contreras and ruter 2011). genome size can be estimated by measuring chromosomal data. therefore, chromosome size is directly related to evolution (mehra and bawa 1972; contreras and ruter 2011; esra et al. 2020). the results of our study show that the examined species have 2n = 18 chromosome numbers and the basic chromosome numbers are x = 9. different researchers have suggested x = 8, 9 and 17 as the most common primary and secondary base numbers for the genus nepeta (gill 1972, 1979; aryavand 1977; saggoo 1983; bir and saggoo, 1984; hasaninejad et al. 2020). the previous studies support the results of our study (kaczmarek 1957; gill 1979, 1984; ghaffari and kelich 2006; saggoo et al. 2011; kharazian et al. 2013; payandeh et al. 2015; akbarpur mamagani et al. 2016; hasaninejad et al. 2020), reporting the base chromosome number, x = 9 for nepeta as a common number. the studied species in this research had small chromosomes according to the classification of lima-defaria (1980), with mean chromosome lengths (clm) ranging from 1.37 to 1.83 μm (table 3). whereas baden (1983) argued that the karyotype details studies are difficult because of the small size of chromosomes. although the chromosome number of all studied nepeta species was the same (2n = 18), their karyotype formulas were different, 9 sm of n. cephalotes and n. eremokosmos and 9m of n. mahanensis and 8sm+m, 6m+3sm, 5m+4sm, 7sm+2m and 7sm+2m of n. denudate, n. gloeocephala, n. hormozganica, n. ispahanica and n. pungens, respectively. baden (1983) reported the metacentric and submetacentric karyotype formula for n. sibthorpii benth. and kharazian et al. (2013), suggested the metacentric, sub-metacentric and metacentric point karyotype formula, which confirms our results. n. cephalotes is distinguished by having the highest ar and the lowest ci values, and n. mahanensis by 59chromosome counts of eight iranian endemic species of nepeta l. (lamiaceae) having lowest ar, tcl and cl values, n. isphanica by having the highest tcl and cl values; n. denudata by having the lowest ar value (table 2). it was found that all studied nepeta species are in classes 1a, 2a and 3a based on stebbins classification. 3a species are more asymmetric or more advanced than class 1a species. thus, n. cephalotes and n. denudate are more symmetric and n. hormozganica and n. mahanensis are more asymmetric. this study suggested that tf% varied from 31.41 to 42.79. n. mahaensis was distinguished by having the highest tf%, n. cephalotes by having the lowest tf% (table 3). kharazian et al. (2013) reported 2n = 22 for n. pungens, which is in line with the previously reported base numbers (chen et al. 2018). in our study, the chromosome number of n. pungens was counted 2n = 18, which is contrary to the previous reports. in this case of variability, gill (1979) reported the intra-specific races for some of nepeta species, or the case may be incorrect identification of the studied specimen. moreover, n. mahanensis was reported with 2n = 18 by payandeh et al. (2015). in our report, the basic chromosome number is x = 9, which is fully in agreement with the results of our study for this species. all studied species are either iranian or regional endemics and showed chromosome numbers of x = 9. srivastava (2012) believed that, there is a probability of base number x = 9 at the phylogenetic root of the nepeta, but annual species are considered to be the most evolved species in the genus (jamzad et al. 2003b). as it is shown here for four annual nepeta species (n. ispahanica, n. mahanensis and n. hormozganica), the base number is x = 9, which does not support sirvastava’s idea. previous literatures indicate that the genus nepeta has a heterogeneous set of chromosome numbers. considering the close phylogenetic relationship among the studied species (jamzad et al. 2003b), it may be inferred that the similar chromosome numbers approve their close phylogenetic relationships. future comprehensive cytotaxonomic studies and inferring the results on the nepeta phylogenetic tree may elucidate the evolutionary trends in the genus and lead us to better understanding of the evolutionary values of chromosome numbers. most frequent count of the base chromosome number in nepeta is x = 9. whereas, in most species of lamiaceae, the base chromosome numbers are different. the chromosome number as 2n = 30 is typical in some genera including origanum, clinopodium l., micromeria benth., satureja l., thymus etc. (esra et al. 2020). in genus caryopteris bunge the chromosome number was reported as 2n = 26 and x = 16 in genus chelonopsis miq. was (chen et al. 2018). huang et al. (1996) reported that the basic chromosome number was x = 8 in eriophyton benth.. phlomoides moench is known to have a base chromosome number of x = 11, which is distinct from the base number x = 10 in phlomis l. (fang et al. 2007). scutellaria is one of the largest genera within lamiaceae that also has a complex chromosomal variation as at least 14 different chromosome numbers have been found for the genus 2n = 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 44, 60, 88. the basic chromosome number x = 13 in east and southeast asia, x = 12 in america and x = 11 in north africa and eurasia are predominating (ranjbar and mahmoudi 2013). whereas all studied species were homoploid, but according to previous studies (gill 1972; bir and saggoo 1979, 1984; saggoo 1983; chen et al. 2018; hasaninejad et al. 2020), aneuploidy and dysploidy changes had role in taxa evolution. variation in the chromosome numbers is one of the important factors in the process of evolution (srivastava 2012). however, all these species were not affected by chromosome number variation. the results of this study provided a considerable contribution to the cytotaxonomic data of the genus nepeta. references acar m., ozcan t., satil f., dirmenci t. 2011. a comparative anatomical study on two endemic nepeta l. species (n. baytopii and n. sorgerae). biological diversity and conservation. 4(3):58-70. akbarpur mamagani a., mahmoodi kordi f., mohajjel shoja h. 2016. new chromosome counts in nepeta crassifolia an endemic medicinal plant from iran. 14th internatianl and iranian genetics congress. aryavand a. 1977. in iopb chromosome number reports lvii. taxon. 26: 443-452. asgarpanah j., sarabian s., ziarati p. 2014. essential oil of nepeta genus (lamiaceae) from iran: a review. the journal of essential oil research. 26(1):1-12. baden c. 1983. chromosome numbers the nepeta sibthorpii group (lamiaceae). willdenowia. 13:337340. bentham g. 1848. labiatae, in a. candolle (eds), prodromus systematis naturalis regni vegetabilis. vol. 12, p. 27-603. treuttel and wurtz, paris. bir s.s., saggoo m.i.s. 1984. cytological studies on the family labiatae from gharwal himalayas. in: g.s. paliwal (eds), the vegetational wealth of the himalaya. pp. 471-482. cantino pd., harley rm., wagstaff sj. 1992. genera of labiatae: status and classification. in r.m. harley 60 maryam hasaninejad, ziba jamzad, saeid afsharzadeh, hojjatollah saeidi and t. reynolds (eds), advances in labiate science. p.511–522. royal botanic gardens, kew. london. celenk s., dirmenci t., malyer h., bicakci a. 2008. a palynological study of the genus nepeta l. 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secondarily, mice migrated from the korean peninsula and replaced the distributions of the former mice, considering the distribution patterns of mtdna haplotypes 82 hikari myoshu, masahiro a. iwasa (yonekawa et al. 1988; terashima et al. 2006; nunome et al. 2010, 2013; suzuki et al. 2013; kuwayama et al. 2017; myoshu and iwasa 2018). nuclear genome analysis has shown evidence of the introgression and replacement (kuwayama et al. 2017). additionally, many studies suggest recent migrations by stowaway introduction in several areas, including non-port areas (miyashita et al. 1985; yonekawa et al. 2000; tsuda et al. 2001, 2002; terashima et al. 2006; nunome et al. 2010; kodama et al. 2015; kuwayama et al. 2017; myoshu and iwasa 2018). on the other hand, the distribution of nuclear dna types is not always congruent with that of mtdna haplotypes in the japanese house mice. the sequence of the musculus lineage is observed in all targeted regions of the nuclear genome, or relatively short segments of the castaneus lineage are observed in some targeted regions, although its samples were obtained from a locality where the cas type is exclusively observed (kuwayama et al. 2017). in addition, our previous study (myoshu and iwasa 2018) shows that observed external characteristics sometimes do not coincide with the subspecific characteristics estimated by the mitochondrial haplotypes. thus, these incongruences between nuclear traits and mtdna traits suggest complicated hybridization and/ or replacement process, regarding different progress between biparental and maternal elements. to elucidate the process of replacement by the korean mice, it is necessary to comprehensively investigate the biparental element by a marker distinguishing the korean mice. cytogenetically, variations in the c-band patterns have been well studied in wild house mice (dev et al. 1973, 1975; miller et al. 1976; moriwaki and minezawa 1976; ikeuchi 1978; moriwaki et al. 1985, 1986; moriwaki 2010; yonekawa et al. 2012; myoshu and iwasa 2016). by evaluating the c-band patterns, we can confirm whether the japanese house mice have experienced hybridization and/or replacement with the mice that introduced from northern china and the korean peninsula, or not. according to these previous studies, the c-banding patterns of house mice can be roughly categorized into two patterns. the european, central and southern asiatic, and laboratory mice show a monomorphism of c-band sizes in a homologue; almost all chromosomes carry smaller centromeric c-bands (hereinafter called a “monomorphic pattern”). on the other hand, northern chinese and/or korean mice show a polymorphism of c-band sizes in a homologue; a few chromosomes carry larger centromeric c-bands, and most of the residual chromosomes carry no c-band (hereinafter called a “polymorphic pattern”). the c-banding patterns of the japanese house mice are visually categorized as the latter (dev et al. 1973, 1975; moriwaki and minezawa 1976; ikeuchi 1978; moriwaki et al. 1985, 1986, 2009; moriwaki 2010; yonekawa et al. 2012), polymorphic states of c-bands (myoshu and iwasa 2016). the mice in southern china and/or southeastern asia, which primarily migrated to the japanese islands (suzuki et al. 2013; kuwayama et al. 2017), shows the former type of c-band pattern (moriwaki et al. 1986; yonekawa et al. 2012). in addition, the c-band patterns of f1 offspring reveal the inheritance states of the c-band size, because the c-band size does not vary over a generation (dev et al. 1973, 1975; miller et al. 1976). thus, the c-band pattern is a useful marker to comprehensively investigate the biparental element. in this study, we statistically characterized the c-band patterns of house mice from four areas in hokkaido and honshu of the japanese islands that we previously analysed for mtdna haplotypes and morphological characteristics (myoshu and iwasa 2018). according to results of previous mtdna analysis and the present analysis, we elucidated the migration, hybridization, and replacement processes of maternal and biparental elements in the four areas. materials and methods mouse samples wild-caught mice of the japanese islands (mus musculus) were collected (n = 31; table 1 and figure 1) in the sorachi and iburi areas of hokkaido (n = 3; hkd1, including bbi and hyk; table 1, 1 and 2 in figure 1(a)), the hidaka area of hokkaido (n = 8; hkd2, including mid, kb1, kb2, and nk2; table 1, 3 to 6 in figure 1(a)), iwate and miyagi prefectures in honshu (n = 3; hon1, including syg, tns, and ftk; table 1, 7 to 9 in figure 1(b)) and kanagawa and chiba prefectures in honshu (n = 11; hon2, including kzk, kmn, ohb, and cgs; table 1, 10 to 12 in figure 1(c)) using sherman traps baited with oatmeal. we used the same division names and abbreviations of areas and localities in this study as in myoshu and iwasa (2018). in addition, a wild-caught mouse (m. musculus) collected in seongmodo island, neighboring the korean peninsula, was used (n = 1; smd in table 1; 14 in figure 1(d)). a laboratory mouse (c57bl/6, japan slc inc.) was also used for the analysis as a standard. moreover, hybrid mice from a cross experiment using a female wild-caught mouse from kanagawa prefecture (specimen nos.: mai-1239, 1306 and 1308) and a male c57bl/6n were analyzed (n = 3; table 1) to confirm intermediate c-banding patterns from their parents. 83differences in c-band patterns between the japanese house mice (mus musculus) in hokkaido and eastern honshu c-banding for somatic cells chromosome preparations were performed from bone marrow cells. bone marrow cells were cultured in mem including 15% calf serum containing colchicine (final concentration: 0.025 µg/ml) at 37 ˚c for 40 min. these cells were treated in 0.075 m kcl at 37 ˚c for 20 min as a hypotonic treatment. subsequently, the cells were fixed with modified carnoy’s fixative (methanol : acetic acid = 3 : 1) three times. then air-dried cells were primarily g-banded using the asg technique (sumner et al. 1971) to identify each chromosome by committee of standardized genetic nomenclature for mice (1972) and cowell (1984). after destaining using carnoy’s fixative, the cells were subsequently c-banded using the bsg technique by summer (1972). quantification of the c-band pattern photographs of somatic metaphases were obtained using a digital camera under a microscope (olymtable 1. house mouse samples examined in this study. collecting locality (code*) specimen no. (sex) wild caught mice sorachi and iburi areas, hokkaido, japan (hkd1) koshunai-cho, bibai, hokkaido (bbi, 1) mai-1919 (f ) hayakita-tomioka, abira-cho, yufursu-gun, hokkaido (hyk, 2) mai-1895 (f ), 1915 (m) hidaka area, hokkaido japan (hkd2) midorimachi, hidaka-cho, saru-gun, hokkaido (mid,3) mai-1837 (m), 1840 (f ) kabari, hidaka-cho, saru-gun, hokkaido (kb1, 4) mai-1916 (m), 1917 (m) kabari, hidaka-cho, saru-gun, hokkaido (kb2, 5) mai-1913 (m), 1918 (f ) bansei, niikappu-cho, niikappu-gun, hokaido (nk2, 6) mai-2004 (f ), 2016 (m) northeastern honshu area, japan (hon1) shimoyahagi, rikuzentakata, iwate pref., honshu (syg, 7) mai-1289 (m) takinosato, rikuzentakata, iwate pref., honshu (tns, 8) mai-1293 (m) futaki, sendai, miyagi pref., honshu (ftk, 9) mai-1843 (m) central honshu area, japan (hon2) kohzaki, katori-gun, chiba pref., honshu (kzk, 10) mai-1991 (m), 1992 (f ) kameino, fujisawa, kanagawa pref., honshu (kmn, 11) mai-1114 (f ), 1120 (f ), 1443 (m), 1444 (f ) ohba, fujisawa, kanagawa pref., honshu (ohb, 12) mai-1239 (f ), 1306 (f ), 1308 (f ), 1309 (m) akabane, chigasaki, kanagawa pref., honshu (cgs, 13) mai-1548 (f ) korea seongmodo is., ganghwa-gun, incheon-gwangyeoksi, korea (smd, 14) heg007-97 (m) laboratory mouse c57bl/6n mai-1301 (m) hybrid mice (wild caught mice x laboratory mouse) mai-1239 x 1301 mai-1450 (f ), mai-1452 (f ) mai-1306 x 1301 mai-1379 (f ) mai-1308 x 1301 mai-1563 (m) *code numbers are corresponding to those in figure 1. fig. 1. collection localities of the house mice examined in this study. locality code numbers correspond to those in table 1. 84 hikari myoshu, masahiro a. iwasa pus bx41). to correct errors caused by variations in the extension condition of chromosomes among metaphase plates, we calculated the relative lengths of the c-bands. primarily, the boundary between negatively and positively stained regions was identified at the proximal region of the long arm in the no. 2 chromosomes according to myoshu and iwasa (2016). subsequently, the distance from the distal end of the long arm to the proximal boundary of the negatively stained region was measured in one of the no. 2 chromosomes as a control length for all relative lengths on its metaphase using adobe illustrator cc. in addition, the lengths of all of positively c-banded regions on all chromosomes were measured by the same method. finally, the relative lengths of the c-bands on each chromosome were calculated using following formula: the length of the positively stained c-band region of each chromosome / the control length of the no. 2 chromosome in its metaphase plate. when a positively stained c-band region was not observed in a chromosome or when the entire chromosome was stained lightly, as seen in the y chromosome (figure 2), we considered relative length of the chromosome to be zero. the relative lengths of c-bands for all chromosomes were calculated in five or more metaphase plates per individual. all of the relative lengths of c-bands were categorized as classes by 0.1. the mean number of chromosomes of each class in an individual was calculated using following formula: the observed number of chromosomes included in each class in an individual / the number of observed metaphase plates in an individual. regarding the set of all mean numbers in each class for an individual as the c-band pattern of the individual, we performed a clustering analysis for the c-band patterns of all individuals to estimate analogies among them. we first calculated euclidian distances using all sets of the mean numbers for each class from all mouse samples. then we performed a clustering analysis using the ward method based on these distances. results in the karyotypes of wild-caught mice, the hybrid individuals and c57bl/6, ty pical examples of the c-banded metaphases (samples prepared from wildcaught mice in smd, hon1, hon2, nk2, and hkd1, and from c57bl/6n and the hybrid mice) were shown in figure 3. these c-banding patterns showed the presence of chromosomes with null c-bands in the mouse samples without c57bl/6n carrying the y chromosome negatively stained by c-banding (figures 2 and 3). the c-band patterns of the hybrid individual seemed to be inherited from those of the parents (a wild-caught mouse and c57bl/6n) as their intermediate type (figure 2). in addition, size variations of c-bands seemed to be confirmed in all of the mice excluding c57bl/6 (figures 2 and 3). the mean numbers of chromosomes with a c-band and with a null c-band were classified into classes based on the relative length; 0.01–0.10, 0.11–0.20, 0.21–0.30, 0.31–0.40, 0.41–0.50, and >0.51 as in table 2, and the fig. 2. typical c-banded karyotypes of a wild-caught mouse (a, mai-1239), a hybrid mouse (b, mai-1452), and a c57bl/6n mouse (c, mai-1301). stars indicate chromosomes with null c-bands. asterisks indicate crossing of chromosomes. 85differences in c-band patterns between the japanese house mice (mus musculus) in hokkaido and eastern honshu histograms of these mean numbers were indicated in figure 4. on the basis of our observation and calculation of c-bands on a metaphase plate of c57bl/6 (table 2 and figure 3(d), upper histogram in figure 4), we defined the c-bands (0.01–0.30 in relative length) as “smaller c-bands”. in contrast, we defined the c-bands that is never observed in c57bl/6 (> 0.31 in relative length) as ‘larger c-bands’. furthermore, the result of clustering analysis showed clear discriminations with three major clades consisting of the hon1/hon2 mice and the smd mouse, hkd1/hkd2 (without nk2) mice and the hybrid mice, and hkd2 (nk2) mice and c57bl/6 (figure 5). the hon1/hon2 mice and the smd mouse in a major clade (figure 5) showed high frequencies of null c-band chromosomes (the mean numbers ranged from 23.6 to 29.5 per metaphase plate, table 2). in addition, there were residual chromosomes carrying positively stained c-bands showing variable sizes, including larger c-bands with relative lengths of not only 0.31–0.50 but also > 0.51 (sums of the mean numbers of chromosomes with relative lengths of > 0.31 ranged from 1.4 to 7.0 per metaphase). on the other hand, the mean numbers of smaller c-bands were lower (sums of the mean numbers of chromosomes with relative lengths of 0.01– 0.30 ranged from 5.2 to 14.4 per metaphase). of these, smaller c-bands with relative lengths of 0.21–0.30 were observed much more than those with relative lengths of 0.11–0.20 in hon2; however, individuals from hon1 showed the highest number of c-bands with relative lengths of 0.11–0.20 (table 2 and figure 4). moreover, the larger c-bands on the no.2 chromosomes, which were usually observed in the hon1/hon2 mice (figure 3(b) and 3(c), respectively) were not observed in the smd mouse (figure 3(a)). furthermore, hybrid mice and the hkd1/hkd2 (without nk2) mice belonged to the other major clade (figure 5). the mean numbers of null c-bands were apparently lower (the mean numbers ranged from 14.2 to 19.4 per metaphase, table 2) than those from hon1/ hon2 and smd (the mean numbers ranged from 23.6 to 29.5 per metaphase, table 2) and higher than those of c57bl/6n and nk2 of hkd2 (the mean numbers ranged from 1.2 to 9.0 per metaphase, table 2). additionally, the mean numbers of the smaller c-bands (sums of the mean numbers with relative lengths of 0.01–0.30 ranged from 17.8 to 23.8 per metaphase, table 2) was also intermediate between those from hon1/ hon2 and smd (5.2~14.4) and that from c57bl/6n (38.8). moreover, there was lower number of larger c-bands, at least one chromosome per metaphase (sums of the mean numbers with relative lengths of > 0.31 ranged from 0.4 to 1.0 per metaphase, table 2). the third major clade consisted of c57bl/6 and mice from nk2 of hkd2 (figure 5). c57bl/6 showed smaller c-bands in all of the chromosomes (sum of the mean numbers of chromosomes with relative lengths of 0.01–0.30 was 38.8 per metaphase, table 2). in addition, c57bl/6 showed no larger c-band with relative lengths of > 0.31 and lower numbers of null c-bands (the mean number was 1.2 per metaphase, table 2). meanwhile, individuals from nk2 of hkd2 (specimen nos. mai2004 and mai-2016) carried a pattern similar to that of c57bl/6, especially in terms of the higher number of appearances of smaller c-bands (sums of the mean numbers of chromosomes with relative lengths of 0.01– 0.30 were 36.2 and 30.0 per metaphase in mai-2004 and mai-2016, respectively, table 2). moreover, they carried no more than a chromosome with a larger c-band (sums of the mean numbers of chromosomes with relative lengths of > 0.31 were 0.2 and 1.0 per metaphase in mai-2004 and mai-2016, respectively, table 2) and several chromosomes with null c-bands (the mean numbers were 3.6 and 9.0 per cell in mai-2004 and mai2016, respectively). fig. 3. typical examples of c-banded metaphase plates of polymorphic patterns: seongmodo is. (a, heg007-97), ohba (b, mai1306), and takinosato (c, mai-1293); monomorphic patterns: c57bl/6n (d, mai-1301) and niikappu-2 (e, mai-2004); and intermediate patterns: a hybrid mouse (f, mai-1379) and hayakita (g, mai-1839). 86 hikari myoshu, masahiro a. iwasa discussion the polymorphic pattern in two areas geographically isolated in the japanese islands, hkd1/hkd2 (without nk2) and hon1/hon2, are categorized into two groups (figure 5), which include the smd and hybrid mice, respectively. on the other hand, the monomorphic pattern consists of many smaller c-bands without a null c-band and a larger c-band (table 2 and figure 4) and belongs to the cluster including c57bl/6n (figtable 2. mean numbers of chromosomes with null c-band and c-band classified into each class of relative length. specimen null c-band classification smaller c-band relative length of c-band larger c-band relative length of c-band 0.01~0.10 0.11~0.20 0.21~0.30 total 0.31~0.40 0.41~0.50 >0.51 total hkd1 mai-1919 19,0 0 10,4 7,6 18,0 2,8 0,2 0 3,0 mai-1895 18,2 1,4 11,2 7,6 20,2 1,6 0 0 1,6 mai-1915 18,2 0,2 10,6 7,8 18,6 3,0 0,2 0 3,2 hkd2     mai-1837 15,8 1,2 13,4 5,6 20,2 2,4 1,4 0 3,8 mai-1840 17,4 0 9,0 8,8 17,8 2,6 1,4 0 4,0 mai-1916 19,4 0,8 13,0 5,6 19,4 1,2 0 0 1,2 mai-1917 18,2 0,4 10,4 8,2 19,0 2,6 0,2 0 2,8 mai-1913 16,6 0 13,3 7,9 21,2 1,9 0,3 0 2,2 mai-1918 18,4 0,8 15,6 4,8 21,2 0,2 0,2 0 0,4 mai-2004 3,6 1,2 31,4 3,6 36,2 0,2 0 0 0,2 mai-2016 9,0 3,2 23,4 3,4 30,0 0,6 0,4 0 1,0 hon1     mai-1289 25,0 0 7,4 5,2 12,6 2,0 0,4 0 2,4 mai-1293 23,6 0,4 10,4 3,6 14,4 1,2 0,6 0,2 2,0 mai-1843 27,6 0,6 2,8 4,4 7,8 2,6 2,0 0,0 4,6 hon2     mai-1991 28,0 0 1,0 4,6 5,6 3,4 2,6 0,4 6,4 mai-1992 27,3 0 3,0 5,0 8,0 3,3 0,9 0,6 4,7 mai-1114 29,5 0 1,2 4,0 5,2 2,0 1,8 1,5 5,3 mai-1120 29,2 0,2 0,8 4,2 5,2 3,2 1,5 1,0 5,7 mai-1443 27,8 0 5,2 5,6 10,8 0,8 0,6 0 1,4 mai-1444 29,2 0 0 3,8 3,8 2,8 2,6 1,6 7,0 mai-1239 28,2 0,2 1,8 5,4 7,4 3,0 1,0 0,4 4,4 mai-1306 24,8 1,3 6,2 3,1 10,7 2,4 1,9 0,2 4,6 mai-1308 28,0 0,2 3,8 4,2 8,2 2,7 1,0 0,2 3,8 mai-1309 28,0 0 2,4 3,6 6,0 2,5 2,2 1,3 6,0 mai-1548 29,2 0 3,6 3,8 7,4 2,8 0,6 0 3,4 korea     heg007-97 25,2 0,2 9,6 3,4 13,2 1,0 0,6 0 1,6 labolatory mouse     mai-1301 1,2 0,2 34,8 3,8 38,8 0 0 0 0 hybrid mice     mai-1450 14,4 0 16,6 7,2 23,8 0,8 0,6 0,4 1,8 mai-1452 14,4 0,2 13,2 9,0 22,4 1,4 1,0 0,8 3,2 mai-1379 15,8 1,4 16,2 4,0 21,6 0,8 1,6 0,2 2,6 mai-1563 14,2 1,6 16,2 4,4 22,2 2,8 0,6 0,2 3,6 87differences in c-band patterns between the japanese house mice (mus musculus) in hokkaido and eastern honshu ure 5). our previous study (myoshu and iwasa 2018) indicates the occurrences of three cytb haplotypes that are confirmed in the same areas of the japanese islands, as shown in table 1. in the areas showing the polymorphic pattern similar to smd, only the subspecific castaneus (cas) type and only the subspecific musculus (mus) type occur in hon1 and hon2, respectively. additionally, only the cas type and multiple haplotypes including the cas, mus, and the subspecific domesticus (dom) types occur in the area showing the polymorphic pattern similar to that of hybrid mice, hkd1, and hkd2 without nk2, respectively. on the other hand, not the mus type but rather the cas and dom types occur in nk2 showing the monomorphic pattern. according to these results, a concordant combination between biparental c-band pattern and maternal cytb haplotype is shown in hon2 and hkd2 but is not shown in hon1 and hkd1. fig. 4. histograms showing mean numbers of chromosomes without c-bands or with c-bands per metaphase, classified into each class of relative length with 0.01–0.10, 0.11–0.20, 0.21–0.30, 0.31–0.40, 0.41–0.50, and >0.51. 88 hikari myoshu, masahiro a. iwasa the present c-band results can agree with the finding of cytb properties in hon2/hkd2 and the potential founders, mice in northern china and/or the korean peninsula (yonekawa et al. 1988; terashima et al. 2006; nunome et al. 2010, 2013; suzuki et al. 2013), carry the polymorphic c-bands and the mus type as in the hon2 mice (myoshu and iwasa 2018; figures 3, 4, 5). on the basis of these results, it is a feasible explanation from both viewpoints by chromosome and mtdna traits (myoshu and iwasa 2018; figures 3, 4, 5) that the hon2 mice have maintained the traits of potential founders. in hkd2, on the basis of a simple inheritance of the c-band size (dev et al. 1975; figure 2), the hybridization between mice carrying a combination of the monomorphic pattern and the cas or dom type, and mice carrying a combination of the polymorphic pattern and the mus type from northern china and/ or the korean peninsula is concordantly estimated by both viewpoints from these analyses (myoshu and iwasa 2018; figures 3, 4, 5). the monomorphic pattern of nk2 is in accordance with our previous results showing not only the occurrence of cytb haplotypes (the cas and dom types) but also their morphological characteristics (external body dimensions and coat coloration) (myoshu and iwasa 2018). there is a possibility that the distribution in the japanese islands of the predominant mice which were introduced from northern china and/ or the korean peninsula (yonekawa et al. 1988; terashima et al. 2006; nunome et al. 2010, 2013; suzuki et al. 2013) has not yet expanded into nk2. however, putting emphasis on their larger head and body length as the subspecies m. musculus domesticus (myoshu and iwasa 2018) and the occurrence of the “intact” stowaway haplotype of nuclear dna (nunome et al. 2010; kodama et al. 2015) and mtdna (yonekawa et al. 2000; tsuda et al. 2001, 2002) in the japan islands, a more feasible explanation is that a relatively recent introduction(s) has led stowaway mice to this locality. in the areas with exclusive occurrence of the cas type from southern china and/or southeastern asia (suzuki et al. 2013; kuwayama et al. 2017), hkd1 and hon1, there are discordances between the both viewpoints mentioned above (myoshu and iwasa 2018; table 2; figures 3, 4, 5). in contrast to the cytb finding, the c-band patterns of the hkd1 mice and the hon1 mice are estimated to have been affected by the introgression from northern chinese and/or korean mice carrying the polymorphic patterns (yoshida and kodama 1983; moriwaki et al. 1985, 1986; moriwaki 2010; yonekawa et al. 2012). in addition, although the cas type occurs exclusively in both areas, the c-band patterns are not categorized into the same groups (figure 5). specifically, the numbers of null c-bands (23.6–27.6) and smaller c-bands (7.8-14.4) of the hon1 mice are especially more similar to those in the hon2 mice (24.8-29.5 null c-bands; 3.8-10.8 smaller c-bands) than those in the hkd1 mice (18.0-20.2 null c-bands; 18.6-20.2 smaller c-bands). moreover, the sizes of the smaller c-bands differ between hon1 (more frequent relative length: 0.11-0.20, table 2 and figure 4) and hon2 (more frequent relative length: 0.21-0.30, table 2 and figure 4). these results suggest that the c-band patterns of hon1 and hkd1 are not genetically identical. several studies using biparental markers, which are the haplotypes on the haemoglobin bate chain (hbb) locus (minezawa et al. 1979; miyashita et al. 1985; kawashima et al. 1991, 1995; ueda et al. 1999; sato et al. 2006, 2008; yonekawa et al. 2012), eight (nunome et al. 2010) and seven (kodama et al. 2015) linked nuclear genes, would provide a suggestion for why the difference in the c-band pattern has been caused in the same mtdna occurrence areas. according to these previous studies, the distributions of the polymorphic c-band patterns (moriwaki and minezawa 1976; moriwaki et al. 1985, 1986; yonekawa et al. 2012; myoshu and iwasa 2016; table 1 and figures 4, 5) overlap the distributions of the p (minezawa et al. 1979; miyashita et al. 1985; kawashima et al. 1995; ueda et al. 1999; yonekawa et al. 2012) and the mus-ii haplotype groups (nunome et al. 2010; kodama et al. 2015) which are derived from the fig. 5. cluster analysis considering the mean numbers of chromosomes without c-bands or with c-bands in each class. top values indicate euclidian distances. 89differences in c-band patterns between the japanese house mice (mus musculus) in hokkaido and eastern honshu musculus lineage. the other hapolotype groups, which are recognized as the d (minezawa et al. 1979; miyashita et al. 1985; kawashima et al. 1995; yonekawa et al. 2012) and the recombinant haplotype groups (nunome et al. 2010; kodama et al. 2015) mainly derived from the castaneus lineage, have been observed in northern honshu and hokkaido localities. however, a survey of the hbb allele reveals the difference in p frequency between the northeastern area of honshu (37.0% in minezawa et al. 1979) and hokkaido (7.9% in minezawa et al. 1979). thus, the introgression of the lineage with mus-ii and p haplotypes, which has been derived from eurasian mice carrying the polymorphic c-band patterns, has strongly affected the hon1 mice more than the hkd1 mice. in addition, since male mice have larger dispersal areas than female mice (pocock et al. 2005), expansions of maternal genetic traits would be later than those of the paternal and biparental genetic traits. on the basis of this dispersal pattern, it is estimated that the polymorphic patterns as a biparental trait primarily expand into populations including both male and female mice with monomorphic patterns and the cas type, by male mice considering the larger dispersal potential. therefore, the polymorphic patterns from male mice have less affected mice in hkd1 than in hon1, based on the sign of hybridization in its c-band patterns is significantly observed in hkd1. in hon1, where the c-band patterns are similar to those in hon2 and smd, the mice may have been replaced completely by the male mice carrying the polymorphic patterns. otherwise, the hon1 mice have hybridized and/or maintained the traits of mice carrying a discordant combination, for example the polymorphic pattern and the cas type, which may have been caused in other places as suggested in searle et al. 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p. 94–113. yoshida mc, kodama y. 1983. c-band patterns of chromosomes in 17 strains of mice. cytogenet cell genet. 35: 51–56. caryologia international journal of cytology, cytosystematics and cytogenetics volume 72, issue 2 2019 firenze university press karyotype analysis of a natural lycoris double-flowered hybrid jin-xia wang1, yuan-jin cao1, yu-chun han1, shou-biao zhou1,2, kun liu1,* insights on cytogenetic of the only strict african representative of genus prunus (p. africana): first genome size assessment, heterochromatin and rdna chromosome pattern justine germo nzweundji1, marie florence sandrine ngo ngwe2, sonja siljak-yakovlev3,* assessment of cytotoxicity and mutagenicity of insecticide demond ec25 in allium cepa and ames test arzu özkara cytogenetic effects of fulvic acid on allium cepa l. root tip meristem cells özlem sultan aslantürk evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of allium test ioan sarac1, elena bonciu2,*, monica butnariu1, irina petrescu1, emilian madosa1 fluorescence in situ hybridisation study of micronuclei in c3a cells following exposure to elf-magnetic fields luc verschaeve1,2,*, roel antonissen1, ans baeyens3, anne vral3, annemarie maes1 phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities nicoleta anca şuţan1,*, irina fierăscu2, radu fierăscu2, deliu ionica1, liliana cristina soare1 telomeric heterochromatin and meiotic recombination in three species of coleoptera (dorcadion olympicum ganglebauer, stephanorrhina princeps oberthür and macraspis tristis laporte) anne-marie dutrillaux, bernard dutrillaux* a whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of populus trichocarpa l. subjected to different stresses alberto vangelisti#, gabriele usai#, flavia mascagni#, lucia natali, tommaso giordani*, andrea cavallini differences in c-band patterns between the japanese house mice (mus musculus) in hokkaido and eastern honshu hikari myoshu, masahiro a. iwasa* karyotypic description and repetitive dna chromosome mapping of melipona interrupta latreille, 1811 (hymenoptera: meliponini) natália martins travenzoli1, ingrid cândido de oliveira barbosa2, gislene almeida carvalho-zilse2, tânia maria fernandes salomão3, denilce meneses lopes1,* caryologia. international journal of cytology, cytosystematics and cytogenetics 74(2): 3-9, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-840 caryologia international journal of cytology, cytosystematics and cytogenetics citation: matthew jo, faluyi jo (2021) chromosomal analysis of eight cultivars in three species of cultivated yam (dioscorea l.) species in nigeria. caryologia 74(2): 3-9. doi: 10.36253/caryologia-840 received: january 02, 2020 accepted: april 26, 2021 published: october 08, 2021 copyright: © 2021 matthew jo, faluyi jo. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid mj: 0000-0003-3401-9327 chromosomal analysis of eight cultivars in three species of cultivated yam (dioscorea l.) species in nigeria joshua matthew1,2,*, julius oloaye faluyi1 1 botany department, obafemi awolowo university, ile-ife, nigeria 2 national horticultural research institute, ibadan, nigeria *corresponding author, jolumatthew@gmail.com abstract. the genus dioscorea comprises of economically-important plant species known for their starch throughout the world; it is also a major source of food and income in africa. the most important dioscorea species cultivated and consumed in the west africa belt include d. cayenensis, d. rotundata and d. alata. the plant materials used in this study were collected from omu-ekiti, oye local government of ekiti state, nigeria using the on farm participatory method (ofpm). mitotic chromosome studies were carried out on three species viz, dioscorea alata (‘ewura’), d. cayenensis (‘igangan’) and d. rotundata (‘areyingbakumo’, ‘gaungaun’, ‘ikumo’, ‘ogunmole’ and ‘sandpaper’). mitotic chromosome studies were carried out on each of the cultivars using the root tip squash method made in modified orcein stain (flp-orcein). dormant tubers were cut to mini-setts and placed in carbonised rice husk for rooting. this study reports the basic chromosome number of x = 8, i.e. 2n = 4x =32 (d. alata), 2n = 4x =38 (d. rotundata) and 2n = 8x = 68 (d. cayenensis) for dioscorea suggesting that both d. rotundata and d. cayenensis are aneuploids. keywords: dioscorea, chromosomes, aneuploids, polyploids, mixoploidy. introduction the genus dioscorea l. belongs to the family dioscoreaceae which includes about 90% of the species in the family (murti 2001). the genus dioscorea is principally tuber-bearing and has great economic value in the tropics as food, pharmaceutical, starch, socio-cultural uses and source of income to farmers in west and central africa (asiedu et al. 1998; séka et al. 2009). the most important dioscorea species cultivated and consumed in the west africa belt include d. cayenensis lam., d. rotundata poir. and d. alata l. (iita 2009). west african countries produce over 90% of world dioscorea, of which, nigeria is the largest producer of dioscorea in the world, producing over 60% of the world dioscorea (fao 2016; 2017). dioscorea has presented a challenge to systematists for many years due to its great morphological diversity, its reproductive biology: dioecy, small flowers, low or no seed set and tuber propagation (wilkin et al. 2005). nor4 joshua matthew, julius oloaye faluyi man et al. (2012) has reported the difficulty of chromosome studies in yams (dioscorea spp.) due to the small dot-like chromosomes and few dividing cells in the root tips. also, mixoploidy has been reported to be characteristic of many highly productive commercial cultivars with small chromosome sizes (kunakh 2005; kunakh et al. 2008). mixoploidy was reported by baquar (1980) in some dioscorea species studied which he tagged “odd chromosome numbers” dioscorea alata was reported to have the highest diversity in polyploidy ranging between 2n = 30 and 2n = 80 (sharma and deepesh 1956; franklin and oritz 1963; egesi et al. 2012). the ploidy levels of d. cayenensis have been reported to range between a tetraploid (2n = 4x = 40) and an octoploid (2n = 8x = 80) on a basic chromosome number of 10 (baquer 1980; gamiette et al. 1999; dansi et al. 2001). a basic chromosome number of 20 has been suggested using d. cayenensis (dora et al. 2005) and d. alata (arnau et al. 2009). flow cytometry has offered some advantages in ploidy level analysis (babil et al. 2010). two ploidy levels (4x and 8x) were detected by flow cytometry in two populations of d. cayenensis and d. rotundata cultivars (dansi et al. 2000; babil et al., 2010). however, this technique has failed to detect aneuploidy in this population. therefore, babil et al. (2010) recommended the use of classical chromosome studies to determine ploidy levels and solve the complication of mixoploidy in the genus dioscorea. babil et al. (2010) then recommended that determination of the basic chromosome number of dioscorea spp. requires further investigations. norman et al. (2012) advocated the need for chromosome studies which is necessary to clarify the structure, function, organisation and evolution of yam genomes. the aim of this study was to investigate the chromosome number of the cultivars in the three major dioscorea species that are present in nigeria using the squash technique. the results presented will be useful both in the identification and understanding of the phylogenetic relationship among the major cultivars in the three major species of dioscorea. methodology the plant materials used in this study were collected from omu-ekiti (n 07.90497’ e 005.39092’) in the oye local government of ekiti state, nigeria. this community typifies an epicentre of loss of genetic resources as a result of mass adoption of introduced dioscorea cultivars by migrant farmers from the middle belt of nigeria in the last twelve years. mitotic chromosome studies were carried out on seven cultivars in three species: dioscorea alata, d. cayenensis and d. rotundata. dormant tubers were cut into mini-setts placed in carbonised rice husk for rooting. the root tips were harvested between 10.30– 11.30 am, rinsed in water and transferred into 1:3 acetic ethanol fixative. this was left on the working bench for 24 h at room temperature before keeping in the refrigerator for future usage. the root tips for examination were hydrolysed in 18% hcl for 10 min, squashed and stained with flp-orcein. photomicrographs of the good mitotic chromosome spreads were documented under oil immersion (x1000) objective magnification using bk series phase contrast microscope (pw-bk 5000t) equipped with a dcm510 5 megapixel camera. the chromosome numbers were based on five consistent counts. results phenotypic variation in leaf characters table 1 and figure 1 show the forms and shapes of the leaves of some of the dioscorea species studied. all the cultivars studied had simple, glabrous, cordate leaves. the petiole of d. alata (‘ewura’) had purple wing which was absent in other species. the leaves of d. rotundata (‘areyingbakumo’) and d. cayenensis (‘igangan’) cultivars had orbiculate (broad cordate) leaves. table 1. leaf characteristics of the yam cultivars studied. species local name foliar description dioscorea alata ewura green colour, ovate shape, acuminate apex, cordate base and winged petiole. dioscorea cayenensis igangan light green colour, orbicular shape, acuminate apex and cordate base. dioscorea rotundata areyingbakumo green colour, orbicular shape, acuminate apex and cordate base. gaungaun green colour, ovate shape, acuminate apex and sagittate base. ikumo green colour, orbicular shape, acuminate apex and cordate base. ogunmole green colour, ovate shape, acuminate apex and cordate base. sandpaper dark green colour, long ovate shape, acuminate apex and sagittate base. 5chromosomal analysis of eight cultivars in three species of cultivated yam (dioscorea l.) species in nigeria however, the leaf colour of d. cayenensis was light green while that of d. rotundata were dark green. only, d. rotundata (‘sandpaper’) had long cordate leaves. the cultivars had acuminate leaf apices. the leaf bases observed in d. rotundata, (‘gaungaun’ and ‘sandpaper’ cultivars) were sagittate while other d. rotundata cultivars studied had cordate leaf bases. chromosome number and morphology the mitotic chromosome counts observed in the dioscorea species studied were shown in figures 2 and 3. a mitotic chromosome count of 2n = 32 was observed in d. alata cultivar while a mitotic chromosome count of 2n = 68 was observed in the d. cayenensis cultivar (figure 2). the mitotic chromosome count observed in all the d. rotundata cultivars was 2n = 38 (figures 2 and 3). table 2 shows the mitotic chromosome counts in this study and the previous chromosome counts. the morphology of the chromosomes could not be ascertained in this study because of their small sizes. discussion the findings of this study revealed morphological variations between and within the leaves of the cultivars of the dioscorea species studied. as established in table 1 and figure 1, the distinct by its winged petiole of d. alata distinguishes it from delimit the d. rotundata and d. cayenensis cultivars while can be. also, leaf colour figure 1. leaf form and shape in some the cultivars of dioscorea studied. (a) d. cayenensis (b) d. rotundata (c) d. rotundata (d) d. rotundata 6 joshua matthew, julius oloaye faluyi serves as a delimiting character, d. cayenensis has lightgreen leaves while d. rotundata has dark-green leaves and d. alata leaves are green. the orbicular leaf shape of d. cayenensis distinguish it from some d. rotundata and d. alata cultivars. however, in the morphological characterisation of dioscorea, there is possibility of overlap of characters, therefore, the use of multiple delimiting features is important for their characterisation. this study established a chromosome number of 2n = 38 for the four cultivars of dioscorea rotundata, 2n = 32 for d. alata and 2n = 68 for d. cayenensis. none of the mitotic counts observed in this study is in agreement with the previous mitotic chromosome numbers reported for yam (table 1). (bousalem et al. 2006) reported that the dot-like and varying chromosome sizes that occurred in the mitotic cells of dioscorea made the definite determination of chromosome numbers difficult. the mitotic chromosome counts reported in this study were smaller compared to the mitotic chromosome numbers earlier reported. asiedu et al. (1998) had reported the occurrence of smaller chromosome numbers and polyploidy levels in the species of dioscorea from asia and africa. this study reports the basic chromosome number of x = 8 in d. alata (2n = 4x =32), d. rotundata, (2n = 4x =38) and d. cayenensis (2n = 8x = 68). the x = 8 basic chromosome number agrees with the findings of (dansi et al. 2001). there was no mixoploidy observed. baquar (1980) reported odd chromosome numbers that were not direct multiples of their basic chromosome numbers which he tagged “odd chromosome numbers”. the study cytogenetics of dioscorea using roots from tubers could give a better result in terms chromosome morphology and stainability compared to vines generated roots. the findings indicate that d. rotundata (2n = 38) and d. cayenensis (2n = 68) are distinct species. this figure 2. mitotic metaphase spreads in the dioscorea species studied. a. d. alata (ewura), 2n = 32 (arrows show mitotic chromosome overlaps); b. d. alata (ewura), 2n = 32; c. d. cayenensis (igangan), 2n = 68 (arrow shows mitotic chromosome overlap); d. d. cayenensis (igangan), 2n = 68; e. d. rotundata (ikumo), 2n = 38 (arrows show mitotic chromosome overlap); f. d. rotundata (ikumo), 2n = 38. 7chromosomal analysis of eight cultivars in three species of cultivated yam (dioscorea l.) species in nigeria study affirms that d. cayenensis is a distinct species from d. rotundata thus corroborating the results of bressan et al. (2014) who classified the two species separately through isozymatic analysis. d. cayenensis might be a speciated polyploid of d. rotundata based on the leaf morphology and the chromosome count reported in this study. dioscorea is principally propagated vegetatively, hence, d. cayenensis could have arose through the process somatic cell divisions and polyploidy. the probability that this occurrence could have been as a result of abnormality in the somatic cell divisions of the planting materials (sharma and deepesh 1956; stebbins 1971; baquar 1980) is considered remote. polyploidy has been reported in domesticated plants which include dioscorea species (lewis 1980; leitch and leitch 2008; jeredi et al. 2012). based on a basic number of x = 8 (dansi et al., 2001), d. alata (2n = 4x =32) is a tetraploid and d. rotundata (2n = 38) can only be an aneuploid trisomic for six linkage groups while d. cayenensis (2n = 68) would be an octaploid with four trisomic sets. conclusion the chromosome numbers reported in this work are based on five consistent counts for all the cultivars. it is difficult to agree that mixoploidy is an issue in the chromosome numbers of the cultivars studied because the analysable cells did not show wide variations in chromosome number. on the other hand, for a crop that is maintained by clonal propagation, the occurrence of multiple chromosome numbers is not impossible, especially since a cultivar is not a taxonomic hierarchy. rather, it is characterized by a cluster of valuable food and agronomic attributes that have distinguished it for selection and conservation through generations of cultivation by peasant farmers. acknowledgements the authors are grateful to dr s.o. azeez (botany dept. oau) and mr o.f. oyelami (iita, ibadan) for the information and suggestions provided in the course of this study. figure 3. mitotic metaphase spread in dioscorea species studied. a. d. rotundata (gaungaun), 2n = 38 (arrows show chromosome overlaps); b. d. rotundata (gaungaun), 2n = 38; c. d. rotundata (sandpaper), 2n = 38 (arrows show chromosome overlaps); d. d. rotundata (ogunmole), 2n = 38 (arrows show chromosome overlaps); e. d. rotundata (areyingbakumo), 2n = 38; d. rotundata (areyingbakumo), 2n = 38 (arrow shows chromosome overlap) 8 joshua matthew, julius oloaye faluyi references abraham, k., nair, p. g., 1991. polyploidy and sterility in relation to sex in dioscorea alata l. 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(amaryllidaceae). caryologia 72(4): 105-119. doi: 10.13128/caryologia-670 published: december 23, 2019 copyright: © 2019 c.f. crane. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. megagametophyte differentiation in zephyranthes drummondii d. don and zephyranthes chlorosolen (herb.) d. dietr. (amaryllidaceae) charles f. crane usda-ars crop production and pest control research unit, west lafayette, in 47907 and department of botany and plant pathology, purdue university *corresponding author: charles.crane@ars.usda.gov; ccrane@purdue.edu abstract. megagametophyte differentiation was examined in cleared ovules from emergent buds and open flowers of zephyranthes drummondii d. don and z. chlorosolen (herb.) d. dietr., two highly apomictic species that exhibit the antennaria type of megasporogenesis and hemigamy. stages from binucleate megagametophytes through early endosperm divisions were sampled. a large central vacuole appears after the megasporocyte divides mitotically, and this vacuole persists until the endosperm becomes cellular. upon cellularization of the egg and antipodal apparati, a central column of cytoplasm develops longitudinally across the central vacuole, and both polar nuclei move into it before moving in unison to the chalazal end. the mature megagametophyte is organized conventionally, with one egg, two synergid, two polar nuclei, and three antipodal cells. endosperm development is helobial, but there are few divisions in the chalazal chamber of the endosperm. the behavior of fertilized and unfertilized ovules was also studied in response to pollination. although synergids degenerate autonomously, pollination accelerates synergid degeneration in unfertilized as well as fertilized ovules relative to unpollinated flowers of the same age. tabulation of numerical abnormalities suggests that progressive imprinting is involved in megagametophyte differentiation; the data do not support a strictly zonal specification of nuclear fate, but instead a role for nuclear polarization before mitotic divisions. this study demonstrated the value of cleared ovules in gathering statistically and temporally meaningful observations of megagametophyte differentiation, relating in particular to the movement of polar nuclei and the response of the megagametophyte to pollination. keywords. megagametophyte, embryo sac, apomixis, hemigamy, polar nuclei. introduction zephyranthes drummondii d. don and z. chlorosolen (herb.) d. dietr. are two congeneric amaryllid species that share apomictic reproduction and a flowering response to rainfall, hence the common name “rain lilies”. the buds differentiate within the bulb for several months and then emerge 106 charles f. crane in response to wetting of the roots. the flowers open at sunset, most frequently on the fourth day following rainfall. the flowers remain open for one or two days, depending on temperature. in nature, self-pollination usually occurs soon after the anthers dehisce inside the bud at mid-morning on the day of anthesis. the incongruous combination of large, showy, sweetly scented flowers and self-pollination has motivated several studies of reproduction in these species and the related habranthus tubispathus (l’her.) traub (pace, 1913; brown, 1951; coe, 1953). these studies indicate obligate apomixis by mitotic megasporogenesis, i.e., the antennaria type (first described in antennaria alpina (l.) gaertn. by juel, 1900), and hemigamy (synonym: semigamy; battaglia, 1945), which is development of the zygote after plasmogamy but without fusion of egg and sperm nuclei within the cytoplasm of the egg cell. various details of the stages from the free-nuclear embryo sacs through fertilization remain to be documented, such as the path taken by the polar nuclei to reach the chalazal end of the central cell and whether the central cell experiences triple fusion to initiate the endosperm. many apomictic species require pollination for seed set, but the mechanisms vary. in potentilla (gustafsson, 1946, p. 31), the embryo can begin to develop autonomously in unpollinated flowers, and a sperm nucleus fertilizes only the central cell, thus initiating endosperm development. the exact fate of the other sperm nucleus is rarely known; in the ranunculus auricomus species complex it has been reported also to fertilize the central cell frequently (nogler, 1972), leading to expectedly 6n endosperm with the 2:1 maternal to paternal genome ratio usually found in sexually produced endosperm. facultative double fertilization of the central cell has also been indicated with f low cytometry in apomictic crataegus (talent and dickinson, 2007). in most apomictic panicoid and eragrostoid poaceae (brown and emery, 1957; voight and bashaw, 1972), there is but one polar nucleus, and fusion with only one sperm would produce the 2:1 maternal:paternal ratio. nevertheless, bashaw and hanna (1990) reported frequently observing a sperm in the central cell of the panicoid grass cenchrus ciliaris l., but none near the egg, possibly indicating that both sperms usually enter the central cell but only one fuses with the lone polar nucleus. in most species with nucellar (adventitious) embryos, such as apomicts in the genus citrus, the megagametophyte is reduced and sexual fertilization is more or less unaffected; the nucellar embryos then outcompete the sexually produced embryo (gustafsson, 1946, p. 35; nygren, 1967, p. 559). as defined above, nearly obligate hemigamy is known in nature only in certain species of rudbeckia (asteraceae; battaglia, 1945) and habranthus and zephyranthes (sister genera in the amaryllidaceae). also, a dominant, incompletely penetrant hemigamous mutant se has been recovered in cotton (turcotte and feaster, 1969); unlike hemigamy in zephyranthes, it readily produces maternal haploid, paternal haploid, and hybrid sectors in chimeric embryos or maternal-paternal twin embryos. similar behavior has been observed at ca. 1% frequency in wild-type theobroma cacao, where it has been exploited as a source of haploids (lanaud, 1988). in contrast to broadly descriptive classical studies, modern research (mostly in arabidopsis) has taken advantage of a battery of transposon insertion mutants, a finished genome sequence, fluorescent reporter molecules, and informatic tools, to accumulate a body of concepts and literature dealing with signaling and gene interactions during fertilization in sexual species (zhou and dresselhaus, 2019). no form of apomictic reproduction is understood in comparable detail, although apomictic behaviors can spur insights into aspects of sexual reproduction. for example, the facultative double fertilization of the central cell in crataegus (talent and dickinson, 2007) suggests that the polar nuclei briefly remain attractive or receptive to a second sperm nucleus after fusing with the first one, that the central cell can attract both sperm nuclei, and that the egg more strongly attracts one and only one sperm nucleus (else triploids and haploids result); once framed, all three of these hypotheses can be tested experimentally in an amenable species. unfortunately, to date there apparently is no reported arabidopsis mutant that exactly duplicates the hemigamous behavior seen in zephyranthes. previous embryological studies of apomictic zephyranthes and habranthus (pace, 1913; brown, 1951; coe, 1953) have produced limited evidence about megasporogenesis, because this stage occurs within the bulb where the bud length is not visible without destroying the plant and each plant produces zero to five buds per year. the evidence in favor of the antennaria type is mostly negative: dyads expected from first-meiotic restitution (the taraxacum type) or complete omission of the first meiotic division (the blumea type [chennaveeriah and patil, 1971] or the syndrome in elymus rectisetus (nees in lehm.) a. love et connor [crane and carman, 1987]) have not been observed, while enlarging, vacuolate, undivided megasporocytes are frequent. the positive evidence is a single image of a mitotic metaphase in an enlarged, vacuolate megasporocyte in habranthus tubispathus (brown, 1951). indirect evidence is the occurrence of the ordinary, monosporic polygonum type in sexual zephyranthes candida (ao et al., 2016), which militates against the occurrence of the ixeris type (mei107megagametophyte differentiation in zephyranthes otic first-division restitution in the tetrasporic fritillaria type) in zephyranthes. the present study took advantage of the readily accessible later stages in bud development to understand the maturation of the megagametophyte in two apomictic species of zephyranthes, with particular interest in three processes shared with related sexual species: the movements of polar nuclei, the senescence of the megagametophyte with and without fertilization, and the regulation of nuclear fates as the megagametophyte matures. these are universal aspects of angiosperm reproduction that are easily observed in apomictic zephyranthes. materials and methods the ovules of maturing flowers in zephyranthes are particularly suited for clearing studies. at this stage, the ovules are easily removed from the ovary before fixation, and the cleared ovules are readily pipetted onto a microscope slide. the ovules are flat and thus present the embryo sac in sagittal optical section. the embryo sac is very large and thus the egg apparatus and polar nuclei are not close to the plane of overlying and underlying nucellar cells. the nucellus is free of birefringent calcium oxalate crystals at the stages examined, facilitating differential interference-contrast microscopy. finally, the refractive index of methyl salicylate is close to optimal to resolve nuclei and yet see through many cell layers, which has not been the case in other species like nothoscordum bivalve (l.) britton in n.l.britton & a.brown (crane, 1978) and elymus rectisetus (nees) a.love & connor (crane and carman, 1987). flowers were examined in z. drummondii d. don and z. chlorosolen (herb.) d. dietr. at stages from emergence from the bulb through 48 hours post-anthesis. zephyranthes drummondii was collected from two sites in austin, texas: at the intersection of 27th street and speedway, and at a hilltop on the east side of interstate 35 just south of its interchange with u.s. 183. zephyranthes chlorosolen was collected along the entrance ramp of u.s. 183 onto southbound interstate 35. excised ovules of both species were fixed overnight in fpa50, which is 37% formalin: glacial propionic acid: 50% ethanol, 1:1:18 v:v:v (herr, 1971), and dehydrated through 70%, 95%, and absolute ethanol. the ovules were infiltrated with methyl salicylate in three steps: 2:1 absolute ethanol: methyl salicylate, 1:2 absolute ethanol: methyl salicylate, and pure methyl salicylate. the dehydration and infiltration steps were minimally one hour. ovules were viewed under differential interference contrast (nomarski) as whole mounts in methyl salicylate with cover slips at the side to support an overlying cover slip. variations of this method have been used subsequently by young et al. (1979), stelly et al. (1984), and zeng et al. (2007); a recent application appeared in kwiatkowska et al. (2019). buds of z. drummondii were collected during the spring of 1976 at stages from emergence through early endosperm development. buds of z. chlorosolen were collected in triplicate in seven specific groups from 7 july 1976 through 10 july 1976 after rainfall on 4 july 1976, in order to survey development in pollinated and unpollinated flowers before, during, and shortly after the usual time of self-pollination. the z. chlorosolen groups are detailed in table 1 (below). all the flowers in the first six rows of table 1 were emasculated at sunset one or two days before opening. pollinations were performed within an hour with pollen from opening flowers elsewhere in the population. the stigma was removed from unpollinated flowers to prevent pollination. the stigma was slightly exserted above the anthers in the three flowers of the seventh row in table 1, and these flowers were self-pollinated upon anthesis. after a delay of 48 to 72 table 1. batches of z. chlorosolen flowers used in this study. codea emasculation date pollination status date of anthesis date picked age relative to anthesis when picked -2e+72 7 july unpollinated 9 july 10 july +1 day -2pol+72 7 july pollinated 9 july 10 july +1 day -1e+48 7 july unpollinated 8 july 9 july +1 day -1pol+48 7 july pollinated 8 july 9 july +1 day -1e+72 7 july unpollinated 8 july 10 july +2 days -1pol+72 7 july pollinated 8 july 10 july +2 days 0pol+48 8 july pollinated 8 july 10 july +2 days acodes consist of number of days relative to anthesis (0, -1, -2), pollination status (pollinated or emasculated and unpollinated, and the approximate number of hours after pollination or emasculation when the flower was picked for fixation. 108 charles f. crane hours, picked flowers were brought indoors, with ovule excision and fixation commencing immediately for the first flower processed. the other two flowers per treatment were held at 4c until earlier flowers had been processed. about 30 of the 50 to 80 total ovules were randomly sampled per flower. mature embryo sacs of z. chlorosolen were classified as normal or abnormal on the basis of nuclear and cell count. a normal embryo sac consisted of one egg, two synergids, three antipodals, and a binucleate central cell whose nuclei ultimately fused. abnormal embryo sacs differed in count, usually as a result of non-division of a nucleus at an earlier stage. synergids were classified as having a filiform apparatus, which usually coincided with a micropylar-end position of their nucleus and a chalazal-end vacuole. the egg did not have a filiform apparatus and had a chalazal or lateral nuclear position and a micropylar-end vacuole. free nuclei in the central cell were classified as polar nuclei. sometimes the antipodal cells resembled a second egg apparatus in nuclear and vacuolar positions, such that one antipodal had a nucleus closer to a polar nucleus. abnormalities were tabulated in an attempt to discern if there was a pattern of successive determination of nuclear fates. synergid and egg degeneration were also followed in relation to ageing and pollination. degeneration was indicated by cytoplasmic collapse, nuclear shrinkage, and general loss of visible cellular content. results gametophytic maturation in zephyranthes drummondii most fertile ovules had reached the four-nucleate stage as the bud emerged from the neck of the bulb at the soil surface, but a few were still binucleate. the embryo sac was discoid at this stage, and its micropylar end directly abutted the nucellar epidermis. there was a large central vacuole, which persisted throughout further development until the endosperm cellularized in fertilized ovules. a tapering cytoplasmic strand, narrowest at the middle, traverses the vacuole after the first mitosis, but this strand disappears before the second mitosis. a prominent hypostase was fully developed at the chalazal end of the ovule, and it persisted well into endosperm development. four-nucleate embryo sacs (fig. 1a) and eight-nucleate embryo sacs lacked any visible cytoplasmic strands that span the central vacuole. in most ovules, the last mitosis occurred on the third day before flower opening. mitosis at the chalazal end of a four-nucleate embryo sac was observed to precede mitosis at the micropylar end, but it is not known if this is generally the case. the posttelophase daughter nuclei at the micropylar end already occupied the positions expected of nuclei in the egg apparatus (fig. 1b), and their flattened shape indicated that the two mitotic spindles had been perpendicular to each other. the prospective egg nucleus and micropylar polar nucleus were already larger than the prospective synergid nuclei on the second day before flower opening. meanwhile, the divisions at the chalazal end of the embryo sac were more difficult to see because of the thick, birefringent walls of the hypostase and the presence of up to 20 cell layers in the light path. nevertheless, the last mitotic spindles there appeared to be mutually perpendicular as they were at the micropylar end, figure 1. early development and migration of polar nuclei in z. drummondii. the micropylar end is to the left or down in each picture. a. tetranucleate embryo sac with nuclei side by side at each end. b. flattened nuclei soon after telophase at the micropylar end, indicating orthogonal spindles; e, predicted egg nucleus; mp, predicted micropylar polar nucleus; s, predicted synergids, based on frequently occurring positions in the mature egg apparatus. c. more mature, fully cellular egg apparatus with egg nucleus (at left) unusually close to the micropylar end of the egg cell. the filiform apparatus of a synergid (right) appears feltlike or fibrillar. d. mature antipodal apparatus in contact with the hypostase, whose walls are thickened and birefringent. the migrated but unfused polar nuclei lie immediately to the left. e. inception of the central column. there are also smaller, variously oriented cytoplasmic strands that appear (with light microscopy) to have intruded into a previously uninterrupted central vacuole. the egg apparatus of this embryo sac appeared in c. f. the central column has reached full thickness. g. the micropylar polar nucleus has entered the central column first. h. both polar nuclei have entered the central column. i. striations between the approaching polar nuclei are possibly cytoskeletal elements. j. the polar nuclei can meet near the egg apparatus, or move there temporarily after meeting at the center. 109megagametophyte differentiation in zephyranthes and a candidate chalazal polar nucleus was evident farthest from the hypostase before cellularization. cellularization occurred by the day before flower opening. the filiform apparatus began to develop (fig. 1c) in the synergids. the egg nucleolus developed a nucleolar vacuole (fig. 1c). after cellularization, one of the antipodal nuclei was larger than the other two (fig. 1d), just as the egg nucleus was larger than the synergid nuclei, and this distinction was even more evident in unfertilized ovules post-anthesis. the polar nuclei migrated on the day before opening, after the initial cellularization of the egg and antipodal apparati. before migration, both polar nucleoli had formed a nucleolar vacuole. thin strands of cytoplasm began to intrude into the central cell vacuole (fig. 1e), and the central strand soon spanned the vacuole. the central strand continued to thicken and developed a granular appearance (fig. 1f). the polar nuclei migrated into the central strand; in fig. 1g the micropylar nucleus has moved first. the polar nuclei approached each other in the strand (fig. 1h), and faint striations indicated the presence of cytoskeletal elements (microtubules and/ or microfilaments) between them (fig. 1i). although the polar nuclei could meet relatively near the egg apparatus (fig. 1j), they finally moved in unison to the chalazal end of the central cell (fig. 1d). then the central cytoplasmic strand began to separate into separate strands and disappear (figs. 2a and 2b; also fig. 1d) on the day of flower opening. over time the polar nuclei became appressed (fig. 2c) and then fused, as indicated by fusion of their nucleoli (fig. 2d). in one abnormal case with extra polar nuclei in lieu of synergids, all four polar nuclei became trapped within the middle of the central strand and maintained their distinctness through three days past flower opening (fig. 2e). the abnormality with four trapped polar nuclei was seen several times also in cleared ovules of hippeastrum xjohnsoni (h. reginae (l.) herb. x h. vittatum (l’her.) herb.). fertilization occurred on the second day post-opening. figure 2f possibly depicts plasmogamy of the egg and sperm cells. later on the smaller sperm nucleus was visible within the egg, and it could divide before being walled off from cb of the bicellular embryo (figs. 2g and 2i). some eggs partially collapsed after plasmogamy (fig. 2h), but most maintained an expanded, semicircular shape leading to elongation toward the chalaza. the initial division plane was usually transverse. the pollen tube was sometimes visible. in one ovule that had been punctured during excision and handling, where the surrounding synergid debris had been lost, there was an apparent pore at the hooked pollen tube tip where sperms had been released. karyogamy appeared to be necessary for endosperm development. in one abnormal instance, karyogamy was incomplete on the tenth day after floral opening, and the partially decondensed, distended sperm nucleus was still appressed to the polar fusion nucleus, which had not divided. in another ovule, the sperm and polar fusion nucleus were close to each other, and both were degenerating. further evidence for the necessity of karyogamy in hemigamous zephyranthes and habranthus comes from the results of interspecific pollinations, which often result in an increased frequency of endosperm failure and empty seeds. in h. tubispathus x z. candida (lindl.) herb., for example, all the seeds were empty in spite of seed setting of more than 95%. the primary endosperm nucleus divided transversely and thus produced a small chalazal cell covering the antipodals and a far larger figure 2. later development in z. drummondii, except j, which is from habranthus robustus pollinated with z. macrosiphon. a, b. dissipation of the central column, which splits into parallel strands. c. appressed polar nuclei next to antipodals. d. completely fused polar nuclei with fused nucleoli. e. persistent central column in abnormal embryo sac with four polar nuclei and no synergids. f. egg cell during or shortly after plasmogamy with a sperm cell. g. bicellular embryo; one of two sperm-derived daughter nuclei (arrow) lies below the maternal nucleus in cb. h. zygote shrinkage or degeneration. i. both sperm-derived nuclei (s) have been walled off from cb in the same embryo as g. j. intranuclear metaphase of endosperm nucleus in the micropylar chamber of the helobial endosperm. k. highly endopolyploid, multinucleolate nucleus in failing endosperm. l. sterile ovule with uninucleate embryo sac whose nucleus resembles a polar nucleus and occupies the chalazal position of migrated polar nuclei. 110 charles f. crane micropylar cell containing the rest of the volume of the central cell. free-nuclear divisions ensued in both cells, but no more than five nuclei were observed in the chalazal cell. abundant free-nuclear mitoses in the micropylar cell resulted in a multinucleate shell that laid down cell walls centripetally as it began to fill in the central vacuole. the first mitoses in the micropylar cell were synchronized and resulted in 2, 4, 8, 16, or 32 nuclei at a time before synchrony broke down, whereas mitoses in the chalazal cell were not synchronized. the early endosperm nuclei were large enough to hold an entire spindle apparatus within the old nuclear membrane (fig. 2j). the nuclei of mature, fully cellular endosperms were smaller and spherical. lobate, multinucleolate endosperm nuclei sometimes appeared, but they seemed to be associated with endosperm failure. they appeared to be under traction by attached spindle fibers (fig. 2k; this example is from habranthus robustus herb. ex sweet pollinated with z. carinata herb.). the fate of unfertilized embryo sacs was also examined. neither the egg nucleus nor the fused polar nuclei ever divided. both synergids usually degenerated one or two days before the egg did. the degenerating egg apparatus lost evident vacuoles as the nuclei faded out. the cytoplasm collapsed, i.e., the egg shrank and its boundary became crenulate. the antipodals degenerated at the about same time as the synergids, and they could be crushed by proliferating nucellar cells near the incompressible hypostase. the central cell nucleus was usually the last nucleus to degenerate in the embryo sac. from five to 20% of the ovules were sterile, lacking a mature embryo sac. although both integuments were of normal size, the nucellus was smaller. sterile ovules fell into four main types: those with a uninucleate embryo sac in a hypodermal position, those with a slightly enlarged megasporocyte in a hypodermal or subhypodermal position, those with no enlarged cells at all, and those with the megasporocyte surrounded by thickened cell walls within the hypostase. sterile ovules were most frequent toward the base of the ovary, but they could occur anywhere. figure 2l shows a uninucleate embryo sac with an enlarged nucleus and prominent nucleolar vacuole. pollination response and senescence in zephyranthes chlorosolen the experimental layout appears in table 1. observations centered on timing of fertilization relative to pollination, synergid degeneration, sequence of degeneration of cells in unfertilized embryo sacs, timing of first division in the egg versus the endosperm, frequency of spermatic division in the egg, and numerically abnormal embryo sacs. in total, 796 ovules were examined. they were classified as unfertilized normal, fertilized normal, unfertilized abnormal, or sacless, depending on presence of an embryo sac, number of components in the embryo sac, and evidence of fertilization such as dividing endosperm or presence of a pollen tube at the micropyle or presence of a sperm nucleus within the egg. there were 524 unfertilized normal, 122 fertilized normal, 63 unfertilized abnormal, and 87 sacless ovules in all. table 2 gives the numbers of instances for all conditions of embryo sacs and their components versus treatment. supplemental table 1 gives the same information divided among the 21 individual flowers sampled. no embryo or endosperm developed in emasculated, unpollinated flowers. pollen tubes reached the ovules about 48 hours after pollination, but their growth rate depended on the time of pollination, and their arrival continued over a period of many hours. thus in the “egg” rows of table 2, most of the fertilizations occurred between 48 and 72 hours after pollination in buds pollinated the day before natural anthesis, whereas most of the fertilizations had occurred less than 48 hours after pollination in flowers pollinated upon opening. no pollen tubes reached the ovules after pollination two days before opening. in spite of the slower growth rate, pollen tubes reached the ovules sooner after early pollination, as evidenced by the presence of multicellular embryos only after early pollination. at the times sampled, 110 ovules had received one pollen tube, seven had received two, and one had received three; the number was not noted or pollen tubes were not seen in the other evidently fertilized ovules. if only the early pollinated buds collected 72 hours later are considered, these totals became 65, two, and one. when more than one pollen tube had reached an ovule, they followed different paths toward the embryo sac. no instances of two sperm nuclei were seen in undivided egg cells. the synergids degenerated in both fertilized and unfertilized ovules, but pollination accelerated degeneration (table 2, “synergid” rows). at least one of the synergids had begun to degenerate in every fertilized embryo sac, whereas both synergids were intact in a substantial minority of embryo sacs in unpollinated flowers of the same age. synergids were more degenerated in the unfertilized ovules of pollinated flowers than they were in unpollinated f lowers. although the two synergids generally did not degenerate exactly synchronously, the combination of intact and fully degenerated synergids was relatively uncommon and appeared mostly in fertilized ovules. the independence of degeneration was tested with the chi-squared test. for ovules collected from 111megagametophyte differentiation in zephyranthes table 2. classification of z. chlorosolen embryo sacs and their components, merged by time of emasculation, pollination, and collection. treatmenta -2e -2pol -1e -1pol -1e -1pol 0pol hours post 72 72 48 48 72 72 48 unfertilized 96 91 94 91 96 22 33 fertilized 0 0 0 6 0 70 47 abnormal 9 8 15 4 3 7 17 sacless 8 13 12 3 9 10 32 total 113 112 121 104 108 109 129 frac.abnorm.b 0.08 0.071 0.124 0.038 0.028 0.064 0.132 frac.sacless 0.071 0.116 0.099 0.029 0.083 0.092 0.248 frac.(ab+sa) 0.15 0.188 0.223 0.067 0.111 0.156 0.38 unf unf unf unf fert unf unf fert unf fert egg ok 95 89 87 83 5 91 20 59 33 47 embryo 0 0 0 0 0 0 0 8 0 0 deging 1 0 2 5 1 4 1 3 0 0 deg 0 2 5 3 0 1 1 0 0 0 synergids ok-ok 22 16 19 16 0 10 4 0 0 0 ok-+/-ok 4 4 11 8 0 6 2 0 3 0 ok-deging 10 2 2 4 0 7 0 1 1 3 ok-deg 9 13 4 2 0 3 0 9 1 11 +/-ok-+/-ok 3 5 17 11 0 7 0 0 2 1 +/-ok-deging 5 6 6 8 1 5 1 1 4 1 +/-ok-deg 9 7 10 14 2 12 2 9 1 5 deging-deging 9 10 3 4 0 11 5 2 6 2 deging-deg 8 4 7 8 1 16 0 14 8 9 deg-deg 17 24 15 16 2 19 8 34 7 15 polar nuclei oop 5 8 0 1 0 0 0 0 0 1 deging 9 10 2 3 0 11 0 1 3 6 ch appress 63 54 69 69 3 12 2 0 4 7 ch fusing 14 11 19 13 2 28 6 5 10 5 ch fused 5 5 4 4 1 45 14 9 15 17 mult. pen 0 0 0 0 0 0 0 3 0 5 >=2 endosp 0 0 0 0 0 0 0 50 0 6 1 mc + 1 ch 0 3 0 1 0 0 0 1 1 0 antipodals 3: all ok 75 72 59 68 4 47 12 18 9 13 3: all+/-ok 5 1 3 2 0 7 1 7 1 2 3: all deging 2 0 1 2 0 2 3 9 5 4 3: all deg 1 1 0 0 0 3 2 16 4 13 2+/-ok 1deging 3 6 7 7 0 11 3 4 5 4 1+/-ok 2deging 3 1 5 2 0 11 1 9 2 2 5 any 0 1 0 2 0 0 0 0 0 0 4 any 3 4 3 2 1 2 0 1 0 1 2 any 3 3 14 5 0 8 0 3 5 4 1 any 1 0 1 1 1 5 0 3 2 3 none seen 0 2 1 0 0 0 0 0 0 1 code uuuu 16 13 11 9 0 6 3 0 0 0 uduu 59 56 50 55 5 44 9 13 10 13 112 charles f. crane unpollinated flowers 24 hours after natural opening (the first and third columns of table 2, “synergid” rows), there were 76 in which neither synergid had greatly degenerated, 55 in which only one synergid had degenerated, and 59 where both had degenerated. this gave a probability of 0.5447 of not having degenerated versus 0.4553 of having degenerated. if degeneration occurred at random, one would expect 56.373 with neither synergid degenerated, 94.241 with one degenerated, and 39.387 with both degenerated. the resulting chi-squared value was 32.94 with two degrees of freedom, and thus the synergids did not degenerate independently. the polar nuclei had failed to migrate in six embryo sacs, and they were out of their usual chalazal-end position in 15 more embryo sacs (table 2, “polar nuclei” rows). eighteen of these instances occurred in flowers emasculated two days before opening, even though the flowers were collected at the same age as those emasculated one day before opening. most polar nuclei were appressed at 24 hours after natural opening. while the polar nuclei were aligned along the long axis of the embryo sac during migration, they assumed other alignments upon reaching the chalazal end, and about a 20:4 ratio of perpendicular to parallel alignments was observed relative to the long axis. most polar nuclei had begun to fuse or had completely fused, as indicated by complete fusion of their nucleoli, at 48 hours past natural opening. pollination accelerated nuclear and nucleolar fusion of polar nuclei in both fertilized and unfertilized ovules. the primary endosperm nucleus developed multiple nucleoli before dividing. the large number of multinucleate endosperms after pollination one day early further indicated the earlier time of fertilization in that group. one or two extra antipodal cells occurred in 18 embryo sacs, but the divisions that produced them were not seen (table 2, “antipodal” rows). fertilization, especially endosperm development, accelerated antipodal degeneration. the unfertilized ovules of pollinated flowers also had more degenerated antipodals than did ovules in unpollinated flowers of the same age. asynchronous degeneration was more likely to be observed in unpollinated flowers. the condition of each embryo sac was encoded in table 2 in four letters, all either u for intact or d for degenerating and degenerated. the letters respectively denoted the egg, synergids, polar nuclei, and antipodals, and any instance of degeneration merited a “d” for synergids and antipodals. completely intact embryo sacs were most common at 24 hours past natural opening, and pollination two days early did not affect them. later pollination accelerated synergid and antipodal degeneration. completely intact embryo sacs were never seen in fertilized ovules, since at least one synergid had degenerated. antipodal degeneration usually began soon after synergid degeneration had begun. in 20 ovules, the entire egg apparatus was degenerating before the polar nuclei or antipodals began to degenerate. the egg and the polar nuclei were about equally likely to be the last intact component in senescing embryo sacs. however, all possible degeneration sequences were observed multiple times. endosperm division clearly preceded egg division (table 2, ninth column). the egg usually divided after at least eight nuclei existed in the micropylar chamber of the helobial endosperm. in four out of 122 fertilized ovules, the egg degenerated without dividing; this is a typical frequency of mature seeds with plump endosperm but no embryo in z. chlorosolen. the number of sperm nuclei was tabulated in 83 fertilized egg cells. three of them had experienced division of both sperm treatmenta -2e -2pol -1e -1pol -1e -1pol 0pol udud 9 7 19 11 0 28 7 51 20 27 uddu 1 7 2 0 0 2 0 0 1 0 uddd 4 1 0 0 0 7 0 2 1 6 uuud 2 2 4 5 0 3 1 0 0 0 uudu 2 1 1 2 0 0 0 0 0 0 uudd 2 0 0 1 0 1 0 0 1 0 dduu 1 2 4 8 1 3 0 2 0 0 duuu 0 0 3 0 0 0 0 0 0 0 ddud 0 0 0 0 0 2 2 1 0 0 atreatment and “hours post” refer to pollination versus emasculation and the number of hours after either when picked. bleft column abbreviations are: frac.sacless, fraction of all ovules that lacked an embryo sac; frac.abnorm, fraction of all embryo sacs that were abnormal; frac.(ab+sa), sum of frac.sacless and frac.abnorm.; deg, degenerated; deging, degeneration in progress; ok, intact; oop, out of position; ch, chalazal; mc, micropylar; mult. pen, multinucleolate primary endosperm nucleus; appress, appressed; any, any condition. the ud codes follow a system given in the results section in reference to table 2. 113megagametophyte differentiation in zephyranthes and egg nuclei. the remainder contained only one sperm nucleus. there were 63 embryo sacs that differed from the conventional organization of one egg, two synergids, two polar nuclei, and three antipodals. these are described and enumerated in table 3, whose columns give the row number in the table, the number of instances observed, the total number of nuclei, the counts of eggs, synergids, free nuclei in the micropylar part of the central cell, free nuclei in the chalazal part of the central cell, and antipodals. because most of the embryo sacs were examined after the polar nuclei had met and migrated into the chalazal end of the central cell, the last two columns give a best guess as to the numbers of free nuclei at each end of the central cell prior to migration. most abnormal embryo sacs contained fewer than eight nuclei. the most common abnormalities were misdifferentiation of an antipodal nucleus as a third polar nucleus (row 1), and absence of the egg apparatus (rows 10 and 15). there were 13 embryo sacs with only one antipodal cell, and in four of these (rows 8 and 17) there appeared to be only two chalazal-end nuclei prior to cellularization. the number of functioning polar nuclei ranged from one to four; the latter occurred when both expected synergid nuclei instead became polar nuclei and behaved as in fig. 2e. although no instances were observed in z. chlorosolen, four polar nuclei and no synergids (row 5) were seen multiple times in hippeastrum xjohnsonii. eight embryo sacs could not be described as combinations of egg, synergids, polar nuclei, and antipodals. in the first, the antipodal nuclei were not walled off from the central cell in an otherwise normal embryo sac. instead, they remained separate from the two fused polar nuclei. in the second and third, the synergids lacked a filiform apparatus and the polar nuclei were still separate at the ends of the central cell 24 hours after normal anthesis. in the fourth, only the cell walls of the egg apparatus persisted, and all nuclei had degenerated. in the fifth, there was but one antipodal, and it was unusually large. the two polar nuclei flanked this antipodal, and the egg apparatus had degenerated. in the sixth, a transverse partition near the micropylar end divided the sac into two cells, each with four free nuclei seemingly randomly arranged near the partition. in the seventh, the synergid and egg nuclei occupied the same, egglike cell. in the eighth, the egg nucleus had been walled off partially, but then the egg nucleus had migrated to the chalazal end of the central cell as a polar nucleus. the embryo sac was otherwise normal. discussion development of the embryo sac from the megasporocyte in apomictic zephyranthes is similar to development from the surviving megaspore in ornithogalum caudatum (tilton and lersten, 1981). the biggest differences are the larger size and more discoid shape of the embryo sac and its central vacuole in zephyranthes, such that the four-nucleate stage has two nuclei side-by-side at each end, rather than arranged linearly as is usual in ornithogalum. a second difference is the stated movement of only the micropylar polar nucleus through the central column to the chalazal end in ornithogalum, versus migration of both polar nuclei into the column in zephyranthes before migration of both to the chalazal end. otherwise, tilton and lersten (1981) observed the initiation, thickening, and splitting of the column table 3. component counts in numerically abnormal embryo sacs in z. chlorosolena. row n total eggs syn cent mc cent ch antip inf mc inf ch 1 7 8 1 2 0 3 2 1 2 2 1 8 0 2 0 3 3 2 1 3 1 8 1 1 0 3 3 2 1 4 1 8 1 1 0 4 2 2 2 5 0 8 1 0 0 4 3 3 1 6 2 7 1 2 0 1 3 0 1 7 1 6 1 0 0 2 3 1 1 8 3 6 1 2 0 2 1 1 1 9 1 6 0 2 0 2 2 1 1 10 6 5 0 0 0 2 3 1 1 11 1 5 1 0 0 1 3 0 1 12 2 5 1 2 0 0 2 0 0 13 1 5 1 0 1 2 1 2 1 14 2 5 1 2 0 1 1 1 0 15 4 4 0 0 0 1 3 0 1 16 2 4 0 0 0 2 2 0 2 17 3 4 1 0 0 2 1 1 1 18 1 4 1 2 0 1 0 1 0 19 2 3 0 0 0 1 2 0 1 20 4 3 0 0 0 3 0 1 2 21 1 3 0 0 0 2 1 1 1 22 1 3 1 0 1 1 0 1 1 23 3 2 0 0 0 1 1 0 1 24 3 2 1 0 0 1 0 1 0 25 1 1 0 0 0 1 0 0 1 asyn, synergids; cent mc, free nuclei in micropylar half of the central cell; cent ch, free nuclei in chalazal half of the central cell; antip, antipodal cells; inf mc, inferred to come from the micropylar end of the central cell; inf ch, inferred to come from the chalazal end of the central cell. 114 charles f. crane much as in zephyranthes, although they did not specifically order these stages in time. tilton and lersten mentioned other examples of the central column from various taxa, including crocus, hordeum, and in general any species with helobial endosperm. they also cited the notion, from a 1902 paper by ikeda, that at least one polar nucleus moves through the column. more recently, zeng et al. (2007) and hu et al. (2009) also observed in rice the migration of polar nuclei through a transient, de-novo formed cytoplasmic column, where they met at the center and then moved to the micropylar end of the central cell. tilton and lersten (1981) suggested that in ornithogalum the second sperm nucleus might reach the fused polar nuclei via the central column, but in zephyranthes drummondii this column has dissipated prior to arrival of the pollen tube at a synergid, and therefore the sperm must take some path through the peripheral cytoplasm of the central cell. embryo sac development in emergent buds of z. drummondii and z. chlorosolen is more rapid than in the related zephyranthes candida, at four days in the former and up to seven days in the latter. as in z. drummondii, ao (2018) reported (on the basis of 10-μm paraffin sections) the formation of a central cytoplasmic column that spanned the central cell and conveyed the micropylar polar nucleus to the chalazal end. however, ao (2018) reported that the column persisted until fertilization and that sometimes the polar nuclei and sperm nucleus fused within the chalazal part of it, as depicted in his figure 2d. ao did not distinguish a stage where both polar nuclei moved into the column and met near its midpoint. ao (2018, 2019) also noted that the antipodal cells developed callosic walls when the polar nuclei approached them, that the antipodal cells later fused when the callose had disappeared, and that the antipodal nuclei ultimately fused into one. in contrast, in z. drummondii no callose or cellular fusion was observed in the antipodal apparatus before it degenerated. also, ao (2019) noted a nuclear endosperm in z. candida, and this in conjunction with the claimed fusion of antipodal cells and claimed increase in antipodal nuclei to nine is consistent with a misinterpretation of a helobial endosperm where the small chalazal chamber has been mistaken for fused antipodal cells. the inception, thickening, and eventual dissipation of a central column differ markedly from the description of polar-nuclear migration in arabidopsis thaliana. there the polar nuclei are depicted as traveling through a peripheral cytoplasmic shell surrounding the central vacuole, and they migrate while the egg and antipodal apparati form cell walls (yadegari and drews, 2004). in rice, the photographs of zeng et al. (2007) and hu et al. (2009) were not suitable to detect incipient cell walls, since the nuclei were stained preferentially, but it is possible that the migration began before cellularization began. the egg apparatus appears to develop complete cell walls in zephyranthes. this is supported by fig. 1c, which is the egg apparatus from the embryo sac whose pre-migration central cell appears in fig. 1e. a zephyranthes egg cell tends to maintain its hemispherical shape even when plasmolysis pulls the central cell cytoplasm away from it, which is consistent with a rigid egg-cell wall. also, tilton and lersten (1981) noted the appearance of a complete cell wall around the egg in ornithogalum caudatum, but commented that the chalazal end of the wall might be reticulate at ultrastructural magnification. synergid degeneration appears to be autonomous in zephyranthes, but pollination accelerates degeneration in unfertilized as well as fertilized ovules. degeneration of one synergid is positively correlated with degeneration of the other synergid. although up to three pollen tubes were observed at the micropyle, there is no evidence in this study that intact synergids attracted pollen tubes. it is possible that penetration necessarily destroyed the receiving synergid. the situation is concordant with the conclusions of leydon et al. (2015) and cited references therein, that synergids can degenerate autonomously, that approach of a pollen tube promotes synergid degeneration, and that discharge of a pollen tube is observed only in a degenerated synergid. the embryo-sac abnormalities observed in zephyranthes are not consistent with a random assignment of function at the eight-free-nucleate stage, since combinations such as three egg cells or one synergid and three micropylar-end polar nuclei were not observed. on the other hand, there was insufficient evidence to support a rigidly progressive imprinting of nuclear fate, with consistent defaults for undivided nuclei from each of the three rounds of mitosis. for example, embryo sacs with only one synergid were rare (two out of 63 abnormals), and thus the nuclei that normally become synergid nuclei tended to share the same fate, either becoming synergids or becoming extra, functioning polar nuclei. for this reason, one might conclude that the commitment to becoming synergid nuclei has already happened at the four-nucleate stage of the embryo sac and that it is distinct from the commitment to divide. yet the combination of no synergids and three polar nuclei was not observed, so the usual fate of an undivided synergid precursor is unclear. for another example, an undivided micropylar end nucleus (one having skipped the second and third mitotic divisions) tended to function 115megagametophyte differentiation in zephyranthes as a polar nucleus, moving to the chalazal end of the central cell upon cellularization. for that matter, even an undivided megasporocyte nucleus could develop the chalazal position and single, large, vacuolated nucleolus typical of migrated polar nuclei (fig. 2l). in contrast, if the second round of mitosis were skipped, each daughter of the third round could form a polar nucleus and one of something else. furthermore, failure to wall off one antipodal resulted in an extra free nucleus that was not observed to become appressed to or fuse with the legitimate polar nuclei. thus very careful, systematic evaluation of a much larger sample of properly timed, abnormal embryo sacs, with attention to the filiform apparatus, nuclear position, and cellular and nucleolar vacuolization, will be needed to establish if the spectrum of abnormalities can support the inference of checkpoints and default fates in differentiating embryo sacs. the spectrum of abnormalities varies among plant taxa. yudakova (2009) observed a 10to 100-fold higher frequency of extra eggs and polar nuclei in embryo sacs of the hieracium type in poa chaixii and poa pratensis than in embryo sacs of the taraxacum (or possibly elymus rectisetus) type in poa badensis. yudakova (2009) also stated that, “no significant cases of extra polar nucleus formation from the synergid nuclei were recorded among numerous analyzed embryo sacs of the studied bluegrass species, while female gametophytes with additional egg cells instead of synergids occurred in all studied plants.” this situation markedly contrasts with the greater frequency of expected synergid nuclei becoming extra polar nuclei in zephyranthes and hippeastrum. in semifertile hybrids of indica × japonica rice, zeng et al. (2007) observed a wide range of abnormalities over stages from degenerating megaspores through mature embryo sacs. at maturity, frequent abnormalities included small size, a misplaced (lateral) egg apparatus, absence of the egg apparatus, misplaced (lateral) antipodals, and migration of polar nuclei to the chalazal end (which would be normal in zephyranthes). if the egg apparatus was absent, the micropylar-end polar nucleus was either present or absent. otherwise, zeng et al. (2007) did not detail numerical abnormalities in the manner of table 3. the failure to observe multiple egg cells in zephyranthes (table 3) could reflect observational bias, since the filiform apparatus develops over time and is not always clearly visible in whole mounts, and the position of the nucleus varied from lateral to chalazal-end within egg cells. an indirect line of evidence comes from twin embryos. spontaneous diploid-haploid twins are relatively easily found in lilium (cooper, 1940) and in grasses, where they are a source of spontaneous haploids apart from indeterminate gametophyte mutants. within zephyranthes, identical (maternal) twins are about 10 times more frequent in z. pulchella j.g. smith than in z. chlorosolen. an extra egg would be the most facile source of such twins, although postzygotic cleavage is also possible, and experimental distinction would be difficult in an apomictic species. an extra egg could account for frequent maternal haploid production from indeterminate gametophyte (ig) mutants in maize and rice (evans, 2007; zhang et al., 2015), although ig maize also produces androgenetic haploids when used as a female parent with other maize genotypes (kindiger and hamann, 1993). yudakova (2009) followed the four-zone hypothesis of enaleeva (2002) to account for abnormalities in poa embryo sacs. from the micropylar end, the consecutive zones are synergid, egg, central, and antipodal. yudakova (2009) conjectured that the zones were established in response to signals from adjacent nucellar cells and that each zone determined the fate of its contained nuclei. abnormalities would arise when a free nucleus was situated out of its proper zone, and a piece of evidence for this was the prevalence of an extra egg rather than polar nucleus in place of a synergid, since the synergid and central zones are not adjacent. however, in zephyranthes and hippeastrum the relative abundances are reversed, with a tendency of both prospective synergid nuclei to function as extra polar nuclei. also, in zephyranthes, the egg and synergid nuclei can begin at the same distance from the micropylar extremity of the embryo sac (fig. 1b). furthermore, in rice (zeng et al., 2007) and habranthus tubispathus (pace, 1913), an otherwise normal egg apparatus sometimes appears on the side of the embryo sac rather than at the micropylar end. a lateral egg apparatus would require that the zones would respond to particular cells or cell groups in the nucellus and that free nuclei would move in response to some attraction from those nucellar cells. alternatively, a lateral egg apparatus could easily arise from a misoriented spindle of the first mitotic division, if the nuclei move only slightly thereafter. nuclear movement seems to occur only at particular times in the differentiation of embryo sacs, first after the division of the megasporocyte (which functions as the surviving megaspore in apomictic zephyranthes), then upon migration of the polar nuclei, then upon fertilization, and finally during the first few rounds of mitosis in the developing endosperm. the nuclei appear to remain nearly stationary apart from these four episodes. the nature of the fertilization block is particularly interesting in zephyranthes, since the sperm nucleus can approach within its own diameter the egg nucleus with116 charles f. crane out fusing with the egg nucleus. also remarkable is how the other sperm nucleus moves or is moved across more than 100 micrometers of the peripheral cytoplasm of the central cell to reach the fused polar nuclei. karyogamy of the sperm and fused polar nuclei appears to be necessary for endosperm development in z. drummondii and z. chlorosolen, as evidenced by an invariably undivided central cell nucleus in unfertilized ovules and the similar types of endosperm failures in interspecific crosses of these species and interspecific crosses of sexual z. traubii (w. hayw.) moldenke. however, this necessity contrasts with the situation described by tandon and kapoor (1962) in zephyranthes cv. ‘ajax’ (z. candida (lindl.) herb. x z. citrina baker), where 4n chromosome counts were reported for 366 out of 430 endosperm metaphases after self-pollination. this suggests that hemigamy also operates in the central cell, assuming that this seed-propagatable cultivar is apomictic like its z. citrina parent (howard, 1996). apomictic zephyranthes possibly vary in their requirement for karyogamy for endosperm inception. if so, there is a very unanswered question as to how the usual maternalpaternal imprinting mechanism (endosperm balance number) is circumvented in z. citrina and z. pulchella. the problem merits further investigation with flow cytometry and paternal markers in all of these species. study location the z. drummondii flowers were collected at latitude 30°17’37.5”n, longitude 97°44’11.5”w (30.293736, -97.736538). the z. chlorosolen f lowers were collected at latitude 30°20’16.4”n, longitude 97°42’04.4”w (30.337887, -97.701208). both populations have been destroyed by subsequent building and road construction. acknowledgements i performed the experiments in partial fulfillment of the requirements for the ph.d. degree at the university of texas at austin. i thank my now deceased major professor, walter v. brown, for stimulation and tolerance of this and related projects during my graduate research. i thank stefan kirchanski for suggesting methyl salicylate as a clearing agent. i thank edwin t. bingham, david a. sleper, john g. carman, david m. stelly, and stephen b. goodwin for support over subsequent decades. finally, i thank my wife, yan m. crane, for reformatting the photomicrographs and for her steadfast support. declaration the author declares no conflicts of interest regarding this research. references ao, c.q. 2019. the endosperm development and the variations of structures of embryo sacs: unraveling the low fecundity of zephyranthes candida (amaryllidaceae). plant biosystems. 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rice. journal of experimental botany 66: 99-112. zhou, l.z., and dresselhaus, t. 2019. friend or foe: signaling mechanisms during double fertilization in flowering seed plants. current topics in developmental biology 131: 453-496. 118 charles f. crane su pp le m en ta l t ab le 1 . c la ss ifi ca tio n of z . c hl or os ol en e m br yo s ac s an d th ei r co m po ne nt s, it em iz ed in c ol um ns b y in di vi du al fl ow er . c od es a pp ea r as in t ab le 2 , e xc ep t f ix d , w hi ch is tim e of d ay w he n ov ul es w er e fix ed . c o n d -2 e7 2 -2 e7 2 -2 e7 2 -2 p7 2 -2 p7 2 -2 p7 2 -1 e4 8 -1 e4 8 -1 e4 8 -1 p4 8 -1 p4 8 -1 p4 8 -1 e7 2 -1 e7 2 -1 e7 2 -1 p7 2 -1 p7 2 -1 p7 2 0p 48 0p 48 0p 48 fi x d 20 :0 0 22 :0 0 1: 00 21 :0 0 0: 00 2: 00 22 :0 0 22 :0 0 23 :0 0 22 :0 0 23 :0 0 20 :0 0 23 :0 0 1: 00 21 :0 0 23 :0 0 1: 00 23 :0 0 u n fe 33 33 30 28 30 33 32 31 31 33 31 27 31 33 32 11 8 3 12 7 14 fe r t 0 0 0 0 0 0 0 0 0 0 1 5 0 0 0 21 22 27 7 23 17 a bn o 5 3 1 5 3 0 5 6 4 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3 0 1 1 1 1 1 u n se 0 0 0 0 2 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 st at us u u u u 3 8 5 2 6 5 7 2 2 1 2 0 6 0 0 1 5 0 0 3 0 0 0 0 0 0 0 0 0 u d u u 22 18 19 15 18 23 9 23 18 18 23 1 14 4 17 11 16 4 1 4 9 1 3 4 1 2 4 4 8 u d u d 5 2 2 5 1 1 7 6 6 7 3 0 1 0 9 14 5 5 19 1 11 1 21 6 4 4 14 10 9 u d d u 0 1 0 4 1 2 2 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 1 0 0 0 0 0 u d d d 1 2 1 0 0 1 0 0 0 0 0 0 0 0 3 3 1 0 0 0 1 0 1 0 1 1 5 0 0 u u u d 0 0 2 1 0 1 4 0 0 3 0 0 2 0 0 0 3 0 0 0 0 1 0 0 0 0 0 0 0 u u d u 0 2 0 0 1 0 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 u u d d 2 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 d d u u 0 0 1 1 1 0 0 0 4 2 2 0 4 1 1 1 1 0 1 0 1 0 0 0 0 0 0 0 0 d u u u 0 0 0 0 0 0 2 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 d d u d 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 2 0 0 0 0 1 0 0 0 0 0 0 substantia an international journal of the history of chemistry vol. 2, n. 1 march 2018 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 74(1): 23-31, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-597 caryologia international journal of cytology, cytosystematics and cytogenetics citation: s. dehury, s. kumar dehery, a. bandhu das (2021) karyotype variation in eight cultivars of indian dessert banana (musa acuminata l.) of section eumusa from odisha, india. caryologia 74(1): 23-31. doi: 10.36253/ caryologia-597 received: april 03, 2020 accepted: february 05, 2021 published: july 20, 2021 copyright: © 2021 s. dehury, s. kumar dehery, a. bandhu das. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid abd: 0000-0002-1816-4850 karyotype variation in eight cultivars of indian dessert banana (musa acuminata l.) of section eumusa from odisha, india shomina dehury1, subrat kumar dehery1, anath bandhu das1,2,* 1 molecular cytogenetics laboratory, department of botany, utkal university, vani vihar, bhubaneswar 751004, odisha, india 2 centre of excellence for north east india studies, (under rusa 2.0 programme), new academic block, utkal university, vani vihar, bhubaneswar 751004, odisha, india. *corresponding author. e-mail: abdas.uubot@gmail.com; a_b_das@hotmail.com; abdas.uubot@utkaluniversity.ac.in abstract. banana (musa spp.) cultivars especially dessert banana are important cash crop with high market demand all over the world as an integral part of the diet. the need for assessment of cytogenetic characters in musa cultivars is inevitable as out of thousands of cultivars, cytogenetic characterization of most of them remains unresolved due to difficulties like small chromosome size, diversity in ploidy levels and high cultivar diversity which behave differently to standardized cytogenetic protocols. in this report, somatic chromosome number, detailed karyotype analysis including total chromosome length, volume, form percentage, interphase nuclear volume (inv) were accessed on eight dessert type of musa accessions from different places of odisha. all the cultivars studied were found triploid (2n = 33) with a basic chromosome number of x=11. the karyotype formulae were assigned to each cultivar by grouping the chromosome according to their shared characteristics. the total chromosome length ranged from 54.95 µm in cv. robusta to 81.5 µm in cv. kathia with symmetric karyotype in all the studied cultivar. karyotype formula revealed structural alteration of chromosome with total form percentage (tf%) variation from 35.65% in cv. amritapani to 41.68% in cv. patakpura that confirms more number of nearly median constricted chromosome as compared to sub-median chromosome. the total chromosome volume recorded from 10.78 µm3 in cv. robusta to 15.99 µm3 in cv. khatia and the inv varied from 1336.44 µm3 in cv. dwarf cavendish to 2048.37 µm3 in cv. patakpura. the recorded structural variation might be due to differential genome specific condensation of chromosome. chromosome length and volume found statistically significant among the cultivars. keywords: chromosome number, genome analysis, ploidy, table-top banana, total form percentage. introduction banana (musa spp.) belongs to family musaceae is an important monocot plant used as staple food and cash crop for millions of people that provide 24 shomina dehury, subrat kumar dehery, anath bandhu das nutrition and minerals with high calorific value. cultivated banana are distinguished into dessert or simply called banana and cooking banana or plantain. banana is cultivated primarily for its highly nutritious fruit beside it has good fiber content obtained from its pseudostem, leaves are used as disposable leaf plates and inflorescence are used for food with high potassium (50.08 mg g-1), calcium (3.78 mg 1-1) and phosphorus (3.66 mg g-1) content in dry weight basis (fingolo et al. 2012). there are over a thousand domesticated musa cultivars with a very high genetic diversity (stover and simmonds 1987; perrier et al. 1990). however, due to difficulty of genetic makeup, and sterility of the crop, the development of new varieties through hybridization, mutation or transformation was not very successful in musa till date (heslop-harrisons and swarzacher 2007). the ploidy level determination of different varieties of musa is economically important as well as preliminary requisite to facilitate breeding programme from existing genetic diversity of the country for future quantitative and qualitative morphological trait targeted breeding programme. inter and intra specific hybridization of two wild diploid (2n = 2x = 22) musa species, musa acuminata (aa) containing ‘a’ genome and musa balbisiana (bb) having ‘b’ genome gave rise to most of the natural banana cultivars with different genomic and ploidy levels i.e. aa, aaa, aab, abb, aaab, aabb and abbb. the cultivated banana are mostly triploid (2n = 3x = 33) with a limited varieties/species with diploid or tetraploid constituents. various cultivars of banana have been originated from independent sources in the wild, so the hybridization events and mutations giving rise to seedless and parthenocarpic characters have occurred many hundreds of times (simmonds and shepherd 1955; heslop-harrisons and swarzacher 2007). where fertile plants occur together, hybridization continues to produce new diversity (pollefeys et al. 2019) and parental combinations, hence, structural analysis of chromosome is important. simmonds (1962) considered five plant characteristics that lead to farmers for picking plant vigour, yield, seedlessness, hardiness and fruit quality, the first four of which are related to polyploidy (triploidy). karyotype analysis provides valuable information related to the mechanisms of genome evolution. several types of banana out of thousands of cultivars are adapted to the agro-climatic condition of odisha. traditionally the economically important cultivars grown in the state are silk (patkapura), poovan (champa), cavendish group. recently, there has been a trend towards the cultivation of amritpani due to high productivity and consumer acceptability (maharana et al. 2017). some of the earlier reports confirmed chromosome number with karyotypes, still data are scanty for different cultivars of banana (cheesman and larter 1935; das and das 1997). in this study, a detailed karyotype analysis and chromosome number determination has been carried out for further structural analysis of chromosome which is the prerequisite for localization of specific marker gene of interest on to the chromosome through fluorescence in situ hybridization (fish) for genome analysis in eight triploid cultivars of dessert banana cultivated in different parts of odisha. materials and methods eight cultivars of m. acuminata namely cv. amritapani, cv. champa, cv. chini champa, cv. dwarf cavendish, cv. grand naine, cv. kathia, cv. patkapura, cv. robusta were collected from different parts of odisha and maintained in green house of department of botany, utkal university, bhubaneswar (table 1). actively growing root tips were pre-treated in half saturated para dichlorobenzene (pdb) and aesculin mixture (1:1) for 3½ h at 18oc in refrigerator and then fixed in 1:3 acetic acid : ethanol overnight at room temperature. fixed roots were treated in 45% glacial acetic acid for 15 min. chromosome staining of fixed roots were done with 2% aceto-orcein preceded by cold hydrolysis with 5n hcl at 4oc for 5 min. chromosome squash preparation were made using 45% glacial acetic acid. squashed slides were observed under olympus bx-53 microscope and number of chromosomes were calculated. digital microphotographs were taken in micro publisher 5.0 rtv camera observed under olympus bx-53 microscope for detail analysis of chromosomes and karyotype. total chromosome length was estimated by adding the length of all chromosomes in the karyotype and total chromosome volume by applying formula πr2h, where ‘r’ is the radius and ‘h’ is the length of the chromosome respectively. analysis of the chromosome type was conducted according to the classification system of levan et al. (1964), and that of the karyotype in accordance with the classification standard of stebbins (1971) modified by das and mallick (1993). form percentage (f %) of individual chromosome was calculated. interphase nuclear volume (inv) was calculated following the formula of sphere i.e. 4/3πr3, where r is the radius of interphase nucleus. results were analysed from 5-6 well spread metaphasic plates each obtained from the eight musa cultivars. in order to ascertain the significant differences of different genomic parameters among eight cultivars of banana, if any, the one-way anova test (sokal and rohlf 1973) was carried out with tukey’s 25karyotype and chromosome number in desert banana of odisha honest significant difference (hsd) test among the cultivars (tukey 1949). correlation co-efficient ‘r’ of different chromosomal parameters were made following ‘t’ test to compare the significant cytological variation, if any, among the studied cultivated desert banana cultivars. results chromosome numbers of all the eight cultivars found to be 2n = 3x = 33. the chromosome size varied from small to large. all the somatic chromosomes are classified as type a with comparatively large chromosomes having nearly median (nm) primary and secondary constrictions. type b with medium to large sized chromosomes having nearly sub-median (nsm) primary constriction and nearly sub terminal (nst) secondary constriction. type c with medium size chromosome having nearly median primary constriction (nm) and type d with small to medium size chromosomes having nearly sub-median (nsm) primary constriction (fig. 1). although all the cultivars showed 2n = 33 chromosomes, the number variation of different types of chromosomes in the karyotype formulae were found among the genotypes showing definite differences in their chromosome structure (figs. 2, 3, tables 2, supplementary table 1). the total chromosome length ranged from 55.68 µm in cv. chini champa to 81.50 µm in cv. kathia. predominance of nearly median chromosomes is a characteristic table 1. list of the eight cultivars of dessert banana (musa acuminata) germplasm collected from different parts of odisha. cultivar/accession number 2n genome constitution place of collection district latitude/longitude amritapani (mu-90) 33 aaa ouat, bhubaneswar khurda 20.26° n, 85.81°e champa (mu-107) 33 aab ches, bhubaneswar khurda 20.24°n, 85.78°e chini champa (mu-133) 33 aab tangi-chaudwar cuttack 20.55°n, 85.99°e dwarf cavendish (mu-53) 33 aaa rprc, bhubaneswar khurda 20.27°n, 85.79°e grand naine (mu-60) 33 aaa nimapada puri 20.05°n, 86.00°e kathia (mu-38) 33 abb kapilas dhenkanal 20.69°n, 85.74°e patakpura (mu-44) 33 aab chandanpur puri 19.88on, 85.81oe robusta (mu-137) 33 aaa ramagarh cuttack 20.55°n, 85.98°e ches = central horticultural experimental station, bhubaneswar, rprc regional plant resource centre, bhubaneswar, ouat = orissa university of agriculture and technology, bhubaneswar. figure 1. standard karyotype of desert banana (m. acuminata). figure 2a-h. metaphase plates of eight cultivars of desert banana of odisha; (a) cv. amritapani, (b) cv. champa, (c) cv. grand naine, (d) cv. patakpura, (e) cv. dwarf cavendish, (f ) cv. kathia, (g) cv. chini champa, (h) cv. robusta. magnification bar = 10 µm. 26 shomina dehury, subrat kumar dehery, anath bandhu das of the eight studied cultivars in which the tf% varied from 35.65% in cv. amritapani to 41.68% in cv. patakpura. the total chromosome volume was found lowest in cv. robusta (10.78 µm3) and highest in cv. kathia (15.99 µm3). the interphase nuclear volume ranged from 1336.44 µm3 in cv. dwarf cavendish to 2048.37 µm3 in cv. patakpura. presence of secondary constricted chromosomes varied among cultivars from 12 in cv. amritapani to 3 in cv. grand naine and robusta. karyotype of all the cultivars showed type a secondary constricted chromosome except cv. patakpura while b type secondary constricted chromosomes were found in cv. amritapani and cv. patakpura. other related cytological parameters against each cultivars have been given in table 2. statistical analysis showed significant differences among the cultivars of banana (table 3). the chromosome length and volume found significantly correlated with a coefficient value of r = 0.99. however, chromosome length and volume have no such significant correlation with nuclear volume which were -0.287 and -0.288 respectively. tukey’s honest significant difference (hsd) test confirmed that significant differences of total chromosome length and inv were recorded among the studied varieties (data not shown). the chromosome volume varied significantly among the varieties without having any significant variations between cv. chini champa and cv. champa, cv. dwarf cavendish and cv. grand naine, cv. champa, cv. chini champa and cv. robusta (table supplementary 2). no significant variation of tf% was also observed between cv. champa and cv. chini champa, cv. kathia and cv. champa or cv. chini champa following trukey hsd test (table supplementary 2). discussion cultivated bananas are scientifically interesting, as there is no genetic exchange during reproduction and selection is mostly depends on random mutations (oladosu et al. 2016). knowledge of chromosomal characters of the edible cultivars is valuable in order to know banana genetics in details. edible bananas have 2n = 2x 22, 33 or 44 chromosomes for diploid, triploid and tetraploid cultivars respectively (stover and simmonds 1987). these cultivars have a wide range of genome permutafigure 3. comparative karyogram of different cultivars of banana of the corresponding metaphase plates. table 2. detail karyotype analysis of the eight banana cultivars with different chromosomal parameters. variety genome somatic chromosome number (2n=3x) karyotype formula nsc+ total chromosome length (µm±se) total f% total chromosome volume (µm3±se) inv++ (µm3±se) amritapani aaa 33 9a+3b+15c+6d 12 75.60±1.23 35.65 14.83±0.13 1604.66±3.32 champa aab 33 6a+18c+9d 6 58.35±0.98 39.29 11.45±0.23 1526.50±5.58 chini champa aab 33 9a+15c+9d 9 55.68±1.45 39.36 10.93±0.34 1352.80±2.91 dwarf cavendish aaa 33 9a+12c+12d 9 64.52±0.56 35.82 12.66±0.22 1336.44±2.74 grand naine aaa 33 3a+18c+12d 3 63.76±1.25 38.20 12.52±0.15 1401.46±4.19 kathia aab 33 9a+21c+3d 9 81.50±2.12 39.10 15.99±0.34 1437.33±5.16 patakpura aab 33 6b+24c+3d 6 69.08±1.34 41.68 13.56±0.16 2048.37±6.21 robusta aaa 33 3a+21c+9d 3 54.95±0.67 40.18 10.78±0.09 1443.50±3.17 + nsc = number of secondary constricted chromosome; ++inv = interphase nuclear volume. 27karyotype and chromosome number in desert banana of odisha tions, including aa, ab, bb, aaa, aab, abb, aaab, abbb, and aabb. simmond and shepherd (1955) differentiated 5 genomic groups viz. aa, ab, aaa, aab, and abb based on the scoring of morphologically diagnostic characters relating to the two wild species m. acuminata and m. balbisiana. within each group, related clones are associated in a subgroup. cytological studies of indian species, varieties and cultivars are very scanty except some recent reports (ghosh et al. 2013; das et al. 2020; dehery et al. 2020) and molecular marker analysis (venkatachalam et al. 2008), though there are many biodiversity hotspots of banana in north east india exists which need to be explored. banana varieties of odisha have remarkable popularity in the locality and the cytogenetics of some of the varieties like cv. amritpani, cv. champa, cv. patakpura, cv. kathia has not been extensively covered and reported before. musa cultivars were studied and no numerical changes in the somatic chromosomes was observed in the genome that reconfirmed x = 11 (table 2). majority of the chromosomes in each karyotype were found to be in the group of the medium-sized chromosome with median primary constriction. all the 4 types of chromosomes were present in cv. amritapani whereas rest of cultivars has only 3 types of chromosomes. type c and d were common in all the cultivars with different doses whereas type b was present in cv. patakpura only and rest cultivars had type a chromosomes. the dose of nearly median constricted chromosomes were found more in all the cultivars except cv. dwarf cavendish and cv. grand naine that showed 12 type d chromosomes in the karyotype. numbers of secondary constricted chromosomes found variable among the cultivars. the total chromosome length varied from 54.95 µm in cv. robusta to 81.50 µm in cv. kathia and tf% varied from 35.65% in cv. amritapani to 41.68% in cv. patakpura among the studied cultivars. chromosome volume also found significantly different among the cultivars ranged from 10.78 µm3 in cv. robusta to 15.99 µm3 in cv. kathia that might be due to genome specific differential condensation of the heterochromatin and euchromatic region of the chromosomes during metaphase. thus, variety specific chromosome condensation and volume variation might be an indication of genome size variation which need further experimentation. differences in chromosome length or chromosome volume may be due to differential condensation and spiralization of the chromosome arms. in addition, the species-specific compaction of dna threads along with nucleosomes with altered non-histone proteins (das and mallick 1989). the alteration in the tf% might be due to chromosomal alteration due to break and reunion of the chromosome arms in early stages of evolution in the genome rather than the methodological defect of chromosome squash preparation. furthermore, translocation mediated structural alteration played a crucial role in chromosome evolution (lysak et al. 2006; luiz et al. 2009) besides heteromorphicity in centromeriac position among the chromosomes of allium localizing gcand at-rich repeats by cmaand dapi-banding patterns (mahbub et al. 2014). the dissymmetrical coefficient of the karyotype through fish in hibiscus mutabilis f. mutabilis, l. confirms relatively advanced type over plants with symmetrical chromosomes of the primitive type with respect to evolution (li et al. 2015). duplication of chromosomes or translocation between the chromosomes with or without secondary constrictions at a very early stage of evolution might be the reason for the structural alteration of the chromosome morphology as well as the variation of secondary constricted chromosomes in the above cultivars (das and das 1994; rai et al. 1997; ghosh et al. 2013; das et al. 2015, 2020; dehery et al. 2020). cultivars with reported aaa genome like cv. amritapani, cv. dwarf cavendish, cv. grand naine and cv. robusta found to have type a, c and d found common with 12 numbers of type d chromosomes each of cv. dwarf cavendish and cv. grand naine and 3 type a each of cv. grand naine and cv. robusta showing interrelationships among them having close affintable 3. analysis of variance (anova) of different genomic parameters among the eight cultivars of m. acuminata. source df ss ms f total chromosome length between cultivars 7 42.682 6.097 62.214* within cultivars 32 3.153 0.098 total 39 total chromosome volume between cultivars 7 32.127 4.589 57.362* within cultivars 32 2.563 0.080 total 39 total form % (tf%) between cultivars 7 422.256 60.322 105.458* within cultivars 32 18.334 0.572 total 39 total inv between cultivars 7 5267.365 752.480 442.895* within cultivars 62 105.34 1.699 total 69 * significant at p ≥ 0.001 level. df, degrees of freedom; ss, sum of squares; ms, mean squares; f, variance ratio 28 shomina dehury, subrat kumar dehery, anath bandhu das ity which need further investigation applying different dna markers. however, cv. amritapani had 9 type a with 3 numbers of type b of chromosomes with less numbers of type d chromosomes i.e. small sized submedian primary constriction. less number of secondary constricted chromosomes in cv. grand naine and cv. robusta genome might be more stable with less chances of chromosomal alteration due to break and reunion of the chromosomes in karyotypes during micro-evolution. but cv. amritapani differs from others with the presence of more number of secondary constriction and the karyotype is comparatively more fragile and karyotype asymmetry analysis might through some light on karyotype evolution in banana (dehery et al. 2020). cultivars recorded aab genome types like cv. champa, cv. cheni champa, cv. patakpura and cv. kathia with 3 types of chromosomes where type c and d were common in all the 4 cultivars. in this genotypic group cv. patakpura showed 6 type of b chromosomes. in contrary, cv. chini champa and cv. kathia showed each of 9 type a chromosomes that with less number of type d chromosomes in cv. kathia than cv. chini champa. evidently, all the members of aab group might close genetic relationship and decrease of median constricted type c chromosomes and increase of type d chromosomes in cv. champa and cv. chini champa clearly indicates their close genetic affinity in this genotypic group. high tf% in all the cultivars except cv. amritapani indicate the alteration of chromosome structure in the genome. these factors indicate greater genome stability conferring resistance to the cultivars against biotic or abiotic environmental stresses which is a characteristic feature of cultivars with b genome that need to confirm in future by fluorescent in situ hybridization (fish) or genomic in situ hybridization (gish) as shown in other cultivars of banana using bac clones (d’hont et al. 2000; doležel et al. 2004; d’hont 2005; jeridi et al. 2011). chromosomes with median, nearly median, submedian or nearly sub-median position of centromere are prevalent in karyotypes reported in this work. significant variations in the chromosome were not noted while analyzing the karyotypes of the eight cultivars studied as the eight triploid varieties known to have been derived from hybridization of the wild species have almost similar combinations of chromosomes with median and sub-median constrictions, with minute variations. although a significant variation in genome length, volume and inv was recorded (table 3). the small size of the chromosomes and the difficulty in obtaining a sufficient number of cells containing metaphase chromosomes makes it tedious rather difficult for the studies of the karyotype of bananas and plantain represented by many cultivars and subgroups in nature need to be analyzed with fish applying genome specific probes of transposable element for evolution among the cultivars. a positive high correlation was noted between chromosome length and chromosome volume (r = 0.99) that might be due to genome specific genetic control of chromosome condensation and packaging of histone protein. evolution of karyotype in species of identical chromosome number belongs to a distinct phylogenetic group is a long-standing issue that could be addressed by comparative chromosome painting to reconstruct karyotype evolution as evident in crucifer species of brassicaceae (mandáková and lysak 2008) and orchidaceae (medeiros-neto et al. 2017). acknowledgments the authors are thankful to the head of the botany, utkal university for providing administrative and microscopic facilities developed under dsr-iii, university grant commission, and fist programme, govt. of india to carry out the research. abd acknowledge the financial assistance received from council of scientific and industrial research, human resource development group, sanction no. 21(1107)/20/emr-ii), government of india, new delhi. funding this work was supported financially by the department of biotechnology, ministry of science and technology, government of india [project no dbt-ner/ agri/33/2016 (group-i, application no. 02)] is highly acknowledged. references cheesman ee, larter lnh. 1935. genetical and cytological studies of musa. iii. chromosome numbers in musaceae. j. genetics 30: 31–52. d’hont a. 2005. unraveling the genome structure of polyploids using fish and gish; 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chromosomal evolution in higher plants. london: edward arnold. stover rh, simmonds nw 1987. classification of banana cultivars. in: stover rh and simmonds nw (ed.) bananas, 3rd edn. wiley, new york, pp.97-103. tukey j. 1949. comparing individual means in the analysis of variance. biometrics. 5 (2): 99–114. venkatachalam l, sreedhar rv, bhagyalakshmi n 2008. the use of genetic markers for detecting dna polymorphism, genotype identification and phylogenetic relationships among banana cultivars. mol phylogenet evol 47:974–985. 30 shomina dehury, subrat kumar dehery, anath bandhu das supplementary table 1. detailed karyotype analysis of the eight dessert banana cultivars. chromosome types number of chromosomes total chromosome length (µm) length of short arm (µm) f% nature of constriction 1. m. acuminata cv. amritapani a 9 26.27 8.95 34.06 comparatively large chromo-some with nm primary and nsm secondary constrictions.7.28 27.71 b 3 7.48 2.19 29.27 nsm primary and secondary constriction. 1.62 21.65 c 15 28.22 12.30 43.58 medium size chromosomes with nm primary constriction. d 6 11.67 3.81 34.64 small size chromosomes with nsm primary constriction. 2. m. acuminata cv. champa a 6 11.88 4.15 34.93 comparatively large chromo-some having nm primary and nsm secondary constriction.2.80 23.56 c 18 24.77 11.02 44.48 medium size chromosomes with nm primary constriction. d 9 21.70 7.24 33.36 medium to small size chromosome with nsm primary constrictions. 3. m. acuminata cv. chini champa a 9 16.71 5.69 34.04 comparatively large chromo-some having nm primary and nsm secondary constrictions.4.60 27.52 c 15 25.67 11.55 45.0 medium size chromosomes with nm primary constriction. d 9 13.3 4.48 33.7 medium to small size chromo-some with nsm primary constrictions. 4. m. acuminata cv. dwarf cavendish a 9 22.85 8.83 36.49 comparatively large chromo-some having nm primary and nsm secondary constrictions.6.20 27.13 c 12 25.58 10.38 40.57 medium size chromosomes with nm primary constriction. d 12 14.84 5.10 34.36 medium size chromosomes with nsm primary constrictions. 5. m. acuminata cv. grand naine a 3 6.81 2.58 37.88 comparatively large chromo-some having nm primary and nsm secondary constrictions respectively.1.95 27.60 c 18 33.59 14.81 44.09 medium size chromosomes with nm primary constriction. d 12 23.36 7.29 31.20 medium size chromosomes with nsm primary constrictions. 6. m. acuminata cv. kathia a 9 24.68 8.99 36.42 comparatively large chromo-some having nm primary and nsm secondary constrictions.7.06 28.60 c 21 45.6 19.79 43.40 medium size chromosomes with nm primary constriction. d 3 11.21 3.72 33.18 medium size chromosomes with nsm primary constrictions. 7. m. acuminata cv. patakpura b 6 14.55 4.17 28.65 comparatively large chromo-somes with nsm primary and secondary constriction.3.61 24.81 c 24 47.76 21.03 44.03 medium size chromosomes with nm primary constriction. d 3 6.77 2.46 36.33 medium size chromosomes with nsm primary constrictions. 8. m. acuminata cv. robusta a 3 6.46 1.9 29.41 comparatively large chromo-some having nsm primary and secondary constrictions.1.5 23.22 c 21 32.02 14.6 45.6 medium size chromosomes with nm primary constriction. d 9 16.47 5.22 31.70 medium size chromosomes with nsm primary constrictions. nm = nearly median, nsm = nearly sub median, nst = nearly sub terminal. 31karyotype and chromosome number in desert banana of odisha supplementary table 2. mean difference of different cytological parameters among different varieties of m. acuminata and their significant level after tuky’s test. champa chini champa dwarf cavendish grand naine kathia patakpura robusta chromosome length amritapani 17.25* 19.92* 11.08* 11.84* 5.9* 6.52* 20.65* champa 2.67* 6.17* 5.41* 23.15* 10.73* 3.4* chini champa 8.84* 8.08* 25.82* 13.4* 0.73* dwarf cavendish 0.76* 16.98* 4.56* 9.57* grand naine 17.74* 5.32* 8.81* kathia 12.42* 26.55* patakpura 14.13* chromosome volume amritapani 3.38* 3.9* 2.17ns 2.31ns 1.16ns 1.27ns 4.05* champa 0.52ns 1.21ns 1.07ns 4.54* 2.11ns 0.67ns chini champa 1.73ns 1.59ns 5.06* 2.63ns 0.15ns dwarf cavendish 0.14ns 3.33* 0.9ns 1.88ns grand naine 3.47* 1.04ns 1.74ns kathia 2.43ns 5.21* patakpura 2.78* total form percentage (tf%) amritapani 3.64* 3.71* 0.17* 2.55* 3.45* 6.03* 4.53* champa 0.07ns 3.47* 1.09* 0.19ns 2.39* 0.89* chini champa 3.54* 1.16* 0.26ns 2.32* 0.82* dwarf cavendish 2.38* 3.28* 5.86* 4.36* grand naine 0.90* 3.48* 1.98* kathia 2.58* 1.08* patakpura 1.50* interphase nuclear volume (inv) amritapani 78.16* 251.86* 268.22* 203.2* 167.33* 443.71* 161.16* champa 173.7* 190.06* 125.04* 89.17* 521.87* 83.0* chini champa 16.36* 48.66** 84.53* 695.57* 90.7* dwarf cavendish 65.02* 100.89* 711.93* 107.06* grand naine 35.87* 646.91* 42.04* kathia 611.04 6.17* patakpura 604.87* * significant at p ≥ 0.001 level. caryologia. international journal of cytology, cytosystematics and cytogenetics 73(3): 55-63, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-646 caryologia international journal of cytology, cytosystematics and cytogenetics citation: m. rojas-gómez, a. garcíapiñeres, p. bolaños-villegas, g. arrieta-espinoza, e.j. fuchs (2020) genome size and chromosome number of psidium friedrichsthalianum (o. berg) nied (“cas”) in six populations of costa rica. caryologia 73(3): 55-63. doi: 10.13128/caryologia-646 received: october 03, 2019 accepted: may 31, 2020 published: december 31, 2020 copyright: © 2020 m. rojas-gómez, a. garcía-piñeres, p. bolaños-villegas, g. arrieta-espinoza, e.j. fuchs. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. genome size and chromosome number of psidium friedrichsthalianum (o. berg) nied (“cas”) in six populations of costa rica mónica rojas-gómez1,*, alfonso garcía-piñeres2, pablo bolaños-villegas3,4, griselda arrieta-espinoza5, eric j. fuchs6 1 centro nacional de innovaciones biotecnológicas (cenibiot), cenat-conare,1174-1200 san josé, costa rica 2 centro de investigación en biología celular y molecular and escuela de química, universidad de costa rica, 11501-2060 san josé (costa rica) 3 fabio baudrit agricultural research station, universidad de costa rica, la garita, alajuela 20101, costa rica 4 lankester botanical garden, university of costa rica, p.o. box 302-7050, cartago, costa rica 5 centro de investigación en biología celular y molecular (cibcm), universidad de costa rica 11501-2060 san josé, costa rica 6 escuela de biología, universidad de costa rica, 11501-2060 san josé, costa rica *corresponding author. e-mail: morogo27@gmail.com; mrojas@cenat.ac.cr. abstract. psidium friedrichsthalianum (o. berg) nied is a species found from southern mexico, central america; and there are reports that it is also found in venezuela and ecuador. it is a common fruit component of the costa rican diet, and it is valued industrially for its high content of polyphenols, mainly proanthocyanidins (pacs). this crop is not completely domesticated and there are no improved varieties produced through plant breeding. genome size or ploidy levels have not been investigated in costa rican populations of psidium friedrichsthalianum. information about chromosome number and genome size is paramount for plant breeding strategies. therefore, the main objective of our study was to determine chromosome number using pollen meiocytes and genome size by flow cytometry in six populations of p. friedrichsthalianum in costa rica. we found x = 11 bivalent chromosomes in all meiocytes analysed, classifying these populations as diploid. all populations had an average nuclear dna content of 2c = 1.960 ± 0.005 pg. no statistically significant differences in nuclear dna content were found among populations. we conclude that the consistency in chromosome number and genome size among populations suggests a common origin among them. our estimates of the number of chromosomes and genome size of p. friedrichsthalianum determined in this study will be essential for future breeding programs, hybridization practices and development of qtl (quantitative trait loci). keywords: ploidy, fluorescent microscopy, 2c nuclear dna, flow cytometry, costa rican guava, plant breeding. 56 mónica rojas-gómez et al. introduction psidium friedrichsthalianum (o. berg) nied is a tropical species in the family myrtaceae, subfamily myrtoideae, tribe myrteae (lucas et al. 2019); commonly known as “cas”, “sour guava” or “costa rican guava”. it is a medium-sized tree with reddish branches and abundant foliage of intense green color. flowers are perfect, possibly allogamous and pollination is performed by bees and occasionally by hummingbirds (barahona and rivera 1995). fruits are fleshy globose berries, between 5 and 10 cm in diameter with a greenish to yellow exocarp and a very distinct soft and acidic pulp. in addition, it is presumed that its center of origin is in costa rica (barahona and rivera 1995; rojasrodríguez and torres-córdoba 2013). “cas” fruits are characterised by abundant polyphenol content, mainly proanthocyanidins (pacs); these metabolites have important antioxidant, anti-inflammatory, antimicrobial and vasodilatory properties (cuadrado-silva et al. 2017; flores et al. 2013; rojas-garbanzo et al. 2019; granados-chinchilla et al. 2016; gonzález et al. 2012). vasconcelos et al. (2019) described the chemical composition and allelopathic properties of essential oils extracted from p. friedrichsthalianum, suggesting that this oil may be used as a natural weed control comparable in efficacy, to synthetic herbicides. this fruit is considered an important resource due to its photochemical properties; however, few studies have been conducted on this tropical fruit. the germplasm of p. friedrichsthalianum in costa rica has not yet been genetically characterized, however, srivastava (1977), reported this species as diploid (2n = 2x = 11), while hirano (1967) reported tetraploid and hexaploid individuals in central america samples. the diversity in chromosomal number previously reported for p. friedrichsthalianum may be a consequence of its ongoing domestication process. information on chromosome numbers in the myrtaceae is generally scarce, the fairly small chromosomes found in this taxonomic group, which rarely exceed 2 mm (costa 2004), may curb chromosome determination. presently, genome sizes have been reported for psidium acutangulum, psidium cattleianum, psidium guajava l. (white cultivar), psidium guajava l. (red cultivar), psidium guineense and psidium grandifolium (costa and forni-martins 2006b, costa et al. 2008; machado-marques et al. 2016; coser et al. 2012; souza et al.2015) (table 1). however, the genome size or the 2c value of p. friedrichsthalianum have not been analyzed yet. estimates of the number of chromosomes and genome size for p. friedrichsthalianum are essential for the design of effective improvement strategies, such as hybridization practices, the development of qtl (quantitative trait loci), as well as to better understand the effects of inbreeding and heterosis (birchler 2013; washtable 1. chromosomes number and genome size from different species of psidium and eucalyptus (myrtaceae) determined in previous studies. the content of holoploid nuclear dna (2c, pg dna) and the content of monoploid dna (1c, pg dna) are also provided. species 2n ploidy level nuclear dna content reference 2c (pg) 1c (pg) 1c (mbp)* genus eucalyptus eucalyptus microcorys 22 2x 1.040 0.520 508.56 almeida-carvalho et al.(2017) eucalyptus botryoides 22 2x 1.350 0.675 660.15 almeida-carvalho et al.(2017) genus psidium psidium guajava 22 2x 0.950 0.475 464.55 machado-marques et al.(2016); coser et al. (2012) psidium guajava (purple) 18 2x 0.990 0.495 484.11 souza et al.(2015) psidium guajava (“paluma”) 22 2x 1.020 0.510 498.78 souza et al.(2015) psidium guajava (white cultivar) 22 2x 0.507 0.253 247.43 coser et al. (2012) psidium guajava (red cultivar) 22 2x 0.551 0.275 268.95 coser et al. (2012) psidium grandifolium var. cinereum 44 4x 1.280 0.640 625.92 costa and forni-martins (2009) psidium grandifolium var. argenteum 44 4x 0.820 0.410 400.98 costa and forni-martins (2009) psidium cattleianum 44 4x 1.053 0.526 514.42 costa and forni-martins (2006b) psidium cattleianum 44 4x 1.990 0.995 973.11 souza et al.(2015) psidium guineense 44 4x 2.020 1.010 987.78 souza et al.(2015) psidium guineense 44 4x 1.850 0.925 904.65 machado-marques et al.(2016) psidium acutangulum 44 4x 1.167 0.583 570.17 costa and forni-martins (2009) *1pg dna = 978 mbp (dolezel et al. 2003; bennett et al. 2000). 57genome size and chromosome number of psidium friedrichsthalianum (o. berg) nied (“cas”) in six populations of costa rica burn and birchler 2014). additionally, with the current development of second and third generation sequencing techniques (ngs), information on genome size or c values are essential to establish appropriate experimental conditions, to effectively prepare genomic libraries and sequencing of complete genomes (leitch and leitch 2008). the c-values reported here may be used as a tool for genomic analysis in this species, which should benefit genetic improvement practices in this species. therefore, given the absence of information on ploidy level and nuclear dna content in populations of psidium friedrichsthalianum; we aimed to determine the chromosome number via fluorescent dapi stain and flow cytometry to determine the nuclear dna content of this tropical fruit in six populations of costa rica, its likely centre of origin. materials and methods sample collection we analysed individuals from six populations of p. friedrichsthalianum in costa rica. samples were collected from local small-scale plantations from different regions in the country (table 2). plantations were located at different elevations ranging from sea level to over 1500 m asl (metres above sea level). samples were always taken from reproductive trees and care was taken to collect samples from individuals that were separated by at least 10 meters to avoid collecting possible genets. chromosomal count using dapi stain chromosome counts were performed on pollen mother cells in meiotic metaphase. at least seven flower buds were collected from each of six populations; flower buds ranged between 0.7 cm and 0.8 cm in length. flower buds were fixed in faa solution (96% ethanol, 5% glacial acetic acid and 40% formaldehyde) for 24 hours. as suggested by dyer (1979), flower buds were dissected to 3/4 of their final bud size. anthers were placed on slides and subjected to mechanical disaggregation (macerating anthers with a thin spatula) adding occasional drops of acetic acid to prevent desiccation. macerated anthers were stained with dapi fluorochrome (sigma-aldrich, ilinois, usa) and were incubated in the dark for 5 min. we used an epifluorescence microscope (olympus bx50, olympus corporation, tokyo, japan) to visualize and photograph stained cells. only cells that were in a state of meiotic metaphase were photographed. for each population at least five slides were evaluated and at least one cell was recorded in a state of meiotic metaphase. finally, to facilitate chromosome counts, image color adjustments were performed with adobe photoshop cs82 (adobe systems, san jose, ca) and the imagej software (us national institutes of health, 2007) was used to count chromosomes. flow cytometry estimates of dna content we used flow cytometry to estimate genome size on p. friedrichsthalianum. we collected fruits from at least 10 individuals per population. seeds were manually extracted, washed with tap water and dried in open air for a week to remove moisture. after one week, seeds were sown in pots with vermiculite soil and placed in a greenhouse for 12 weeks until seedling emergence. ten seedlings per population were analyzed in a bd facscalibur tm (becton dickinson, san jose, ca, usa) flow cytometer. after initial parameter adjustment in the flow cytometry equipment, samples were prepared following the protocol by dolezel et al. (2007) with some modifications. we used 1 mg of leaf sample from glycine max as a reference (2c = 2.50 pg) (dolezel et al. 2007) and 5 mg of leaf tissue from p. friedrichsthalianum. leaves were table 2. collection sites of “costa rican guava” used to determine genome size and chromosomal number. ppt: mean annual precipitation (mm); samples cmf: number of seedlings used for flow cytometry; samples nm: number of samples used to determine chromosomes count. population name geographical coordinates altitude (m a.s.l.) ppt (mm) temp (°c) samples cmf samples nm cervantes 09°53´28.3˝n 83°47´24.0˝w 1465 2500 24 10 5 guápiles 10°13´42.1˝n 83°46´06.3˝w 262 4535 27 10 5 tacacorí 10°03´07.3˝n 84°12´52.3˝w 952 2100 23 10 5 ciruelas 09°59´05.7˝n 84°15´26.3˝w 910 1900 23 10 5 batán 10°04´34.4˝n 83°22´37.2˝w 114 3567 28 10 5 escazú 09°55´08.3˝n 84°07´42.6˝w 2428 1929 24 10 5 58 mónica rojas-gómez et al. placed in a petri dish on ice, then 1 ml of otto-i lysis buffer (0.1 m citric acid, 0.5% (vol / vol) tween 20) supplemented with 2 mm dithiothreitol (dtt) was added to the leaf cutouts (otto 1990). subsequently, leaves were cut with a razor blade until homogenization. the extract was filtered through a 41µm nylon mesh, onto a 2.0 ml microcentrifuge tube. the filtrate was centrifuged at 10,000 rpm for 5 minutes. the supernatant was discarded and the pellet was resuspended in 100 µl of ottoi lysis buffer and incubated for 15 min at 4 ° c. after incubation and prior to analysis in a flow cytometer, 300 µl of otto-ii buffer (0.4 m na 2 hpo 4 · 12 h 2 o) (otto 1990), 20 µl of propidium iodide (50 µg / ml) and 2 µl of rnase (50 µg / ml) were added to the mixture. all measurements were based on the fluorescence of at least 5000 total events (total nuclei). we analyzed two independent replicas of each sample on different days and estimated an average nuclear dna content. mean fluorescence intensity (mfi), number of events per peak and variation coefficient were all calculated using the fcs express 4 flow cytometry software (de novo software, los angeles, ca). finally, the nuclear dna content was calculated according to dolezel et al. (2007) as follows: a = (b × c) d where a = 2c (pg) nuclear dna content concentration of p. friedrichsthalianum; b = mean fluorescence intensity (mfi) of the g0 / g1 peak of p. friedrichsthalianum; c = 2c (pg) nuclear dna content of the internal standard; d = mfi of the g0 / g1 peak of the internal standard. genome size was estimated from dna content as 1 picogram (pg) of dna being equivalent to 978 megabase pairs (mbp) (bennett et al.2000; dolezel et al. 2003). the 2c nuclear dna content data of all individuals was compared among populations with a one-way anova, followed by tukey’s test to determine individual differences (p <0.05). statistics were done using r 3.5.0 software (r core team 2018). results and discussion we consistently found 11 bivalent chromosomes in all meiocytes from psidium friedrichsthalianum (figure 2) across all populations (table 2). taking into account that the basic chromosome number of the myrtaceae figure 1. (a) seedlings of p. friedrichsthalianum used to measure genome size by flow cytometry. (b and c) flower buds of p. friedrichsthalianum used for cytogenetic observations. 59genome size and chromosome number of psidium friedrichsthalianum (o. berg) nied (“cas”) in six populations of costa rica family is x = 11 (atchison 1947; raven 1975) we classified all costa rican samples of p. friedrichsthalianum as diploid (2n=2x=22). the diploid nature of the costa rican guava mirrors the results from srivastava (1977), who similarly found a 2n = 2x = 22 diploid chromosome count in different genotypes of psidium friedrichsthalianum. costa and forni-martins (2006a, b, 2007) also described chromosome numbers for 50 species in the myrtaceae, and found a predominance of 2n = 2x = 22 diploid species. naitani and srivastava (1965), coser et al. (2012), éder-silva et al. (2007), and souza et al. (2015), all found predominantly diploid species in the psidium genus, such as in psidium chinense and psidium guajava. previous results reported p. friedrichsthalianum individuals with 2n=4x=44 and even 2n=6x=66 (hirano 1967), suggesting that this species may have tetraploid and hexaploid members. these results clearly indicate that there may be variation in ploidy levels among populations of p. freidrichsthalianum in different areas. in contrast, our results show that at least in costa rica, cultivated populations are consistently diploid. this chromosomal uniformity may be the result of a common historical origin among populations. alternatively, our results may also be a consequence of artificial selection by farmers who selected cytotypes with specific homogenous traits of interests such as fruit size of pulp content. multiple cytotypes have also been found in other psidium congeners, for example in psidium cattleyanum the cytotypes 2n = 44, 66, 77 and 88 have been described (costa and forni-martins 2006a; costa 2009). multiple cytotypes have been also found in populations of psidium guineense and psidium guajava (srivastava 1977; costa and forni-martins 2006a; éder-silva et al. 2007; souza et al. 2015). polyploidy is recognized as one of the main evolutionary forces in angiosperms (soltis et al. 2015); and it is frequently associated with interspecific hybridization followed by chromosomal duplication to restore hybrid fertility (soltis et al. 2009). results from congeners suggests that p. friedrichsthalianum may also have the potential to create other cytotypes may represent important prospects for future breeding programs. our study found bivalent and univalent chromosomes in meiocytes of p. freidrichsthalianum (figure 2a-2c). chromosomes were also observed in a trivalent state (figure 2d) and this is consistent with previous observations by srivastava (1977) in this species. univalent chromosomes are frequently observed in plants; these can arise through three different ways: (i) when a chromosome is not matched completely in zygotene stage; (ii) when paired bivalents separate in diplotene because robust chiasmata have not yet formed between them; (iii) due to premature disjunction of the bivalents during anaphase (pires-bione et al. 2000). the premature migration of univalent chromosomes to the poles during cell division is common in plants, giving rise to micronuclei (pagliarini 1990; pagliarini and pereira 1992; consolaro et al. 1996). alternatively, univalent chromosomes may occasionally occur in plants due to environmental factors such as temperature fluctuations (heilborn 1934; katayama 1935). some of our sites differ drastically in climatic conditions, however, further studies are needed to better understand the cytology of this species. our flow cytometry estimates were very consistent across all plant samples. our coefficients of variation were all less than 5% (table 3), which confirms that our suspensions had a sufficient number of stoichiometrically stained and intact nuclei. additionally, dtt used in nuclei suspensions proved to be effective inhibiting cytosolic interfering compounds which resulted in clear histograms. dtt is commonly used in flow cytometry studies because of its broad antioxidant activity (dolezel et al. 2007). in our study, dtt was very efficient because many woody species in the myrtaceae, as is the case of p. friedrichsthalianum, contain abundant secondary metabolites that may interfere with dna content staining (loureiro et al.2006) (ohri and kumar 1986). we determined a mean nuclear value of 2c = 1.960 ± 0.005 pg for p. friedrichsthalianum, equivalent to figure 2. bivalent chromosomes of p. friedrichsthalianum in meiotic metaphase, stained with dapi, scale bar 10um. (a, b and c) samples from the populations of cervantes, tacacorí and escazú respectively, showing 11 bivalent chromosomes. (d) image showing chromosomes in trivalent, bivalent and univalent states. 60 mónica rojas-gómez et al. 1916.88 mbp (bennett et al. 2000) (figure 3, table 3). nuclear dna content did not statistically vary among all six populations (f=0.29; df=5; p = 0.917). leitch et al. (1998) and soltis et al. (2003) classified species with 1c ≤ 1.4 pg content as species with a very small genomes compared to other angiosperms. therefore, given our 1c estimates (1c = 0.98 ± 0.005 pg) (table 3) the costa rican guava should also be classified as a small genome species. consistently, machado-marques et al. (2016) found that psidium guajava and psidium guineense, also have very small genomes as 1c = 0.475 pg and 1c = 0.925 pg, respectively (table 1). almeidacarvalho et al. (2017) determined that 25 species of the genus eucalyptus (myrtaceae), all had 1c values between 1c=0.40 pg and 1c=0.75 pg which may indicate that the myrtaceae family may typically contain species with smaller genomes. on the other hand, our 2c estimates (2c =1.960 ± 0.005 pg) are within the range described by souza et al. (2015), who also used flow cytometry on different species of psidium and found 2c values that ranged between 2c=0.99 pg and 2c=5.48 pg. however our estimates are significantly higher than those found for different varieties of psidium guajava; for example, coser et al. (2012) found 2c = 0.507 pg for the white varieties, and 2c = 0.551 pg or 2c = 0.950 pg for the red varieties; while souza et al. (2015) found 2c = 0.990 pg and 2c = 1.020 pg in purple and “paluma” varieties respectively (table 1). these differences in genome size may be due to (i) natural or bred adaptations of these species to different environmental conditions (cavallini and natali 1990), for example, to new cultivation environments; (ii) hybridization events, or (iii) changes in repetitive dna sequences (martel et al.1997). several authors have suggested that transposable elements (te) may be important in the evolution of genome sizes in plants (wang et al. 2016; wendel et al. 2016; zhao et al. 2016). for example, almeida-carvalho et al. (2017) compared the genome size of two eucalyptus species, e. botryoides (2c=1.350 pg) and e. microcorys (2c=1.040 pg); and found big differences in genome size between them, although both had the same chromosome number (2n = 2x = 22) (table 1). they argued that variations in 2c values in these eucalyptus were caused by chromosome rearrangement and possibly te elements. therefore, our results on ploidy level and genome size of p. friedrichsthalianum, contribute to the cytogenetic characterization of this economically important fruit species. this information may be used to design regional conservation strategies that preserve local genetic resources. flow cytometry may be used to assess ploidy level in in vitro propagated plants (ochatt et al. 2011), to screen for plants with higher ploidy levels, which may have new features of economic interest such as increased fruit size, or better juicing capabilities. additionally, results from our study could aid the taxonomic definition of p. friedrichsthalianum species and the understanding of phylogenetic relationships among other members in the genus psidium. conclusions populations of psidium friedrichsthalianum from six different regions of costa rica, the likely centre of origin table 3. parameters obtained by flow cytometry to determine the genome size of psidium friedrichsthalianum. ne: number of events obtained; cv: coefficient of variation obtained; 2c (pg): holoploid nuclear dna content obtained; 1pg dna = 978 mbp (dolezel et al. 2003; bennett et al. 2000). species ne cv 1c (pg) 2c (pg) mbp psidium friedrichsthalianum 2452 ± 0.001 2.95 ± 0.007 0.980 ± 0.005 1.960 ± 0.005 1916.88 glycine max (standard) 2756 ± 0.005 3.01 ± 0.005 figure 3. relative fluorescence intensity (propidium iodide (pi)) histogram obtained after a simultaneous cytometric analysis of nuclei of reference standard (glycine max, 2c=2.50 pg of dna) and psidium friedrichsthalianum (2c= 1.960 ± 0.005 pg). 61genome size and chromosome number of psidium friedrichsthalianum (o. berg) nied (“cas”) in six populations of costa rica of this species, have a chromosome number equal to 2n = 2x = 22, indicating that cultivated populations in costa rica, are all consistently diploid. furthermore, these populations have an average 2c nuclear dna content of 1.960 ± 0.005 pg. the uniformity found across populations in terms of chromosomal number and nuclear dna content, suggests a common origin among them. acknowledgements we acknowledge the help of estación experimental agrícola fabio baudrit moreno (universidad de costa rica); centro nacional de innovaciones biotecnológicas (cenibiot) at cenat-conare; finca los diamantes at instituto nacional de innovación y transferencia en tecnología agropecuaria (inta). this work would not have been possible without local “cas” farmers who provided free samples from their farms: david badilla, gregorio menocal, juan pablo orozco, alfonso ruíz, josé fuentes. this work was conducted under permit # 1332018 from the comisión de biodiversidad-ucr at universidad de costa rica. geolocation data geolocation data is found on table 2. funding details this work was supported by the vicerrectoría de investigación de la universidad de costa rica (ucr) under grant 111-b7-261; consejo nacional de rectores (conare) under grant fees-15-2019. references almeida-carvalho gm, carvalho cr, ferrari-soares fa. 2017. flow cytometry and cytogenetic tools in eucalypts: genome size variation × karyotype stability. tree genetics & genomes. 13: 106 atchison e. 1947. chromosome numbers in the myrtaceae. american journal of botany. 34: 159-164. barahona m, rivera g. 1995. development of jocote (spondias purpurea l.) and cas (psidium friedrichsthalianum niedz) in the premontano humid forest of costa rica. mesoamerican agronomy. 6: 23-31. bennett md, bhandol p, leitch ij. 2000. nuclear dna amounts in angiosperms and their modern uses 807 new estimates. annals of botany.86: 859-909. birchler ja. 2013. genetic rules of heterosis in plants. in: polyploid and hybrid genomics. wiley. pp 313-321 cavallini a, natali l. 1990. nuclear dna variability within pisum sativum (leguminosae): cytophotometric analyzes. plant systematics and evolution. 173: 179–185. consolaro mel, pagliarini ms, chaves lj. 1996. meiotic behavior, pollen fertility and seed production in brazilian populations of centella asiatica (l.) urban (umbelliferae). cytologia. 61: 375-381. coser sm, ferreira mfs, ferreira a, mitre lk, carvalho cr, clarindo wr. 2012. assessment of genetic diversity in psidium guajava l. using different approaches. scientia horticulturae. 148: 223–229. costa ir, dornelas mc, forni-martins er. 2008. nuclear genome size variation in fleshy-fruited neotropical myrtaceae. plant systematics and evolution. 276: 209-217. costa ir, forni-martins er. 2006a. chromosome studies in brazilian species of campomanesia ruiz & pávon and psidium l. 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(online). cartago, cr, institute of technology of costa rica. esc. of forest engineer. accessed: may 15, 2013. vasconcelos lg, from souza santos e, from oliveira bernardes c, from silva ferreira mf, ferreira a, tuler ca, macedo carvalho ja, fontes pinheiro p, pracafontes mm. 2019. phytochemical analysis and effect of the essential oil of psidium l. species on the initial development and mitotic activity of plants. environmental science and pollution research. 43: 1-13. washburn jd, birchler ja. 2014. polyploids as a ‘‘model system’’ for the study of heterosis. plant reprod. 27:1–5. wang k, huang g, zhu y. 2016. transposable elements play an important role during cotton genome evolution and fibre cell development. sci chinalifesci. 59: 112-121. wendel jf, jackson sa, meyers bc, wing ra. 2016. evolution of plant genome architecture. genome biol. 17:37. zhao d, ferguson aa, jiang n. 2016. what makes up plant genomes: the vanishing line between transposable elements and genes. bbageneregulmech. 1859: 366-380. caryologia. international journal of cytology, cytosystematics and cytogenetics 72(2): 21-27, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-698 citation: a. özkara (2019) assessment of cytotoxicity and mutagenicity of insecticide demond ec25 in allium cepa and ames test. caryologia 72(2): 21-27. doi: 10.13128/caryologia-698 published: december 5, 2019 copyright: © 2019 a. özkara. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. assessment of cytotoxicity and mutagenicity of insecticide demond ec25 in allium cepa and ames test arzu özkara department of molecular biology and genetic, faculty of arts and sciences, afyon kocatepe university, afyonkarahisar/turkey e-mail: ozkara@gmail.com abstract. the mutagenicity and cytotoxicity of demond ec25, a synthetic pyrethroid insecticide, was assessed using two standard genotoxicity assays of the salmonella typhimurium mutagenicity assay (ames test) and allium cepa test. cytogenetic effects of demond ec25 were evaluated in the root meristem cells of allium cepa. the test concentrations of compounds were selected by determining ec50 of the allium root growth and onion seeds were exposed to demond ec25 (50, 100, and 200 ppm) for 24, 48, and 72 hours. the concentrations demond ec25 was compared with the value for the negative control using dunnet-t test, 2 sided. the results indicated that mitotic index was clearly decreased with increasing the concentration of demond ec25 in each treatment group as compared to the controls. demond ec25 was tested for mutagenicity in bacterial reversion assay systems with two strains (ta98 and ta100) of salmonella typhimurium absence and presence of s9 fraction. the doses of demond ec25 were 50, 100, 200, 400, 800 µg/plate and test materials were dissolved in dmso. our results show that demond ec25 was found to be mutagenic in 800 and 400 μg/plate doses of ta98 in the without s9 mix and 800 μg/plate in the with s9 mix. in ta100, demond ec25 was found to be mutagenic only 800 μg/plate doses without s9 mix. the other doses of this insecticide was not found to be mutagenic in both test strains. keywords. allium test, ames test, cytotoxicity, demond ec25, mutagenicity, pesticide. indroduction pyrethroids are among the most commonly used insecticides in agriculture; they are also widely used indoors in pet shampoo, lice treatment, and even insect repellent (saillenfait et al. 2015). they are therefore frequently present in food, air and dust of dwellings and thus can lead to both dietary and non-dietary exposure (morgan 2012). pyrethroids are botanical insecticides which are synthetic derivatives of pyrethrins and have been used for many years. however, most of pyrethroids are defined as moderately hazardous (class ii) by the world health organization (who 2009) (jensen et al. 2011). the residues of pyrethroids have been detected in fruits, vegetables, 22 arzu özkara tea, pasteurized milk and porcine muscle (nakamuraet al. 1993). wider use of pyrethroids posed a serious risk to environment and human. therefore, it may be an urgent need to evaluate the possible adverse effects of their use (miao et al. 2017) pyrethroid pesticides disrupt the nervous system of insects and, to a lesser degree, of mammals, and thus raise human health concerns. (oulhote and bouchard 2013; viel et al. 2015). pyrethroid residual insecticides exert their toxic effects by targeting the nervous system of insects. pyrethroids interfere with sodium channels in nerve fiber membrane and organophosphates bind to inhibit the activity of ache found in the synaptic junction. both actions result in continued nerve signaling and over-stimulation of nerve cells. poisoned insect exhibits tremors and convulsions, eventually leading to death (atsdr 2003; valles and koehler 2003). it is essential to carefully study and analyze the hazards of pyrethroids on human health including their genotoxic and cytotoxic properties. hereby, it can be take adequate measures to prevent humans from potential mutagenic and carcinogenic effects. (nagy et al. 2014). deltamethrin is a synthetic pyrethroid insecticide, sold by safa tarım limited with trade names demond ec 25 in local market. to our knowledge, there is no study mutagenicity of demond ec 25 except in the present paper. the aim of this experiment was to evaluate both the mutagenic and cytotoxic effects of different doses of demond ec 25 by the bacterial reverse mutation assay in s. typhimurium ta98 and ta100 strains with or without s9 mix and allium cepa test, respectively. material method chemicals the test substance demond ec25 was purchased from a local market in afyonkarahisar/turkey and dissolved in sterille distilled water. allium cepa onion bulbs, 25–30 mm diameter, were obtained from a local market without any treatments. the other chemicals were obtained from merck and riedel. test strains the lt-2 ta98 and ta100 histidine demanding auxotrophs of s. typhimurium were kindly obtained from prof. b.n. ames (university of california, berkeley). these strains were incubated for 16h in liquid nutrient broth and kept at -80°c. their genetic markers and other properties, such as the numbers of spontaneous revertants and responses to positive controls, were controlled as described by maron and ames (1983). allium test ec50 determination and mitotic index analysis the procedure of the root inhibition test as described by fiskesjo (1985) was followed with some modifications. the allium root inhibition test was carried out to determine suitable concentrations for the genotoxicity assay. the outer scales of the bulbs and the dry bottom plate were removed without destroying the root primordia. the onions were grown in freshly distilled water for the first 24h and afterwards exposed for 96h to the demond ec25 solutions (12.5, 25, 50, 100, and 200 ppm, respectively). in order to determine the ec50 values, the roots from each bundle were cut off on the fifth day and the length of each root was measured from both the demond ec25 exposed bulbs and the control group. the ec50 value was considered as the concentration which retards the growth of the root 50% less when compared to the control group. the ec50 value for demond ec25 was approximately 100 ppm. in order to demonstrate possible concentration-dependent effects of this pesticide, the root tips were treated with 50 ppm (ec50/2), 100 ppm (ec50), 200 ppm (ec50x2) concentrations of demond ec25, and all application groups were tested 24, 48, and 72h treatment periods. additionally we also used positive control group by using methyl methanesulfonate (mms). after the treatment, the roots were washed in distilled water and fixed in 3:1 ethanol: glacial acetic acid for 24h and then the roots were transferred into 70% alcohol and stored at +4°c. the root tip cells were stained with feulgen and five slides were prepared for each test group. ames salmonella/microsome assay the mutagenicity of the demond ec25 was determined using the standard plate incorporation assay. salmonella typhimurium strains ta98 and ta100 were used with or without s9 mix in this test (ames et al. 1975; maron and ames 1983). the tester strains were tested for the presence of the strain-specific markers as described by maron and ames (1983). the cytotoxic doses of the demond ec25 (800, 400, 200, 100, 50 µg/ plate) were determined by the method of dean et al. (1985). the stock solutions of the test materials were dissolved in sterile distilled water and stored at 4°c. the s. typhimurium strains were incubated in nutrient broth at 37°c for 16h with shaking. the positive controls were 23assessment of cytotoxicity and mutagenicity of insecticide demond ec25 in allium cepa and ames test 4-nitro-o-phenylenediamine (npd) for the ta 98 and sodium azide (sa) for the ta100, used without metabolic activation, and 2-aminofluorene (af) for ta 98 and 2-aminoanthracene (2aa) for the ta 100 used with metabolic activation. the test plates for the assays without the s9 mix were prepared by adding 0.1 ml of the test suspension for each concentration, 0.1 ml bacterial suspension from an overnight culture, and 0.5 ml phosphate buffer to 2 ml top agar (kept in 45°c water bath). the mixture was shaken for 3 s using a vortex mixer and then poured into the minimal agar. the test plates with the s9 mix were prepared by adding 0.5 ml of s9 mix instead of the phosphate buffer. all the test plates were incubated for 72h at 37°c, and then the revertant colonies on each plate were counted. the experiments were run in triplicate for each concentration and all the results from the two independent parallel experiments were used for the statistical analysis. statistical analysis the data obtained for the root length, mi, and mitotic phases were expressed as percentages. the levels of difference in the treatment groups were analyzed statistically by using the spss 15.0 version for windows. in the analyses, the dunnett-t test (2 sided) was performed on both the allium and ames tests. results allium root growth test results are summarized in table 1 and table 2 gives the effect of demond ec25 on mi and mitotic phase in the root meristematic cells of a. cepa treated for 24, 48 and 72h. the effective concentration (ec50) was determined as 100 ppm in allium test. at all concentrations treated in the incubations of root decreased mi compared to negative control at all exposure time. the reduced of mi results (p<0.05) were found statistically significant with all concentrations and all treatment time. all doses of demond ec25 applied in the experiment caused changes in the percentage of particular phases’ distribution in comparison to the control. table 1. allium root growth inhibition test. test substance concentrations (ppm) mean of root length±sd negative control 3.57±0.24 positive control 1.03±0.15* demond ec25 12.5 3.12±0.42* 25 2.02±0.15* 50 1.68±0.41* 100 1.45±0.23* 200 1.12±0.22* *significantly different from negative control (p<0.05 dunnet-t test, 2-sided), sd: standart deviation. table 2. the effects of demond ec25 on mi and mitotic phases in the root cells of a. cepa. concentration (ppm ) treatment time counted cell number mitotic index ± sd mitotic phases (%) ± sd prophase metaphase anaphase telophase negative control 24 hour 4965 82.45±6.71 79.12±9.42 1.80±0.32 1.12±0.32 0.92±0.57 positive control 5001 68.78±5.46* 34.24±4.62* 0.48±0.70* 0.49±0.54* 0.52±0.81 50 4889 51.48±4.09* 34.05±2.17* 1.00±0.72* 0.79±0.21* 1.10±0.62 100 4963 46.25±4.74* 32.54±4.20* 0.94±0.42* 0.68±0.34* 1.03±0.42 200 5013 45.21±2.69* 30.21±2.54* 0.82±0.40* 0.52±0.21* 0.59±0.74 negative control 48 hour 5007 67.28±3.47 70.11±6.74 1.62±0.21 1.27±0.24 1.21±0.26 positive control 4997 63.11±3.14* 31.75±3.45* 0.45±0.31* 0.52±0.16* 0.65±0.13 50 5101 52.25±3.45* 30.26±3.35* 0.71±0.92* 0.62±0.21* 1.19±0.21 100 5051 42.42±3.65* 28.45±2.70* 0.68±0.40* 0.54±0.02* 1.06±0.32 200 5113 36.20±1.25* 24.45±2.92* 0.52±0.32* 0.45±0.18* 0.89±0.14 negative control 72 hour 5142 38.21±2.65* 57.52±3.41 1.43±0.41 1.21±0.21 1.09+±0.52 positive control 5123 26.36±3.02* 29.04±2.28* 0.31±0.43* 0.60±0.42* 0.68±0.32 50 5263 19.23±1.75* 25.45±3.85* 0.58±0.23* 0.52±0.45* 0.49±0.41 100 5047 14.12±2.42* 21.42±2.56* 0.49±0.41* 0.46±0.71* 0.50±0.72 200 4985 13.21±2.21* 16.47±2.31* 0.34±0.42* 0.39±0.43* 0.56±0.61 * significantly different from negative control (p< 0.05 dunnet-t test. 2-sided) sd: standart deviation. 24 arzu özkara the results of the ames test are shown in table 3. in this experiment, first, the cytotoxic doses of demond ec25 were determined. as seen in table 3, spontaneous revertants were within the normal values in all the strains examined. all of the doses with and without s9 mix in ta98 and ta100 slightly increased when compared to the negative control. on the other hand, the plates containing positive control mutagens displayed very significant increases in the spontaneous mutation rate in two strains tested. most of the results, whether increasing or decreasing relative to the negative control group, were not statistically significant at p<0.05 (dunnett-t test, 2 sided) in the examined strains, except for in the 800 and 400 μg/plate doses of the demond ec25 in the ta98 without s9 mix and 800 μg/plate doses with s9 mix. additionally it was obtained mutagenic in the ta100 without s9 mix 800 μg/plate doses. discussion pyrethroid insecticides are commonly used in agriculture, veterinary medicine, and to control insect pests in human dwellings because of their high selective toxicity for insects and relatively low acute toxicity to mammals (casida and quistad 1998). these insecticides are favored because of their effective role and have replaced organophosphorus pesticides in many areas of applications (ministry of the environment in japanese 2011). because of their advantages, pyrethroid insecticides including demond ec25 are becoming widespread and, therefore, studies on the biological effects of these pesticides are of immediate concern. numerous studies on their toxicity, both in insects and mammals, have been reported in the literature. although pyrethroid insecticides have consistently shown negative results in microbial genotoxicity tests, the outcome of other assays has been variable and it has not been possible to draw definite conclusions about the genotoxicity of this group of pesticides (grossman 2007; surralles et al. 1995). in determining mutagenicity of chemicals, the ames test has shown a variety of chemicals to be either mutagenic or anti-mutagenic, and has been shown to be over 90% accurate in predicting genotoxicity (weisburger 2001). in the ames test, s. typhimurium strains that have a mutation in the his-operon are used to detect the mutagenicity of chemicals (maron and ames 1983). in the present study, demond ec25 was studied for its mutagenic activity with the ames test and results can be concluded that demond ec25 induced mutations in the 800 and 400 μg/plate doses of the ta98 without s9 mix and 800 μg/plate doses with s9 mix and in the ta100 without s9 mix. under our experimental conditions, demond ec25 showed to produce point mutations in the ames test, both in the absence and presence of the s9 metabolic activation system in high concentrations of both test strains. in order to characterize the possible mechanism of mutagenicity, the important bacterial strains, sensitive to different mutational events due to their specific genotypes, were used. particularly, s. typhimurium ta98 is characterized by the -1 frameshift deletion hisd3052, which affects the reading frame of a nearby repetitive –c–g– sequence table 3.the mutagenicity assay results of demond ec25 for s. tyhimurium ta98 and ta100 strains test substance concentration (µg/ plate) no of his+ revertants/plate, mean±sd ta98 ta100 s9 + s9 s9 + s9 demond ec25 800 95.32±5.41* 116.42±5.52* 206.45±9.44* 215.52±12.85 400 88.04±4.13* 102.21±3.96 178.42±7.45 203.12±10.25 200 68.12±4.63 92.54±4.25 142.45±6.74 184.32±9.54 100 52.09±3.86 78.09±4.52 121.22±6.61 168.35±8.65 50 47.31±3.38 56.24±4.45 102.10±5.08 123.09±6.85 neg. control 100 36.07±3.36 49.14±3.70 90.10±13.42 114.23±7.38 sa 10 2965.56±56.35* 2aa 5 2628.42±60.41* 2af 200 1002.40±16.65* npd 200 1575.50±24.56* *mean statistically significant at p<0.05 (dunnett t-test), sa:sodium azide, npd: 4-nitro-o-phenylendiamine, 2af: 2-aminofluorene, 2aa: 2-aminoanthracene, sd: standard deviation, negative control: distilled water. 25assessment of cytotoxicity and mutagenicity of insecticide demond ec25 in allium cepa and ames test and can be reverted by frameshift mutagens. ta100 contains the marker hisg46, which results from a base-pair substitution of a leucine (gag/ctc) by a proline (ggg/ ccc): this mutation is reverted by mutagens causing base substitutions at g-c base pairs (di sotto et al. 2008). taking into account these bacterial features, our results highlighted that the demond ec25 mutagenicity, in the absence and presence of s9 in ta98, was likely due to frameshift mutations, and in the absence s9 in ta100 due to base-change mechanisms. the data reported on the genotoxicity of synthetic pyrethroids are rather controversial, depending on the genetic system used (akintonwa et al. 2008; saleem et al. 2014). studies have shown an important relationship between a substance’s chemical structure and its biological activity (oztas¸ 2005) chlor. several factors, including rings, the functional groups, and the positions of binding locations in the chemical structure may affect a chemical’s binding ability. mitotic index proved to be a useful parameter that allows one to detect the frequency of the cellular division (marcano et al. 2004). the estimation of the potential cytotoxicity of the compounds is generally related to the inhibition of the mitotic activities (smaka-kincl et al. 1996). in this study, the used concentrations of demond ec25 also caused significant inhibition of the mitotic index. the significant decline in the mitotic index could be due to the inhibition of the dna synthesis or the blocking of the g1 suppressing the dna synthesis or effecting the test compound at the g2 phase of the cell cycle (sudhakar et al. 2001; majewska et al. 2003). when a pesticide penetrates the cells and reaches a critical concentration, it could be in an active form, causing lesions during several following cellular cycles (marcano et al. 2004). the decrease of the mitotic index in our study can be related to this. in this study, all the concentrations of demond ec25 caused the changes in the percentage of the particular phases’ distribution when compared to the control group. pesticides accumulate in the cell due to this substance not being able to emerge out of the cell easily after once penetrating the cell and it may be highly toxic in the cell (antunes-madeira and madeira 1979). deltamethrin, the active ingredient in demond ec25 has immunosuppressive (lukowicz and krechniak, 1992), reproductive effects on sperm cells (bhunya and pati 1990; carrera et al. 1996) and developmental toxicity (martin 1990). deltamethrin is reported to cause chromosomal damage in allium cepa (chauhan et al. 1986), chromosomal aberrations and micronucleus formation in bone marrow cells of mice exposed in vivo (chauhan et al. 1997; gandhi et al. 1995). saxena et al. (2009) evaluated of cytogenetic effects of deltamethrin in root meristem cells of allium sativum and allium cepa and cells analyzed immediately after the exposure showed a significant, concentration-dependent inhibition of mitotic index (mi) and induction of mitotic and chromosomal aberrations in both the test systems. additionally, in vitro exposure of deltamethrin is reported to cause dna damage in comet assay in human peripheral blood leukocytes (villarini et al. 1998). in the present study with allium cepa root tip meristem cells however, the three concentrations of demond ec25 tested induced genotoxicity thus corroborating the findings of these studies. in contrast to our results, no genotoxic response of deltamethrin was observed in salmonella typhimurium and v79 chinese hamster ovary cells (pluijmen et al. 1984). data on the genotoxicity and carcinogenicity of deltamethrin are rather controversial, depending on the genetic system or the assay used (shukla and taneja, 2000). the safety evaluation of a fragrance material includes a broad range of toxicological information, both for the compound itself and for structurally related chemicals belonging to the same chemical group (bickers et al., 2003). among toxicological information, genotoxicity is a systemic consideration, as it can be related to carcinogenicity (di sotto et al. 2008). normally, to evaluate a potential genotoxic risk due to a chemical exposition, in vitro assays for detecting point mutations (ames test) and extended treatment (e.g., micronucleus assay, allium test, single cell gel electrophoresis assay or comet assay) are used in the first instance (emea 2008; di sotto et al. 2013). if the results of these studies are positive, in vivo studies, for example a mammalian cytogenetic study, are performed (efsa 2014). the tested substances with different test systems can be genotoxic or not genotoxic depending on a number of factors such as chemical structure and biological activity, having rings in the structure and the positions of the binding location (kutlu et al. 2011). in addition to these, it might be related to differences in test conditions, such as exposure time, cell types, concentrations of substances, the dispersal of the materials and physicochemical characteristics of the compounds (ema et al. 2012).therefore, it could be explained why some studies find an increase of genetic damage while in others result as negative. in conclusion, demond ec25 was found to be cytotoxic due to decreasing of mi in allium test and showed mutagenic activity at some doses in the ames test. demond ec25 had clear cytotoxic effects and may pose a genotoxic risk for humans. for this reason, further 26 arzu özkara investigations are needed to determine the toxicity of this compound using other in vivo and in vitro biological test systems. a single test system is not enough to determinate a compound whether it is toxic or non-toxic. in this study we performed two different test methods. 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effects of fulvic acid on allium cepa l. root tip meristem cells özlem sultan aslantürk evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of allium test ioan sarac1, elena bonciu2,*, monica butnariu1, irina petrescu1, emilian madosa1 fluorescence in situ hybridisation study of micronuclei in c3a cells following exposure to elf-magnetic fields luc verschaeve1,2,*, roel antonissen1, ans baeyens3, anne vral3, annemarie maes1 phytochemical analysis and in vitro assessment of polystichum setiferum extracts for their cytotoxic and antimicrobial activities nicoleta anca şuţan1,*, irina fierăscu2, radu fierăscu2, deliu ionica1, liliana cristina soare1 telomeric heterochromatin and meiotic recombination in three species of coleoptera (dorcadion olympicum ganglebauer, stephanorrhina princeps oberthür and macraspis tristis laporte) anne-marie dutrillaux, bernard dutrillaux* a whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of populus trichocarpa l. subjected to different stresses alberto vangelisti#, gabriele usai#, flavia mascagni#, lucia natali, tommaso giordani*, andrea cavallini differences in c-band patterns between the japanese house mice (mus musculus) in hokkaido and eastern honshu hikari myoshu, masahiro a. iwasa* karyotypic description and repetitive dna chromosome mapping of melipona interrupta latreille, 1811 (hymenoptera: meliponini) natália martins travenzoli1, ingrid cândido de oliveira barbosa2, gislene almeida carvalho-zilse2, tânia maria fernandes salomão3, denilce meneses lopes1,* caryologia. international journal of cytology, cytosystematics and cytogenetics 73(2): 99-110, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-573 citation: r. tabaripour, m. sheidai, s. mehdi talebi, z. noormohammadi (2020) population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies. caryologia 73(2): 99-110. doi: 10.13128/caryologia-573 received: july 26, 2019 accepted: april 16, 2020 published: july 31, 2020 copyright: © 2020 r. tabaripour, m. sheidai, s. mehdi talebi, z. noormohammadi. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies raheleh tabaripour1,*, masoud sheidai1, seyed mehdi talebi2, zahra noormohammadi3 1 faculty of biological sciences and biotechnology, shahid beheshti university, tehran, iran 2 department of biology, faculty of science, arak university, arak, iran 3 biology department, islamic azad university, sciences and research branch, tehran, iran *correspondence author. e-mail: raheleh.tp@gmail.com abstract. ziziphora clinopodioides lam., (lamiaceae), is a perennial herb which is used as traditional medicine in iran. different authors disagree on the number of subspecies. in general, taxonomic and biosystematic studies of ziziphora clinopodioides have been limited and no molecular phylogenetic or biogeographic study of the species has been carried out. therefore, the aims of this study were (1) to determine the number of subspecies, (2) to produce information on the species’ genetic structure and intra-specific genetic variability, and (3) to produce data on the probable date of appearance of ziziphora clinopodioides in iran. we used a combination of morphological and molecular data to study plants randomly collected from 5 geographical regions. both analyses revealed a high level of within population variability and grouping of the studied provinces produced an admixture that indicated the absence of any subspecies within the species. structure analysis and k-means clustering identified two gene pools within the country. the probable date of divergence obtained was 5-10 mya for the appearance of this species in the mountainous regions of qazvin and mazandaran. keywords: biogeography, genetic diversity, structure analysis, subspecies delimitation, ziziphora clinopodioides. introduction species and subspecies delimitation is a difficult and somewhat subjective task in a species complex and in species with several overlapping geographical populations (wiens 2007). in general, the recognition of a species or subspecies is based on morphological observations. different species can be delimited by a few distinct morphological characteristics that show no overlap with other species. this criterion is very tra100 raheleh tabaripour et al. ditional yet it makes sense biologically, which suggests that there is no gene flow between the species (based on some assumptions; for example any morphological difference has a genetic basis) (wiens 2007). however, this approach can either fail to discriminate species and mask the presence of cryptic species or discriminate different species while in reality there is only one. in these situations, it is suggested that different and combined approaches such as morphological, molecular, cytological, and other approaches are used to determine species boundaries (duminil and di michele 2009; carstens et al. 2013). in some cases, incongruence may occur across the results from different methods. this may be due to either introgression or a difference in the power to detect cryptic lineages across one or more of the approaches used (carstens et al. 2013). in recent years, parallel developments in the analyzing power of both molecular phylogenetic and population genetic methods as well as their use in combination have resulted in more powerful species delimitation strategies. one of the most striking examples of a joint population genetics and phylogenetic approach is the use of the multispecies coalescent model to estimate phylogeny (edwards 2009; kingman 1982). this is further strengthened by development of new algorithms for detecting population genetic structure (pritchard et al. 2000; huelsenbeck and andolfatto 2007; huelsenbeck et al. 2011). the procedure usually involves comparing clusters obtained on the basis of observed polymorphism in both morphological and molecular characters to test if they are in agreement. in case of infra-specific taxon identification (e.g. subspecies), the occurrence of discontinuity in both datasets can be suggestive (seif et al. 2012; koohdar et al. 2015). k nowles and carstens (2007) addressed how molecular data (i.e., gene trees from dna sequence data) can be used in species delimitation. they proposed a new method which uses coalescent simulations to test hypotheses about species limits. their method is particularly valuable in that it can incorporate data from multiple loci and does not require species to have diverged to the point of being reciprocally monophyletic. similarly, medrano et al. (2014), applied population genetics methods to the species delimitation problem in narcissus (amaryllidaceae) using amplified fragment length polymorphism (aflp) molecular markers. ziziphora clinopodioides lamarck (lamiaceae) is a perennial herb with the common persian name “kakutie kuhi”. it is used as a traditional medicine in iran to treat diseases such as the common cold, gastrointestinal disorders and inflammation (naghibi et al. 2010). controversy exists as to the number of subspecies that should be recognized. for example, there are nine subspecies native to iran according to flora iranica (rechinger 1982), but in the flora of iran (jamzad 2012), no subspecies are considered. ziziphora clinopodioides has prostrated to erect stems and mainly branches at the base. the leaves vary in size and shape. the flowers are light to dark purple and white, with or without a peduncle, gathered in a compact capitulum. it is distributed in the eastern balkan peninsula, south east asia and central asia to the pamir-altay mountains and the himalayas (iran, iraq and central and eastern parts of turkey) as well as in africa (beikmohammadi 2011). in iran it grows on rocky slopes, low hills and grasslands. in general, there has been no detailed study looking at the taxonomy, molecular phylogeny and biogeography of this species. therefore, the aims of this study were (1) to determine the number of subspecies, (2) to produce information on the species genetic structure and intraspecific genetic variability and (3) to produce data on the probable date of appearance in iran of z. clinopodioides and its ancestral area of distribution. material and methods plant materials in the present study, 69 plant specimens from 19 populations of z. clinopodioides were randomly collected from five geographical localities (five provinces) of iran. these populations occur from northern to eastern parts of the country and have almost a continous pattern of distribution. (table 1, fig. 1). voucher specimens are deposited in the herbarium of shahid beheshti university (hsbu). morphological studies in total 29 morphological (5 qualitative, 24 quantitative) characters were studied. these characters include both vegetative and reproductive (f loral) variables (tables 2, 3). molecular studies for molecular analyses, we used both multilocus molecular markers of inter-simple sequence repeats (issrs) as well as the chloroplast rpl16 region. for issr analysis we used 69 specimens (1-6 samples from each 101population genetic and phylogeographic analyses of ziziphora clinopodioides population) and for cpdna analysis, we used a subset of 15 randomly selected plants (1-3 samples) from the studied populations of five provinces (table 1). both markers are widely used for species diversity analysis and phylogeny (weising et al. 2005; sheidai et al. 2014). issrs are particularly suitable markers for infra-specific studies and can reveal genetic discontinuities among populations (sheidai et al. 2012; sheidai et al. 2013). dna extraction, amplification and issr assay genomic dna was extracted using a ctab (cetyl trimethyl-ammonium bromide) activated charcoal protocol (sheidai et al. 2013). the quality of extracted dna was examined by running on a 0.8% agarose gel. 10 issr (inter simple sequence repeat) primers, (agc)5gt, (ca)7gt, (agc)5gg, ubc810, (ca)7at, (ga)9t, ubc807, ubc811, (ga)9a and (gt)7ca, were used (university of british columbia). pcr reactions were performed in a 25μl volume containing 10 mmtris-hcl buffer at ph 8, 50 mm kcl, 1.5 mm mgcl2, 0.2 mm of each dntp (bioron, germany), 0.2 μm of each primer, 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). the reactions were performed in a techne thermocycler (germany) with the following program: 5 min initial denaturation step at 94 °c, followed by 40 cycles of 45s at 94 °c; 1 min at 60 °c and 1min at 72 °c. the reaction was completed with a 7 min extension step at 72 °c. the amplification products were visualized by running on 2% agarose gels. the fragment size was estimated using a 100 bp molecular size ladder (fermentas, germany). in order to identify reproducible bands, the experiment was replicated 3 times. table 1. locality information for populations of z. clinopodioides sampled, including herbarium vouchers for specimens used for morphological and issr analyses and genbank accession numbers for specimens used for cp-dna analysis. pop no. province elevation (m) longitude latitude number of specimens sampled voucher no. genbank accession no.issr & morphology cpdna 1 razavi khorasan 2042 352715.9 595344.9 4 1 hsbu2014413  (1) mg738475 2 razavi khorasan 1976 353559.4 5839.7 4 2 hsbu2014414 (2) mg738476 hsbu2014427 (3) mg738477 3 mazandaran 1039 363610.3 534952.7 4 3 hsbu2014415 (4) mg738478 hsbu2014428 (10) mg738484 hsbu2014429 (11) mg738485 4 mazandaran 2597 368309 511855 6 2 hsbu2014421 (5) mg738479 hsbu2014430 (6) mg738480 5 tehran 2978 354349 521384.8 4 1 hsbu2014425 (7) mg738481 6 qazvin 1400 362765.5 501711.4 5 2 hsbu2014431 (8) mg738482 hsbu2014432 (9) mg738483 7 mazandaran 2225 363107 5456 3 2 hsbu2014426 (12) mg738486 hsbu2014433 (13) mg738487 8 ardebil 1493 381209.9 483909.2 6 1 auh522 (14) mg738488 9 ardebil 1389 381235 481757.3 3 1 aluh526 (15) mg738489 10 tehran 2308 355775.2 512954.5 2 hsbu2014424 11 qazvin 2750 392936 573450 4 hsbu2014416 12 qazvin 1333 363155.1 509824 5 hsbu2014417 13 razavi khorasan 2652 362319.7 5959.5 6 hsbu2014412 14 mazandaran 2103 362633.5 512838 1 hsbu2014419 15 mazandaran 2299 355514 521172.8 2 hsbu2014420 16 mazandaran 2341 355293.4 528320 4 hsbu2014423 17 mazandaran 1510 311137.1 523006.6 1 auh529 18 tehran 3245 362229.8 512628.2 3 hsbu2014422 19 tehran 2398 354636.4 515869.6 2 hsbu2014418 102 raheleh tabaripour et al. chloroplast dna the intron in the gene for ribosomal protein l16 (rpl16) located in the chloroplast genome was amplif ied and sequenced with universal primers following the methodology of shaw and small (2005) and timmer et al. (2007). the rpl16 forward primer was 5´-gtaagggtcatttagtaggtcgttt -3´ and the reverse primer 5´-tccttaccattaagttgatc -3 .́ each 20 µl pcr tube contained 10 µl of 2x pcr buffer, 0.5 mm of each primer, 200 mm of each dntp, 1 unit of taq dna polymerase (bioron, germany), and 1 µl of template genomic dna at 20 ng µl-1. the amplification reaction was performed in a techne thermocycler (germany) with the following program: 2 min initial denaturation step at 94°c, followed by 35 cycles of 5 min at 94°c; 1.30 min at 62°c and 2 min at 72°c. the reaction was completed by a final extension step of 7 min at 72°c. pcr products were visualized on 2.5% agarose gels with gelred™ nucleic acid gel staining. fragment sizes were estimated using a 100 bp size ladder (thermo fisher scientific, waltham, ma usa). data analyses morphometry morphological characters were first standardized (mean = 0, variance = 1) and used to establish euclidean distances among pairs of taxa (podani 2000). for grouping of the plant specimens, the upgma (unweighted pair group method with arithmetic mean) and ordination method of pca (principal components analysis) were used (podani 2000). a pca (principal components analysis) biplot was used to identify the most variable morphological characters among the studied populations (podani 2000). past version 2.17 (hammer et al. 2012) was used for multivariate statistical analyses of morphological data. table 2. qualitative morphological characters studied in z. clinopodioides populations. character state of character and their codes vegetative form straight (1), geniculate (2) basal vegetative form woody (1), dense woody (2), sparse woody stacked (3) stem leaf shape lanceolate (1), lanceolate-ovate (2), multiform (3) calyx hair frequency frequent (1), sparse (2), very sparse (3) calyx pedicle present (1), not present (2) table 3. quantitative morphological characters studied in z. clinopodioides populations. no. characters 1 plant length (cm) 2 leaf length of stem(mm) 3 leaf width of stem(mm) 4 stem leaf length / width ratio 5 petiole length(mm) 6 inflorescence leaf length (mm) 7 inflorescence leaf width (mm) 8 inflorescence leaf length/ width ratio 9 pedicle length (mm) 10 calyx length(mm) 11 calyx width(mm) 12 calyx length/ width ratio 13 calyx teeth length(mm) 14 calyx teeth width(mm) 15 calyx teeth length/ width ratio 16 inflorescence length(cm) 17 inflorescence width(cm) 18 inflorescence length/ width ratio 19 corolla length(mm) 20 corolla tube length(mm) 21 petal length(mm) 22 corolla tube length/petal length 23 stamen length(mm) 24 style length(mm) figure 1. distribution map of the studied provinces. 103population genetic and phylogeographic analyses of ziziphora clinopodioides issr analyses issr bands obtained were coded as binary characters (presence = 1, absence = 0). for grouping of the studied provinces, issr bands obtained were coded as binary characters (presence = 1, absence = 0). for grouping of the studied provinces, pco plot (principle coordinate analyses) was used (noormohammadi et al. 2011). the mantel test was performed to check correlation between geographical distance and genetic distance of the studied provinces (podani 2000). the past ver. 2.17 (hammer et al. 2012) program was used for these analyses. amova (analysis of molecular variance) based on fst and nei’s gst as implemented in genalex 6.4 (peakall and smouse 2006) was used to reveal genetic difference of the studied provinces. in order to determine the genetic structure of geographical provinces, we used two different approaches. first, bayesian model based structure analysis (pritchard et al. 2000), and second, the maximum likelihood based method of k-means clustering. for structure analysis with 105 permutations, data were scored as dominant markers (falush et al. 2007). we performed k-means clustering in genodive ver. 2. (2013). two summary statistics, 1) pseudo-f, and 2) the bayesian information criterion (bic), provide the best fit for k in the k-means clustering method (meirmans 2012). the population assignment test was performed using the maximum likelihood method as implemented in genodive (meirmans and van tienderen 2004). in order to identify agreement between the genetic tree and the morphological tree, we obtained a consensus tree using darwin ver.5 (2012). cp-dna sequence analyses and estimation time of divergence the intron in the gene for ribosomal protein l16 (rpl16) was aligned with muscle (robert, 2004) implemented in mega 5. the molecular clock test was performed as implemented in mega 5 (tamura et al. 2011). the test was done by comparing the ml value for the given topology with and without the molecular clock constraints under the tamura and nei (1993) model. using the parsimony method of templeton et al. (1992), implemented in tcs 1.13 program (clement et al. 2000). before estimating time of divergence, we used mega 5 to test the molecular clock and to find the best substitution model for the given sequences. the equal evolutionary rate of the studied sequences was rejected at a 5% significance level and therefore we used the relaxed molecular clock model in further analyses (drummond et al. 2006). moreover, hky was the best substitution model identified by model test as implemented in mega 5 (posada and crandall 1998). beast v1.6.1 (drummond et al. 2010a; drummond et al. 2010b) was used for the bayesian mcmc inferred analyses of the nucleotide sequence data (drummond and rambaut 2007). lallemantia baldschuanica gontscharow, l. iberica fisch. & c.a. mey. and l. royleana bentham were used as outgroups. beauti (bayesian evolutionary analysis utility version) v1.6.1 (drummond et al. 2010a, 2010b) was utilized to generate initial xml files for beast. a yule process of speciation (a ‘pure birth’ process) was used as a tree prior for all the tree model analyses. the yule tree prior is widely recognized as giving the best-fit model for trees describing the relationships between different species (drummond et al. 2010a, 2010b) and can be regarded as explaining the net speciation rate (nee 2006). for the mcmc analyses, the chain length was 10000000. after discarding 100 trees representing the burn-in, 10000 trees were used for the analyses. the beauti xml file was run in beast v1.6.1 (drummond et al. 2010a, 2010b). because no fossils are available for the studied species, we assumed a rate of evolution of the plastid sequence (u = 1.0 x 10 -9 s s-1 year-1) (zurawski et al. 1984; minaeifar et al. 2016). this was included in the option of molecular clock model in beauti v1.6.1. the normal distribution (mean = 0, standard deviation = 1) was used for priors. tracer v1.5 (drummond and rambaut 2007) was used to examine sampling and convergence. tree annotator v1.6.1 (drummond and rambaut 2007) was used to annotate the phylogenetic results generated by beast to form a single ‘target’ tree (maximum clade credibility tree, mcc) including summary statistics. figtree v1.3.1 (rambaut 2009) was used to produce the annotated beast mcc tree (fig. 6). biogeography the distribution range of ziziphora clinopodioides studied was divided into 5 areas (provinces): a (razavi khorasan), b (ardebil), c (mazandaran), d (qazvin) and e (tehran). we used s-diva (statistical dispersalvicariance analysis) and bbm (bayesian binary method) analyses implemented in rasp to reconstruct the possible ancestral ranges on the phylogenetic trees (yu et al. 2010, yu et al. 2015). in these methods, the frequencies of an ancestral range at a node in ancestral reconstructions are averaged over all trees (yan et al. 2010). we used initially the tree obtained from the beast 104 raheleh tabaripour et al. analysis (mcc tree), followed by rasp analysis. the final tree for the area ancestry determination was based on the majority rule consensus tree. results systematics morphometry the mean values and standard errors for the quantitative morphological characters are provided in table 4. the anova test revealed significant difference in stem leaf length (p = 0.01), inflorescence leaf length / width ratio (p = 0.01) and corolla tube length/petal length ratio (p = 0.02). different clustering and ordination methods produced similar results, therefore only the pca plot of the studied provinces based on the morphological data is provided (fig. 2). the studied provinces were placed inter-mixed, thus there is no support for morphological divergence among provinces. there appears to be some morphological differentiation between province 2 (razavi khorasan) and all other provinces which is plausible as it is the most geographically separated (fig. 2). pca analysis of morphological characters revealed that the first three pca components comprised 70% of the total variability. morphological traits (stem leaf shape, petiole length and style length) showed the highest level of correlation with the first pca component (>0.65), while characters 6 and 7 were highly correlated with the second pca component (>0.62). therefore, these are the most variable morphological characters among the five studied provinces. the pca biplot, (not shown) revealed that morphological characters 3 and 10 differentiate mainly province 2 (razavi khorasan), while character 26 differentiates province 5 (ardebil) from the others. issr analysis issr analysis of the studied provinces produced 97 reproducible bands. the pco plot (fig. 3) revealed that plants from different provinces were grouped together due to genetic similarity, for example those from provinces 2, 3 and 4. therefore, issr data do not differentiate the studied provinces. this is in agreement with our morphometric analyses. table 4. the mean value and standard error of quantitative morphological characters. character qazvin razavi khorasan mazandaran tehran ardebil 14 specimens 14 specimens 21 specimens 11 specimens 9 specimens leaf length of stem(mm) 15.50 ±0.73 8.00 ±0.49 12.30 ±1.16 15.18 ±0.74 10.07 ±0.36 leaf width of stem(mm) 4.50 ±2.00 3.42 ±0.27 3.60 ±0.23 3.80 ±0.35 4.00 ±0.44 stem leaf length / width ratio 3.48 ±0.16 2.41 ±0.13 3.32 ±0.12 4.18 ±0.28 2.90 ±0.26 petiole length(mm) 1.75 ±0.23 2.85 ±0.77 1.60 ±0.13 1.40 ±0.13 1.73 ±0.87 inflorescence leaf length (mm) 7.04 ±1.03 4.94 ±0.44 6.74 ±0.62 6.77 ±0.84 7.35 ±0.63 inflorescence leaf width (mm) 2.40 ±0.20 2.50 ±0.20 3.00 ±0.22 2.52 ±0.31 3.25 ±0.27 inflorescence leaf length/ width ratio leaf width 3.31 ±0.31 2.50 ±0.11 2.23 ±0.11 2.80±0.21 2.30 ±0.31 pedicle length (mm) 1.33 ±0.13 0.49 ±0.10 1.40 ±0.07 1.38 ±0.06 1.51 ±0.07 calyx length(mm) 4.07 ±0.13 4.58 ±0.27 5.24 ±0.15 8.25 ±3.48 5.17 ±0.18 calyx width(mm) 1.05 ±0.08 1.15 ±0.10 1.20 ±0.05 1.25 ±0.08 1.25 ±0.02 calyx length/ width ratio 4.08 ±0.30 4.29 ±0.22 4.41 ±0.15 3.83 ±0.17 4.11 ±0.13 calyx teeth length(mm) 0.78 ±0.07 0.96 ±0.06 0.93 ±0.06 0.81 ±0.09 0.82 ±0.05 inflorescence length(cm) 1.63 ±0.06 1.33 ±0.09 1.37 ±0.08 1.29 ±0.13 1.45 ±0.17 inflorescence width(cm) 1.84 ±0.05 1.82 ±0.06 1.62 ±0.07 1.59 ±0.09 1.53 ±0.14 inflorescence length/ width ratio 0.86 ±0.03 0.82 ±0.05 0.83 ±0.03 0.83 ±0.08 0.94 ±0.05 corolla length(mm) 5.87 ±0.27 6.21 ±0.33 6.28 ±0.27 6.12 ±0.26 6.21 ±0.45 corolla tube length(mm) 3.48 ±0.16 3.57 ±0.19 3.80 ±0.18 3.43 ±0.17 4.03 ±0.38 petal length(mm) 2.25 ±0.17 2.60 ±0.20 2.48 ±0.12 2.69 ±0.12 2.18 ±0.09 corolla tube length/petal length 1.54 ±0.10 1.48 ±0.13 1.56 ±0.08 1.26 ±0.06 1.84 ±0.13 stamen length(mm) 1.19 ±0.16 2.07 ±0.28 1.78 ±0.22 0.68 ±0.21 1.99 ±0.26 style length(mm) 4.78 ±0.23 4.81 ±0.41 5.13 ±0.28 4.59 ±0.28 4.54 ±0.29 mean ± standard error. 105population genetic and phylogeographic analyses of ziziphora clinopodioides moreover, the consensus tree of morphological and genetic features did not differentiate the plants collected in the studied provinces (fig. not given), only distinguishing plant numbers 6 and 7 of qazvin province (province 1), and plants 46 and 47 of mazandaran province (province 3). this result suggests that morphological variation in the studied provinces is not in agreement with their genetic features. therefore, the present study does not support the idea that z. clinopodioides contains any subspecies in iran. this conclusion is further supported by haplotype networking of cp-dna (fig. 4). the studied plants differed in cp-dna sequences. the haplotype network separated outgroups from the studied ziziphora clinopodioides plants. moreover, it revealed large-scale within-province cp-dna variation. for example, plants studied in mazandaran, ardebil and razavi-khorasan provinces were widely scattered on the network. provincial genetic diversity analyses genetic diversity parameters from the studied provinces are presented in table 5. the highest value of genetic polymorphism in province 3 (79.38%) and the highest value of nei gene diversity occurred in province 2 (0.158), while the lowest value of the same parameters was observed in province 5 (45.36% and 0.123, respectively). this indicates that province 5 has a lower degree of within province genetic variability. amova and gst results revealed significant difference among the studied provinces. amova produced a phipt value of 0.068 (p = 0.01), while the gst value was 0.065 (p = 0.01). pair-wise analysis of fst and gst revealed significant difference between provinces (table 6). amova revealed that 93.2% of total genetic variability occurred due to within province diversity and 6.87% due to among province diversity. this is in agreement with pco plot of issr data presented before; the provinces were not differentiated. migration analysis of genetic data in all populations of five provinces produced a mean nm value of 6.45 and fig 4. haplotype network of cp-dna data in the studied ziziphora clinopodioides provinces. figure 2. pca plot of ziziphora clinopodioides provinces based on 29 morphological characters. figure 3. pco plot of ziziphora clinopodioides provinces based on issr data. table 5. genetic diversity parameters in the studied provinces based on issr data province n na ne i he uhe %p hs qazvin 14 1.340 1.191 0.227 0.134 0.139 67.01 0.218 razavi khorasan 14 1.464 1.227 0.262 0.158 0.163 73.20 0.251 mazandaran 21 1.588 1.193 0.246 0.142 0.145 79.38 0.234 tehran 11 1.361 1.197 0.242 0.143 0.149 68.04 0.242 ardebil 9 0.907 1.192 0.195 0.123 0.130 45.36 0.185 n = no. plants, na = no. alleles, ne = no. effective alleles, i = shanon information index, he = nei gene diversity, uhe = unbiased gene diversity, %p = percentage of genetic polymorphism, and hs = genetic diversity due to population. 106 raheleh tabaripour et al. a gst value of 0.07. these values indicate a high degree of gene flow among the studied populations. moreover, structure analysis based on a genetic admixture model also revealed a high degree of genetic admixture among the studied provinces as they had very similar allele combinations (similarly colored segments). these common shared alleles are either ancestral shared alleles or occurred due to ongoing gene flow among the populations. the evanno test identified two gene pools. the province assignment test revealed that gene flow occurred between all provinces but was higher between plants in provinces 1, 3 and 4. province 5 had the lowest degree of gene flow as revealed by the lowest within province genetic variability as stated above. this province had limited gene flow with provinces 3 and 4. the pseudo-f value of k-means clustering and evanno test of structur e revealed two genetic groups. when we performed the structure analysis for k = 2 (fig. 5), it revealed that provinces 1 and 5 formed the first genetic group, while provinces 2-4 comprised the second genetic group. therefore, we have two gene pools in iran for this medicinal plant that can be used in germplasm conservation and future medicinal evaluation. the mantel test produced significant correlation (r = 0.184, p = 0.01) between geographical distance and genetic distance of the studied provinces. this means that ibd (isolation by distance) has occurred in z. clinopodioides provinces and the neighboring provinces can exchange genes more frequently compared to those that are further from each other. this could be the reason for the higher degree of genetic similarity observed between provinces 2, 3 and 4. divergence time estimation cp-dna haplotypes can be considered as good molecular markers for investigating probable dates of appearance of populations and their paths of distribution in the country (minaeifar et al, 2016). beast and rasp analyses (figs 6, 7) suggested that the oldest cpdna haplotype of z. clinopodioides appeared sometime around 5-10 mya in mazandaran province (province 3). this suggests that z. clinopodioides could possibly have appeared in the northern regions of the country during the miocene era, with plants subsequently dispersing towards the north-eastern (razavi khorasan), northwestern (ardebil) and central parts of iran (tehran and qazvin). discussion in the present study molecular markers such multilocus issrs and cp-dna sequences and morphological variables were used for genetic diversity, species and subspecies delimitation of z. clinopodioides. according table 6. pair-wise analysis of fst in the studied provinces based on issr data. qazvin razavi khorasan mazanda ran tehran ardebil qazvin 0.000 razavi khorasan 0.075 0.000 mazandaran 0.035 0.030 0.000 tehran 0.057 0.054 0.028 0.000 ardebil 0.099 0.156 0.123 0.132 0.000 figure 5. structure plot of ziziphora clinopodioides provinces based on k = 2. (provinces 1-5 are: 1qazvin, 2razavi khorasan, 3mazandaran, 4tehran, and 5ardebil). figure 6. chronogram from beast analysis of the studied provinces for ziziphora clinopodioides based on the cp-dna dataset (rpl16), showing 95% highest posterior density bars (hpd) in purple. numbers on nodes are clade credibility values. (provinces 1-5 are: 1qazvin, 2razavi khorasan, 3mazandaran, 4tehran, and 5ardebil). 107population genetic and phylogeographic analyses of ziziphora clinopodioides to several evaluations, phylogenetic markers (its and cpdna) and issr molecular techniques are useful for genetic diversity, species and subspecies delimitation for different taxa such as, diospyros l. (li et al. 2018), marrubium l. (salehi et al. 2018), carum l. (papini et al. 2015), acer velutinum boiss (siahkolaee 2017), cycas diannanensis z. t. guan & g. d. tao (jian et al. 2015) and petunia axillaris (lam.) britton, sterns & poggenb (turchetto et al. 2014). both molecular markers (multilocus issrs as well as cp-dna sequences) and morphological characters produced similar results and showed a lack of province discontinuity within z. clinopodioides. therefore, our data do not suggest the presence of subspecies in the studied populations of this species. jamzad (2012) in the flora of iran, after thorough morphological investigation in z. clinopodioides, suggested that due to a high degree of morphological variability and co-occurrence of many subspecies in one location, she could not be sure about the number of subspecies within z. clinopodioides and suggested the use of molecular studies to solve this problem. moreover, hig h mor pholog ica l, pa ly nolog ica l and molecular diversity exist among ziziphora taxa (tabaripour et al. 2018; tabaripour et al. 2019) and the genus shows very variable chromosome number along a descending dysploidy line starting from 2n = 16 to 2n = 34 (taarna 1973; selvi et al. 2013), but z. clinopodioides proved to has 2n=18 (selvi et al. 2013). in a similar study, subspecies determination was conducted in the western australian species pityrodia scabra a.s. george. (lamiaceae) using a combined approach with non-coding chloroplast gene regions and morphological data (shepherd et al. 2013). they observed that some morphological features varied among the populations and provided some evidence for cryptic taxa. furthermore, molecular phylogenetic analyses revealed genetic distinctiveness between the wyalkatchem (type) population and the southern cross and lake lefroy populations. this evidence, when used in conjunction with the morphological differences, provided support for the recognition of the new subspecies described as pityrodia scabra subsp. dendrotricha k.a. sheph. subsp. nov. population genetics studies are an important step in planning genetic and breeding programs for crop and medicinal plants. they provide data on genetic variability, gene flow versus population genetic isolation, population genetic fragmentation, alongside the role of genetic drift, bottlenecks and other evolutionary forces acting on population divergence (sheidai et al. 2013, 2014). with increases in sizes of human populations, crop plants and medicinally important plant taxa are consumed and destroyed faster than before. medicinal plants such as z. clinopodioides are extensively used by locals and therefore potentially threatened in their natural habitats. therefore, to design an effective conservation strategy, knowledge of genetic diversity in the target species is important. the present study revealed a high level of morphological and genetic variability both within and among provinces of z. clinopodioides. amova revealed that 93% of total genetic variability occurred due to within province diversity and 7% due to among province diversity. this could be due to the out-crossing nature of this species. these plants are usually cross pollinated in nature by insects, which can result in high within population genetic variability. we can exploit this variability in future hybridization and breeding strategies. assessments of levels of withinand among-population genetic variations have been used to prioritize populations for conservation efforts (petit et al. 1998), with (all else being equal) more weight given to populations exhibiting higher levels of within-population variation and to those that are more genetically divergent. the structure plot and province assignment revealed some degree of genetic admixture among the fig 7. cp-dna rasp analysis based on beast tree (mcmc) showing probable ancestral area distribution for ziziphora clinopodioides provinces. razavi khorasan, bardebil, cmazandaran, d qazvin, etehran and flellemanthia (outgroup). 108 raheleh tabaripour et al. studied z. clinopodioides provinces. gene flow is also important in conservation contexts, particularly for species with local populations. fortunately, z. clinopodioides provinces showed high within-province genetic variability and high among province gene flow. gene flow among local populations could mitigate losses of genetic variation caused by genetic drift in local populations and potentially save them from extinction (sheidai et al. 2014; safaei et al. 2016). the mantel test revealed isolation by distance in the studied z. clinopodioides provinces. in plant species that form geographical populations, as geographical isolation increases, a reduction in both seed dispersal and pollen flow will result in decreased gene flow between distantly located populations (freeland et al. 2011). this explains why the evanno test and k-means clustering identified two different gene pools for z. clinopodioides within the country. beast and rasp results suggested that z. clinopodioides haplotypes appeared around 5-10 mya in the mountainous regions of qazvin and mazandaran. active divergence occurred between 1-5 mya in these mountains due to their reactions to pleistocene glaciations. our mean date of 7 mya is in agreement with the study of drew and sytsma (2012). acknowledgment we thank the iran national science foundation (insf) with 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(plantaginaceae) saeed mohsenzadeh*, masoud sheidai, fahimeh koohdar a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding timir baran jha1,*, partha sarathi saha2, sumita jha2 chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india harminder singh2, jaswant singh1,*, puneet kumar2, vijay kumar singhal1, bhupendra singh kholia2, lalit mohan tewari3 population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies raheleh tabaripour1,*, masoud sheidai1, seyed mehdi talebi2, zahra noormohammadi3 induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions girjesh kumar, shefali singh* karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(2): 39-49, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-791 citation: a. iannucci, s. cannicci, z. lin, k.w.y. yuen, c. ciofi, r. stanyon, s. fratini (2020) cytogenetic of brachyura (decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species. caryologia 73(2): 39-49. doi: 10.13128/caryologia-791 received: december 23, 2019 accepted: april 6, 2020 published: july 31, 2020 copyright: © 2020 a. iannucci, s. cannicci, z. lin, k.w.y. yuen, c. ciofi, r. stanyon, s. fratini. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. cytogenetic of brachyura (decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species alessio iannucci1, stefano cannicci1,2,*, zhongyang lin3, karen wy yuen3, claudio ciofi1, roscoe stanyon1, sara fratini1 1 department of biology, university of florence, via madonna del piano 6, 50019 sesto fiorentino, italy. e-mail: alessio.iannucci@unifi.it; claudio.ciofi@unifi.it; roscoe.stanyon@ unifi.it; sarafratini@unifi.it 2 the swire institute of marine science and division of ecology and biodiversity, the university of hong kong, pokfulam road, hong kong sar. e-mail: cannicci@hku.hk. 3 the school of biological sciences, the university of hong kong, pokfulam road, hong kong, hong kong s.a.r. e-mail: wzylin@connect.hku.hk; kwyyuen@hku.hk. *corresponding author. abstract. brachyura is one of the most specious infra-order belonging to decapoda and it plays a central role from an ecological and economic point of view. despite its importance, cytogenetic studies on brachyura (decapoda) are extremely limited due to the difficulties in obtaining chromosome preparations of good quality. molecular cytogenetic have proven to provide basic knowledge on the genome organization of species and in methods for manipulating genomes. it is also very useful to support aquaculture. in this study we focused on six semi-terrestrial mangrove crab species to test several variations of technical steps to produce chromosome preparations in brachyura. the best results were obtained using cells from early stage embryos incubated with 0.0005% nocodazole or 0.05% colchicine and hypotonized with 0.075 m kcl solution. the best method to analyze the chromosomes was the use of confocal microscope after dapi staining. we recorded a high chromosome number for the six study species. similar chromosome morphology was recorded for allied species likely due to phylogenetic relationship. variable results with cytogenetic treatments in different species suggest that there may be a species-specific response to the techniques we tested. chromosome number ranges reported in this study will be useful in future genome sequencing studies, i.e. to assess the quality of sequencing assemblies. keywords: aquaculture, chromosomes, confocal microscope, mangrove crabs, dapi, metaphase. introduction decapoda represents the most species-rich crustacean order with more than 2,700 genera and 17,000 species inhabiting marine, intertidal, freshwater and terrestrial ecosystems (de grave et al., 2009). the infra-order brachyura is particularly species-rich (about 6,800 species in 1270 genera) 40 alessio iannucci et al. and includes all the extant true crabs (ng et al., 2008; ahyong et al., 2011; tsang et al., 2014). the importance of this group is unquestionable both from an ecological and economic point of view. many species of brachyuran crabs are edible, being extensively fished and eaten worldwide. according to recent data from the food and agriculture organization (fao 2019), the species of the genus portunus, such as p. trituberculatus (miers, 1876) and p. pelagicus (linnaeus, 1975) represent the most fished crabs together with callinectes sapidus rathbun, 1896, cancer pagurus linnaeus, 1758 and species belonging to the genus scylla de haan, 1833. information about species karyotypes is a fundamental prerequisite for many advanced and applied studies. in genomics, for example, knowledge about species chromosome numbers is critical to assess the quality of assemblies and have an idea of the genome organization (e.g. sharakhov et al., 2014). moreover, comparing the sequence and structure of genes and their organization into chromosomes is now the best approach to understand genome evolution and consequently organism evolution (see coghlan et al., 2005). moreover, cytogenetic information is necessary in methods for modifying and manipulating genomes (see abdelrahman et al., 2017). aquaculture can also greatly benefit from improved cytogenetic analysis. it is fundamental in mapping loci involved in disease resistance and to improve commercial stocks by selecting cloned lines of aquacultured species (see gui and zhu 2012). it is necessary for controlling sex and inter-specific hybridization (see colombo et al., 1998; bartley et al., 2001; shpak et al., 2017). despite their ecological and economic importance, little is known about the karyology of brachyuran crabs. cytogenetics studies of brachyurans are relatively few, probably because they are technically more difficult than in other decapods as well as in mollusks and fishes (e.g. sola et al., 1981; galetti et al., 2000; coluccia et al., 2004; thiriot-quievreux 2002, 2003; scalici et al., 2010; salvadori et al., 2012, 2014; torrecilla et al., 2017; guo et al., 2018). brachyurans have a high number of chromosomes that are usually very small (e.g. niiyama 1959; lécher et al., 1995; lee et al., 2004; tan et al., 2004). in addition, despite cell culture might provide better and more abundant materials for karyological analyses, the few attempts to establish cell cultures in this taxon have not meet with great success and, thus, chromosome preparations are usually obtained directly from living tissues (e.g. in toullec 1999; sashikumar et al., 2008; zeng et al., 2010; hong et al., 2013). for these reasons, the preparation of good quality karyotyping and chromosome banding in brachyura has been never obtained, the only works being restricted to descriptions of chromosome numbers (see lécher 1995). moreover, most of karyological studies on brachyurans are decades old (niiyama 1942, 1959, 1966; mittal and dhall 1971; vishnoi 1972; trentini et al., 1989, 1992; lécher 1995 and references therein), while recent works are scarce and mostly related to species of economic importance (lee et al., 2004; zhu et al., 2005; swagatika and kumar 2014; cui et al., 2015). these recent papers reported that the mitten crabs eriocheir japonica (de haan, 1835) and e. sinensis (h. milne-edwards, 1853) have a diploid chromosome number of 2n = 146 (lee et al., 2004; cui et al., 2015), and the karyotype of portunus pelagicus includes 51 pairs of chromosomes (jazayeri et al., 2010), whereas the congeneric p. trituberculatus has 53 pairs (zhu et al., 2005). recently, swagatika and kumar (2014) recorded that the mud crab scylla serrata (forsskål, 1775) and the blue crab p. pelagicus have 2n = 106 and 2n = 98 chromosomes, respectively. the present study aims to contribute a step forward in the crab cytogenetic methods by comparing different variables necessary to obtain chromosome preparations from live tissues. we selected six crab species, from four different brachyuran families, commonly found in the mangrove forest of the south china sea, for which we systematically tested different technical variations in order to obtain metaphase chromosomes. the key elements for obtaining a high number of mitotic cells were scrutinized. materials and methods study species about 5 adult males and 5 females, including 2 ovigerous, from six species of hong kong semi-terrestrial and mangrove crabs were collected at low tides, in october 2017. in particular, we collected parasesarma bidens (de haan, 1835) and metopograpsus frontalis (miers, 1880) at tung chung mangroves (lantau island); chiromantes haematocheir (de haan, 1833) and gelasimus borealis (crane, 1975) at uk tau (new territories), and austruca lactea (de haan, 1835), g. borealis and metaplax tredecim tweedie, 1950 at starfish bay (new territories). these species, belonging to four different brachyuran families, are common inhabitants of lowland forests, mangrove forests and adjacent mudflats. they are all active during low tide, despite occupying different supratidal and intertidal habitats. all are also sold in pet trade for aquariophily (e.g. mong kok market, hong kong). taxonomical and ecological information concerning the studied species are summarized in table 1. 41cytogenetic of brachyura (decapoda) within few hours from collection, crabs were transported to laboratories of the school of biological sciences, the university of hong kong, divided according to their species and accommodated in terraria containing mangrove mud and sea water. in case of herbivore species (i.e. p. bidens and c. haematocheir) fresh kandelia obovatae (sheue, liu and yong, 2003) leaves (i.e. the dominant tree in their original habitats) were provided as food items. each terrarium was also provided with stones and pieces of mangrove wood and bark as hiding places. animals were kept at room temperature (around 22° c) and at natural light conditions. dna barcoding identification of species was made based on morphological traits and verified by dna barcoding analysis performed on an individual per species. dnas were extracted from muscle tissue, removed from one pereiopod, using the puregene kit (gentra system), then resuspended in distilled water and stored at -20°c. a fragment of the cytochrome oxidase subunit i (coxi), corresponding to the barcoding region and consisting of 656 base pairs (bp), was amplified using polymerase chain reaction (pcr) with the following primers: col6b 5’-acaaatcataaagatatygg-3’ (schubart and huber 2006) and hco2198 5’-taaacttcagggtgaccaaaaaatca-3’ (folmer et al., 1994). the amplifications were performed in a perkin elmer 9600 thermal cycler with the following pcr conditions: 40 cycles of denaturation for 45 s at 94°c, annealing for 1 min at 48°c, extension for 1 min at 72°c, preceded by an initial denaturation for 10 min at 94°c followed by a final extension for 10 min at 72°c. subsequently, pcr products were visualized on an agarose gel, purified by precipitation with sure clean (bioline) and then resuspended in water. the sequence reactions were performed with the big dye terminator mix (big dye terminator1v 1.3 cycle sequencing kit; applied biosystems) followed by electrophoresis in an abi prism automated sequencer (abi prism™ 310 genetic analyzer; applied biosystems). the sequences were corrected manually with the program chromas v. 1.55 (technelysium pty ltd, queensville, australia). we then used the software blast (available on the website of the national center for biotechnology information ncbi, https://blast.ncbi.nlm.nih.gov/blast.cgi) to compare our sequences to sequence databases and calculate the statistical significance of matches. we also compared the obtained sequences to our own reference sequences. cytogenetic analysis the general workflow used to obtain chromosome preparation is as follows: metaphase blocking, tissue preparation, hypotonization and fixation, slides preparation and staining. for all these steps several tests were performed. a schematic representation of the experimental plan is provided in figure 1. tissue preparation adult tissues: after injection or incubation with the metaphase blocking agent (colchicine or nocodazole, see below), animals were anesthetized for 10 min at -20°c, and then sacrificed. gonads, gills and hepatopancreas were dissected and placed in a small petri dish with 1-2 ml of hypotonic solution. tissues were either kept intact, or mashed by rubbing against a stainlesssteel grid with curved forceps, and then transferred to a 15 ml tube containing the pre-warmed (28°c, i.e. the average environmental temperature during this season) hypotonic solution. hemolymph samples were also collected by extraction of 0.5-1.0 ml of liquid with a 6 mm insulin syringes in proximity of the coxa of the fourth pair of legs, and directly placed in pre-warmed hypotonic solution. table 1. biological and ecological information on the six mangrove crab species. species family habitat max cw (in mm) aquariophily gelasimus borealis ocypodidae mud flat, sublittoral fringe 28.1 yes austruca lactea ocypodidae sand flat, eulittoral 16.4 yes metopograpsus frontalis grapsidae mud flat, sublittoral fringe 26.0 no metaplax tredecim varunidae mud flat, sublittoral fringe 19.0 no chiromantes haematocheir sesarmidae lowland forests, supralittoral 38.0 yes parasesarma bidens sesarmidae mangrove forests, eulittoral 28.5 yes data shown are: family; mangrove habitat occupied by adult populations (personal data); max cw, maximum male carapace weight (aiyun and siliang 1991); presence in the pet trade for aquariophily. 42 alessio iannucci et al. fertilized eggs: clusters of fertilized eggs were removed from ovigerous females by cutting the proximal part of pleopods. for g. borealis, eggs at two different embryonic stages were used, stage i and v (simoni et al., 2011). for all other species, we utilized embryos at stage v of development. eggs were incubated in metaphase blocking agent (see below), hypotonized, and then either fixed directly on slides or minced. mincing of eggs was performed with needles in a small petri dish in 1-2 ml of fixative. metaphase blocking two different metaphase blocking agents were tested to arrest the mitotic spindle and visualize chromosomes: nocodazole (15 mg/ml in dimethyl sulfoxide, dmso) and colchicine (powder). metaphase blocking agents were diluted/dissolved in sterile sea water to obtain different final concentrations and applied to tissues and eggs via injection and incubation respectively. injection: animals were injected in proximity of the coxa of the fourth pair of legs. both agents were tested for the amount of 0.2 and 2 µg/g crab weight. animals were kept in terraria at room temperature for 8, 16 or 24 hours and then dissected as described above. incubation: eggs were transferred to 15 ml tubes and incubated in 10 ml of 0.0005, 0.005 or 0.05% of colchicine or nocodazole solution for 2, 4, 8, 16 or 24 hours at 28°c, and then transferred to pre-warmed hypotonic solutions. to guarantee eggs an appropriate level of oxygenation, tubes were kept without lids and gently stirred during incubation process. hypotonization tissues (intact or mashed) and eggs were incubated in hypotonic solution to achieve a good cell swelling and metaphase spreading. to facilitate the access of the hypotonic solution to the embryos, part of the eggs was punctured before incubation. two different solutions were tested, 0.1% sodium citrate and 0.075 m potassium chloride, with incubation times of 15, 30 or 45 min. after centrifuge at 150 rcf for 10 min, hypotonic solution was removed from mashed tissues, while it was removed from intact tissues and eggs by gently pipetting out the liquid. fixation after hypotonic removal, tissues and eggs were fixed by applying 3–5 ml of cold, freshly prepared fixative (3 parts methanol or absolute methanol: 1 part glacial acetic acid). eggs were then minced as described above and transferred to a 15 ml tube. tubes containing cell suspensions from eggs and mashed tissues were centrifuged at 200 rcf for 10 min. the supernatant was removed, and fresh fixative was added. this step was repeated three times. intact tissues were left in 15 ml tubes containing fixative solution for 20 min, then fixative was changed, and tissues stored at -20°c, until slides preparation. part of hypotonized eggs were also transferred directly to a clean slide and macerated using a thin needle. after the maceration, we applied a fixation solution of 3:3:4 ethanol:acetic acid:distilled water three times, followed by a fixation solution of 1:1 acetic acid:ethanol and finally a few drops of glacial acetic acid. between each application of the fixation solutions, the excess was removed with the aid of a filter paper. the slides were left to dry at room temperature. slides preparation intact tissues were macerated with the help of two needles directly on slides. few drops of glacial acetic figure 1. schematic representation of the experimental plan. crabs representations modified from www.fiddlercrab.info. 43cytogenetic of brachyura (decapoda) acid were used to help maceration. slides were then air dried. cell suspensions obtained from eggs and mashed tissues were gently shacked and left decanted to separate cells from egg chorion and largest pieces of tissues. the upper part of decanted preparation was transferred to a 2 ml tube and centrifuged at 1,200 rcf for 10 min. fixative was removed and resuspended cell pellet was used to drop slides. few drops of acetic acid were added to part of the cell suspensions to improve chromosome spreading. for cell suspensions, three different protocols for slides preparation were tested: air dried: cell suspension was dropped with a siliconized pasteur pipette from a height of about 10 cm onto a pre-cleaned microscope slide and dried at room temperature before staining. hot dried: the above procedure was applied, but slide was pre-heated and dried at 50°c. humid dried: the cell suspension was dropped from a height of about 10 cm onto a pre-cleaned microscope slide chilled to -20°c. after a short drying period at room temperature in which the fixative was partially evaporated, the slides were held two to three times briefly into water steam. the slides were then dried on a metal block which was half submerged in a 75°c water bath. after drying, the slides were stained with giemsa 10% solution for 20 min or 4’,6-diamidino-2-phenylindole (dapi) and mounted. image capture and chromosomes counting metaphases were observed under optical, fluorescence and confocal microscopes. leitz dialux 20 optical microscope was mounted with moticam pro 205b. zeiss axio imager.d2 f luorescence microscope was mounted with zeiss axiocam 503 mono. zeiss lsm 710 nlo confocal microscope was mounted with airyscan module for super resolution. images were edited with adobe® photoshop® cs5 extended (adobe systems inc., san jose, california, usa). the mode of diploid chromosome numbers was calculated, using excel, after counting 42, 18, 13, 21, 19 metaphases of g. borealis, a. lactea, m. frontalis, m. tredecim and c. haematocheir respectively. results barcoding analysis the pcr successfully amplified the mtdna coxi gene in the six species, resulting in sequences about 600 bp long, excluding the primer. all the sequences have an a-t rich nucleotide composition as expected for the mitochondrial dna of arthropods (simon et al., 1994). the dna barcoding confirmed the morphological identification of the six species. the sequences have been deposited in genbank ((access numbers: mt265074-79). tissue preparation injection of the metaphase blocking agents did not cause any visible damage to the animals, and individuals of all species survived the treatments. no metaphases were observed in cytogenetic preparations obtained from mashed and intact tissues, regardless of the concentrations and exposure times to metaphase blocking agents. slides obtained from intact tissues presented well preserved nuclei at different cell cycle stages. preparations obtained from cell suspensions of mashed tissue showed well separated cells, indicating that manipulation and maceration procedures were correctly performed. metaphases were observed in the cell suspensions obtained from eggs of all the six species (fig. 2). the highest number of metaphases was observed in preparations obtained from embryonic stage i eggs (fig. 2a). both nocodazole and colchicine were effective in metaphase arrest of embryonic cells. optimal results were obtained with colchicine and nocodazole at 0.05% and 0.0005%, respectively. no visible differences were observed between colchicine and nocodazole treatments (i.e. chromosome condensation or metaphase spreading). preparations obtained with higher concentrations of nocodazole did not show any metaphases. on the other hand, few metaphases were also detected in preparations obtained with lower concentrations of colchicine. same hypotonization treatments gave different level of spreading in the six species, as described below. no relevant differences were registered between the two hypotonic solutions, and the three incubation times. however, results obtained with 0.075 m potassium chloride solution, with an incubation time of 30 min produced better spreading metaphases in m. tredecim as described below (figs. 2j, k, l). no differences were registered between punctured and unpunctured eggs. an increase of incubation time with hypotonic solution up to 3 hours did not affect or improve metaphase quality. chromosome preparation obtained from mashed eggs suspensions showed well separated cells and very few residues of chorion, indicating that the manipulation and fixation procedures were adequate, and the decantation step was useful. no metaphases were observed in preparations obtained from eggs directly fixated on slides. moreover, cells were sparse and clustered, preventing an accurate observation of the preparation. 44 alessio iannucci et al. figure 2. metaphase spreads, obtained from eggs incubated with 0.05% colchicine and hypotonized with 0.075 m kcl of gelasimus borealis (a, b, c), austruca lactea (d, e, f), metopograpsus frontalis (g, h, i), metaplax tredecim (j, k, l), chiromantes haematocheir (m, n, o), parasesarma bidens (p, q, r). slides were either stained with giemsa and observed under an optical microscope (a, d, g, j, m, p) or stained with dapi and observed under a fluorescent (b, e, h, k, n, q) and confocal microscope (c, f, i, l, o, r). photos by stefano cannicci. 45cytogenetic of brachyura (decapoda) slides dropping methods were all successful, and no differences were registered among the three methods. the addition of a few drops of acetic acid to cell suspensions visibly increased the spreading of chromosomes. metaphases were better detected when dapi staining was applied as, contrastingly to giemsa, it did not stain organic and inorganic residual material, making it easier to detect metaphases. confocal microscopy gave better resolution of chromosome morphology and heterochromatic regions were visible. chromosome number and morphology we obtained metaphase chromosomes for all the studied species but p. bidens, for which only broken metaphases were observed with all treatments (figs. 2p-r). we analyzed an average of 50 metaphases for each species. in g. borealis the number of chromosomes per cell ranged between 30 and 60 with a mode at 48. chromosomes morphology was hardly distinguishable due to their small size. metaphase spreading was limited (figs. 2a-c), however a high number of metaphases per slides was observed when preparations were made with eggs at embryonic stage i (fig. 2a). chromosome morphology and metaphase spreads in a. lactea were very similar to preparations obtained from the eggs of the allied species g. borealis (figs. 2d-f). in this ocypodid the number of chromosomes per cell ranged between 56 and 74 with a mode at 60. we obtained a very low number of metaphases for m. frontalis, and the hypotonic treatment was less successful in this species than in the other ones (figs. 2g-i). the number of chromosomes per cell ranged between 38 and 62 with a mode at 55. the hypotonization treatment gave the best results for the varunid m. tredecim, despite its chromosomes appeared very small (fig. 2j-l). the number of chromosomes per cell ranged between 78 and 108 with a mode at 80. for t he two sesarmid species concerned, we obtained reliable results for c. haematocheir only. chromosomes of c. haematocheir were larger than those of the other species, and their morphology could be better observed (figs. 2m-o). confocal images showed that most chromosomes were biarmed, and dapi staining revealed at-rich pericentromeric regions (fig. 2n, o). the number of chromosomes per cell ranged between 58 and 74 with a mode at 66. discussion this study, reporting the results of several cytogenetic technical trials on six semi-terrestrial and mangrove crab species selected as representatives of the infraorder brachyura, provides insights on the technical aspects necessary to obtain chromosome preparations in brachyuran crabs. the best results were obtained with pre-hatching embryos incubated with colchicine or nocodazole at 0.05% and 0.0005%, respectively, hypotonized in 0.075 m kcl solution for 30 min and fixed in freshly prepared fixative. the best method to analyze the chromosomes resulted to be the use of confocal microscope after dapi staining. embryos and post-hatched larvae of decapods contain rapidly growing tissues with a high mitotic activity (anger 2001). thus, these life stages represent an optimal material to obtain metaphases for karyological studies. optimal results for chromosome preparations using fertilized eggs have already been obtained by campos-ramos (1997) for another suborder of the decapoda, the dendrobranchiata. in agreement with the present study, this author tested colchicine concentrations from 0.006% to 0.1% in penaeus vannamei (boone, 1931) and p. californiensis (holmes, 1900) eggs and obtained optimal results using 0.05% colchicine, with no differences in chromosomes condensation at variable colchicine concentrations. recently martin et al., (2016) also obtained optimal chromosome preparations from procambarus virginalis (lyko, 2017) (decapoda: pleocyemata: astacidea) eggs using 0.05% colchicine. larvae at early zoeal stages were used by cui et al. (2015) who obtained good chromosome preparations for the chinese mitten crab eriocheir sinensis. in the present study, clear differences were obtained using embryos of g. borealis at different stages, with early stage embryos presenting the highest number of metaphases with respect to the late stage ones. this suggests that the highly dividing tissues of embryos at initial stage are even more suitable for chromosome preparation, despite the abundance of yolk, which reduces the cleanliness of the preparations. absence of metaphases in preparations obtained with higher concentrations of nocodazole is likely due to the toxicity of dmso present in nocodazole solution, which caused an arrest of cell cycle in embryos (moralli et al., 2011). the hypotonic treatment was the most critical phase as several metaphases did not spread sufficiently and overlapping of the chromosomes made it difficult to make reliable chromosome counts. the best results were obtained with 0.075 m potassium chloride as hypotonic 46 alessio iannucci et al. solution, which is the most commonly used in decapods (e.g. in salvadori et al., 2012; cui et al., 2015; martin et al., 2016; torrecilla et al., 2017). however, it was also evident that the same hypotonization treatments yielded different level of chromosome spreading in each of the six analyzed species. such differences may be due to differences in the characteristics of the chorion, or in the osmotic concentration and physiological characteristics of the embryos of different species. our target species, in fact, occupy different habitats in hong kong, from the lowland forests, in the case of c. hematocheir, to the true mangrove forests, such as p. bidens and m. frontalis, to the lower intertidal sand and mud flats, such as a. lactea, g. borealis and m. tredecim. indeed, the permeability and osmotic characteristics of their chorions, as well as the osmotic and physiological traits of their embryos are adapted to different conditions in terms of salinity, temperature, submersion and water availability. it is known that eggs of semi-terrestrial and intertidal families are permeable to air-borne gasses and can intake oxygen from air, while the embryos of marine species can only rely on water (cannicci et al., 2011; simoni et al., 2011). moreover, the osmolarity of tissues of brachyuran crabs is strictly related to their microhabitat, since they are osmoconformers (charmantier 1998). it is plausible that the differences we obtained for the six species using the same treatments may be related to differences in permeability to solutes of their chorion and in osmoregulation mechanisms of their embryos, which may have influenced the response of cells to hypotonization. the best spreading results were obtained for m. tredecim which is the only species colonizing the lower intertidal belt. we failed to obtain metaphases from adult tissues. in decapods, there are a few cytogenetic studies using gills and hepatopancreas as tissue of choice for chromosome preparation (e.g. indy et al., 2010; salvadori et al., 2012, 2014; gonzález-tizón et al., 2013). a few other studies concluded that testes were a suitable tissue for chromosome preparations (e.g. lee et al., 2004; tan et al., 2004; awodiran et al., 2016; milnarec et al., 2016). however, the inactivity of testes in species with seasonality of reproductive activity (commonly described in male crustaceans, especially in representatives from colder regions: adiyodi 1988) may lead to a scarcity of dividing cells in this organ, and thus to the lack of metaphases. this could be the case in our samples, whose sperm ducts appeared reduced in size as expected during the “resting” reproductive phase. this result is plausible since our sampling was performed at the very end of the reproductive period for hong kong crab species, when only very few females were still ovigerous. we recorded a high chromosome number for our species, as known for other brachyuran crabs and decapoda in general (niiyama 1959; lécher et al., 1995; lee et al., 2004; cui et al., 2015). the highest chromosome numbers were recorded for the varunid m. tredecim, with a mode value of 80. this is indeed lower than what reported for other two varunid species, the mitten crabs eriocheir japonica and e. sinensis whose diploid chromosome number is 2n = 146. while we recorded different chromosome numbers for g. borealis and a. lactea (numbers ranging between 30-60 and 56-74, respectively), their chromosomes are more similar to each other than to the rest of the study species. this similarity is likely due to their close phylogenetic relationship: the two species being part of the same ocypodid subfamily gelasiminae (shih et al., 2016). the wide range of chromosome number registered in our study species can be attributed to a poor metaphase spreading. this was mainly due to an ineffective hypotonic treatment, a step that proved to be one of the most crucial ones, as previously stated. a further evidence of this comes from the fact that the numerical counts of chromosomes registered for metaphases analyzed under the confocal microscope are greater than the mode (all these values fall into the right tail of the frequency distributions). indeed, the higher resolution of confocal microscope allowed a better visualization of the smallest chromosomes when the metaphase was poorly spread, resulting in a higher chromosome number (e.g. fig 2e, f). the issue of poor spreading of metaphases is known to affect the counts of chromosomes in crustaceans, and some authors suggested to use the scanning electron microscope (sem) to gain more resolution for a better analysis of chromosomes (lee et al., 2004, 2008). however, this technology is very laborious and thus not very effective. the wide variability in distribution of chromosome numbers as well as the small size of chromosomes prevented us from proposing a reference karyotype for the species. such problems were also registered for the other species described so far, for which authors did not provide a karyotype. to our knowledge, the only karyotypes available for brachyura are those of scylla serrata and portunus pelagicus (swagatika et al., 2014). analyses under a confocal microscope gave the best resolution, allowing discernment of chromosome morphology and revealing the presence of at-rich pericentromeric regions in c. haematocheir. this is the first observation of this kind for crab chromosomes, albeit being a common feature of eutherian species (sumner 2008). nonetheless the lack of a reference karyotype for any of the study species, our results on species’ chro47cytogenetic of brachyura (decapoda) mosome numbers will be extremely valuable in future genomic studies, i.e. for assessing genome assembly quality. it is known, in fact, that the comparison between the number of final scaffolds in assemblies and the chromosome number range of a given species may provide a clear indication of the level of fragmentation of the assembly. in particular, when the number of final scaffolds is much higher than the chromosome number mode, the assembly needs more refinement; while if the number of final scaffolds is much lower than the mode, the assembly presumably includes chimeric scaffolds and thus needs to be revised (ma et al., 2012; burton et al., 2013; sharakhov et al., 2014). conclusions brachyura are undoubtedly an ecologically and economically important taxon, however, so far, very few studies have targeted their karyology, with information generally limited to the description of chromosomes numbers. many authors report difficulties in obtaining cytogenetic information in this taxon due to high number and small size of chromosomes (lécher et al., 1995; lee et al., 2004; tan et al., 2004). our results corroborate the presence of such methodological issues and stress the fact that several improvements are still needed to reach the quality standard needed for molecular cytogenetic researches. this study also underlines that ecological and physiological adaptations of a species can affect its responses to the sequential steps of karyotyping analysis. this outcome makes the design of a standard protocol for cytogenetic analyses in brachyurans even more difficult. however, our comparative approach highlighted the critical steps that must be improved to obtain high quality material in true crabs. we believe therefore that this study provides a step forward in the cytogenetic of brachyurans and represents an important basis for further cytogenetic methods in this taxon. acknowledgements we thank lok yi cheng, pedro juliao-jimenez, martina scigliano and sandra colombo for helping us to collect animals. we also thank aline ferreira de quadros and elisabetta rovida for the useful suggestions in the preparation of chromosomes and image acquisition. marta svartman provided precious comments to the last version of the manuscript: we are greatly thankful to her support. the 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portunus trituberculatus. j fish china. 25:649-653. caryologia international journal of cytology, cytosystematics and cytogenetics volume 73, issue 2 2020 firenze university press the first molecular identification of egyptian miocene petrified dicot woods (egyptians’ dream becomes a reality) shaimaa s. sobieh*, mona h. darwish gene flow patterns reinforce the ecological plasticity of tropidurus hispidus (squamata: tropiduridae) fernanda ito, danielle j. gama-maia, diego m. a. brito, rodrigo a. torres* the technique of plant dna barcoding: potential application in floriculture antonio giovino1,*, federico martinelli2,*, anna perrone3 cytogenetic of brachyura (decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species alessio iannucci1, stefano cannicci1,2,*, zhongyang lin3, karen wy yuen3, claudio ciofi1, roscoe stanyon1, sara fratini1 comparison of the evolution of orchids with that of bats antonio lima-de-faria identification of the 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(plantaginaceae) saeed mohsenzadeh*, masoud sheidai, fahimeh koohdar a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding timir baran jha1,*, partha sarathi saha2, sumita jha2 chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india harminder singh2, jaswant singh1,*, puneet kumar2, vijay kumar singhal1, bhupendra singh kholia2, lalit mohan tewari3 population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies raheleh tabaripour1,*, masoud sheidai1, seyed mehdi talebi2, zahra noormohammadi3 induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions girjesh kumar, shefali singh* karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(2): 127-143, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-579 citation: e. martin, t. dirmenci, t. arabaci, t. yazici, t. özcan (2020) karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy. caryologia 73(2): 127-143. doi: 10.13128/caryologia-579 received: july 31, 2019 accepted: april 27, 2020 published: july 31, 2020 copyright: © 2020 e. martin, t. dirmenci, t. arabaci, t. yazici, t. özcan. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 1 department of biotechnology, faculty of science, necmettin erbakan university, konya, turkey 2 department of biology education, necatibey education faculty, balıkesir university, balıkesir, turkey 3 department of pharmaceutical botany, faculty of pharmacy, i̇nönü university, malatya, turkey *corresponding author. e-mail: dirmenci@balikesir.edu.tr abstract. in this study, chromosome numbers and structures of some origanum l. taxa growing in turkey were identified. using the image analysis system, the complements of plant accessions belonging to eight sections, namely amaracus (gleditsch) vogel, anatolicon benth., brevifilamentum ietsw., longitubus ietsw., chilocalyx (briq.) ietsw., majorana (miller) ietsw., origanum, and prolaticorolla ietsw. were determined, by classification with the cytogenetic method. the chromosome number of all taxa except o. sipyleum l. (2n = 28) and o. rotundifolium boiss. (2n = 28) is 2n = 30. in addition, the hybrids and their parental species have 2n = 30 chromosome numbers. also, the smallest chromosome length is 0.32 μm in o. sipyleum. the largest chromosome length is 2.02 μm in o. minutiflorum o.schwarz & p.h.davis. the smallest total haploid length is 10.08 μm in o. vulgare subsp. hirtum (link) a.terracc. and the largest value is 22.00 μm in o. haussknechtii boiss. the smallest mean length is 0.33 μm in o. vulgare l. subsp. hirtum and o. saccatum p.h.davis. the largest mean length is 0.74 μm in o. sipyleum l. the chromosome numbers obtained in this study support the speciation of origanum members via homoploid hybridization. finally, the somatic chromosome numbers of 10 taxa (including 2 hybrids), chromosome measurements of 22 taxa (including 2 hybrids), and ideograms of 19 taxa (including 2 hybrids) were for the first time performed in this study. keywords: chromosome, hybrid, karyotype, lamiaceae, origanum. introduction the genus origanum l. is placed in the family lamiaceae, subfamily nepetoideae, tribe mentheae, subtribe menthineae, contains 43 species and 20 hybrids (ietswaart 1980, 1982; govaerts et al. 2013; dirmenci et al. 2018a, 2018b, 2019). approximately, 21 species (24 taxa, including 13 endemic) and 128 esra martin et al. 13 hybrids are reported from the turkey (ietswaart 1982; davis et al. 1988; duman 2000; dirmenci et al. 2018a, 2018b, 2019). majority of the species are found in mediterranean basin and 75% of them are only found in eastern mediterranean region. some species are distributed in the middle east (syria and lebanon), north africa (algeria, libya and morocco) and the canary islands (ietswaart 1980, 1982). the genus origanum contains ten sections, eight of them are distributed in turkey (ietswaart 1980). the species are mostly distributed along the taurus mountains in turkey. recently, the use of the image analysis system in karyotyping of plant taxa having small and indistinguishable somatic chromosomes (fukui 1986, 1998; fukui and iijima 1991; iijima et al. 1991) has drawn the attentation for study chromosome morphology in origanum. literature studies belonging to the genus origanum have revealed that there are too few karyotype analyses of this genus. the lack of sufficient data on the karyomorphology of the genus is probably due to the small size of its chromosomes. the chromosome numbers are 2n = 28, 30 and 32 (ietswaart 1980; gill 1970, 1981, 1981a 1984; saggoo 1983; magulaev 1984; ayyangar and vembu 1985; krasnikov and schaulo 1990; wentworth et al. 1991; khatoon and ali 1993; stepanov and muratova, 1995; dobea et al. 1997; kıtıkı 1997; balım and kesercioğlu 1998; albers and pröbsting 1998; lövkvist and hultgård 1999; yıldız and gücel 2006; dirmenci et al. 2018a, 2018b, 2019) in the genus origanum. the hybridization is a widespread phenomenon among origanum species. it usually occurs in the regions where the distribution of the species overlaps. the overlappings can occur in natural habitat or botanical gardens. because of interspecific hybridization, so far 19 hybrids have been identified in the genus. it was reported that the chromosome number of some hybrids was 2n = 30 (bakha et al. 2017; dirmenci et al. 2018a, 2018b, 2019). these results support the idea that the origin of the hybridization in origanum is probably homoploidy. the main aims of this study are to contribute to the cytotaxonomy of origanum with the following marks: 1to define the karyotypes of some origanum specimens for the first time, 2to verify the homoploidy in the origanum genus, 3to present the chromosomal differences of the origanum species, 4to observe the chromosome structure of the hybrids and their parents in origanum. materials and methods pla nt materia ls used in t his study were collected between 2013 and 2017 and collected specimens were deposited in balıkesir university. voucher numbers and collection information of the examined specimens were given in the appendix section. the seeds were germinated at room temperature. all kar yological obser vations were carried out on root tips. firstly, the root tips were pretreated for 16 h in α-monobromonaphthalene at 4°c, fixed in 3:1 absolute alcohol/glacial acetic acid, then hydrolyzed with 1 n hcl for 12 min at room temperature and stained with 2% aceto-orcein for 3 h at room temperature. stained root tips were squashed in a drop of 45% acetic acid, and permanent slides were made by mounting in depex (martin et al. 2016). the five metaphase plates from each species were used to obtain chromosomal data using an olympus microscope and the chromosomal data were measured with software image analyses (bs200prop). chromosome lengths were made by nomenclature following levan et al. (1964). results sect. amaracus (gleditsch) benth. the chromosome numbers of origanum ayliniae dirmenci & yazıcı, o. boissieri ietsw., o. saccatum p.h.davis, and o. solymicum p.h.davis are 2n = 30 (figure 1a-d, table 1). the smallest chromosome length is 0.39 μm in o. saccatum (no. td4522). the largest chromosome length is 1.94 μm in o. boissieri (no. td4319). the smallest total haploid length is 10.13 μm in o. saccatum (no. td4522), and the largest value is 19.74 μm in o. boissieri (no. td4319). the chromosome morphologies of origanum ayliniae, o. boissieri, o. saccatum, and o. solymicum are described for the first time in this study. sect. anatolicon benth. the chromosome numbers of origanum hypericifolium p.h.davis is 2n = 30, and o. sipyleum l. has two different counts as 2n = 28 and 2n = 30 (figure 1e-f, table 1). the smallest chromosome length is 0.32 μm in o. sipyleum (no. td4308). the largest chromosome length is 2.00 μm in o. sipyleum (no. td4517). the smallest total haploid length is 10.46 μm in o. sipyleum (no. td4522) and the largest value is 20.98 μm in o. sipyleum (no. td4308). the chromosomes morphologies of origanum hypericifolium are described for the first time. the chromosome number of origanum sipyleum is mostly 2n = 30 in a biosystematic study performed by 129karyotype studies on the genus origanum (lamiaceae) species and some hybrids defining homoploidy kıtıkı 1997 (table 2). the diploid number of no. 4308 is 2n = 28 as different from the literature (table 2). while the number of chromosomes of o. sipyleum is included in the literature, the chromosome morphologies of the species have been demonstrated for the first time in this study. sect. brevifilamentum ietsw. the chromosome numbers of origanum acutidens (hand.-mazz.) ietsw. (no. td4956a), o. brevidens bornm. dinsm. (no. td4331), o. haussknechtii boiss. (no. td2824), o. husnucan-baseri h.duman, aytaç & a.duran, (no. td4528), and o. leptocladum boiss. (no. td4345), 2n = 30 (figure 1g-k, table 1). the chromofigure 1a-i. somatic metaphase chromosomes of origanum taxa: a) o. ayliniae (td4516) b) o. boissieri (td4319); c) o. saccatum (td4732); d) o. solymicum (td4347); e) o. hypericifolium (td4357); f) o. sipyleum (td4727); g) o. acutidens (td4956a); h) o. brevidens (td4331); i) o. haussknechtii (ta2824). scale bar: 10 µm. 130 esra martin et al. some number of o. rotundifolium boiss. (no. td3943), 2n = 28 (figure 1l, table 1). the smallest chromosome length is 0.49 μm in o. leptocladum. the largest chromosome length is 2.00 μm in o. haussknechtii. the smallest total haploid length is 14.67 μm in o. brevidens and the largest value is 22.00 μm in o. haussknechtii. the chromosomes morphologies of origanum acutidens, o. brevidens, o. haussknechtii, o. husnucan-baseri, o. leptocladum, and o. rotundifolium are described for the first time. sect. chilocalyx (briq.) ietsw. the chromosome numbers of origanum bilgeri p.h.davis (no. td4343), o. minutiflorum o.schwarz & p.h.davis (no. td4348) and o. vogelii greuter & burdet (no. td4509) are 2n = 30 (figure 1m-o, table 1). the smallest chromosome length is 0.38 μm in o. bilgeri. the largest chromosome length is 2.02 μm in o. minutiflorum. the smallest total haploid length is 14.25 μm in o. bilgeri and the largest value is 19.01 μm in o. minutiflorum. table 1. chromosome counts and size of species and hybrids of origanum determined in the study. section taxa chromosome number (2n) chromosome length (µm) min-max total haploid length (µm) mean chromosome length (µm) amaracus o. ayliniae td 4516 30 0.83-1.72 18.35 0.61 o. boissieri td 4319 o. boissieri td 4501 30 30 0.78-1.94 0.47-1.61 19.74 14.48 0.65 0.48 o. solymicum td 4347 o. solymicum td 4520 30 30 0.45-1.59 0.40-1.26 16.22 12.16 0.54 0.40 o. saccatum td 4342 o. saccatum td 4522 o. saccatum td 4732 30 30 30 0.53-1.75 0.39-1.12 0.60-1.46 17.17 10.13 14.77 0.55 0.33 0.49 anatolicon o. hypericifolium td 4357 30 0.34-1.36 13.80 0.40 o. sipyleum td 4308 o. sipyleum td 4352 o. sipyleum td 4534 o. sipyleum td 4517 o. sipyleum td 4727 28 30 30 30 30 0.79-2.00 0.53-1.59 0.39-1.52 0.32-1.11 0.49-1.08 20.98 14.19 13.32 10.46 11.26 0.74 0.47 0.44 0.34 0.37 brevifilamentum o. brevidens td 4331 o. haussknechtii td 2824 o. husnucan-baseri td 4528 o. leptocladum td 4345 o. rotundifolium td 3943 30 30 30 30 28 0.50-1.63 0.99-2.00 0.56-1.79 0.49-1.63 0.57-1.82 14.67 22.00 14.88 15.27 16.497 0.49 0.73 0.49 0.50 0.58 chilocalyx o. bilgeri td 4343 o. minutiflorum td 4348 30 30 0.38-1.58 0.69-2.02 14.25 19.01 0.47 0.63 longitubus o. amanum td 4514-a 30 0.73-1.91 16.92 0.56 majorana o. majorana td 3984 o. majorana td 4356 o. majorana td 4346 30 30 30 0.50-1.58 0.431.84 0.43-1.54 17.25 17.35 14.44 0.57 0.57 0.48 o. onites td 4355 o. onites td 4725 o. onites td 4532 30 30 30 0.50-1.77 0.63-1.27 0.43-1.26 15.93 15.14 13.45 0.53 0.50 0.44 o. syriacum subsp. bevanii td 4336 o. syriacum subsp. bevanii td 4330 30 30 0.531.58 0.49-1.13 16.26 11.02 0.54 0.36 origanum o. vulgare subsp. hirtum td 4733 o. vulgare subsp. hirtum td 4722 o. vulgare subsp. hirtum td 4359 30 30 30 0.58-1.14 0.49-1.22 0.40-1.07 13.22 13.32 10.08 0.44 0.44 0.33 o. vulgare subsp. vulgare td 4688 30 0.56-1.18 12.73 0.42 prolaticorolla o. laevigatum td 4497 30 0.45-1.96 16.34 0.54 hybrids o. × intermedium td 4726 30 0.46-1.14 11.62 0.38 o. × adae td 4518 30 0.72-1.40 15.47 0.51 131karyotype studies on the genus origanum (lamiaceae) species and some hybrids defining homoploidy table 2. chromosome counts of species and hybrids of origanum according to references. section species chromosome numbers (2n) references amaracus o. ayliniae 30 (dirmenci et al. 2018a) o. boissieri 30 (dirmenci et al. 2018b, kıtıkı et al., 1997) o. calcaratum. 30 (von bothmer, 1970) o. dictamnus 30 (lepper, 1970) o. solymicum 30 (in this study; kıtıkı et al., 1997) o. saccatum 30 (in this study; kıtıkı et al., 1997) anatolicon o. hypericifolium 30 (in this study) o. sipyleum 30 28 (kıtıkı et al., 1997) (in this study) brevifilamentum o. acutidens 30 (dirmenci et al. 2019) o. brevidens 30 (in this study) o. haussknechtii 30 (in this study) o. husnucan-baseri 30 (in this study) o. leptocladum 30 (in this study) o. rotundifolium 28 (in this study) chilocalyx o. bilgeri 30 (in this study) o. minutiflorum 30 (in this study) o. vogelii 30 (in this study ) elongatispica o. elongatum 30 (bastida and talavera, 1994; bakha et al., 2017) o. grosii 30 (bakha et al., 2017) longitubus o. amanum 30 (in this study; lepper 1970) majorana o. majorana 30 (in this study; harriman, 1975; májovský, 1978; fernandes and leitão, 1984; balim and kesercioğlu, 1998) o. onites 30 (in this study; von bothmer, 1970; miege and greuter, 1973; ietswaart, 1980; montmollin, 1986; kıtıkı et al., 1997; bakha et al., 2017) o. syriacum subsp. bevanii 30 (in this study; balim and kesercioğlu, 1998; yildiz and gücel, 2006) origanum o. vulgare 28 (magulaev, 1984) o. vulgare 30 (skalinska et al., 1971; krasnikov and schaulo, 1990; stepanov and muratova, 1995; lövkvist and hultgård, 1999) o. vulgare 32 (ayyangar and vembu, 1985) o. vulgare subsp. hirtum 30 (strid and franzen, 1981; markova and goranova, 1995; dirmenci et al. 2018b) o. vulgare subsp. gracile 30 (astanova, 1981; dirmenci et al. 2019) o.vulgare subsp. virens 30 (fernandes and leitão, 1984; pastor et al. 1990; bakha et al., 2017) o. vulgare subsp. viride 30 (in this study; strid and franzen 1981) o. vulgare subsp. viridulum 30 (strid and franzen, 1981; markova and goranova, 1995) o. vulgare subsp. vulgare 28 (magulaev, 1984) o. vulgare subsp. vulgare 30 (in this study; gill, 1981, 1981a; saggoo, 1983; bir and saggoo, 1984; gill, 1984; krasnikov and schaulo, 1990; wentworth et al., 1991; khatoon and ali, 1993; stepanov and muratova, 1995; dobea et al., 1997; albers and pröbsting, 1998; lövkvist and hultgård, 1999) o. vulgare subsp. vulgare 32 (ayyangar and vembu, 1985) prolaticorolla o. compactum 30 (bakha et al., 2017) o. laevigatum 30 (balim and kesercioğlu, 1998) hybrids o. × adae 30 (dirmenci et al. 2018a) o. × font-queri 30 (bakha et al., 2017) o. × haradjanii 30 (in this study) o. × intermedium 30 (in this study) o. × munzurense 30 (dirmenci et al. 2019) o. × sevcaniae 30 (dirmenci et al. 2018b) 132 esra martin et al. the chromosome morphologies of o. bilgeri, o. minutiflorum, and o. vogelii are described for the first time. sect. longitubus ietsw. sect. longitubus contains only one species. the chromosome number of origanum amanum post (no. td4514a) is 2n = 30 (figure 1p, table 1). ietswaart (1980) reported that the chromosome number of o. amanum was 2n = 30 (table 2). the chromosome figure 1j-r. somatic metaphase chromosomes of origanum taxa: j) o. husnucan-baseri (td4528); k) o. leptocladum (ta4345); l) o. rotundifolium (td3943); m) o. bilgeri (td4343); n) o. minutiflorum (td4348); o) o. vogelii (td4509); p) o. amanum (td4514a); q) o. majorana (td3984); r) o. onites (td4725). scale bar: 10 µm. 133karyotype studies on the genus origanum (lamiaceae) species and some hybrids defining homoploidy lengths range from 0.73 to 1.91 µm. the chromosome morphologies of o. amanum are described for the first time. sect. majorana (mill.) benth. the chromosome numbers of origanum majorana l. (no. td3984), o. onites l. (no. td4725), and o. syriacum l. subsp. bevanii (holmes) greuter & burdet (no. td4336) are 2n = 30 (figure 1q-s, table 1). the smallest chromosome length is 0.43 μm in o. majorana and o. onites (no. 4356, 4346, and 4532). the largest chromosome length is 1.84 μm in o. majorana (no. 4356). the smallest total haploid length is 11.02 μm in o. syriacum subsp. bevanii (no. 4330), and the largest value is 17.35 μm in o. majorana (no. 4356). it was reported that the chromosome number of origanum onites and o. majorana was 2n = 30 (ietswaart 1980; kıtıkı 1997; balım and kesercioğlu 1998) (table 2). the number of chromosomes we obtained overlapped with the literature. in addition to the chromosome numbers of taxa, chromosome measurements are also introduced to the scientific world. sect. origanum the chromosome numbers of origanum vulgare l. subsp. gracile (k.koch) ietsw. (no. td4821), o. vulgare subsp. hirtum (link.) a.terracc. (no. td4507), o. vulgare subsp. viridulum (matrin-dones) nyman (no. td4662a), and o. vulgare subsp. vulgare (no. td4688) are 2n = 30 (figure 1t-w, table 1). the smallest chromosome length is 0.40 μm in o. vulgare subsp. hirtum (no. td4359). the largest chromosome length is 1.22 μm in o. vulgare subsp. hirtum (no. td4722). the smallest total haploid length is 10.08 μm in o. vulgare subsp. hirtum (no. td4359) and the largest value is 13.32 μm in o. vulgare subsp. hirtum (no. td4722). it was reported that the chromosome numbers of origanum vulgare, o. vulgare subsp. hirtum, o. vulgare figure 1s-x. somatic metaphase chromosomes of origanum taxa: s) o. syriacum subsp. bevanii (td4336); t) o. vulgare subsp. gracile (td4821); u) o. vulgare subsp. hirtum (td4507); v) o. vulgare subsp. viridulum (td4662a); w) o. vulgare subsp. vulgare (td4688); x) o. laevigatum (td4497). scale bar: 10 µm. 134 esra martin et al. subsp. viride, and o. vulgare subsp. viridulum are 2n = 28, 30 and 32 (ietswaart 1980; strid and franzen 1981; gill 1981a, 1984; saggoo 1983; bir and saggoo 1984; magulaev 1984; ayyangar and vembu 1985; krasnikov and schaulo 1990; wentworth et al. 1991; khatoon and ali 1993; stepanov and muratova 1995; markova and goranova 1995; dobea et al. 1997; kıtıkı 1997; albers and pröbsting 1998; lövkvist and hultgård 1999; dirmenci et al. 2018b) (table 2). the chromosome morphologies of origanum vulgare subsp. gracile are described for the first time. in addition, the detailed chromosome lengths are given for o. vulgare subsp. gracile, o. vulgare subsp. hirtum, o. vulgare subsp. viridulum, and o. vulgare subsp. vulgare. sect. prolaticorolla ietsw. sect. prolaticorolla contains only one species in turkey. the chromosome number of origanum laevigatum boiss. is 2n = 30 (figure 1x, table 1). the chromosome lengths range from 0.45 to 1.96 µm (no. td4497). the chromosome number of o. laevigatum is given for the first time. the chromosome counts and morphologies of the hybrids and their parents origanum × adae dirmenci & yazıcı (o. ayliniae × o. sipyleum) the chromosome number of origanum × adae is 2n = 30 (table 1). the chromosome lengths range from 0.72 to 1.40 µm. the total haploid length is 15.47 µm (no. td4518). the chromosome lengths of o. ayliniae range from 0.83 to 1.72 µm. the total haploid length is 18.35 µm (no. td4516). the chromosome lengths of o. sipyleum range from 0.32 to 1.11 µm. the total haploid length is 10.46 µm (no. td4517). according to the karyological results, the chromosome numbers of origanum × adae, o. ayliniae, and o. sipyleum are similar with n = 15 for the haplotype (figure 2a-c). karyological analyses support the idea that origanum ×adae, is a natural hybrid, is generated from crossed homoploidy of o. ayliniae and o. sipyleum. the hybrid is generated by homoploid hybridization (all taxa have 2n = 30 chromosomes) (dirmenci et al. 2018a). the chromosome morphologies of origanum ×adae and o. ayliniae, and o. sipyleum are described for the first time. origanum × haradjanii rech.f. (o. syriacum subsp. bevanii × o. laevigatum) the chromosome number of origanum × haradjanii is 2n = 30 (no. td4335). the chromosome lengths of o. syriacum subsp. bevanii range from 0.53 to 1.58 µm. the total haploid length is 16.26 µm. (no. td4336). the chromosome lengths of o. laevigatum range from 0.45 to 1.96 µm. the total haploid length is 16.34 µm (no. td4497). according to the karyological results, the chromosome numbers of origanum × haradjanii, o. laevigatum, and o. syriacum subsp. bevanii are similar with n = 15 for the haplotype (figure 2d-f). karyological analyses support the idea that o. × haradjanii, is a natural hybrid, is generated from crossed homoploidy of o. syriacum subsp. bevanii and o. laevigatum. the hybrid is generated by homoploid hybridization (all taxa have 2n = 30 chromosomes). the chromosome number of o. × haradjanii is first reported. in addition, the chromosome morphologies of o. syriacum subsp. bevanii and o. laevigatum are described for the first time. origanum × intermedium p.h.davis (o. onites × o. sipyleum) the chromosome number of origanum × intermedium is 2n = 30. the chromosome lengths range from 0.46 to 1.14 µm. the total haploid length is 11.62 µm (no. td4726). the chromosome lengths of o. sipyleum range from 0.49 to 1.08 µm. the total haploid length is 11.26 µm (no. td4727). the chromosome lengths of o. onites range from 0.49 to 1.08 µm. the total haploid length is 11.26 µm (no. td4725). according to the karyological results, the chromosome numbers of origanum × intermedium, o. onites, and o. sipyleum are similar with n = 15 for the haplotype (figure 2g-i). karyological analyses support the idea that o. × intermedium, is a natural hybrid, is generated from crossed homoploidy of o. onites and o. sipyleum. the hybrid is generated by homoploid hybridization (all taxa have 2n = 30 chromosomes). the chromosome number and morphologies of origanum × intermedium are described for the first time. origanum × sevcaniae dirmenci, arabacı & yazıcı (o. vogelii × o. vulgare subsp. hirtum) the chromosome number of origanum × sevcaniae is 2n = 30 (no. td4508). according to the karyological results, the chromosome numbers of o. × sevcaniae, o. vogelii and o. vulgare subsp. hirtum are similar with n = 15 for the haplotype (figures 2j-l). karyological analyses support the idea that origanum × sevcaniae, is a natural hybrid, is generated from crossed homoploidy of o. vogelii and o. vulgare subsp. hirtum. the hybrid is generated by homoploid hybridization (all taxa have 2n = 30 chromosomes) (dirmenci et al. 2018b). 135karyotype studies on the genus origanum (lamiaceae) species and some hybrids defining homoploidy figure 2a-l. somatic metaphase chromosomes of hybrids and their parents: a) o. × adae (td4518); b) o. ayliniae (td4516); c) o. sipyleum (td4517); d) o. × haradjanii (td4335); e) o. syriacum subsp. bevanii (td4336); f) o. laevigatum (td4497); g) o. × intermedium (td4726); h) o. onites (td4725); i) o. sipyleum (td4727); j) o. × sevcaniae (td4508); k) o. vogelii (td4509); l) o. vulgare subsp. hirtum (td4507). scale bar: 10 µm. 136 esra martin et al. figure 3a-n. ideograms of origanum taxa: a) o. amanum (td4514a); b) o. bilgeri (td4343); c) o. boissieri (td4319); d) o. brevidens (td4331); e) o. haussknechtii (ta2824); f) o. husnucan-baseri (td4528); g) o. hypericifolium (td4357); h) o. leptocladum (td4345); i) o. majorana (td3984); j) o. minutiflorum (td4348); k) o. rotundifolium (td3943); l) o. saccatum (td4732); m) o. solymicum (td4347); n) o. vulgare subsp. vulgare (td4688). scale bar: 10 µm. 137karyotype studies on the genus origanum (lamiaceae) species and some hybrids defining homoploidy the ideograms, which were drawn by software image analysis (bs200prop) loaded on a personal computer are given in figure 3a-n and figure 4a-f. discussion counting of chromosomes has been a very useful approach (particularly at the generic level) for researchers investigating evolutionary relationships (levin and wilson 1976; stace 1991; goldblatt 2007; guerra 2008; stuessy 2009; contreras and ruter 2011). indeed, the chromosome numbers can affect inbreeding depression and the potential for introgression of traits through interspecific hybridization, among other factors that can alter breeding strategy (fehr 1991; contreras and ruter 2011). measuring of chromosome size correlated with evolutionary age provides to estimate genome size using the chromosomal data (mehra and bawa 1972; contreras and ruter 2011). according to the index to plant chromosome numbers (ipcn, http://w w w.tropicos.org/project/ipcn) (goldblatt and johnson 1979-2017) and chromosome counts database (ccdb, version 1.45, http://ccdb.tau. ac.il/home/) (rice et al. 2015), there are the chromosome numbers of more than 1500 taxa of 140 genera belonging to lamiaceae family (chen et al. 2018). in lamiaceae, the chromosome numbers between genera and even species are highly variable from 2n = 10 to 2n = 240. allopolyploidy and autopoliploidy are the main reasons for variations. the basic numbers are x = 5, 7, 8, and 10. secondly, basic numbers can be assumed to be x = 13, 14, 15, 17, and 19 (singh 1995; mabberley 1997). singh (1995) considered that x = 17 arised as the result of the combination of x = 8 and x = 9 (miura et al. 2005). extensive cytological studies have revealed the presence of diploid, tetraploid, hexaploid and octoploid species in the family lamiaceae. in family, the diversification may be attributed to the presence of polyploidy and aneuploidy (rather et al. 2018). in the genus origanum, it was reported that the main diploid numbers were 2n = 28, 30, and 32 and the figure 4a-f. ideograms of hybrids and their parents: a) o. × adae (td4518); b) o. ayliniae (td4516); c) o. sipyleum (td4517); d) o. × intermedium (td4726); e) o. onites (td4725); f) o. sipyleum (td4727). scale bar: 10 µm. 138 esra martin et al. basic number was x = 15 (ipcn, http://www.tropicos. org/project/ipcn) (goldblatt and johnson 1979-2017) and chromosome counts database (ccdb, version 1.45, http://ccdb.tau.ac.il/home/) (rice et al. 2015). the chromosome counts of the investigated species in present and previous studies are given in table 2. in table 1 and table 2, the chromosome number of all sections is 2n = 30 except some origanum sipyleum specimens (because some of the specimens have 2n = 30) in sect. anatolicon and o. rotundifolium in the sect. brevifilamentum with 2n = 28. the smallest chromosome length is 0.32 μm in origanum sipyleum (no. td4517). the largest chromosome length is 2.02 μm in o. minutif lorum (no. td4348). the smallest total haploid length is 10.08 μm in o. vulgare subsp. hirtum (no. td4359) and the largest value is 22.00 μm in o. haussknechtii (no. ta2824). the smallest mean length is 0.33 μm in o. vulgare subsp. hirtum and o. saccatum (no. td4359; td4522, respectively). the largest mean length is 0.74 μm in o. sipyleum (no. td4308). the chromosome lengths range from 0.75 to 6.00 μm. the chromosome lengths were measured between 0.33 and 0.74 in this karyological study of the genus origanum. in the genus origanum, the centromeric position could not be clearly observed because the chromosomes were generally very small compared to family members. on the other hand, total chromosome length could be measured. the chromosome number as 2n = 30 is typical in some genera of lamiaceae family (clinopodium l., micromeria benth., satureja l., thymus l. etc.). some thymus species have the same chromosome number (jalas & kaleva 1967; vaarama 1948). in addition, some species have different basic numbers as x = 6, 7, 9, 14, 21, 27, and 24 in the genus (darlington and wylie 1955; vaarama 1948). the secondary basic numbers, namely x = 14 and 15 probably originate from x = 7. the most common numbers are 2n = 28, 30, 56, and 60, while 2n = 84 and 90 are rarely. jalas (1967) showed that although the chromosome numbers of distinct thymus subsections were different, both subsections vulgares l. and piperella willk. had the same chromosome number with 2n = 30. in genus micromeria s.str., the chromosome number of most species was reported as 2n = 30 (martin et al. 2011). on the other hand, micromeria s.l. has various somatic chromosome numbers as 2n = 20, 22, 26, 30, 48, 50, and 60. similarly, clinopodium s.str. has various somatic chromosome numbers as 2n = 18, 20, 22, 24, and 48 (ipcn, http://www.tropicos.org/project/ipcn) (goldblatt and johnson 1979-2017) and chromosome counts database (ccdb, version 1.45, http://ccdb.tau. ac.il/home/) (rice et al. 2015). morton (1962) reported that the basic numbers were x = 6, 7, 8, 9, 10, 11, and 15 in satureja. harley and brighton (1977) reported that the genus mentha had various chromosome counts ranging from x = 6 to x = 54. this means that we can see diploid-octoploid members in this genus. as mentioned before, the genus origanum is a member of the mentheae tribe and the menthinae subtribe (harley et al. 2004). the chromosome numbers of menthinae subtribe vary from 2n = 12 (hyssopus) to 2n = 144 (mentha). the chromosome numbers were 2n = 30 in most of the thymbra l., satureja, and micromeria (s.str.) species belonging to the tribe menthinae. in addition, the chromosome numbers of some species in thymus, mentha and prunella l. were 2n = 30 (harley et al. 2004). ietswaart’s hypothesis with regard to the origin of the genus origanum suggests that origanum might have emerged in the pliocene, and that formerly, the tribe saturejeae genera (clinopodium sl., micromeria, satureja, thymus, etc.) were probably the main genera from which origanum could be derived (see ietswaart 1980: pp. 7-14 and 21-24, figure 2). when we analyzed the closest genera mentioned in the discussion part, homoploid hybridization was the main ploidy type in origanum. the emergence of other sections has been diversified by hybridization between the main sections and hybridization of some thymus and origanum species (in part including satureja, micromeria s.str. and clinopodium sl. species) (ietswaart 1982). according to this hypothesis, the genera mentioned above are important regarding chromosome number compatibility. however, thymbra, prunella, and clinopodium (partly) are morphologically very different from origanum species. on the other hand, satureja, thymus, micromeria s.str., and clinopodium (partly) are morphologically closer. it may have been derived from one or more of these genera and then continued to speciation via hybridization in time. the compatibility of chromosome numbers between species supports ietswaart’s hypothesis (1980). although the hybridization is widespread, the generation of a unique and isolated hybrid lineage is probably very rare. new hybrid lineages must establish reproductive isolation and a unique ecological niche to overcome genetic swamping and competition from parental species (goulet et al. 2017). a new hybrid lineage may be formed through allopolyploidy or homoploid hybrid speciation. allopolyploid lineages may be formed by the fusion of unreduced gametes, genome doubling following hybridization, or via a triploid bridge (ramsey and schemske 1998; goulet et al. 2017). homoploid hybrid speciation forms a new unreproductive hybrid lineage 139karyotype studies on the genus origanum (lamiaceae) species and some hybrids defining homoploidy with the same ploidy level with its parents (goulet et al. 2017). it should be shown that both the hybrid origin of the species and the hybridization having reproductive isolation to verify a homoploid hybrid speciation (schumer et al. 2014). while homoploid hybrid speciation is often concentrated on the demonstration of genetic divergence of hybrids and their origin and a pronounced ecological separation, the number of examples showing the strong link between hybridization and isolated species is very few (stebbins 1947; chapman and burke 2007; schumer et al. 2014; yakimowski and rieseberg 2014). hybridization is a common phenomenon in the genus origanum (dirmenci et al. 2018a, 2018b, 2019). the sections amaracus, majorana, and origanum (o. vulgare s.l.) can be considered as the ancestral sections in the genus. later, speciation via homoploid hybridization has an important role in speciation in the genus. this is important evidence that species hybridize easily in different sections, or that species in one section have intermediate characteristics between two different sections. according to ietswaart (1980) and our morphological observations, sect. anatolicon species have transition characters between the species of sect. amaracus and origanum (figures 5a-c, 6a-f). thus, sect. amaracus is characterized by its branches. the firstorder branches are usually present, but the second-order branches are seldom present. leaves are usually leathery, spikes large and usually nodding (figure 5a), bracts partly purple, calyces 1 or 2-lipped, corollas saccate and all stamens greatly exserted from corolla (figure 6a, d). in the sect. anatolicon, branches of the first order are always present, while the second-order branches are sometimes present. leaves may be leathery or not. spikes are medium sized, usually nodding, bracts partly or slightly purple (5b), calyces usually 2-lipped, corollas not saccate and all stamens exserted from corolla (figure 6b, e). the firstand second-order branches of the sect. origanum are always present, while the third-order branches are usually present. leaves are usually herbaceous, spikes dense and small to medium-sized, bracts green or purple (5c), calyces actinomorphic and with 5 equal teeth, corollas not saccate and stamens subincluded or slightly protruding (figure 6e, f). these morphological transitions are important evidence that the genus may have intrageneric speciation via hybridization. besides, recent studies and the chromosome numbers of hybrids and ancestors occurring in this study are the cytological evidence supporting this hypothesis. origanum hybrids originate via homoploidy. the chromosome numbers of origanum × adae, o. × figure 6. flower and calyx of o. ayliniae (a, d), o. sipyleum (b, f), o. vulgare subsp. hirtum (c, f) (figures a, d, b, f from dirmenci et al 2018a and c, f from dirmenci et al. 2018b). figure 5. inflorescence of o. ayliniae (a), o. sipyleum (b), o. vulgare subsp. vulgare (c) (photo by tuncay dirmenci). 140 esra martin et al. intermedium, o. × haradjanii, o. × munzurense sorger & kit tan, o. × sevcaniae, o. × font-queri and their parents were 2n = 30 (bakha et al. 2017; 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origanum ayliniae: aydın: kuşadası, dilek yarımadası nationa l park, radar, 3922 f t, 08.10.2015, dirmenci 4516; origanum bilgeri; antalya: gündoğmuş, between hanboğazı and oğuz yayla, 1. km, 4775 ft, 24.10.2014, dirmenci 4343; origanum boissieri: i̇çel: 15 km çamlıyayla district to saimdibi place, 6045 ft, 18.09.2014, dirmenci 4319, t.arabacı & t.yazıcı; origanum brevidens: osmaniye: 1-2 km from yarpuz to yağlıpınar, 5030 ft, 19.09.2014, dirmenci 4331, t.arabacı & t.yazıcı; origanum haussknechtii; erzincan, 15. km from kemaliye to arapkir, 1000 1100 m, 22.08.2013, t.arabacı 2824; origanum husnucan-baseri; antalya: between alanya and hadim, 1-2 km to gökbel yayla, 4500 ft, 09.10.2015, dirmenci 4528, t.arabacı & t.yazıcı; origanum hypericifolium: denizli: honaz district, honaz mountain, i̇ncekara stream, 1300 m, 07.11.2014, dirnmenci 4357 & t.yazıcı; origanum laevigatum: osmaniye: düziçi, above kuşçu village, 03.10.2015, dirmenci4497 & t.arabacı; origanum leptocladum; karaman: between ermenek and kazancı, above görmeli village, 1. km, 870 m, 24.10.2014, dirmenci 4345, t.arabacı & t.yazıcı; origanum majorana; mersin: güzeldere, 252 m, 13.07.2013, dirmenci 3984, t.arabacı & t.yazıcı; i̇çel: between anamur and gazipaşa, 15-20. km, 300 m 26.10.2014, dirmenci 4356 & t.yazıcı; karaman: ermenek, 2 km from kazancı to abanoz yayla, 1238 m, 24.10.2014, dirmenci 4346, t.arabacı & t.yazıcı; origanum minutiflorum: antalya: kemer, üçoluk, above tülek, 4470 ft, 25.10.2014, dirmenci 4348 & t.yazıcı; origanum onites: denizli: taşocağı, 527 m, 26.10.2014, dirmenci 4355 & t.yazıcı; ibid, 11.10.2015, dirmenci 4532 & t.yazıcı; denizli: between buldan and güney, 13 from road disjunction to güney, dirmenci 4725 & t.yazıcı; denizli: origanum rotundifolium: artvin: between artvin and ardanuç, 600-700 m, 27.08.2013, dirmenci 3943, b.yıldız & t.arabacı; origanum saccatum: antalya, 1 km from gündoğmuş to çayırözü village, 3715 ft, 24.10.2014, dirmenci 4342, t.arabacı & t.yazıcı; antalya: 38 km from alanya to hadim, kuşkayası, 3814 ft, 09.10.2015, dirmenci 4522 t.arabacı & t.yazıcı; ibid., dirmenci 4732 & t.arabacı; origanum sipyleum: denizli: 5 km from serinhisar to denizli, 1066 m, 19.08.2014, dirmenci 4308; ibid., 26.10.2014, dirmenci 4352; denizli: taşocağı, 1808 ft, dirmenci 4534 & t.yazıcı; aydın: kuşadası, dilek peninsuna national park, radar, 3922 ft, 08.10.2015, dirmenci 4517; denizli: denizli: between buldan and güney, 13 from road disjunction to güney, dirmenci 4727 & t.yazıcı; origanum solymicum: antalya: kemer, 4 km from kesmeboğazı to kuzdere village, 1470 m, 25.10.2014, dirmenci 4347 & t.yazıcı; antalya: kemer, 7 km from kesmeboğazı to karçukuru yayla, 1506 ft, 09.10.2015, dirmenci 4520 & t.yazıcı;; origanum syriacum subsp. bevanii: hatay: between antakya and samandağ, around st. symeone church, 20.09.2014, dirmenci 4336, t.arabacı & t.yazıcı; osmaniye: düziçi, between kuşçu village and düldül mountain, 19.09.2014, dirmenci 4330 & t.arabacı; origanum vogelii; niğde: ulukışla, horoz village, fenk boğazı, 4800 ft, 02.10.2015, dirmenci 4509 & t.yazıcı; origanum vulgare subsp. gracile: k.maraş: göksun, between yeşilköy and kınıkkoz villages, 400450 m, 04.08.2017, dirmenci 4821 & t.arabacı; tunceli: 20-21 km from ovacık to tunceli, c. 1200 m, 11.08.2017, dirmenci 4958, t.arabacı & m.açar; origanum vulgare subsp. hirtum: antalya: antalya: between alanya and hadim, 1 km to gökbel yayla, dirmenci 4733, t.arabacı & t.yazıcı; denizli: taşocağı, 527 m, dirmenci 4722 & t.yazıcı; denizli: honaz district, north face of honaz mountain, arpacık yayla road, 4160 ft, 07.11.2014, dirmenci 4359 & t.yazıcı; niğde: ulukışla, horoz village, 143karyotype studies on the genus origanum (lamiaceae) species and some hybrids defining homoploidy fenk boğazı, 4800 ft,, 02.10.2015, dirmenci 4507 & t.yazıcı; origanum vulgare subsp. viridulum: giresun: 33 km from şebinkarahisar to tamdere, nort of eğribel pass, dirmenci 4662a & t.arabacı; origanum vulgare subsp. vulgare: 22 km from şavşat to ardahan, dirmenci 4688 & t.arabacı; origanum × adae: kuşadası, dilek yarımadası national park, radar, 3922 ft, 08.10.2015, dirmenci 4518; origanum × haradjanii: hatay: between antakya and samandağ, around st. symeone church, 20.09.2014, dirmenci 4335, t.arabacı & t.yazıcı; o. × intermedium: denizli: denizli: between buldan and güney, 13 from road disjunction to güney, dirmenci 4726 & t.yazıcı; origanum × munzurense: tunceli: between ovacık and tunceli, 21-22. km, aşağı torunoba village, munzur stream, near bridge, c. 1200 m, dirmenci 4957a, arabacı & açar; origanum × sevcaniae: niğde: u ulukışla, horoz village, fenk boğazı, 4800 ft, 02.10.2015, dirmenci 4508 & t.yazıcı. caryologia international journal of cytology, cytosystematics and cytogenetics volume 73, issue 2 2020 firenze university press the first molecular identification of egyptian miocene petrified dicot woods (egyptians’ dream becomes a reality) shaimaa s. sobieh*, mona h. darwish gene flow patterns reinforce the ecological plasticity of tropidurus hispidus (squamata: tropiduridae) fernanda ito, danielle j. gama-maia, diego m. a. brito, rodrigo a. torres* the technique of plant dna barcoding: potential application in floriculture antonio giovino1,*, federico martinelli2,*, anna perrone3 cytogenetic of brachyura (decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species alessio iannucci1, stefano cannicci1,2,*, zhongyang lin3, karen wy yuen3, claudio ciofi1, roscoe stanyon1, sara fratini1 comparison of the evolution of orchids with that of bats antonio lima-de-faria identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection mehdi zahravi1,*, panthea vosough-mohebbi2, mehdi changizi3, shahab khaghani1, zahra-sadat shobbar4 populations genetic study of the medicinal species plantago afra l. (plantaginaceae) saeed mohsenzadeh*, masoud sheidai, fahimeh koohdar a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding timir baran jha1,*, partha sarathi saha2, sumita jha2 chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india harminder singh2, jaswant singh1,*, puneet kumar2, vijay kumar singhal1, bhupendra singh kholia2, lalit mohan tewari3 population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies raheleh tabaripour1,*, masoud sheidai1, seyed mehdi talebi2, zahra noormohammadi3 induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions girjesh kumar, shefali singh* karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(3): 33-44, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-625 caryologia international journal of cytology, cytosystematics and cytogenetics citation: s. bhadra, z.-q. cai (2020) karyological variability and chromosomal asymmetry in highland cultivars of chenopodium quinoa willd. (amaranthaceae). caryologia 73(3): 33-44. doi: 10.13128/caryologia-625 received: september 17, 2019 accepted: april 27, 2020 published: december 31, 2020 copyright: © 2020 s. bhadra, z.-q. cai. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyological variability and chromosomal asymmetry in highland cultivars of chenopodium quinoa willd. (amaranthaceae) sreetama bhadra1,*, zhi–quan cai1,2,* 1 cas key laboratory of tropical forest ecology, xishuangbanna tropical botanical garden, chinese academy of sciences, mengla 666303, china 2 department of horticulture, foshan university, foshan 528000, china * corresponding authors. e-mail: sreetama.bhadra@gmail.com; zhiquan.cai@126.com abstract. chenopodium quinoa willd. is rapidly gaining importance worldwide as a superfood. however, structural diversity and asymmetry analyses of chromosomes of different cultivars of the species are largely understudied primarily owing to their small chromosomes. in this paper, karyomorphological investigations were performed on 21 cultivars of c. quinoa with varying seed morphology cultivated widely in the highland regions of the andes, which is the center of domestication of this species. somatic chromosome number was found to be 2n= 36 in all cultivars with no occurrence of mixoploidy. lengths of individual chromosomes varied between 0.63–6.53 μm, with their short arms ranging from 0.25–2.95 μm and long arms between 0.38–3.58 μm. types of primary constriction ranged from median to sub–terminal. one pair of chromosome in each complement possessed a secondary constriction. chromosome complements of all cultivars belonged to the asymmetry class 2b with an average asymmetry index value of 3.21±0.61. values of intraand inter-chromosomal asymmetry indices were 0.30±0.02 and 0.20±0.02 respectively across all cultivars. the average coefficients of variation of chromosome lengths was 19.77±2.11 and average centromeric index was 16.28±2.12. arm ratio of the chromosomes varied from 0.34 to 5.76. the mean values of karyotypic asymmetry, symmetry index and karyotype asymmetry index percentage were 17.69±1.53, 147.69±5.54 and 58.71±0.86 respectively. pearson correlation revealed strong correlation within inter– and intra–chromosomal asymmetry indices. our analyses uncovered higher chromosomal variation in quinoa than previously found with high inter–varietal similarities among the studied cultivars, revealed from scattered diagrams between asymmetry indices. keywords: chenopodium quinoa, chromosome, karyotype, chromosomal asymmetry, inter-varietal symmetry. introduction sustainable agricultural systems generally focus on traditional commercial food crops, which might not be adequate in the near future to fulfill the projected increase in global consumption due to increase in population. this, along with rapid climate change, has necessitated the investigation and 34 sreetama bhadra, zhi–quan cai exploitation of alternative crop plants to complement the projected deficit (graf et al. 2015). recent research has focused on the south american crop chenopodium quinoa (quinoa), which the food and agriculture organization of the united nations have touted as a crop that can mitigate global food demand to a large extent (graf et al. 2015). this plant’s adaptability to extreme agro–ecological conditions together with its balanced proportion of amino acids, carbohydrates, lipids, vitamins and minerals, low glycemic acid content and its gluten–free nature, have resulted in expansion of its cultivation outside its native region (ruiz et al. 2014; pereira et al. 2019). improvement programs of the crop include reduction in saponin content of its seed coat, increasing compactness of its inflorescence, high stress tolerance and resistance to pests and pathogens (murphy et al. 2016; jarvis et al. 2017). success of these programs demand identification of existing genetic divergence in the species. an initial idea of genetic variability among plants can be perceived by analyzing cytological characters (guerra 2008). most crop plants have undergone recent polyploidization events (renny-byfield and wendel 2014). polyploidy results in variable chromosome numbers and the ensuing chromosomal changes like translocations and inversions give rise to structural variabilities (leitch and leitch 2008). analyses and comparison of these 2 parameters, viz. chromosome number and structure, among related taxa is considered to be a reliable approach for quantitative interpretation of their similarity or divergence among related plants (bennett and leitch 2005). generally considered an allotetraploid with a basic chromosome number x= 9 (krak et al. 2016; mandák et al. 2016), the reported somatic chromosome counts of quinoa predominantly showed an invariable 2n= 36 (kjellmark 1934; wulff 1936; cárdenas and hawkes 1948; heiser 1963; gandarillas and luizaga 1967; giusti 1970; kolano et al. 2001, 2012; rahiminejad and gornall 2004; bhargava et al. 2006; palomino et al. 2008; mandák et al. 2016). however, there are limited studies on variations in chromosome structure and asymmetry in the species mainly due to their small size, which makes common karyotyping of the species difficult (kolano et al. 2001, 2012). such studies, when present, were mostly limited to examining only 1 or 2 varieties restricting the possibility of understanding inter-varietal chromosomal differences, or, were based on assessments of insufficient asymmetry parameters (kolano et al. 2001, 2012; bhargava et al. 2006; maughan et al. 2006; palomino et al. 2008; yangquanwei et al. 2013). earliest reports of domestication and cultivation of quinoa can be traced back to about 7,000 years ago to the inca dynasty in south american andes, especially near lake titicaca from peru and bolivia that constituted the ancestral highland ecotype of this species (jarvis et al. 2017). although variations in prominent morphological markers for commercial quinoa like seed size and colour, exist within the cultivated varieties of south america, it can be argued that, prolonged farming of a species at and around its center of domestication and diversity might expose them to the risk of genetic erosion, resulting in low genetic variability (gonzalo et al. 2019). existence of such genetic erosion, undesirable for germplasm improvement and conservation programmes, might be realized from cytological studies; however, chromosomal study of quinoa from this region is not forthcoming. the objectives of our work were (1) to analyze the karyotype structure of highland varieties of quinoa, (2) to determine the extent of karyotype differentiation among the varieties, (3) to evaluate chromosomal asymmetry in the varieties. we investigated 21 morphologically variable highland cultivars of quinoa, and, performed karyotypic analyses based on asymmetry parameters that could help in elucidating worthwhile information on the inter–varietal divergence of quinoa. materials and methods materials for the purpose of this study, seeds of white, yellow, red and black–coloured varieties were selected from the highland ecotypes of quinoa, their sizes ranging from 1.5 to 3.0 cm in diameter. these cultivars were collected from peru and bolivia around lake titicaca (figure 1). seeds from at least 5 individuals of each cultivar were germinated on moist filter paper in petri plates, kept in dark for 24–48 h at room temperature for cytological study. growing root tips of freshly germinated seedlings were used for cytological analyses. methods actively growing 1.5–2 cm long root tips of seedlings were harvested after 1 to 2 days of germination initiation, between 10 am to 11 am, for mitotic study. pretreatment of the harvested root tips were performed in aqueous solution of 2 mm 8–hydroxyquinoline for 3 hours at 10°c after which the root tips were fixed overnight in carnoy’s fixative at 10°c. hydrolysis, staining of the root tips and slide preparation were done following the protocol of sun et al. (2017). squashed root tips were observed under olympus bx51 microscope (olympus, germany) at a magnification of 1000x. well scattered 35karyological variability and chromosomal asymmetry in highland cultivars of chenopodium quinoa metaphase plates were photographed with a mounted digital camera using the software scopephoto (ver. 3.0.12.444) (scopetek, hangzhou, china). chromosome numbers were counted from at least 50 metaphase plates prepared from 50 different seedlings for each cultivar of quinoa to ensure the uniformity of somatic chromosome numbers in all the plants. among them, at least 30 best metaphase plates were selected for karyotype analyses with a minimum of 10 seedlings chosen per cultivar. lengths of chromosome arms were measured from at least 5 best metaphase plates for each cultivar using karyotype v2 software (altınordu et al. 2016). the lengths of chromosomes and respective centromeric indices of each metaphase plate, obtained from this data, were used to describe the chromosome morphology following the nomenclatural system of levan et al. (1964), and, to prepare their idiograms with originpro 2018 software (originlab corporation, usa). asymmetry of karyotypes of each cultivar were calculated using stebbin’s classification (1971) and ai (karyotype asymmetry index), intrachromosomal asymmetry indices using a1 (intra-chromosomal asymmetry index), cvci (coefficient of variation of centromeric index), mca (mean centromeric asymmetry), syi (symmetric index), ask% (karyotype asymmetry index percentage) and ar (arm ratio), and inter-chromosomal asymmetry using a2 (inter-chromosomal asymmetry index), cvcl (coefficient of variation of chromosome length) (for details see bhadra and bandyopadhyay 2015). pearson correlation among these intraand inter-chromosomal asymmetry parameters was calculated in spss (version 21) software (münchen, germany). results the chromosomes of studied cultivars of chenopodium quinoa were small, though clearly visible with conspicuous centromeric regions. figure 1. (a) map showing the collection area of the studied cultivars (b) photographs of seeds showing morphological variation (bar=1 cm). 36 sreetama bhadra, zhi–quan cai table 1. chromosome numbers, karyotype formulae, morphometric parameters and values of asymmetry indices studies in 21 chenopodium quinoa varieties. local variety name 2n karyotype formula short arm range (average) in µm long arm range (average) in µm total length range (average) in µm bl71 36 2m+22m+8sm+2st+2m:sm 0.42–1.77 (1.03±0.25) 0.80–2.64 (1.49±0.34) 1.35–4.24 (2.56±0.50) bl72 36 8m+14m+10m+2st+2m:sm 0.42–1.92 (1.04±0.31) 0.50–3.02 (1.52±0.39) 0.92–4.19 (2.59±0.59) bl–d75 36 18m+14sm+2st+2m:sm 0.39–2.15 (1.04±0.26) 0.46–2.83 (1.55±0.40) 0.85–4.51 (2.62±0.57) bl–x3 36 6m+8m+16sm+4st+2m:sm 0.59–2.19 (1.21±0.28) 1.04–3.45 (1.91±0.36) 1.88–4.61 (3.16±0.42) rd14 36 6m+14m+12sm+2st+2m:sm 0.52–1.79 (1.10±0.25) 0.65–2.88 (1.69±0.44) 1.17–3.96 (2.82±0.50) rd31 36 4m+18m+10sm+2st+2m:sm 0.44–2.95 (1.13±0.33) 0.55–3.58 (1.71±0.42) 0.99–6.53 (2.88±0.66) rd36 36 4m+26m+4sm+2m:sm 0.40–1.34 (0.95±0.18) 0.57–2.12 (1.30±0.23) 0.97–3.95 (2.28±0.38) rd47 36 4m+20m+8sm+2st+2m:sm 0.40–1.83 (1.16±0.26) 0.46–2.82 (1.59±0.30) 0.86–4.41 (2.78±0.49) rd56 36 8m+20m+6sm+2m:sm 0.37–1.48 (0.99±0.24) 0.49–2.09 (1.41±0.29) 0.86–3.85 (2.44±0.48) rd58 36 8m+16m+8sm+2st+2m:sm 0.37–2.00 (1.13±0.37) 0.52–3.00 (1.60±0.44) 0.89–4.82 (2.76± 0.70) rd8 36 2m+16m+12sm+4st+2m:sm 0.33–1.93 (1.10±0.30) 0.60–3.66 (1.67±0.44) 0.93–5.45 (2.81±0.64) rd–d88 36 6m+16m+10sm+2st+2m:sm 0.42–1.85 (1.10±0.32) 0.50–2.96 (1.61± 0.35) 0.92–4.44 (2.75±0.58) rd–x2 36 2m+12m+18sm+2st+2m:sm 0.25–1.64 (1.00±0.24) 0.38–2.38 (1.52±0.30) 0.63–3.94 (2.55±0.47) wh32 36 6m+20m+6sm+2st+2m:sm 0.68–2.57 (1.18±0.32) 0.54–2.87 (1.77±0.40) 1.03–5.44 (2.99±0.58) wh82 36 8m+22m+4sm+2m:sm 0.48–1.81 (1.10±0.26) 0.58–2.52 (1.49±0.32) 1.06–4.18 (2.62±0.54) wh–x1 36 2m+18m+12sm+2st+2m:sm 0.35–1.42 (0.85±0.20) 0.38–2.42 (1.26±0.26) 0.73–3.20 (2.14±0.40) yel59 36 8m+18m+6sm+2st+2m:sm 0.49–1.97 (1.16±0.28) 0.73–2.83 (1.63±0.36) 1.22–4.34 (2.82±0.54) yel61 36 4m+16m+12sm+2st+2m:sm 0.42–1.66 (0.97±0.26) 0.47–2.91 (1.54±0.35) 0.92–3.98 (2.55±0.50) yel63 36 6m+22m+4sm+2st+2m:sm 0.47–1.75 (1.06±0.29) 0.59–2.81 (1.59±0.42) 1.06–4.36 (2.69±0.59) yel7 36 2m+22m+10sm+2m:sm 0.44–1.82 (1.16±0.26) 0.53–2.79 (1.70±0.36) 0.97–5.02 (2.92±0.54) yel–x4 36 4m+24m+6sm+2m:sm 0.53–1.68 (1.07±0.24) 0.77–2.61 (1.63±0.31) 1.35–4.50 (2.74±0.42) local variety name centromeric index range (average) (i) karyotype asymmetry class arm ratio range (average) (ar) value of relative chromatin (vrc) total form percentage (tf%) relative length percentage range (rl) bl71 18.79–49.76 (41.17±6.83) 2b 0.70–4.32 (1.51±0.06) 5.11±0.61 41.78±0.96 1.56–4.56 bl72 16.35–49.76 (40.52±7.72) 2b 0.69–5.12 (1.57±0.11) 5.19±0.64 41.35±1.06 0.86–4.06 bl–d75 19.43–49.70 (40.52±6.37) 2b 0.57–4.14 (1.54±0.13) 5.24±0.99 40.80±2.13 0.93–4.46 bl–x3 21.69–49.82 (39.40±7.63) 2b 0.41–3.61 (1.65±0.22) 6.32±0.88 39.76±3.18 1.91–3.64 rd14 14.79–50 (40.01±8.03) 2b 0.54–5.76 (1.62±0.11) 5.65±0.13 40.21±0.36 1.63–3.95 rd31 22.41–49.78 (40.13±6.44) 2b 0.61–3.46 (1.59±0.06) 5.76±1.52 40.33±0.93 1.20–6.74 rd36 31.22–49.76 (42.48±4.17) 2b 0.60–2.20 (1.38±0.02) 4.56±0.47 42.98±0.27 1.26–4.30 rd47 24.31–49.84 (42.36±5.77) 2b 0.77–3.11 (1.43±0.13) 5.56±0.56 42.77±1.88 0.96–4.02 rd56 25.73–49.84 (41.84±5.65) 2b 0.60–2.88 (1.46±0.09) 4.87±0.22 42.26±1.37 0.95–4.29 rd58 15.56–49.81 (41.44±7.46) 2b 0.68–5.42 (1.52±0.10) 5.53±0.89 42.08±0.96 0.89–4.57 rd8 18.16–49.32 (40.02±6.75) 2b 0.55–4.51 (1.59±0.21) 5.63±0.46 40.52±2.49 0.97–4.92 rd–d88 21.32–49.82 (40.35±7.40) 2b 0.39–3.69 (1.57±0.12) 5.49±0.81 41.34±1.44 1.10–5.26 rd–x2 20.92–50 (39.99±6.66) 2b 0.54–3.78 (1.59±0.28) 5.10±0.06 40.48±4.02 0.68–4.24 wh32 19.82–49.64 (40.52±7.08) 2b 0.53–4.04 (1.58±0.09) 5.98±0.45 40.63±1.43 0.88–4.65 wh82 25.00–49.72 (42.38±5.24) 2b 0.70–3.00 (1.38±0.04) 5.25±0.72 43.23±0.71 1.06–4.07 wh–x1 24.37–50 (40.97±5.53) 2b 0.44–3.10 (1.51± 0.20) 4.29±0.37 41.30±2.96 1.01–3.88 yel59 22.09–49.77 (41.92±6.15) 2b 0.76–3.53 (1.46±0.10) 5.64±0.88 42.38±1.52 1.29–3.99 yel61 18.03–49.69 (39.05±7.23) 2b 0.34–4.55 (1.66±0.16) 5.09±0.59 39.69±1.53 1.04–4.49 yel63 17.07–49.83 (40.57±7.38) 2b 0.41–4.86 (1.56±0.10) 5.39±0.53 40.99±2.15 1.04–4.26 yel7 25.69–49.60 (40.97±6.96) 2b 0.67–3.28 (1.51±0.05) 5.84±0.46 41.69±0.48 0.85–4.41 yel–x4 18.03–50 (39.87±6.91) 2b 0.35–4.55 (1.62±0.41) 5.47±0.28 40.49±4.74 1.35–4.51 37karyological variability and chromosomal asymmetry in highland cultivars of chenopodium quinoa local variety name difference of relative length (drl) intra–chromosomal asymmetry index (a1) inter–chromosomal asymmetry index (a2) coefficient of variation of chromosome length (cvcl) coefficient of variation of centromeric index (cvci) bl71 2.52±0.54 0.29±0.02 0.19±0.03 19.46±2.92 16.61±1.14 bl72 2.72±0.30 0.29±0.03 0.22±0.03 22.48±2.72 18.61±1.68 bl–d75 2.92±0.15 0.31±0.06 0.22±0.04 22.35±3.63 15.79±1.83 bl–x3 1.49±0.30 0.33±0.07 0.14±0.03 13.71±3.18 19.58±4.55 rd14 2.18±0.06 0.31±0.01 0.18±0.03 17.65±2.80 20.08±3.43 rd31 4.65±0.95 0.33±0.02 0.23±0.05 23.28±4.90 16.04±2.22 rd36 2.54±0.30 0.26±0.01 0.17±0.01 16.75±1.13 9.80±0.38 rd47 2.48±0.18 0.26±0.05 0.18±0.01 17.78±0.12 13.66±2.14 rd56 2.62±0.38 0.28±0.03 0.20±0.01 19.76±0.73 13.56±3.10 rd58 3.07±0.14 0.27±0.01 0.25±0.02 25.17±2.28 18.04±4.42 rd8 3.06±0.34 0.32±0.06 0.23±0.02 22.78±2.36 17.10±4.25 rd–d88 3.11±1.01 0.30±0.04 0.21±0.05 21.48±5.02 18.46±3.91 rd–x2 2.90±0.59 0.32±0.10 0.18±0.03 18.42±2.72 16.81±2.54 wh32 2.73±0.74 0.31±0.04 0.20±0.03 19.47±2.67 17.54±2.46 wh82 2.56±0.27 0.25±0.02 0.20±0.03 20.35±3.37 12.35±0.94 wh–x1 2.29±0.46 0.30±0.08 0.19±0.02 18.78±1.74 13.68±3.97 yel59 2.34±0.27 0.27±0.03 0.19±0.02 19.44±2.22 14.69±3.76 yel61 2.75±0.95 0.34±0.03 0.20±0.05 20.14±4.61 18.63±5.77 yel63 2.53±0.70 0.30±0.05 0.22±0.04 22.05±4.30 18.24±2.49 yel7 2.89±0.60 0.29±0.02 0.18±0.02 18.32±2.18 14.65±0.44 yel–x4 2.26±0.80 0.32±0.12 0.15±0.03 15.52±3.59 17.93±7.93 local variety name karyotype asymmetry index (ai) mean centromeric asymmetry (mca) symmetric index (syi) karyotype asymmetry index percentage (ask%) bl71 3.21±0.24 16.80±1.39 144.12±5.02 58.22±0.96 bl72 4.20±0.83 17.47±2.05 146.47±6.20 58.65±1.06 bl–d75 3.51±0.51 18.30±3.74 150.64±14.17 59.20±2.13 bl–x3 2.59±0.23 20.43±5.43 157.21±20.92 60.24±3.18 rd14 3.48±0.18 19.08±0.69 154.02±2.09 59.79±0.36 rd31 3.66±0.27 19.46±1.48 153.07±5.90 59.67±0.93 rd36 1.64±0.05 14.55±0.73 136.77±2.15 57.02±0.27 rd47 2.43±0.39 15.04±3.37 138.12±10.76 57.23±1.88 rd56 2.69±0.70 16.18±2.37 142.12±7.60 57.73±1.37 rd58 4.61±1.47 16.34±1.52 141.87±5.68 57.92±0.96 rd8 3.91±1.15 19.02±4.76 152.55±17.95 59.48±2.49 rd–d88 3.97±1.37 18.32±3.14 147.19±9.70 58.66±1.44 rd–x2 3.14±0.94 19.26±7.26 153.63±27.35 59.52±4.02 wh32 3.41±0.60 18.70±2.72 150.94±9.46 59.37±1.43 wh82 2.53±0.57 14.24±1.45 135.69±3.16 56.77±0.71 wh–x1 2.53±0.54 17.70±5.75 147.95±18.65 58.70±2.96 yel59 2.81±0.45 15.52±2.37 140.52±9.14 57.62±1.52 yel61 3.57±0.18 20.85±2.73 158.16±9.74 60.31±1.53 yel63 4.09±1.36 17.98±3.48 149.65±12.98 59.01±2.15 yel7 2.68±0.27 17.03±1.24 145.88±4.32 58.31±0.48 yel–x4 2.66±0.84 19.28±8.95 154.89±32.99 59.51±4.74 38 sreetama bhadra, zhi–quan cai somatic cells of all the cultivars exhibited a uniform chromosome number of 2n= 36 (table 1, figure 2). lengths of chromosomes in all the cultivars varied from 0.63 μm to 6.53 μm, with the highest average chromosome length being present in the cultivar bl–x3 and the lowest in wh–x1 (figure 3 a–f). short arms were 0.25 μm to 2.95 μm in length, while long arms showed a variation of 0.38 μm to 3.66 μm. centromeric indices ranged from 50% to 14.79% resulting in primary constrictions varying from median to sub-terminal types, though one cultivar did not possess any chromosome with median constriction and chromosomes of five cultivars did not exhibit sub-terminal constrictions. number of chromosomes with each type of primary constriction also varfigure 2. somatic metaphase chromosomes of chenopodium quinoa (2n=36): (a) bl71 (b) bl72 (c) bl–d75 (d) bl–x3 (e) rd14 (f) rd31 (g) rd36 (h) rd47 (i) rd56 (j) rd58 (k) rd8 (l) rd–d88 (m) rd–x2 (n) wh32 (o) wh82 (p) wh–x1 (q) yel59 (r) yel61 (s) yel63 (t) yel71 (u) yel–x4 (bar= 5 μm). 39karyological variability and chromosomal asymmetry in highland cultivars of chenopodium quinoa figure 3. idiograms of some cultivars of chenopodium quinoa: (a) rd14 (b) rd31 (c) rd56 (d) bl71 (e) bl72 (f) bl–x3 (g) comparative graphical representation of distribution of constriction types in the cultivars of chenopodium quinoa (m= median constriction, m= nearly median constriction, sm= sub–median constriction, st= sub–terminal constriction, sec= secondary constriction). 40 sreetama bhadra, zhi–quan cai ied among the cultivars. all of the cultivars revealed 2 chromosomes possessing secondary constrictions, with one constriction in median or nearly median region and the other in sub–median region (figure 3 g). the interand intra-chromosomal asymmetry indices were calculated on the basis of chromosome lengths and centromeric indices. stebbins classification (1971) placed the karyotype of quinoa in 2b category while ai ranged from 1.64 in rd36 to 4.61 in rd58. the complementary indices a1 and a2 ranged from 0.25 to 0.34, and, 0.15 to 0.25 respectively. values of cvcl and cvci varied from 15.52–25.17, and, 9.80–20.08 respectively, while that of mca was 14.24–20.85. values of average ar ranged from 1.38 to 1.66, with the lowest recorded in yel61 and highest in rd14. the values of syi and ask% ranged from 135.69 to 158.16, and, 56.77% to 60.31% respectively. discussion domestication of plants has been impacted by polyploidy since the beginning of agriculture, with approximately 40–70% of cultivated plants exhibiting polyploidy (hilu 1993, sattler et al. 2016). polyploids, especially allopolyploids show hybrid vigour with increased growth rates and higher productivity that favours their artificial selection over their diploid progenitors (renny– byfield and wendel 2014). considering goldblatt’s (1980) assumption of polyploid determination, quinoa with the basic chromosome number of x= 9, is a tetraploid with 2n= 36 somatic chromosomes, a condition similar to the closely related species c. hircinum and c. berlandieri (fuentes–bazan et al. 2012; mandák et al. 2016; jarvis et al. 2017). studies have endorsed its allotetraploid nature, hypothesizing possible hybridization between a north american and a eurasian diploid species, whose identities are yet unknown (ward 2000; jarvis et al. 2017). however, polyploid establishment and propagation is often hindered by irregular meiotic segregation of chromosomes that lead to chromosomal abnormality and reproductive sterility (renny–byfield and wendel 2014). on the contrary, stability in the number of somatic chromosomes of this species, especially in those cutivars that were reported from bolivia, chile and peru (cárdenas and hawkes 1948; gandarillas and luizaga 1967; kolano et al. 2012), and the apparent absence of evidences of mixoploidy in the highland varieties investigated in the present study, both failed to give credence to sporadic reports of mixoploidy (kawatani and ohno 1950; gandarillas 1979; wang et al. 1993). this could in effect perhaps lead to the high fertility of quinoa (ward 2000) that, along with its self–pollinating nature, has facilitated its extensive propagation, especially in new regions. karyomorphological trait variations resulting from structural changes of chromosomes, primarily as a result of rearrangement of chromosomal parts including translocation, inversion and/or deletion, is an important character for understanding relationship among closely related taxa (peruzzi and altinordu 2014). however, no published record of structural details of somatic chromosomes of quinoa was available until the beginning of this century. present study recorded small somatic chromosomes with higher length variations, between 0.63–6.53 µm, and the longest chromosome was almost double the length of previously recorded longest chromosome which was 3.30 µm (kolano et al. 2001; palomino et al. 2008; yangquanwei et al. 2013). this difference can be attributed to (a) the variable cytological techniques used in these studies that affect the degree of chromosomal condensation (palomino et al. 2008), and, (b) the use of only 1–2 varieties of quinoa in the previous studies that exempted any scope of understanding existing cytological variations in the species. the latter reason stated here might also be true for the limited variations observed in the position of primary constrictions in the past studies. chromosomes with only nearly median constrictions were observed by palomino et al. (2008) and bhargava et al. (2006), with only a single variety revealing 2 chromosomes with sub-median constrictions being reported by bhargava et al. (2006). this was in contrast with the presence of sub–terminal constrictions in about 75% of the varieties of quinoa investigated in the present study revealing higher degree of intra–chromosomal variations (table 1, figure 3g). the number of chromosomes possessing secondary constrictions in each complement of all the cultivars in the present study, corroborated observations of bhargava et al (2006), though contrasting observations of 2 pairs of secondary constrictions were reported by palomino et al (2008). however, some other studies have reported absence of chromosomes with secondary constrictions (cárdenas and hawkes 1948; gandarillas and luizaga 1967; giusti 1970; gandarillas 1979). chromosomes possessing secondary constriction had median or nearly median primary constriction and secondary constrictions in sub–median position. gross chromosome morphology obtained in the present study, although showed significant variations in both chromosome length and constrictions, did not reveal any characteristic pattern that can be related to the geography or seed morphology of the studied cultivars. study of chromosomal characteristics forms the basis of cytotaxonomy that can be used to understand similarity or divergence among related species, and is based 41karyological variability and chromosomal asymmetry in highland cultivars of chenopodium quinoa on asymmetry indices calculated with the help of chromosome measurements (levitzky 1931; stebbins 1971). since asymmetry is an expression of chromosomal morphology, it is important to compare karyotypes on appropriate statistical grounds and choose correct statistical parameters (peruzzi and altinordu 2014). however, there is a lack of general consensus on the specific karyotype asymmetry parameter to be utilized which necessitates use of multiple available parameters to conclude about the asymmetry status of a taxa and its comparison with relatives. inter-chromosomal asymmetry increases with increasing difference between the lengths of smallest and largest chromosomes of a complement, and, are assessed by calculating a2 and cvcl. intra-chromosomal asymmetry increases with shift in centromeric position from median to terminal constriction in a complement. this is assessed by calculating the values of tf%, syi, ask%, a1, mca and cvci (paszko 2006; peruzzi and eroğlu 2013). in the present investigation, the different cultivars of quinoa showed significantly higher asymmetry than former studies as was evident by the karyotype asymmetry indices (table 1). stebbins asymmetry class, where all the cultivars of the present study were classified as 2b karyotype, endorsed more asymmetric karyotype than 1a and 1b obtained from 7 cultivars by bhargava et al. (2006). ai, with an average value of 3.21±0.62 also indicate the same. however, calculation of these 2 indices are based on a combination of interand intrachromosomal asymmetry, which has been suggested against in recent studies (peruzzi and eroglu 2013). tf% that decreased with increasing asymmetry ranged from 39.69–43.23% with an average of 41.29±0.86%, lower than previously reported values of 43.8–47.4% (bhargava et al. 2006; palomino et al. 2008) affirming higher asymmetry revealed in the present study. similarly, evaluation of ar that ranged from 0.34 to 5.76 in the present study, but showed much less variation (1.00–1.86) revealing lesser asymmetry in the previous studies also imply presence of higher variation among the cultivars assessed in the present study (bhargava et al. 2006; palomino et al. 2008). other asymmetry indices examined in the present study also supported the above contention as was evident by the calculated pearson correlation. inter– chromosomal and intra–chromosomal asymmetry indices revealed strong correlation within themselves (table 2). tf% was found to be negatively correlated with other intra–chromosomal indices of a1 (r= –0.978), cvci (r= –0.770), mca (r= –0.991), syi (r= –0.995) and ask% (r= –1.000) justifying increasing intra-chromosomal asymmetry with decreasing value of tf%. a2 was positively correlated with cvcl (r= 0.992) indicating increasing value with increasing differences in chromosome size. however, inter– chromosomal asymmetry indices were weakly correlated with intra–chromosomal indices, justifying the suggestion of not including both type of indices in a single analysis (peruzzi and eroglu 2013). both interand intra-chromosomal asymmetry are necessary to reveal correlation among related taxa. this is best represented by using bi-dimensional scatter plots that include one inter-chromosomal and one intra-chromosomal parameter in each axis of the plot (peruzzi and eroğlu 2013). restricted distribution of cultivars in the scatter plots generated in the present study comparing the values of a1 versus a2, cvci versus cvcl, and, mca versus cvcl indicated high similarity among the cultivars with respect to chromosomal asymmetry (figure 4). the plot indicated that, although the study revealed consistent differences within the karyotype of each cultivar, the characteristic distinctions were inadequate for cultivar identification, and not related to their geographical origin and seed morphology. overall, high interand intra-chromosomal variability in the quinoa cultivars examined here conclusively exhibited that much variation exists in this species that is yet to be explored exhaustively. during early domestication events, conspicuous divergence of domesticated table 2. values of pearson correlation analysis for intraand inter-chromosomal asymmetry indices   a1 a2 cvcl cvci mca tf syi ask a1 1 a2 –0.071 1 cvcl –0.073 0.992* 1 cvci 0.667* 0.110 0.118 1 mca 0.985* –0.067 –0.070 0.765* 1 tf –0.978* 0.070 0.079 –0.770* –0.991* 1 syi 0.981* –0.121 –0.125 0.745* 0.990* –0.995* 1 ask 0.977* –0.070 –0.079 0.770* 0.991* –1.000* 0.994* 1 *correlation is significant at 0.01 level. 42 sreetama bhadra, zhi–quan cai species from their wild relatives resulted from artificial selection and controlled reproduction, especially cross–fertilization, by human intervention, that ended in genetic bottleneck, thereby reducing genetic diversity of cultivated plants with respect to their wild counterparts (tanksley and mccouch 1997; cornille et al. 2014). along with this, early farmers selected few individuals with more desirable traits for next generation cultivation with little effort of conserving the unselected materials that were being pushed to oblivion, thereby causing severe loss of genetic diversity and limiting the gene pool of present day domesticated plants (pan et al. 2016). in recent years, cultivation of local varieties of quinoa has been largely neglected because of increased pressure on local farmers for production of certified high–yielding quinoa varieties in international quinoa market (salazar et al. 2019). the inter–varietal similarity and stability of chromosomal characteristics observed in our studied quinoa cultivars might be an indication of the bottleneck experienced by the plants due to their long domestication history of over 7,000 years (jarvis et al. 2017; salazar et al. 2019), that had allowed enough time to impart homogeneity and stability in their chromosomal structures. loss of diversity in local quinoa gene pool in this way will pose serious threat in future breeding programs for quality improvement that necessitates an imperative exploration of chromosomal variability (jarvis et al. 2017; salazar et al. 2019). this entails an imperative investigation of inter–varietal chromosomal diversity in quinoa that would include chromosomal f luorescent staining techniques, like fish, incorporating a large number of cultivars encompassing varieties growing on the distinct eco–geographical regions of quinoa cultivation, primarily from the highland and the coastal lowland ecotypes. it will provide a comprehensive basis for evaluation, selection, conservation and maintenance of existing germplasm, thereby assisting in future breeding programs. acknowledgement we thank zhang yh from yunnan normal university in kunming for providing us with the instrumentation facility used in this study. funding this work was supported by the grants from the national natural science foundation of china [31670686], post–doctor program in xtbg and the ‘135’ program of chinese academy of sciences [2017xtbg–t02]. figure 4. scatter diagrams for the chenopodium quinoa cultivars of (a) a1 against a2 (b) cvci against cvcl (c) mca against cvcl. 43karyological variability and chromosomal asymmetry in highland cultivars of chenopodium quinoa references altinordu f, peruzzi l, yu y, he x. 2016. a tool for the analysis of chromosomes: karyotype. taxon. 65:586– 592. bennett md, leitch ij. 2005. nuclear dna amounts in angiosperms: progress, problems and prospects. ann bot. 95:45–90. bhadra s, bandyopadhyay m. 2015. karyomorphological investigations on some economically important members of zingiberaceae from eastern india. caryologia. 68:184–192. bhargava a, shukla s, ohri d. 2006. karyotypic studies on some cultivated and wild species of chenopodium (chenopodiaceae). genet resour crop evol. 53:1309–1320. cárdenas m, hawkes jg. 1948. número de cromosomas de algunas plantas nativas cultivadas por los indios en los andes [number of chromosomes of some native plants grown by the indians in the andes]. rev de agricul bolivia. 4:30–32. spanish. cornille a, giraud t, smulders mjm, roldan–ruiz i, gladieux p. 2014. the domestication and evolutionary ecology of apples. trends genet. 30:57–65. fuentes–bazan s, mansion g, borsch t. 2012. towards a species level tree of the globally diverse genus chenopodium (chenopodiaceae). mol phylogenet evol. 62:359–374. gandarillas h. 1979. botanica. quinua y kaniwa. cultivos andinos [botany. quinoa and kaniwa. andean crops]. in: tapia me, editor. serie libros y materiales educativos [educational books and materials series]. colombia (bogotá): instituto interamericano de ciencias agricolas; 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neethirajan s, karunakaran c. 2013. cytogenetic analysis of quinoa chromosomes using nanoscale imaging and spectroscopy techniques. nanoscale res lett. 8:463. caryologia. international journal of cytology, cytosystematics and cytogenetics 73(4): 39-44, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-776 caryologia international journal of cytology, cytosystematics and cytogenetics citation: l. gonçalves rodrigues, j. de senna pereira, j. mena barreto de freitas, c. ubessi, s. bosio tedesco (2020) antiproliferative analysis of aqueous extracts of cabreúva (myrocarpus frondosus) on the allium cepa cell cycle. caryologia 73(4): 39-44. doi: 10.13128/ caryologia-776 received: december 15, 2019 accepted: december 19, 2020 published: may 19, 2021 copyright: © 2020 l. gonçalves rodrigues, j. de senna pereira, j. mena barreto de freitas, c. ubessi, s. bosio tedesco. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. antiproliferative analysis of aqueous extracts of cabreúva (myrocarpus frondosus) on the allium cepa cell cycle luísa gonçalves rodrigues*, julia de senna pereira, jéssica mena barreto de freitas, cassiane ubessi, solange bosio tedesco biology department, federal university of santa maria, santa maria-rs, brazil *corresponding authore-mail: luisagr.bio@gmail.com abstract. myrocarpus frondosus is widely used in brazilian folk medicine for bronchitis, gastritis, ulcers and wound asepsis. however, there is a scarcity of scientific data proving the safe use of tea of this species or if there is any toxic effect on the human organism. this study aimed to evaluate the antiproliferative effect of aqueous extracts of cabreúva on the allium cepa cell cycle. the aqueous extracts were prepared from leaves, bark and roots (dried material) of the species in two concentrations: 2.5 and 5.0 grams in 250 ml of distilled water. the aqueous extract of the leaves was obtained by infusion and the aqueous extract of the bark and roots by decoction. distilled water was used as negative control and glyphosate 2% as positive control. eight groups of four onion bulbs were evaluated and each group corresponded to one treatment and, in each group, 4000 cells were analyzed and the mitotic index was calculated. the results demonstrated reduction of mitotic indices in all treatments when compared to the negative control in water. the aqueous extracts of cabreúva in the studied concentrations have antiproliferative action on the allium cepa cell cycle, ensuring the safe use of tea of this medicinal species. keywords: myrocarpus frondosus, medicinal plant, mitotic index. 1. introduction plants with medicinal potential have been widely used to treat various diseases and are often the only medicine available to the population (fachinetto and tedesco, 2009). however, indiscriminate use coupled with lack of knowledge about the medicinal species can cause harm to health (frescura et al. 2012). the genus myrocarpus is exclusively south american, being myrocarpus frondosus allemão the only species recorded in southwestern paraguay, northern argentina and southern, southeastern and northeastern brazil (lorenzi and matos, 2008). the species m. frondosus belongs to the family leguminosae, is characterized by being a large tree plant and popularly known as cabreúva (cabrera et al. 2012). cabreúva is considered a relevant species because, besides being used as a medicinal plant, it is also used in reforestation of degraded areas 40 luísa gonçalves rodrigues et al. (sccoti et al. 2011; santi et al. 2017). it is popularly used for diarrhea, gastritis, ulcers, wound asepsis, has expectorant effect, anti-inflammatory and antimicrobial activity (pereira junior et al. 2014; santi et al. 2017). despite its widespread use in folk medicine, there are no scientific studies to prove its effectiveness and / or rule out possible unwanted or adverse effects from its consumption. thus, this fact highlights the need for studies that evaluate the action of aqueous extracts of the species myrocarpus frondosus on organisms. the effects of aqueous extracts can be assessed by testing with bioindicators, which generally include subsystems of a complete organism used to identify a specific target (leme and marin-morales, 2009). from the results obtained, the mitotic index is calculated. mitotic index values are used as indicative of proper cell proliferation and measured by the allium cepa plant test system. this test has been widely used as a genotoxicity bioindicator (tedesco and laghinghouse, 2012). the efficiency of the allium cepa system is due to its proliferation kinetic characteristic, rapid root growth, large number of dividing cells, high tolerance to different cultivation conditions, permanent availability, easy handling, reduced chromosome number (2n = 16) and easy viewing under the microscope (caritá and marin-morales, 2008). and from this test it is possible to monitor the effects of medicinal plant extracts, ensuring their safe use by the population in the treatment of diseases and sporadic consumption (ubessi et al. 2019). considering the medicinal importance of the species myrocarpus frondosus and the lack of information regarding its antiproliferative activity, the effect of aqueous extracts from leaves, bark and roots on the allium cepa cell cycle was evaluated. 2. material and methods 2.1. obtaining plant material the plant material of the species myrocarpus frondosus was collected from a population located in the west of santa maria, rio grande do sul, brazil, under the coordinates 29°41’23.6”s 53°50’45.3”w. the experiment was carried out at the plant cytogenetics and genotoxicity laboratory at the federal university of santa maria (ufsm). 2.2. preparation of aqueous extracts for the preparation of aqueous extracts leaves, bark and roots (dried material) were used in two concentrations: 2.5 and 5.0 grams (g) in 250 ml of distilled water. the leaves were placed in a container containing boiling water, remaining infused for 10 minutes. the extracts of the bark and roots of cabreúva were prepared by decoction in a period of 10 minutes. all extracts after 10 minutes were strained and stored until room temperature. 2.3. allium cepa test the allium cepa test was developed at the plant cytogenetics and genotoxicity laboratory (ufsm) and organized into eight groups of four onion bulbs, which were placed for rooting in distilled water for a period of 72 hours. distilled water was used as negative control and glyphosate 2% as positive control. the evaluated treatments are described in table 1. after rooting, the bulbs remained in contact with the treatments described in table 1 for a period of 24 hours. time lapse mentioned, the roots were detached from the bulbs and fixed in carnoy 3:1 (ethanol: acetic acid) for 24 hours at room temperature. soon after, the roots were placed in 70% ethanol and stored under refrigeration until blades preparation. 2.4. preparation of blades two blades per bulb were made, and 500 cells per blades were analyzed, totaling 4000 cells per treatment. the preparation of the blades was performed according to the crushing technique (guerra and souza, 2002). in this procedure the roots were washed in distilled water and hydrolyzed for 5 minutes in hcl 1n at room temperature. they were then washed again in distilled water with removal of the meristematic region and stained with acetic orcein 2%. the analysis of these blades was performed under a 40x magnification optical microscope, taking into account the phase of the cell cycle in which the cells were present, such as interphase, protable 1. treatments evaluated in the allium cepa test. treatments t1. distilled water negative control. t2. glyphosate 2% positive control. t3. infusion of 2.5 g of dried leaves. t4. infusion of 5.0 g of dried leaves. t5. decoction of 2.5 g of dried bark. t6. decoction of 5.0 g of dried bark. t7. decoction of 2.5 g of dried roots. t8. decoction of 5.0 g of dried roots. 41antiproliferative analysis of aqueous extracts of cabreúva (myrocarpus frondosus) on the allium cepa cell cycle phase, metaphase, anaphase and telophase. to calculate the mitotic index was considered the sum of the number of cells in prophase, metaphase, anaphase and telophase, divided by the total number of cells observed (sehgal et al. 2006; vieira et al. 2009). the result is presented as a percentage. 2.5. statistical analysis the data related to the mitotic index were submitted to the chi-square test (χ2) with the aid of the statistical program bioestat 5.3 (ayres et al. 2007). 3. results the results found for the controls and treatments evaluated in relation to the mitotic index (mi) are presented in table 2. the cells observed for the counting and evaluation of the treatments were in interphase and cell division (figure 1). the negative control (t1) presented higher mi (6.12%), differing significantly from all other treatments evaluated. the positive control (t2) also differed statistically from the negative control, showing lower mi, thus confirming its antiproliferative action. results differed significantly between treatments and controls (table 2). in the comparison between the negative control (t1) (mi= 6.12%) and the positive control (t2) (mi= 4.55%), there was a significant difference and decreased im, which indicates inhibition of cell division. positive control (t2) differed significantly from treatments with bark extract (t5 and t6) and root extract (t7 and t8), and the extracts further inhibited cell division in relation to glyphosate. comparing the treatments of aqueous extracts of leaves at both concentrations (t3 and t4) with glyphosate treatment (t2), there was the same behavior, as the mi did not differ statistically, showing a decrease in cell division. treatments with aqueous extracts of dried bark were significantly different. the extract with higher concentration (t6) strongly inhibited cell division, being the lowest mitotic index observed (2.8%). in relation to treatments with dried root extracts, the two differed statistically from each other, but unlike what occurred with treatments with bark extracts, it was the treatment with the lowest concentration (t7) that most reduced the mi (2.95 %). 4. discussion all treatments differed significantly from the negative water control (t1) demonstrated by the decrease in mi values. this means that there has been a reduction in cell division of the meristematic cells of allium cepa. this decrease indicates antiproliferative activity of aqueous extracts from the leaves, bark and roots of cabreúva at both concentrations used. the observed cells of the onion bulb root that were submitted to the lowest concentrations of extracts prepared by infusion (t3) and decoction (t5) obtained a cellular stimulus, resulting in the increase of the im compared to those with higher concentrations (t4 and t6). this means that the higher concentrations of leaf and bark extracts caused the reduction of mi. in contrast, the aqueous extract from dried roots, at higher concentration (t8), increased the mi compared to the lower concentration treatment (t7). therefore, aqueous extracts of dried leaves and bark table 2. interphase, cell division and mitotic index in allium cepa cells. treatments cells analyzed interphase cells dividing cells mi (%) t1. distilled water 4000 3.755 245 6.12 a* t2. glyphosate 2% 4000 3.818 182 4.55 b t3. 2.5 g de dl 4000 3.811 189 4.72 b t4. 5.0 g de dl 4000 3.816 184 4.60 b t5. 2.5 g de db 4000 3.853 147 3.67 c t6. 5.0 g de db 4000 3.888 112 2.80 d t7. 2.5 g de dr 4000 3.882 118 2.95 d t8. 5.0 g de dr 4000 3.858 142 3.55 c total 32000 30681 1319 *averages followed by the same letter in the column do not differ from each other by the χ2 test at a 5% error probability level. dl= dried leaf; db= dried bark; dr= dried root; mi= mitotic index. figure 1. cell cycle phases of allium cepa with interphase cell and division cells. interphase (a). prophase (b). metaphase (c). anaphase (d). telophase (e). scale 10 µm. 42 luísa gonçalves rodrigues et al. show antiproliferative capacity, especially at high concentrations. by studying luehea divaricata in two populations and at concentrations of 6 g l-1 and 30 g l-1, frescura et al. (2012) also observed this same behavior regarding cell proliferation. the higher concentration led to a decrease in mi values, concluding that there was an increase in antiproliferative capacity with increasing concentration. coelho’s research (2013), analyzing two populations of echinodorus grandif lorus at concentrations of 6 g l-1 and 24 g l-1, also found increased antiproliferative activity at the highest concentration, except for a commercial extract treatment. in this study, regarding the aqueous extracts of leaves, there was no significant difference between the concentrations studied (table 2). similarly, kuhn (2015), when analyzing the peltodon longipes species, observed that leaf extracts at different concentrations (5 g l-1 and 15 g l-1) did not differ significantly. other tree species such as aroeira (myracrodruon urundeuva) (trentin et al. 2013), graviola (annona muricata), ipê-roxo (handroanthus impetiginosus) (melo et al. 2010), angico-branco (anadenanthera colubrina) (lima et al. 2014) and pau-ferro (libidibia ferrea) (guerra et al. 2017) also have antiproliferative activity found in their extracts from various plant parts, such as bark and leaves. some species even belong to the same family as myrocarpus, reaffirming the results obtained in this study. myrocarpus frondosus bark extracts had an mi of 3.67% at a concentration of 2.5 g, while the highest concentration had an mi of 2.8%, reducing cell proliferation when the concentration of the extract increased. there was a significant difference between the two concentrations (table 2). however, other authors, such as frescura et al. (2012), studying the species luehea divaricata, did not observe significant differences between bark extracts at concentrations 32 g l-1 and 160 g l-1. considering the results observed for the root decoctions at different concentrations, there is a difference between the concentrations, because the inhibition of cell division was smaller as the extract concentration increased (table 2). studying aqueous extracts of lavandula angustifolia roots at a concentration of 0.29 g in 250 ml of distilled water, freitas et al. (2016) found antiproliferative potential compared to controls. however, rodrigues et al. (2017), analyzing the same species in higher concentration (3.75 grams in 200 ml of distilled water) observed proliferative potential. the increased concentrations of aqueous extracts of lavandula angustifolia roots induced an increase in the proliferative capacity of the species, a behavior also observed in this study with myrocarpus frondosus roots. when studying the roots of myrocarpus frondosus in vivo and in vitro, bottamedi et al. (2018) found antioxidant and anti-inflammatory activity which were attributed to the presence of flavonoids and phenolics. in addition, the analyzes performed on the essential oil of myrocarpus frondosus leaves showed the presence of α-thujene, α-pinene, sabinene, β-pinene, mircene, p-cymene, limonene, β-bourbonene, β-caryophyllene, dgermacrene, bicyclogermacrene, spatulenol and globulolem, with predominantly β-pinene and bicyclogermacrene (cabrera et al. 2012; santi et al. 2017). in other studies with echinodorus grandiflorus (coelho, 2013), baccharis trimera, baccharis articulata (fachinetto and tedesco, 2009), caesalpinia echinata (bastos et al. 2011) and myracrodruon urundeuva (romano et al. 2013) were also found substances like flavonoids and tannins. these chemical components were attributed to the antiproliferative capacity presented by the researched plants mentioned above. flavonoids exert a broad spectrum of health-beneficial biological activities, including the antiproliferative effect on cancer cells (gibellini et al. 2011; tsai et al. 2016), reaffirming the results obtained with the species myrocarpus frondosus. 5. conclusion the aqueous extracts of leaves, bark and roots of myrocapus frondosus at concentrations of 2.5 and 5.0 grams have antiproliferative effect on the cell division of allium cepa meristematic cells. aqueous extracts from the 5.0 gram myrocapus frondosus bark exhibit high antiproliferative capacity. the antiproliferative activity found in the species myrocapus frondosus may be associated with the presence of flavonoid and phenolic compounds in the plant tissue composition of the species. acknowledgments to cnpq (conselho nacional de desenvolvimento científico e tecnológico) and capes (coordenação de aperfeiçoamento de pessoal de nível superior) for the funding of the research project. references ayres m, ayres júnior m, ayres dl, santos as. 2007. bioestat 5.0: aplicações estatísticas nas áreas das ciências biológicas e médicas. belém, pa, brasil. 43antiproliferative analysis of aqueous extracts of cabreúva (myrocarpus frondosus) on the allium cepa cell cycle bastos ivga, silva gkc, rodrigues gcr, melo cm, xavier hs, souza ia. 2011. estudo fitoquímico preliminar e avaliação da toxicidade aguda do extrato etanólico bruto de caesalpinia echinata lam. rev. bras. farm. 92: 219-222. http://www.rbfarma.org.br/ files/rbf-2011-92-3-23.pdf bottamedi m, nascimento mvps, fratoni e, moon yjk, dalmarco em, mendes bg. evaluation of antioxidant and anti-inflammatory action (in vivo and in vitro) from the trunk barks of myrocarpus frondosus allemão (cabreúva). in: 25° simpósio de plantas medicinais do brasil, são paulo/brasil, 2018. http:// www.eventus.com.br/plantasmedicinais2018/anais_ xxv_simposio_plantas_medicinais_2018.pdf cabrera dc, gomes gls, schmidt n, flach a, costa lama, rosa gr, moura nf. 2012. composição química das folhas da espécie myrocarpus frondosus do sul do brasil. in: 35ª reunião anual da sociedade brasileira de química, são paulo/brasil. http://sec. sbq.org.br/cdrom/35ra/resumos/t0374-1.pdf caritá r, marin-morales ma. 2008. induction of chromosome aberrations in the allium cepa test system caused by the exposure of seeds to industrial effluents contaminated with azo dyes. chemosphere. 72: 722-725. https://doi.org/10.1016/j.chemosphere.2008.03.056 coelho apd. 2013. potencial genotóxico e antiproliferativo dos extratos de echinodorus grandiflorus e sagittaria montevidensis (alismataceae). 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potential of isolated prostate cancer cells. anticancer res. 36(12): 6367-6380. https://doi.org/10.21873/anticanres.11234 ubessi c, tedesco sb, silva cb, baldoni m, krysczun dk, heinzmann bm, rosa ia, mori nc. 2019. antiproliferative potential and phenolic compounds of infusions and essential oil of chamomile cultivated with homeopathy. j. ethnopharmacol. 239: 1-7. https://doi.org/10.1016/j.jep.2019.111907 vieira a, guimarães ma, david gq, karsburg iv, campos anr. 2009. efeito genotóxico da infusão de capítulos florais de camomila. r. trop.: ci. agr. biol. 3: 8-13. https://www.researchgate.net/publication/246044615_evaluation_of_genotoxic_effects_of_ chamomile_floral_chapters_infusion_efeito_genotoxico_da_infusao_de_capitulos_florais_de_camomila caryologia. international journal of cytology, cytosystematics and cytogenetics 74(1): 109-116, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-881 caryologia international journal of cytology, cytosystematics and cytogenetics citation: f. fallah, f. ghahremaninejad (2021) ploidy level determination of hedera (araliaceae) with an emphasis on discussable species (hedera hibernica). caryologia 74(1): 109-116. doi: 10.36253/caryologia-881 received: march 19, 2020 accepted: october 02, 2020 published: july 20, 2021 copyright: © 2021 f. fallah, f. ghahremaninejad. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid ff: 0000-0003-0165-1369 fg: 0000-0001-5860-9976 ploidy level determination of hedera (araliaceae) with an emphasis on discussable species (hedera hibernica) fahimeh fallah, farrokh ghahremaninejad* department of plant sciences, faculty of biological sciences, kharazmi university, tehran, postal code 15719-14911, iran *corresponding author. e-mail: fgh@khu.ac.ir abstract. genome size is a helpful tool for circumscribing taxa at diverse taxonomic degrees (mostly species) and resolving intricate low-level taxonomies. the correct genome size in hedera (araliaceae) has long been discussed, and the ploidy levels of some taxa are still unclear. twelve accessions of hedera were measured via flow cytometry. flow cytometry is a relatively rapid, inexpensive, and credible tool. fresh leaves of hedera samples and internal reference standard parsley (petroselinum crispum) were stained with propidium iodide (pi). flow cytometry measurements showed that for the accessions of 2cv (3.09 6.40 pg), the lowest amount of nuclear dna was 3.09 pg for hedera crebrescens (so), while the highest amount was 6.40 pg for h. hibernica “hamilton,” representing a statistically significant difference. according to this study, the new taxon (h. crebrescens) is a diploid, though this taxon was previously considered h. hibernica (tetraploid). keywords: flow cytometry, petroselinum crispum, genome size, hedera crebrescens. introduction hedera l. is an evergreen woody vine native to europe, asia, and north africa, but it is cultivated worldwide (rose 1996; reichard 2000; ackerfield and wen 2002, 2003). the taxonomic history of the hedera taxa is complicated because of its two-part life cycle, extensive geographic distribution. juvenile phase with palmately lobed leaves on steril stems and adult phase with unlobed cordate adult leaves on fertile flowering stems. the juvenile and adult shoots also differ, the juvenile being slender, pliable and scrambling or climbing with small aerial roots to fix the shoot to the substrate (rock or tree bark), the adult shoots thicker and without aerial roots. (rose 1996; rutherford et al. 1993; ackerfield and wen 2002). taxonomic recognition was first afforded to juvenile and adult plants, which were described by linnaeus (1753) as “h. helix l.” and “h. arborea (l.) walter.” a previous investigation on hedera recognized five species (lawrence and schulze 1942). however, later studies on hedera have suggested that these five species should be subdivided into more species (ackerfield 2001). 110 fahimeh fallah, farrokh ghahremaninejad furthermore, a key to the classification of species and subspecies of hedera has been derived based on trichome and leaf morphology (ackerfield and wen 2002). recently the european garden flora has reported that there are 12 hedera taxa (mcallister and marshall 2017). however, the definition of species and identification of taxa are still disputed (valcárcel and vargas 2010). since ancient times, many hedera cultivars have been used in europe for cover green and garden decorations (rose 1996) and a remarkable number of cultivars have been identified as h. hibernica (g. kirchn.) bean and h. helix subsp hibernica (g. kirchn.) (d.c. mcclinton). based on differences in trichome positioning, leaf form, and chromosome number, h. hibernica has been recognized (mcallister and rutherford 1990) as a distinct species from h. helix. it has been reported (mcallister and rutherford 1990; jacobsen 1954) that h. helix and h. hibernica have different chromosome numbers, with h. helix being diploid (2n=48) and h. hibernica being tetraploid (2n=96). h. hibernica was thought to be a unique tetraploid species, and hence, it was carefully compared with a typical diploid h. helix. however, recent molecular data indicate that h. helix and h. hibernica may represent differrent species (metcalfe 2005). h. helix (diploid) has been the maternal parent for the tetraploid h. hibernica (ackerfield and wen 2003). sometimes, diploid and polyploid species of the hedera genus are barely distinct morphologically or through dna sequencing data (green et al. 2011). the taxonomic and evolutionary significance of variations in genome size has been established, and chromosomal data are extensively used in plant taxonomy (stace 2000; kron et al. 2007; ekrt et al. 2009). increasing ploidy usually results in increased cell size. plants with increased ploidy levels may be apparently distinct morphologically. flow cytometry is a very useful tool for measuring dna content and can be related to the ploidy level for a specified taxon (sharma and sharma 1999). although genome size in hedera has long been disputed, the genomic dna amounts of many taxa are still unknown (domoney and timmis 1980; polito and alliata 1981; konig et al. 1987). however, molecular studies based on flow cytometric measurements have revealed that polyploidy has been significant to the evolution of the hedera species and might have taken place many times independently in various lineages (green et al. 2011). recent reports have indicated that some taxa with different morphological and cytological characteristics are spreading in semi-natural habitats and urban areas that contain escaped gardens (udvardy and bényei-himmer 1999). recently, by studying iv y diversity in hungary, researchers have identified a prominent hedera taxon that has a particular habit, contains a set of distinguishable morphological and phonological features, and has various environmental demands. previously, this was thought to be h. hibernica (bényei-himmer et al. 2017). however, bényei and höhn (2017) recently identified this taxon as h. crebrescens. the purpose of the present study was to use flow cytometry measurements to clarify the situation of the hedera taxa that is spreading in hungary and other countries in central europe, was previously identified as h. hibernica, and has been recently identified as h. crebrescens. materials and methods hedera specimens were collected from different natural habitats in central europe and from the soroksár botanical garden of budapest (fig. 1). genome size was examined by flow cytometery. following ramsey (2007) completely expanded fresh leaf tissues from each sample (0.5 g) were manually chopped with a sharp razor blade for approximately 1 min in petri dish with the same amount of leaf tissue from diploid petroselinum crispum (apiaceae) as an internal reference standard (fig. 2), in 2 ml of isolation buffer (3.6 g hepes, 2 ml of a 0.5 m solution of edta, 6.0 g kcl, 1.2 g na cl, 102.7 g sucrose, 2 ml triton x-100, 1 ml ß-mercaptoethanol, and 0.1 g spermine in 1.0 l distilled water). following that, nuclear suspension was filtered through a nylon mesh (25 μm) to remove debris and then stained with propidium iodide (pi) at a final density 100 μg. ml−1 and complemented with 100 μg. ml−1 ribonuclease a (rnas). 1c-value was calculated based on the converting formula (dolezel et al. 2003) [1pg=978 mega base pairs (mbp)]. the relationship between mean (n=3) 2c-values of leaf samples were processed and the resulting fluorescence histograms were analyzed with flomax software. the total dna amount of a sample was calculated based on the values of the g1 peak means as follows (dolezel et al. 2003, 2007; dolezel and bartos 2005.) (sample 2c dna (pg) content= [(sample g1 peak mean) / (standard g1 peak mean)]×2c dna amount standard). results and discussion as presented in table 1, 12 taxa of hedera genus were evaluated by partech flomax software ver. 2.0.01 in order to assess the nuclear dna contents (pg) and genome sizes (mbp). among 8 diploids examined, the 111ploidy level determination of hedera (araliaceae) with an emphasis on discussable species (hedera hibernica) lowest amount of nuclear dna was 3.09 pg for h. crebrescens (so) and 3.33 pg for the h. crebrescens (jh). as a result, a statistically insignificant difference of 0.24 pg was observed between four tetraploids while a statistically significant difference in 2c-value (0.64 pg) in the range of 5.76-6.40 pg is noticed between h. hibernica ‘‘variegata’’ (5.76 pg) and h. hibernica ‘‘hamilton’’ (6.40 pg). determination of genome size and ploidy level are summarized in (table 1) and results analyzing the amount of nuclear dna are shown in (fig. 3, 4). sometimes in hedera species the related diploid and polyploidy are poorly distinct via morphology and dna sequence data (green et al. 2011). although polyploidy in the hedera genus is common, the occurrence of auto and allopoly-ploidy is poorly understood (yi et al. 2004). trichome morphology and leaf shape analyses revealed that h. hibernica is an allopolyploid from h. helix and h. maroccana (mcallister and rutherford 1990). furthermore, based on nucleotide polymorph isms in nrdna (its) h. hibernica was recognized as an allopolyploid (vargas et al. 1999). it has commonly been believed that internal reduplication is correlated with very small genomes, assuming that minimal dna content is needed for suitable cell functioning. in fact, taxonomists have surely realized that some related species with the same number of chromosomes might be different in terms of dna volume, thus making them easily distinctive by using flow cytometry. recent studies on vascular plants revealed only a weak negative correlation between genome size and degree of polyploidyization (barow and meister 2005). thus, c-values should be treated as a fundamental scal ing factor in living systems (bennett et al. 2000). the specimens examined in the present work by flow cytometry measurements showed that the accessions 2c-value (3.06-6.40 pg) veryfying more than a two-fold variation and showing a corresponding genome size of 1516-3129 mbp. among the eight examined diploids, the lowest amount of nuclear dna was 3.09 pg (for h. crebrescens (so)), while the highest amount was figure 1. images of hedera species analysed in this study: a,b: h. helix, c: h. helix arborescence, d,e: h. hibetnica, f: h. hibernica arborescence, g,h: h. crebrescens, i: h. hibernica “deltoiedea”, j, k: h. hibernica” variegata”, l: h. hibernica “hamilton”. 112 fahimeh fallah, farrokh ghahremaninejad 3.33 pg (for h. crebrescens (jh)). statistically in significant differences were found between four tetraploids. meanwhile, significant differences of 0.64 pg in 2c value (ranging from 5.76-6.40 pg) were recognized for h. hibernica “variegata” (5.71 pg) and h. hibernica “hamilton” (6.40 pg). the results attained by marie and brown (1993) indicate that for h. helix, 2cv=8.18 pg, which is strongly refuted by the data presented in the current study and stands as an uncom mon value for dna amounts (bennett and leitch 1997). petunia hybrid vilm. was used by marie and brown (1993) as a standard reference. h. helix which studied by marie and brown from strasburg-france (latitude n 48°34’50.959”, longitude e 7°45’49.623”) is not very far from to region we were collected h.helix, according to the present study, the data for h. helix (2cv=4.6 pg) reported by domoney and timmis (1980) remains unsuported. as reported previously, h. helix 2cv=2.95 (konig et al. 1987), h. helix 2cv=2.80 (obermayer and greilhuber 2000) and h. hibernica 2cv= 6.00 (zonneveld et al. 2005), which are close to the present results. the mor phometric analyses of h. helix, h. hibernica “hamilton” and the new taxon (h. crebrescens) were based on vegetative and generative organs and were conducted to distinguish the new taxon from h. helix and h. hibernica (clarke et al. 2006). the genome size, which represents an inherent attribute, is a supportive feature for circumscribing taxa of various taxonomic ratings (mainly species) and resolving intricate low-level taxonomies (loureiro et al. 2006). the analysis based on flow cytometry indicated that h. crebrescens can be considered a distinct taxon among the diploid ivies. according to the present study, which is strongly supported by the notion of a newly identified diploid taxon (hedera crebrescens) (bényei and höhn 2017), it should be emphasized that this taxon which was previously treated as h. hibernica is not identical to the tetraploid taxon. table 1. samples, locality and determination of genome size and ploidy level. samples locality latitude ( n ) longitude ( e ) ploidy level mean 2c value level (pg± se) mean 1c value (pg) mean 1c value (mbp)* % cv h. helix soroksár (hungary) n 47°23’ 18.644” e 19°9’ 2.165” 2x 3.16 ± 0.05 1.58 1545.24 0.02 h. helix arborescence gellért hill (hungary) n 47°29’ 12.898” e 19˚2’ 40.23” 2x 3.16± 0.04 1.58 1545.24 0.01 h. hibernica gellért hill (hungary) n 47°29’ 12.898” e 19˚2’ 40.23” 4x 6.23 ± 0.30 3.11 3041.58 0.05 h. hibernica arborescence soroksár (hungary) n 47°23’ 18.644” e 19°9’ 2.165” 2x 3.17 ± 0.07 1.57 1535.46 0.02 h. hibernica “deltoiedea” soroksár (hungary) n 47°23’ 18.644” e 19°9’ 2.165” 4x 6.33± 0.12 3.16 3090.48 0.02 h. hibernica “hamilton” gellért hill (hungary) n 47°29’ 12.898” e 19˚2’ 40.23” 4x 6.40± 0.07 3.2 3129.6 0.01 h. hibernica “variegata” soroksár (hungary) n 47°23’ 18.644” e 19°9’ 2.165” 4x 5.76± 0.11 2.88 2816.64 0.02 h. crebrescens (v) vienna (austria) n 48°12’ 31.307” e 16°22’ 21.702” 2x 3.19 ± 0.01 1.59 1555.02 0.01 h. crebrescens (jh) jános hill (hungary) n 47°29’ 52.836” e 19˚2’ 23.675” 2x 3.33 ± 0.12 1.66 1623.48 0.04 h,crebrescens (sze) szeged (hungary) n 46°10’ 18.332” e 19˚25’ 22.52” 2x 3.22 ± 0.05 1.61 1574.58 0.02 h. crebrescens (so) soroksár (hungary) n 47°23’ 18.644” e 19°9’ 2.165” 2x 3.09± 0.07 1.54 1506.12 0.02 *1 pg = 978 mbp27 figure 2. the histogram for analysis of the amount of nuclear dna in leaves: parsley (p. crispum) reference standard (2c dna=4.50 pg). 113ploidy level determination of hedera (araliaceae) with an emphasis on discussable species (hedera hibernica) figure 3. the histogram for analysis of the amount of nuclear dna in leaves: the left peaks refer to the unknown hedra samples and the right peaks to the known parsley (p. crispum) reference standard (2c dna = 4.50 pg). a: h. crebrescens (v) = 3.19 pg. b: h. crebrescens (jh) = 3.33 pg, c: h crebrescens (sze) = 3.22 pg. d: h. crebrescens (so) = 3.09 pg. e: h. crebrescens (gh) 5 = 3.16 pg. f: h. helix=3.16 pg. g: h. helix arborescence=3.16 pg, h: h. hibernica arborescence=3.17 pg, diploids hedera. 114 fahimeh fallah, farrokh ghahremaninejad conclusions most of the recently reported new incidences of h. helix from the lowland of hungary (see bartha and király 2015) refer to h. crebrescens. h. crebrescens is the most invasive ivy taxon in hungary and probably most of the countries in central europe. according to flow cytometry results, h. hibernica arborescence is diploid, whereas h. hibernica is tetraploid. therefore, this name is not correct for this taxon, and due to its morphological features, it is probably a subspecies of h. crebrescens. studies that include species from the eastern part of the distribution range of the hedera genus in iran and the caucasus (formerly mentioned by k. koch and g. woronow) are necessary in order to conduct a more thorough survey of hedera’s diversity and relationships. acknowledgements we would like to thank prof. ghasem karimzade, the head of the laboratory biotechnology, faculty of agriculture, tarbiat modares university, tehran, iran for his technical assistance. we would also like to extend our gratitude to prof. jaroslav doležel, the head of the laboratory of molecular cytogenetic and cytometry, institute of experimental botany, sokolovská 6, 456 olomouc, czech republic, for providing seeds of dna reference standards and all his papers. references ackerfield j. 2001. trichome morphology in hedera (araliaceae). edinburgh journal of botany 58(2): 259-267. figure 4. the histogram for analysis of theamount of nuclear dna in leaves: the left peaks refer to the known parsley (p. crispum) reference standard and right peaks to the unknown hedera samples. a: h. hibernica = 6.23 pg, b: h. hibernica “deltoiedea” = 6.33 pg, c: h. hibernica “variegata” =5.76 pg, d: h. hibernica “hamilton” =6.40 p. 115ploidy level determination of hedera (araliaceae) with an emphasis on discussable species (hedera hibernica) ackerfield j, wen j. 2002. a morphometric analysis of hedera l. 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10.13128/caryologia-759 published: december 13, 2019 copyright: © 2019 a. ždralović, a. mesic, i. eminović, a. parić. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. cytotoxic and genotoxic activity of plantago major l. extracts amira ždralović, aner mesic, izet eminović, adisa parić* department of biology, faculty of science, university of sarajevo, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina *correspondence to: e-mail: adisa.p@pmf.unsa.ba abstract. plantago major l. is a perennial, wild plant that belongs to the plantaginaceae family, and is used as a good indicator in the assessment of destructive anthropogenic impact on the environment. the aim of the present study was to evaluate cyto/ genotoxic effects of methanol extracts of plantago major, collected from two locations (tetovo and smetovi), using allium cepa test. we demonstrated that the highest concentration of p. major extracts from both locations reduced the mitotic index, while the lowest increased mitotic index value comparing to the positive control. as for genotoxic effects of extract from tetovo, all concentrations increased the frequency of sticky chromosomes and chromosome missegregations in comparison with both controls, and frequency of multipolar anaphases when compared to the negative control. higher number of cells with vagrants in comparison with positive control was detected after the treatment with 0.005 and 0.02 mg/ml concentrations. p. major extract from smetovi (0.005 and 0.01 mg/ml) induced an increase in the number of vagrants as compared to the positive control, and frequency of sticky chromosomes when compared to both controls (0.01 mg/ml). exposure to extract (0.005 and 0.02 mg/ml) caused increased number of multipolar anaphases in comparison with negative control. apoptosis were not detected for p. major extract from smetovi, while its highest concentration (0.02 mg/ml) induced increase in the frequency of necrosis as compared to the positive control. our results demonstrated that methanol extracts of p. major, collected from tetovo and smetovi, showed cyto/genotoxic effects on a. cepa meristem cells. keywords. plantago major, cyto/genotoxicity, allium cepa test, heavy metals introduction plantago major l. is herbaceous, perennial wild plant from the family plantaginaceae that is distributed throughout the world (samuelsen 2000; thome et al. 2012). low growth and growth in the form of rosettes make this type well-adjustable on trampling, grazing and mowing (thomet 1978). p. major contains biologically active compounds such as polysaccharides, lipids, phenols, flavonoids, iridoid glycosides, terpenoids (samuelsen 2000; chiang et al. 2003), benzoic compounds (chiang et al. 2003), tannins, saponins and sterols (jurišić grubešić et al. 2005). recent research shows that plants used in traditional medicine consumption exhibit mutagenic, geno36 amira ždralović et al. toxic and cytotoxic effects in vitro and in vivo (higashimoto et al. 1993; schimmer et al. 1994; kassie et al. 1996; aşkin çelik and aslantürk 2007). many chemical plant constituents have the ability to react with the dna molecule, and may cause damage in dna structure and/or disruption of biochemical reactions (sofradžija et al. 1989). in order to reduce the risk of application of natural agents, plants and their parts, as well as plant extracts, it is necessary to assess their ability to induce cytotoxic, genotoxic and mutagenic effects (askin celik 2012). an indispensable aspect that must be noted in the modern use of plants for medicinal purposes is the increasing pollution of the environment by human activity. plants actively participate in the circulation of nutrients and gases such as carbon dioxide, oxygen and also provide a big surface area for absorption and accumulation of air pollutants reducing the level of pollutants in the environment (escobedo et al. 2008). numerous studies have shown that p. major l. is good indicator of the degree of destructive anthropogenic impact on the environment (montacchini and siniscalco 1979). furthermore, it has been demonstrated that methanol extracts of p. major  demonstrated cytotoxic activity in different cancer cell lines (kartini et al. 2017). allium cepa assay has been described as an efficient test used for genotoxicity assessment of potential genotoxic agents in the samples taken from the environment, due to its sensitivity and good correlation with mammalian test systems in vitro (firbas and amon 2014; prajitha and thoppil 2016). therefore, the aim of this research was to examine the cyto/genotoxic effects of methanol extracts of p. major sampled from tetovo (polluted location) and smetovi (control location) using allium cepa test. materials and methods plant material plantago major was collected on two locations: tetovo, which is near ironworks arcelormittal (exposed to daily air pollution, dust, sulfur dioxide and other pollutants) and smetovi, located at 1.025 meters above sea level and is a popular resort and hiking destination. plant samples were collected in october 2015. for testing were used leaves of the plant, which were carried out on the same day at both sites in pvc packaging bags and within 3 hours of collection delivered in the laboratory of plant physiology, department of biology on faculty of science, university of sarajevo. plant material was dried at room temperature, away from direct light and stored at + 4°c until analysis. voucher specimens (no. lrper 383-384) were deposited in the laboratory for research and protection of the endemic gene pool. extraction procedure to prepare the plant extracts we used dry plant material and 80% methanol as solvent. 1 g of plant material was chopped and mixed with 40 ml of methanol. incubation period was 24 h at 4°c. after filtration, supernatant was evaporated to a dry residue which was re-dissolved in 80% methanol in three concentrations: 0.02; 0.01; 0.005 mg/ml. along three concentrations of extracts (from each location), we tested two controls: positive (80% methanol) and negative (distilled h2o). allium test for detection of cyto/genotoxic effects of p. major l. extracts, allium cepa bulbs were used. onion bulbs were grown in the glass vessels filled with tap water and left for germination for 48 h at room temperature with water injection as needed. we selected four bulbs for each treatment and measured their root length as previously described by fiskesjo (1993). in this sense, the length of the root bundles from each onion bulbs was measured. the measure is taken from the point where the roots sprout, down to where the most root tips end their growth. afterward, the bulbs were treated with p. major l. extracts for 24h at room temperature. at the end of the exposure period the root lengths of the bulbs were measured. for each treatment the bulb root is removed and placed into the appropriate labeled tubes containing ethanol/glacial acetic acid (3:1, v/v) fixative and kept for 24h at 4°c. cytogenetic analysis a. cepa roots were hydrolyzed in 1m hcl solution for 15 minutes at room temperature. after that, the roots were transferred to distilled water. the apical 2 mm of the root were cut and placed in one drop of 2% acetorcein and squashed. microscope slides were analyzed under the light microscope with a magnification of 400x. all photographs were made by use of sony cyber shoot iso 3200 camera. to calculate the mitotic index values, 1000 cells per slide was analyzed. chromosomal aberrations were analyzed on 100 cells in division per treatment. counting 1000 interphase cells for each concentration, the frequency of micronuclei was determined. apoptosis and necrosis were analyzed at 1000 interphase cells per slide. 37cytotoxic and genotoxic activity of plantago major l. extracts statistical analysis to evaluate differences between tested concentrations and controls, for all analyzed parameters, student’s t-test was used. all statistical analyses were conducted by use of microsoft excel 2007 (microsoft corporation) and spss 20.0 software (spss, chicago, il). p values less than 0.05 were considered statistically significant. results the most important macroscopic parameter in allium test is a root length (fiskesjö 1985). different concentrations of p. major extracts had different effects on root growth (table 1). the extracts of p. major from polluted location inhibited root growth. statistically significant effect had the highest concentration (0.02 mg/ml) of extract from mentioned location when compared to the control plants. concentration of the extract of p. major (0.005 mg/ml) significantly stimulated root growth as compared to the negative control. in order to evaluate the effect of p. major extracts, mitotic activity of a. cepa meristem cells was expressed as a percentage of cells in division in relation to the total number of analyzed cells. statistically significant difference compared to control, was observed in the treatment with p. major extract from smetovi (0.02 mg/ml, and 0,005 mg/ml) (table 2). the highest concentrations of p. major extracts (0.02; 0.01 mg/ml) from tetovo lead to the reduction in mitotic activity compared to the control, but the difference was not statistically significant. the results of the genotoxic and cytotoxic effects of different concentrations of the plantago major extract from two locations are presented in table 3. several genotoxic effects were observed, such as micronuclei, sticky chromosomes, anaphase bridges, vagrant chromosomes, chromosome missegregation, multipolar anaphases, as well as apoptotic and necrotic cells as cytotoxicity endpoints. p. major extract from tetovo (0.005 mg/ml) caused the highest number of chromosome aberrations. similar effects were observed for extract from same location in concentration of 0.02 mg/ml. in this sense, micronuclei (fig. 1a), multipolar anaphase (fig. 1b), sticky chromosomes (fig. 1c), anaphase bridges (fig. 1d), vagrant chromosomes (fig. 1e), chromosome missegregation, were observed. the highest number of apoptotic and necrotic cells (fig. 1f ) is observed after the treatment with the highest concentration (0.02 mg/ml) from this location, while apoptotic cells were not detected at the lowest concentration. unlike tetovo, the frequency of chromosomal aberrations on meristem cells that were treated with extract of p. major from smetovi (non-polluted place), was much lower. chromosome aberrations that have reached statistical significance are: sticky chromosomes, vagrant chromosomes and multipolar anaphases. on table 1 roots length of allium cepa (mean ± sd) before and after the treatment with different concentrations of the tested p. major extracts. concentration (mg/ml) tetovo smetovi methanol h2o roots lengtha roots lengthb roots lengtha roots lengthb roots lengthc roots lengthd roots lengthc roots lengthd 0,005 1.90±0.52 1.87±0.47 1.52±0.15 1.62±0.12* 0,01 2,02±0.17 2,02±0.17 1.87±0.68 1.92±0.65 1,56±0,56 1,56±0,56 1.80±0.29 2,50±0.33 0,02 1.72±0.25 1.67±0.18* 1.70±0.73 1.70±0.74 *statistically significant difference compared to the negative control (p < 0.01). values are expressed in centimeters. sd: standard deviation. a root length in the first 48 h before treatment with different concentrations of the extracts. b root length in the next 24 h after treatment with different concentrations of the extracts. c root length of the control group in the first 48 h. d root length of the control group in the following 24 h. table 2 the mitotic index of allium cepa meristematic cells (mean ± sd) exposed to various concentrations of the samples concentration (mg/ml) tetovo smetovi methanol h2o 0,005 2,10±0.69 4.57±0.49** 0,01 1.27±0.63 2.92±0.94 2.05±0.51 2.65±2.14 0,02 1.65±1.02 0.95±0.65* **statistically significant difference compared to the positive control (methanol) (p < 0.001). values are expressed as a percentage. sd: standard deviation. mi: mitotic index. *statistically significant difference compared to the positive control (methanol) (p < 0.05). 38 amira ždralović et al. the meristem cells treated with p. major extract from this location, apoptotic changes were not observed, while necrotic cells were few, but the numbers were statistically significant only for 0,02 mg/ml concentration (because of low number of necrotic cells compared to the positive control). table 3 the results of the genotoxicity and cytotoxicity in allium cepa (mean ± sd) exposed to various concentrations of the p. major extract. tested extracts (mg/ml) micronuclei sticky chromosomes abnormal ana/telophases apoptotic cells necrotic cells bridges vagrant chromosome missegregation multipolarity tetovo 0,005 0.75±0.95 5±3.16 **2 2±1.41*1 2.5±1.29** 4±1.41**2 9.25±2.873 n.o. 2±1.82 0,01 n.o. 4.50±3*1 0.25±0.50 2.75±2.98 3±1.15**2 7.75±3.502 0.50±1 1.75±1.25 0,02 0.50±0.57 2±1.15**2 1.25±1.25 1.50±1.29* 2.50±1**2 7.50±2.513 0.50±0.57 7±2.16 smetovi 0,005 n.o. 0.50±0.57 0.75±0.95 2.50±1.29* 0.25±0.50 5.25±1.503 n.o. 1.75±2.36 0,01 n.o. 2.75±2.21*1 1±1.41 2.50±1.91* 1.50±0.57 5.50±0.57 n.o 1±2 0,02 0.25±0.50 0.75±0.95 0.50±0.57 3±3.82 1±0.57 7.75±6.021 n.o. 0.25±0.50* methanol n.o. n.o. n.o. n.o. 0.25±0.5 4.25±3.30 n.o. 4.50±3.10 h2o n.o. n.o. n.o. 1±0.81 0.25±0.5 n.o. n.o. 5.25±4.57 statistically significant difference compared to the negative control (h20): 1 p < 0.05, 2 p < 0.01; 3 p < 0.001. sd: standard deviation. statistically significant difference compared to the positive control (methanol) after the t-test: * p < 0.05; ** p < 0.01. fig. 1 genotoxic effects of allium cepa meristem cells treated with p. major extracts from tetovo: amicronucleus, bmultipolar anaphase, csticky chromosomes, danaphase bridge, evagrant chromosomes, fnecrotic cells. 39cytotoxic and genotoxic activity of plantago major l. extracts discussion plant species are an excellent source of biologically active substances, whose effects on genetic material are largely unknown (barnes 2003). toxicity is easy to see in inhibition of root growth, while mutagenicity correlates with chromosomal aberrations (fiskesjö 1985). inhibition of root growth is always parallel with the decline in cell division (fiskesjö 1997), and can be caused by heavy metals in the plant extract. it has been found that the toxicity of extracts from plants which contain heavy metals, such as manganese, cadmium and lead is often associated with these pollutants (boroffice 1990; fiskesjö 1997). recent study from locations tetovo and smetovi demonstrated that p. major is exposed to the negative impact of heavy metals, particularly in the area of tetovo (muratovic 2016). heavy metals imply inhibition of a. cepa root growth. early researches of plant tolerance to the heavy metals have shown that root growth is particularly sensitive to the presence of metal toxins. as a result of root growth cytokinesis, cell differentiation and extensions, metal induced inhibition of root growth is a result of toxic influences, acting on any of the three processes (baker and walker 1989). rajeshwari et al. (2015) proved that aluminum nanoparticles increased the number of chromosomal aberrations in the a. cepa root tip cells and similar results were observed for other heavy metals such as cu, pb, fe, cd, ni, zn etc. (olorunfemia et al. 2015). therefore, our results suggest that inhibited growth of roots that were treated with p. major extract from polluted area of tetovo could be due to presence of the heavy metals in plants. the cytotoxicity of some chemical component, or plant extracts can be determined based on the increase or decrease in the mitotic index (smaka-kincl et al. 1996). reduction of mitotic activity can arise as a result of inhibiting the synthesis of dna molecules in cells or by stopping the g2 phase of the cell cycle through the action of various toxic substances present in plant extracts (sudhakar et al. 2001). in this regard, it is important to accentuate that reduction in mitotic activity is parallel with the root growth inhibition of a. cepa meristem cells, which were observed after exposure to p. major extracts (0.02; 0.01 mg/ml) from tetovo. similar results were observed by askin çelik & aslantürk (2006) with reduction of mitotic index induced by plantago lanceolata l. extracts, which indicates that the substance in the aqueous extract can have a cytotoxic effect. it is proven that extract of p. major reduce cell proliferation in vitro (samuelsson 2004). extracts of plantago species have a cytotoxic effect on different tumor cell lines (richardson 2001) due to the presence of luteolin 7-o-β-glucoside, as the main flavonoid present in most plantago species (galvez et al. 2003). comparing the genotoxic effects of these two locations, we can see that the total number of chromosomal aberrations was higher in cells treated with extract of p. major from tetovo, which was expected because of air pollutants source. smetovi is well known as an excursion site, and perceived aberrations (vagrant and sticky chromosomes) on this location could be explained by gasses from motor vehicles and the presence of waste material. the toxicity of metals in the plant can be manifested with few biological markers that can be detected and analyzed at different levels of the organization, morphology of the plant as well as at the biochemical and molecular level. therefore, they are very useful for plant monitoring and assessment of the environmental pollution (olorunfemia et al. 2015). among various biological effects which could be consequence of environmental pollution, genotoxicology is one aspect that is related to dna damage and genome. according to this, our results are of great value in terms of use of plantago major as an indicator of environmental pollution with heavy metals and other toxic substances. in conclusion, the results of the present study revealed that p. major extracts from polluted location tetovo reduced root growth and mitotic activity of a. cepa meristem cells, and that possess significant cyto/ genotoxic potential. 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www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-856 caryologia international journal of cytology, cytosystematics and cytogenetics citation: a. horozal, ö. aksoy (2020) evaluation of the genotoxicity of some standart and eco-friendly detergents with vicia faba. caryologia 73(4): 129-139. doi: 10.13128/caryologia-856 received: february 17, 2020 accepted: september 30, 2020 published: may 19, 2021 copyright: © 2020 a. horozal, ö. aksoy. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. evaluation of the genotoxicity of some standart and eco-friendly detergents with vicia faba aylin horozal, özlem aksoy* department of biology, faculty of science and literature, university of kocaeli, kocaeli, turkey *corresponding author. e-mail: ozlem.aksoy@kocaeli.edu.tr abstract. the increasing use of detergents as a result of rapid increase of population and irregular industrialization has a big role in the environmental pollution. determination of genotoxic effects of some standart and eco-friendly detergents on vicia faba was aimed in this study. faba beans were treated with detergents for 24, 48 and 72h by using ec50 and 2xec50 values and analysed by mitotic index (mi) and comet assay. according to the results of root inhibition test, ec50 value for the detergent a1 was 20ml/l, a2 was 50ml/l, b1 was 40ml/l, b2 was 60ml/l respectively. the least decrease in the mitotic index was found in detergent b2 (eco-friendly laundry) and the most decrease was found in detergent a1 (standart dishwasher). various abnormalities were determined in different phases of mitosis. after comet assay the cells were classified as type 0, type 1, type 2, type 3 and type 4 according to the nuclei damage rates. type 3 was the most identified cell damage degree in eco-friendly detergents whereas type 4 was the mostly seen in standard detergents. parameters such as olive tail moment, tail dna percentage and arbitrary units were also identified by comet assay. all the test results showed that standard detergents cause more genotoxic effects than eco-friendly ones. keywords: comet assay, detergents, dna damage, chromosome aberations, mitotic index, vicia faba. introduction hundreds of chemicals continue to be produced to make our lives easier. although they are useful in raising our standard of living, we cannot understand the fate of these chemicals without investigating their effects on the living things. plant biotests have an important role in determining the cytotoxic and genotoxic effects of environmental pollutants, chromosome damage and gene mutations. these studies can contribute to the creation of a database about environmental conditions in various regions of the world. the simple and clear results of the biotests can also be used in public awareness training on the genotoxic effects of environmental pollution. recently, pollution from domestic and industrial wastes is increasing and detergents are replaced by a large percentage of effective pollutants (minareci 130 aylin horozal, özlem aksoy ve diğ., 2008). detergents are mixtures of powder, granule, soft consistency or liquid form, which are preferred for cleaning purposes and which contain anionic surfactants and other cleaning agents as primary cleansers (egemen, 2000). laundary detergents are produced in large quantities and used in daily life and industrial activities (wang et al., 2019). various physical and chemical analyzes are carried out to determine environmental pollution of the detergents, however, these analyzes alone are not sufficient. genotoxicity studies are essential to determine the effects of detergents on organisms. many plants have been used for cytotoxicity testing, recent reports being of allium cepa l. (bonciu et al., 2018), vicia faba l., zea mays (bonea and bonciu, 2017), and drimia indica (roxb.) jessop. (daphedar and taranath, 2018). v.faba root tips, can be easily obtained in a whole year, its development and storage is easy and inexpensive, the root meristem comprises a high proportion of cells undergoing mitosis and the number of chromosomes is low (2n = 12) and appropriate for accurate and complete counting. the v.faba root meristem chromosomal mutation assay is also approved by the international programme on chemical safety (ipcs, who) and the united nations environment programme (unep) as an effective and standard test for the chemical screening (youssef and elamawi, 2020). the most important point in the root growth inhibition test is the determination of the effective concentration (ec50) which reduces the root length by 50% compared to the control group (fiskesjö, 1985). in order to determine the toxic and genotoxic effects of environmental pollutants on chromosomes and cell division ec50 values were calculated in many studies (rank and nielsen, 1998; chauhan et al., 1999, ateeq et al., 2002; saxena et al., 2005; arıkan, 2006). mitotic activity tests are frequently used in chromosome counting and toxicological research. by the application of this test, information can be provided about the effect of chemical treatment on cell division (akı and karabay, 2004). cytogenetic methods are widely used in the biological monitoring of populations exposed to mutagenic and carcinogenic compounds. to date, many methods have been used to detect dna damage. one of them is comet analysis that is widely used because it is a sensitive, fast and reliable method for the detection of various types of dna damages. in addition to visual assessment of dna damage by comet technique, parameters related to tail length, tail moment, and percentage of dna in the tail can be determined using comet image analysis programs (collins, 2002). high toxicity may be the cause of the increase in dna damage. in a study on the genotoxic effects of pollutants in some plants, a relationship between concentration and dna damage has been demonstrated (gichner et al., 2006). there are a few studies that have evaluated the toxicity and genotoxicity of commercial eco-friendly detergents in plant bioassays, their toxic effects assessed generally in  daphnia magna (pettersson et al., 2000), some microalgae (aizdaicher and markina, 2006; azizullah et al., 2011), in  salmonella  test and in human leukocytes (pedrazzani et al., 2012). it was aimed to determine the genotoxic effects of four different dishwasher and laundry detergents, two of which were eco-friendly, on v. faba using the root-tip chromosome aberration test of accepted plant biotests. mitotic index and mitotic abnormalities were investigated with cytotoxicity test and dna damage was investigated with the single cell gel electrophoresis (scge) method which is also called as comet assay. thus, it will provide new sights to the environmental pollution and indirectly find out the damage caused to the other organisms via the food chain. materials and methods v. faba (2n = 12) seeds were used as research material obtained from local market. the commercial dishwasher (a1 and a2) and laundry (b1 and b2) detergents, two of which were standart named as a1 and b1 and two of which were eco-friendly named as a2 and b2, were used. chemical content information about detergents was shown in table 1. germination of v. faba seeds and determination of ec50 concentrations surface sterilization was performed by selecting dimensionally and morphologically uniform v. faba seeds. the seeds were shaken in 70% alcohol for 1 minute, after 15 minutes in a 3% sodium hypochlorite containing 2 drops of tween-20 for 200 ml, and then washed 4 times with sterile distilled water. sterilized seeds were kept in sterile distilled water for 12 hours to swell before they were germinated (hamdy and hattori, 2006). petri dishes used to germinate the seeds were first sterilized in autoclave and then exposed to uv light in a sterile cabinet for 15 minutes. sterile whatman papers treated with detergent concentrations were placed into sterile petri dishes placed at regular intervals. 20 pieces of seed were placed in each petri and covered with 131evaluation of the genotoxicity of some standart and eco-friendly detergents with vicia faba whatman papers treated with detergent concentrations and then put into 24 °c incubator. the detergent concentrations used in determination of ec50 values for each detergent was listed in table 2. the root length of the seeds, which were germinated in the incubator for 7 days, were measured for root inhibition test. at the end of one week, the averages of the root lengths were calculated and the decrease in the root lengths were compared with the control group. the test set-ups for calculating the ec50 value were performed in 3 replicates. after the ec50 and 2xec50 values were calculated, all experiments were carried out on these values. chromosomal abberation test at the end of 24, 48 and 72 hours, the roots that were approximately 1-2 cm long were cut with the help of sterile scalpel and transferred to carnoy (3 parts 70 % alcohol, 1 part 45 % glacial acetic acid) fixation fluid. after 24 hours, the root tips were put in 70 % ethyl alcohol and then hydrolised in 1 ml 1 n hcl with the help of the burner flame. the roots that were hydrolyzed in the watch glass were stained with 1-2 drops of 2 % aceto orsein (merck, germany). the cover glass was covered on the stained root tip and the crush-smear preparation technique was applied. photographs of the cells were taken with 40x and 100x lenses to determine chromosome abnormalities with olympus bx51 photomicroscope. in the calculation of the percentage of mitotic index, 500 cells were counted in each of the 10 slides were prepared for each concentration and 5000 cells were counted for each concentration (yüksel and aksoy, 2017). the percentage of mitotic indices was then determined by dividing the number of cells divided by the total cell number and multiplying by 100 for each application concentration (ec50 and 2xec50), duration (24, 48, 72 h) and their control groups (souza et al., 2013). the ratios of chromosomal damage such as chromosome bridge, chromosome adhesion, pole shift and laggard chromosome formation in dividing cells were calculated. scge (single cell gel electrophoresis) analysis 400 µl cold tris buffer solution and 5-6 root tips (1-2 cm) of v.faba were put in petri dishes placed on ice cassettes. the roots were cut gently with the aid of a scalpel until the color of the buffer solution was blurred without taking over the ice cassettes. the resulting suspension was transferred to the ependorf tube and left on ice for 15 minutes to allow the nuclei of the meristematic cells to settle down. the slides were coated with 1% nmp (normal melting point) agarose (roth germany 2267, sigma, a9539). 100 µl of the nuclei solution was taken close to the bottom of the ependorf tube, and 100 µl table 1. the chemical contents of the four detergents a1, a2, b1 and b2. detergents chemical content a1 15-30% anionic active substance, polycarboxylate, phosphonate, enzyme (amylase, protease), perfume, paint, preservative. a2 <5% soap, <5-15 anionic substances, <5% nonionic substances, perfumes, methylisothiazolinone, water, raw materials from coconut, palm, wheat and potato. b1 % 5-15 anionic active substance, nonionic active ingredient, <5% phosphonate, soap, enzyme, perfume (alpha ionone, amyl cinnamal, benzyl salicylate, butylphenyl methylpropional, hexyl cinnamal, limonene, linalool), preservative. b2 <5% soap, <5-15 anionic, <5% nonionic substances, perfume, methylchloroisothiazolinone, water, raw materials from coconut, palm, wheat and potato. a1 and b1 are standard detergents. a2 and b2 are classified as eco-friendly detergents. table 2. the detergent concentrations used in determination of ec50 values. detergent concentration (ml/l) detergent concentration (ml/l) a1 1 10 20 30 40 50 100 200 400 b1 1 10 20 30 40 50 100 200 400 a2 1 10 50 100 200 400 b2 1 10 50 60 70 80 90 100 200 400 132 aylin horozal, özlem aksoy of lmp (low melting point) agarose (roth germany, 6351, sigma, a9414) was mixed by pipetting in ependorf tube, allowing the nuclei to adhere to the lmp agarose. nuclei + lmp agarose mixture was poured onto nmp coated slides and closed with lamella immediately. the slides were kept on ice for 10 minutes at +4°c in the refrigerator to stabilize the agarose. cover glass on the slides were gently removed after taken from the refrigerator. the slides were placed in the same direction and adjacent to each other in the horizontal dark electrophoresis tank, which had been pre-cooled to +4°c with the cold water bath. a cold scge buffer was poured slowly from the edges of the tank and the cover of the tank was closed. the slides were stored in the tank for 20 minutes in order to dissolve the dna chain. electrophoresis (cslcom 20, 1000 ml, and cleaver cs-300v power supply) was performed at 27v, 300ma for 25 minutes in order to observe the comets that would occur as a result of different molecular weight dna fragments. at the end of the electrophoresis, the slides were removed from the tank and placed in the trays and kept in cold tris-hcl 3 times each for 5 minutes for neutralization. after the neutralization process, the slides were arranged upright on the drying paper and the excess buffer was allowed to move over the slides. the slides were stained with 100 µl of red safe dye before drying and left for 5 minutes. they were closed immediately with cover glass. slides for comet assay were examined by fluorescence microscope olympus bx51. for each concentration and time, 25 randomly chosen nuclei were analyzed and three slides were evaluated per treatment and the median values of comet parameters for each slide were calculated (türkoğlu, 2012). the averaged median tail length (μm), percentage of tail dna (% of dna in comet tail), olive tail moment (otm in arbitrary unit) values were calculated using the kameram software (argenit, turkey) comet assay analysis program for each treatment group. results as a result of the calculations made respectively; ec50 for a1 was determined as 20 ml/l (fig 1a) and 2xec50 was 40 ml/l; ec50 for a2 was determined as 50 ml/l (fig 1b) and 2xec50 was 100 ml/l; ec50 for b1 was determined as 40 ml/l (fig 1c) and 2xec50 was 80 ml/l; ec50 for b2 was determined as 60 ml/l (fig 1d) and 2xec50 was 120 ml/l. it was observed that the percentage of root lengths decreased gradually compared to control in increasing detergent concentrations. it was also observed that the ec50 values of the eco-friendly detergents were higher than the standard detergents (fig. 1). the effects of the detergents a1, a2, b1, b2 on mitotic frequency and chromosomal abnormality of v.faba root tip cells the mitotic index values were decreased in the treatment groups due to increasing time and concentration at the end of 72 hours. mitotic index values of eco-friendly detergents were higher than the standard detergents (table 3). at the end of the seven days, the mitotic index value for the control group was found to be 14.60%, it was 8,90% at 20ml/l and 7,80% at 40ml/l a1 detergent concentration; the mitotic index value for a2 detergent, decreased to 12.80% in 50 ml/l and to 11.50% at 100ml/l concentration; the mitotic index value for b1 detergent, decreased to 8.30% in 40 ml/l concentration and to 8.10% at 80 ml/l concentration; the mitotic index value for b2 detergent, decreased to 11.90% in 50 ml/l concentration and to 10.60% at 100ml/l concentration. the observed chromosomal abnormalities for all of the detergent treatments of v.faba root tip cells were; laggard chromosome, chromosome adhesion, irregular anaphase, chromosomal bridge, chromosome swelling, micronucleus formation, pole shift, c-mitosis, irregular prophase, irregular metaphase and chromosome stickness (fig 2). the detection of dna damage caused by detergents with scge the degree of dna damage increased in proportion to the concentration and duration, and the degree of dna damage in the eco-friendly detergents was lower than the standard detergents. the classification of nuclei according to the degree of damage with scge yield five types of nucleus; “type 0” means no visible tail, “type 1” means tiny tail , “type 2” means dim tail, “type 3” means a clear tail that is longer than the head diameter and “type 4” means tail is approxiametly three times longer than the head (fig 3). in the control groups, a few dna damaged nuclei, called type 0 and type 1, were observed. ty pe 1 and ty pe 2 grade dna damages were observed at 20ml/l a1 concentration after 24 and 48 hours treatment, and type 3 grade dna damage was observed to occur more than type 2 after 72 hours. type 2 grade dna damages were seen mostly in 24 hours at 133evaluation of the genotoxicity of some standart and eco-friendly detergents with vicia faba a concentration of 40 ml/l a1, while type 2 and type 3 grade damages were observed at 48 hours. type 3 and type 4 grade damages were observed in majority after 72 hours. type 0 and type 1 grade dna damages were detected at 50 ml/l a2 concentration treated for 24 hours. type 1 and type 2 grade dna damages were found at the end of 48 hours, and type 3 grade dna damage was observed when 72 hours were completed. type 0 and type 1 grade dna damages were detected at a concentration of 100 ml/l for 24 hours treatment. type 2 grade dna damages were observed frequently during 48 hours, and nuclei with dna damages in type 2 and type 3 were observed at the end of 72 hours. ty pe 1 and ty pe 2 grade dna damages were observed at 40 ml/l b1concentration for 24 hours, while nuclei with type 2 grade dna damage were observed after 48 hours treatment. type 3 grade dna damage was seen after 72 hours. type 2 grade dna damage was found in 80 ml/l concentration after 24 hours and type 2 and type 3 grade damage was found after 48 hours treatment. at the end of 72 hours, nuclei with type 4 grade dna damage were observed. type 0 and type 1 grade dna damages were determined at 60ml/l b2 concentration treated for 24 hours. type 2 grade dna damage was observed after 48 hours and dna damage level changed to type 3 after 72 hours. type 1 and type 2 grade dna damages were observed at 120 ml/l concentration for 24 hours and type 2 grade dna damage was observed after 48 hours. type 3 grade dna damages were determined after 72 hours. the effects of different concentrations of detergents on dna percentage (tail dna%) were also examined. tail dna percentages for the control group of a1 detergent was 2.25% after 24 hours, 3.6% after 48 hours and 4.2% after 72 hours. at a concentration of 20 ml/l a1 detergent, the value increased from 22.4% after 24 hours to 43.6% in 48 hours, reaching 51.8% at the end of 72 hours. tail dna damage, which was 40.3% at 40 ml/l concentration in 24 hours, reached 52.3% in 48 hours and 66.7% in 72 hours (table 5). the tail dna percentages was 0.9% after 24 hours in the control group of a2 detergent, 0.7% at 48 hours and 1.5% at 72 hours. at a concentration of 50 ml/l, the value of 17.2% in 24 hours increased to 22.9% in 48 hours. at a concentration of 100 ml/l, it was observed that the value of 29.4% in 24 hours, 38.1% in 48 hours and 49.2% in 72 hours (table 5). the tail dna percentages was 1.8% in the control group of b1 detergent after 24 hours which was, reached 2.3% in 48 hours and 2.6% in 72 hours. at a 40 ml/l b1 detergent concentration, the value of tail dna was 26.4% at 24 hours, 40.7% at 48 hours, and 50.5% at 72 hours, at a concentration of 80 ml/l, it was observed that the value was 42.6% in 24 hours reached 62.8% in 48 hours and 66.1% in 72 hours (table 5). the tail dna percentages was 0.65% for b2 detergent in the control group of b1 detergent after 24 hours which was reached 0.8 % in 48 hours and 1.27% in 72 hours. at a concentration of 60 ml/l, the value of 18.5% at 24 hours reached 25.9% at 48 hours, and at 72 hours the value of tail dna was 42.4%. at a concentration of 120 ml/l, it was observed that the value was 32.6% in 24 hours, 46.4% in 48 hours and 50.8% in 72 hours (table 5). for all detergent treatments on v. faba, the values of the tail were calculated as well as the determination of the percentage of tail dna that increased in direct proportion to the concentration and duration. the effect of all detergents on the otm value at varying concentrations is shown in table 6. figure 1. ec50 values determined for the four different detergents, a. detergent a1, b. detergent a2, c. detergent b1, d. detergent b2. arrows show the ec50 values. table 3. mitotic index percentages calculated for the detergents a1, a2, b1 and b2 after 72 h. treatment. detergent concentration(ml/l) mi (%) ± sd* a1 0 20 40 14.60±0.07 8.90±0.08 7.80±0.09 a2 50 100 12.80±0.11 11.50±0.07 b1 40 80 8.30±0.09 8.10±0.05 b2 60 120 11.90±0.04 10.60±0.05 * mi ± sd: average mitotic index percentage±standart deviation. 134 aylin horozal, özlem aksoy figure 2. the chromosomal abnormalities caused by detergents in v. faba meristematic cells a. laggard chromosome in anaphase and chromosome adhesion in metaphase, b. laggard chromosome in anaphase, c. laggard chromosome in telophase, d. irregular anaphase, e. chromosomal bridge and laggard chromosome in anaphase, f. chromosome swelling in metaphase, g. micronucleus formation in cytokinesis, h. laggard chromosome and chromosome adhesion in anaphase, i. chromosomal bridge and laggard chromosome in telophase, j. chromosomal bridge and pole shift in anaphase, k. c-mitosis, l. laggard chromosome in metaphase, m. irregular prophase, n. pole shift and chromosome swelling in metaphase, o. laggard chromosome in anaphase, p. irregular metaphase, q. pole shift in metaphase, r. irregular anaphase, s. c-mitosis and chromosome stickness, t. chromosomal bridge in anaphase, u. laggard chromosome in anaphase, v. c-mitosis and irregular prophase, y. pole shift in telophase, z. chromosomal bridge and laggard chromosome in telophase. 135evaluation of the genotoxicity of some standart and eco-friendly detergents with vicia faba otm values at all detergents showed a signifi cant increase due to time and concentrations. discussion v. faba seeds treated with detergents were allowed to grow and the root inhibition test and the values of the root length halving (ec50) by the end of seven days compared to the control group were determined for each detergent separately. it was found that the standard detergents could reduce the root length in lower concentrations than the eco-friendly detergents. studies on the plant assays revealed that any genotoxic eff ects manifested in a test sample are likely to result in inhibition of root growth (yekeen et al. 2017). ezemonye et al. investigated the genotoxicity of high anionic surfactant-containing detergents on sweet and salt water shrimps (desmoscaris trispinosa and palaemonetes africanus), it has been shown that increasing detergent concentrations increase the mortality rate compared to the control group (ezemonye et al., 2009). wing et al. (2019) demonstrated that both laundry detergents and detergent residue aft er rinsing showed high cytotoxicity on human bronchial epithelial cell culture. pettersson et al. found out the toxic eff ects of 26 laundry detergents and 5 soft eners on daphnia magna, it was reported that the concentration of detergents and the genotoxic eff ects were correlated with each other (pettersson et al., 2000). th e cytotoxic/genotoxic eff ects (by mitotic index and micronuclei frequency) of boron used as detergent additive were reported in root meristems of v. faba (barbefi eri, 2016). the lowest ec50 value was determined in a1 standart detergent that its eff ective concentration was found as 20 ml/l. according to this fi nding, a1 standart detergent is the most toxic from all of the other studied detergents and gave damage to the root mitotic cells in figure 3. classifi cation of nuclei according to the degree of damage in v.faba with comet test a) type 0: undamaged, b) type 1: tail <0,5 c) type 2: tail>0,5 or equal to the head, d) type 3: tail is longer than the head diameter, e) type 4: tail is approxiametly three times longer than the head. table 4. th e eff ect of the detergents on chromosomal abnormality percentages in v.faba detergent concentration (mg/l) duration time(h) chromosomal abnormality(%)±sd* a1 0 24 0.25±0.27 48 1.70±0.48 72 2.70±0.66 20 24 8.0±0.05 48 11.0±0.07 72 14.0±0.07 40 24 23.2±0.06 48 26.2±0.07 72 30.4±0.07 a2 50 24 7.7±0.07 48 9.2±0.05 72 8.9±0.54 100 24 11.8±0.06 48 13.7±0.49 72 14.1±0.07 b1 40 24 9.6±0.07 48 12.2±0.54 72 15.6±0.06 80 24 21.1±0.07 48 32,7±0.05 72 28.3±0.06 b2 60 24 6.6±0.06 48 8.3±0.05 72 9.9±0.06 120 24 10.8±0.05 48 12.7±0.07 72 16.5±0.07 136 aylin horozal, özlem aksoy v. faba. the highest ec50 value was determined in b2 eco-friendly detergent that its effective concentration was found as 60 ml/l. therefore, the lowest amount of damage to root mitotic cells in v. faba was caused by b2 detergent. as a result, the chemicals in standard detergents have negative effects on root growth in v. faba compared to the chemicals in eco-friendly detergents. mitotic index test results showed that there was a reduction in mitoic index percentage with increasing time and concentration in all detergents. the reduction in mitotic index can be explained by the mitodepressive effects of chemicals in detergents, causing errors in normal cell cycle and limiting cell division (özkara et al., 2015). a1 detergent was the detergent that decreased the mitotic index percentage by 10.9% compared to the control, while b2 detergent was the detergent that decreased the mitotic index by 2.4% compared to the control. the mean percentage value of mitotic index was higher in the eco-friendly detergents than the standart detergents. it was concluded that the standard detergents influence cell division negatively when the chromosomal abnormalities in the root tip meristematic cells of v.faba were determined. chromosomal abnormalities occur as a result of inability to repair the fractures in dna double chain (maluszynska and juchimiuk, 2005). in our study, it was determined that chromosomal aberration rate increased significantly in all detergents due to increasing concentration and duration. the most common abnormalies were polar shift, irregular prophase, irregular metaphase, sticky chromosome, c-mitosis and bridge formation. induction of chromosomal aberrations pointed to potential for genotoxicity (yekeen et al., 2017). wang et al. (2019) reported that due to the ability of the detergents that break the lipid-lipid and protein-lipid interactions, membrane proteins and lipids can become soluble in human bronchial epithelial monolayer cell cultures. however, in our study, such a change was not observed visually due to the cell wall in plants but may be observed with cell ultrastructure studies. the highest chromosomal abnormality was observed in a1 detergent at a rate of 30.4% after a period of 72 hours while the lowest chromosomal abnormality was observed in a2 detergent with a value of 14.1%. the standard detergents were found to have more chromosomal abnormalities than eco-friendly detergents. the chromosomal aberrations observed in this assay suggest that all the detergents exert a mutagenic/cytotoxic effect. a wide variety of dna repair mechanisms are available to prevent such damage in the cell nucleus. replication, transcription and protein synthesis inhibition may occur when these repair mechanisms are ineffective or when very severe dna damage occurs. however, chromosomal abnormalities and mutations can be seen in the long term treatments (aksoy, 2017). the damage caused by the genotoxic effects of detergents was also investigated by comet assay test. the damages were classified in five groups as type 0, type 1, type 2, type 3 and type 4 according to their degree of damage. type 3 dna damage was mostly seen in standard detergents at the highest concentrations and durations. in a1 and b1 detergents, dna damage from the head of the comet tail reached almost threefold, while eco-friendly a2 and b2 detergents showed dna damage table 5. the effect of the detergents on tail dna percentages in v. faba detergent concentration (ml/l) duration time/ dna% in tail 24 h 48 h 72 h a1 0 2.25 3.60 4.20 20 22.40 43.60 51.80 40 40.30 52.30 66.70 a2 0 0.90 0.70 1.50 50 17.20 22.90 37.80 100 29.40 38.10 49.80 b1 0 1.80 2.30 2.60 40 26.40 40.70 50.50 80 42.60 62.80 66.10 b2 0 0.65 0.80 1.27 60 18.50 25.90 42.40 120 32.60 46.40 50.80 table 6. the effect of the detergents on otm values in v. faba. detergent concentration (ml/l) duration time/ otm value 24h 48h 72h a1 0 0.75 1.2 1.4 20 7.5 16 17.2 40 14.7 17.5 22.2 a2 0 0.2 0.3 0.57 50 6 8.6 12.6 100 10.8 12.7 16.6 b1 0 0.63 0.8 0.9 40 8.8 14.3 17 80 15.2 20.9 22.3 b2 0 0.21 0.26 0.6 60 7.17 8.7 14.1 120 11.3 15.4 17 137evaluation of the genotoxicity of some standart and eco-friendly detergents with vicia faba of up to twice the size of the tail. as a result, the chemicals in the standard detergents caused much more damage on the dna of v.faba root tip cells than eco-friendly detergents. a1 is the standart detergent that reaches the most intense tail dna percentage while the a2 ecofriendly detergent has the lowest concentration when treated with the highest concentration and time period. studies have also shown dna fragmentation in  v. faba  root apical meristem cells and seedlings exposed to toxic compounds with the highest concentrations and different time periods (arya et al., 2013; liu et al., 2015; ghosh et al., 2016, iqbal, 2016; hu et al., 2017; cortéseslava et al., 2018; youssef and elamawi 2020). as a result, it was determined that the percentage of tail dna in increasing concentrations and durations was higher in standard detergents compared to eco-friendly detergents. the standard detergents caused more genetic damage because they produced more dense dna-containing tails. as in the tail dna percentage, olive tail moment (otm) values showed similar results in terms of increasing concentration and duration. dna tail percentage was studied with otm parameters in a another study on the determination of genetic damage on onion root tips treated with 5-100% concentrations of coal ash (chakraborty et al., 2009). otm and the percentage of tail dna correlate well with the concentrations of chemical substances with genotoxic effect and are parameters that give confidence in comet assay evaluations (kumaravel et al., 2009). in our study, b1 standart detergent had the highest otm value while the a2 eco-friendly detergent was the lowest. as a result otm value of eco-friendly detergents remained lower than standard detergents. sobrino-figueroa stated that since detergents are complex mixtures of different substances, in which additive and/or synergistic effects may occur, the deleterious effect caused by the dishwasher detergent was probably due to the combined effects of the ingredients of the detergent. (sobrino-figueroa, 2013). when all results obtained in the study were evaluated in general, it was observed that eco-friendly detergents produced significantly less genotoxic effects on v.faba root tip cells than standard detergents. this is due to the fact that in the production of eco-friendly detergents that we have chosen to use in the study, a lower proportion of anionic material and nonionic material is used than standard detergents. in addition, the use of raw materials from coconut, palm, wheat and potatoes in production to reduce the proportion of chemicals in its content has also been a supportive factor in creating less genotoxic damage on living beings. the results obtained in this study indicate that detergent wastes that reach foodstuffs through food chain cause serious damage to dna. in light of all these findings, it is obvious that there are serious measures to be taken at the stage of detergent production and use. detergents should be treated more carefully in their production due to the negative effects on the life, development and genetics of living things. detergents that cannot be sufficiently rinsed due to the use of more detergents than necessary are the main reasons that cause residual residues in washing dishes and laundry to cause direct health problems. as it provides only pleasant odor or softness, it should be more sensitive to the consumption of detergents used in addition to cleaning. the amount of waste detergent to be left to the environment should not be increased for such reasons, which is not necessary. being conscious of consumption and having the consciousness of protecting the environment is extremely important for us to leave a cleaner environment for future generations. in addition to the measures that consumers can take, there are some points that manufacturers should take into consideration. the products should be subjected to the necessary tests at every stage of production before they are put on sale. the studies on the determination of the harmful effects of detergents on the environment and the environment and the studies to reduce these damages should be made on the basis of the production of raw materials. particularly in the case of detergents, the amount of phosphate used must be limited and the amounts involved in the water must be removed in phosphate treatment plants. the capacities of the treatment facilities should be increased in order to be able to make a healthier treatment. any industrialization without taking measures to protect nature should be prevented. drink ing and operating water must be checked periodically. water quality parameters should be checked regularly by authorized institutions. acknowledgements this study is a part of the master thesis titled as “determination of genotoxic effects of some detergents on vicia faba l.” and supported by kocaeli university research foundation. references aizdaicher  n, markina v. 2006. toxic effects of detergents on the alga plagioselmis 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and beanmediated green synthesized silver nanoparticles on allium cepa cells. caryologia. 70(4): 366–377. doi: 10.1080/00087114.2017.1370260 youssef ms, elamawi rm. 2020. evaluation of phytotoxicity, cytotoxicity, and genotoxicity of zno nanoparticles in vicia faba. environ. sci. pollut. res. 27: 18972– 18984. https://doi.org/10.1007/s11356-018-3250-1 yüksel b, aksoy ö. 2017. cytological effects of coumarin on the mitosis of lens culinaris medik. fresen. environ. bull. 26(11):6400–6407. caryologia. international journal of cytology, cytosystematics and cytogenetics 74(1): 83-88, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-853 caryologia international journal of cytology, cytosystematics and cytogenetics citation: s. jantarat, s. jumrusthanasan, s. kaewsri, p. supanuam, a. tanomtong (2021) first report of karyological analysis and heteromorphic nucleolar organizer region of black surgeonfish (acanthurus gahhm, acanthuridae) in thailand. caryologia 74(1): 83-88. doi: 10.36253/caryologia-853 received: february 06, 2020 accepted: april 26, 2021 published: july 20, 2021 copyright: © 2021 s. jantarat, s. jumrusthanasan, s. kaewsri, p. supanuam, a. tanomtong. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. first report of karyological analysis and heteromorphic nucleolar organizer region of black surgeonfish (acanthurus gahhm, acanthuridae) in thailand sitthisak jantarat1, sarun jumrusthanasan2, sarawut kaewsri2, praween supanuam3,*, alongklod tanomtong4 1 program of biology, department of science, faculty of science and technology, prince of songkla university, pattani campus, thailand 2 biology program, faculty of science, buriram rajabhat university, buriram, thailand 3 biology program, faculty of science, ubon ratchathani rajabhat university, ubon ratchathani, thailand 4 department of biology, faculty of science, khon kaen university, khon kaen, thailand *corresponding author. e-mail: supanuam@hotmail.com abstract. this research was the first report on karyological analysis and heteromorphic nucleolar organizer region of black surgeonfish (acanthurus gahhm, acanthuridae) in thailand. the 10 male and 10 female specimens were collected from phuket marine biological center, and phang nga coastal research and development center, andaman sea, thailand. mitotic chromosomes were directly prepared from gill and kidney tissues. the chromosomes were stained by conventional giemsa staining and ag-nor banding techniques. results showed that the diploid chromosomes number of a. gahhm was 2n=48, the fundamental numbers (nf) was 54 in both male and female. the karyotype consist of 6 large acrocentric, 20 large telocentric, 18 medium telocentric and 4 small telocentric chromosomes. none of strange size chromosomes related to sex was found. the heteromorphic nucleolar organizer regions (nors) were observed on telomeric short arm of first acrocentric which can defined as 1a1b. there is nor in 1a and not in 1b. the karyotype formula of black surgeon fish was as follows: 2n (48) = la6+lt20+mt18+st4 keywords: acanthurus gahhm, chromosome, karyotype, nors. introduction worldwide there are an estimated 24,000 fish species recorded, thailand is one of the species diversity centers of the world. there are more than 13,000 and 4,000 species of fishes that live in sea and coralline, respectively (tamrongnawasawad et al. 2004). marine fishes are especially important as they provide a high quality source of protein and other nutrients, economically and ecological important, moreover, some species are bioindicator 84 sitthisak jantarat, sarun jumrusthanasan, sarawut kaewsri, praween supanuam, alongklod tanomtong (ohno 1970; le grande and fitzsimons 1988; affonso et al. 2014). the acanthuridae are the family of surgeonfishes, tangs, and unicornfishes. they are well known as ornamental fish. this family includes about 82 extant species in 6 genera, namely acanthurus, ctenochaetus, zebrasoma, paracanthurus, prionurus and naso. for the important character of the family, they have a colorful body. they also have a pair of dangerously precaudal spines. the genus acanthurus has 40 species in worldwide that found in the atlantic, indian and pacific ocean. they are found in tropical oceans, especially near coral reefs, with most species in the indo-pacific but a few are found in the atlantic ocean (monkolprasit et al. 1997; allen et al. 2012). acanthurus gahhm or black surgeonfish is a demersal fish. it lives on reefs and in lagoons and other sandy areas up to 40 meters deep. this species is omnivorous, feeding on algae, zooplankton and other small invertebrates, and detritus. it is active during the day and may swim in groups or remain solitary. it is endemic to the indian ocean. this species is kept in aquaria and harvested for food. this fish reaches up to 50 centimeters in length. it is oval in shape and laterally compressed. the caudal fin has a crescent shape. the mouth is small and pointed. the body is black to dark brown, with a white ring around the base of the tail and a yellow stripe around the eyes. the pectoral fins are tipped with yellow (figure 1). the black surgeonfish are one of the most colorful and economically important fish (carpenter and niem 2001; allen et al. 2012). previous cytogenetic studies of the genus acanthurus stated that their members are only four species, namely a. coeruleus, a. bahianus, a. chirergus and a. triostegus (arai and inoue 1976; ojima and yamamoto 1990; galetti et al 2006; arai 2011; affonso et al. 2014). the two species, a. coeruleus from brazil and a. triostegus from japan show 2n=48. the other species from brazil show the diploid chromosome numbers of 36 and 34 for a. bahianus and a. chirurgus, respectively. nucleolar organizer region (nor) of the species in this family has never been reported. the present study aimed to investigate cytogenetic characterization of the acanthurus gahhm. we exhibit the standardized karyotype and idiogram of the species and also firstly describes the chromosomal characteristics of a. gahhm by means of giemsa conventional staining and ag-nor banding techniques. materials and methods the 10 male and 10 female specimens of black surgeonf ish (acanthurus gahhm) were collected from phuket marine biological center, and phang nga coastal research and development center, andaman sea, thailand. chromosomes were directly prepared in vivo (chen and ebeling 1968; nanda et al. 1995) as follows. the fishes were injected on their abdominal cavity with 0.05% colchicine for 1.0 ml/100 g body weight, then leaved for one hour. chromosome preparation containing gill and kidney tissues were conducted by the colchicine-hypotonic-fixation-air drying technique. the tissues were finely chopped by scissors. the metaphase cell was three times centrifuged at 1,250 rpm for 10 minutes until the white sediment cells were precipitated. the chromosomes were stained with 20% giemsa’s for 30 minutes and identified for nors by ag-nor staining according to howell and black (1980) and verma and babu (1995). chromosomal checks were performed on mitotic metaphase cells under light microscope. the twenty cells of each male and female appeared with clearly observable and well-spread chromosomes were selected and photographed. the length of short arm chromosome (ls) and the length of long arm chromosome (ll) were measured to calculate the length of total arm chromosome (lt, lt = ls + ll). in addition, the relative length (rl), centromeric index (ci), and total arm chromosome (lt) were calculated to classify the type and size of chromosomes based on turpin and lejeune (1965) and chaiyasut (1989). all described parameters were used in karyotyping and idiograming according to tanomtong et al. (2019). for the karyotype formula determination, the chromosomes were classified by size regarding to the symbol “l, m and s” as the representative of large, medium and small chromosomes, respectively. in the same way, the chromosomes were classified by type regarding to the symbol “m, sm, a and t” as the figure 1. general characteristics of black surgeonfish (acanthurus gahhm, acanthuridae) from phuket marine biological center, and phang nga coastal research and development center, andaman sea, thailand (scale bars = 3 cm). 85first report of karyological analysis and heteromorphic nucleolar organizer region of black surgeonfish representative of metacentric, submetacentric, acrocentric and telocentric chromosomes, respectively. the fundamental number (nf) is assigned a value of two for the metacentric, submetacentric and acrocentric chromosomes; however, it is assigned equal to one for the telocentric chromosome. results and discussion this is the first karyological analysis of the acanthurus gahhm. the results showed that the diploid chromosome number was 2n=48 and the fundamental numbers (nf) were 54 for both male and female (figure 2). up to the present, there are only two publications on cytogenetics of the family acanthuridae. affonso et al. (2001) conducted the study on cytogenetics of three species of the family acanthuridae, namely acanthurus coeruleus, a. bahianus and a. chirergus in brazil. they showed the diploid chromosome number of 48 and the fundamental number (nf) of 52 for a. coeruleus. however, they demonstrated the low diploid chromosome numbers (2n) of 36 and 34 and the fundamental numbers (nf) of 52 and 50 for a. bahianus and a. chirergus, respectively. arai and inoue (1976) revealed an establishment of chromosome analysis of a. triostegus which were obtained from yakushima, japan. the karyotype showed 2n=48 and nf=48, like the ancestral perciform karyotype. the present karyotype of a. gahhm consist of 6 large acrocentric, 20 large telocentric, 18 medium telocentric and 4 small telocentric chromosomes. the twenty metaphase cells of each male and female were measured for ls, ll, lt, ci, rl, sd, chromosome sizes and types were showed on table 1. none of the strange in size of chromosome related to sex was observed. the a. gahhm has 6 bi-arm and 42 uni-arm chromosomes. the modal karyotype of ancestral perciformes fish possessing 2n=48, nf=48 and composed all uni-arm chromosomes. the karyotype of a. gahhm indicates that although it has been revealing a model diploid chromosome number of 2n=48, the karyotypes different from the ancestral perciformes pattern have been detected in these studies, indicating pericentric inversion or/ and robertsonian rearrangements as the preferential process in some groups. the karyotype of a. gahhm is quite similar to the a. coeluleus karyotype. the rearrangement mechanism involves to pericentric inversions of 3 uni-arm to 3 bi-arm chromosome pairs from the ancestor. (affonso et al. 2014). the most species of family acanthuridae show the typical perciform karyotype, 2n=48, nf=48, namely a. triostegus, ctenochaetus striatus and prionurus scalprum. the few species show diploid decreasing cause figure 2. metaphase plates and standardized karyotypes of male (a) and female (b) black surgeonfish, acanthurus gahhm, 2n=48 by conventional staining (scale bars = 10 µm). a b 86 sitthisak jantarat, sarun jumrusthanasan, sarawut kaewsri, praween supanuam, alongklod tanomtong by tandem or/ and centric fusion including a. bahianus (2n=36, 16 bi-arm and 20 uni-arm) and a. chirurgus (2n=34, 16 bi-arm and 18 uni-arm) (arai and inoue 1976; ojima and yamamoto 1990; galetti et al 2006; arai 2011; affonso et al. 2014). the karyotype of the family acanthuridae is shown in table 2. moreover, this is the first report on localization of nucleolar organizer regions (nors) of the acanthurus table 1. mean length of the short arm chromosome (ls), long arm chromosome (ll), total arm chromosome (lt), centromeric index (ci), relative length (rl) and standard deviation (sd) of ci, rl from 40 karyotypes of male and female black surgeonfish (acanthurus gahhm), 2n=48. chromosome pair ls (micron) ll (micron) lt (micron) rl±sd ci±sd chromosome size chromosome type 1 0.88 2.57 3.45 0.057±0.001 0.744±0.038 large acrocentric 2 0.76 2.51 3.27 0.054±0.001 0.766±0.036 large acrocentric 3 0.77 2.08 2.85 0.047±0.001 0.729±0.019 large acrocentric 4 0.00 3.37 3.37 0.055±0.003 1.000±0.000 large telocentric 5 0.00 3.19 3.19 0.052±0.001 1.000±0.000 large telocentric 6 0.00 3.13 3.13 0.051±0.002 1.000±0.000 large telocentric 7 0.00 3.06 3.06 0.050±0.002 1.000±0.000 large telocentric 8 0.00 3.01 3.01 0.049±0.002 1.000±0.000 large telocentric 9 0.00 2.93 2.93 0.048±0.002 1.000±0.000 large telocentric 10 0.00 2.66 2.66 0.043±0.000 1.000±0.000 large telocentric 11 0.00 2.62 2.62 0.043±0.000 1.000±0.000 large telocentric 12 0.00 2.55 2.55 0.042±0.001 1.000±0.000 large telocentric 13 0.00 2.49 2.49 0.041±0.001 1.000±0.000 large telocentric 14 0.00 2.41 2.41 0.039±0.001 1.000±0.000 medium telocentric 15 0.00 2.35 2.35 0.038±0.001 1.000±0.000 medium telocentric 16 0.00 2.30 2.30 0.038±0.001 1.000±0.000 medium telocentric 17 0.00 2.13 2.13 0.035±0.000 1.000±0.000 medium telocentric 18 0.00 2.05 2.05 0.033±0.000 1.000±0.000 medium telocentric 19 0.00 1.99 1.99 0.032±0.000 1.000±0.000 medium telocentric 20 0.00 1.92 1.92 0.031±0.000 1.000±0.000 medium telocentric 21 0.00 1.85 1.85 0.030±0.001 1.000±0.000 medium telocentric 22 0.00 1.76 1.76 0.029±0.001 1.000±0.000 medium telocentric 23 0.00 1.70 1.70 0.028±0.002 1.000±0.000 small telocentric 24 0.00 1.50 1.50 0.024±0.001 1.000±0.000 small telocentric table 2. review of cytogenetic publications in the family acanthuridae. species 2n nf karyotype ag-nor locality reference acanthurus triostegus 48 48 48t japan arai and inoue (1976) a. chirurgus 34 50 16bi+18t brazil galetti et al. (2006) 34 18bi+16t pair 8p brazil affonso et al. (2014) a. bahianus 36 52 16bi+20t brazil galetti et al. (2006) 36 18bi+18t pair 8p brazil affonso et al. (2014) a. coeruleus 48 52 4bi+44t brazil galetti et al. (2006) 48 6bi+42t pair 2p brazil affonso et al. (2014) a. gahhm 48 54 6a+42t pair 1p thailand present study ctenochaetus stiatus 48 48 48t japan ojima and yamamoto (1990) prionurus scalprum 48 48 48t japan arai and inoue (1976) remark: 2n = diploid number, nf = fundamental number, bi = bi-arm chromosome, t = telocentric chromosome (uni-arm chromosome). 87first report of karyological analysis and heteromorphic nucleolar organizer region of black surgeonfish figure 3. metaphase plates and standardized karyotypes of male (a) and female (b) black surgeonfish, acanthurus gahhm, 2n=48 by agnor banding (scale bars = 10 µm). chromosome pair 1 show heteromorphic nor in 1a. arrows indicate nors. a b figure 4. standardized idiogram of black surgeonfish, acanthurus gahhm, 2n=48 by conventional staining. figure 5. standardized idiogram of black surgeonfish, acanthurus gahhm, 2n=48 by ag-nor banding. chromosome pair 1 show heteromorphic nor in 1a. arrows indicate nors. 88 sitthisak jantarat, sarun jumrusthanasan, sarawut kaewsri, praween supanuam, alongklod tanomtong gahhm. one pair of the short arm of the largest chromosome 1 showed clearly observable nors. the first record on heteromorphism of nors in the a. gahhm (figure 3) was also reported here. this finding indicates the presence of heteromorphic of chromosome pair 1 (1a1b). nors were found in 1a, but not in 1b. the three acanthuridae species, namely a. coeluleus, a. bahianus and a. chirurgus show the single nucleolar organizer regions on the short arms of the largest subtelocentric pairs (affonso et al. 2014). the idiogram shows gradually decreasing length of the chromosomes. the largest chromosome shows two times larger than the smallest chromosome. an important karyotype trait is the presence of an asymmetrical karyotype pattern. there were only two types of chromosomes found, the acrocentric and telocentric chromosomes. the standardized conventional and ag-nor idiograms of acanthurus gahhm are shown on figure 4 and 5, respectively. the karyotype formula of black surgeonfish (acanthurus gahhm) can be deduced as: 2n (48) = la6+lt20+mt18+st4 funding this research was financially supported by the government budget grant, prince of songk la university, thailand, in fiscal year 2017, research code: sat600116s. references affonso, pram, fernandas ma, almeida js, molina wf. 2014. sequential steps of chromosomal differentiation in atlantic surgeonfishes: evolutionary inferences. the scientific world journal. 2014: 825703. doi: 10.1155/2014/825703. allen g, steene r, humann p, deloach n. 2012. reef fish identification: tropical pacific. 5th ed. florida: new world publications. arai r. 2011. fish karyotypes a check list. japan: springer. arai r, inoue m. 1976. chromosomes of seven species of pomacentridae and two species of acanthuridae from japan. bull. natn. sci. mus. tokyo, (a). 2: 73–78. carpenter ke, niem vh. 2001. the living marine resources of the western central pacific. rome: food and agriculture organization of the united nations. chaiyasut k. 1989. cytogenetics and cytotaxonomy of the genus zephyranthes. bangkok: department of botany, faculty of science, chulalongkorn university. thailand. galetti jpm, molina wf, affonso, pram, aguilar ct. 2006. assessing genetic diversity of brazilian reef fishes by chromosomal and dna markers. genetica. 126:161–177. howell wm, black da. 1980. controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. experientia. 36:1014–1015. le grande, wh, fitzsimons jm. 1988. chromosome numbers of some gulf coast sciaenid fishes. copeia. 2: 491–493. monkolprasit s, sontirat s, vimollohakarn s, songririkul t. 1997. checklist of fishes in thailand. bangkok: office of environmental policy and planning. ohno s. 1970. evolution by gene duplication. new york: springer-verlag. ojima y, yamamoto k. 1990. cellular dna contents of fishes determined by flow cytometry. la kromosomo, ii. 57:1871–1888. phuket marine biological center. 2007. a guide to reef fishes of the andaman sea, thailand. phuket: phuket marine biological center, dpartment of marine and coastal resources. thai. tanomtong a. 2019. cyogenetics. bangkok; chulalongkorn university press. thailand. turpin r, lejeune j. 1965. les chromosomes humains. paris: gautier-villars. french. verma rs, babu a. 1995. human chromosomes principles and techniques. 2nd ed. new york: mcgraw-hill inc. caryologia. international journal of cytology, cytosystematics and cytogenetics 72(3): 105-115, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-772 citation: a. teixeira mesquita, m.v. romero-da cruz, a.l. sousa azevedo, e.r. forni-martins (2019) chromosome number and genome size diversity in five solanaceae genera. caryologia 72(3): 105-115. doi: 10.13128/ caryologia-772 published: december 13, 2019 copyright: © 2019 a. teixeira mesquita, m.v. romero da cruz, a.l. sousa azevedo, e.r. forni martins. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. chromosome number and genome size diversity in five solanaceae genera amanda teixeira mesquita1,*, marìa victoria romero-da cruz1, ana luisa sousa azevedo2, eliana regina forni-martins1 1 departamento de biologia vegetal, universidade estadual de campinas, rua monteiro lobato 255, cep: 13.083-970 campinas (sp), brasil 2 embrapa gado de leite, empresa brasileira de pesquisa agropecuária (embrapa), rua eugênio do nascimento 610, cep: 36.038-330 juiz de fora (mg), brasil *corresponding author: mesquita.at@gmail.com abstract. sixteen species of solanaceae, belonging to five genera, were studied karyologically through chromosome counting, chromosomal measurement, and karyotype symmetry. genome size (gs) estimation was performed on fifteen species using flow cytometry. the chromosome number 2n=24 was found in all solanum species and acnistus arborescens, 2n=22 was found in brunfelsia uniflora, and 2n=16 in cestrum representatives. physalis pubescens was the only specie with evidence of polyploidy, showing 2n=4x=48 chromosomes. the chromosome numbers of s. adspersum, s. inodorum, s. flaccidum, s. sanctae-catharinae, and b. uniflora were reported for the first time. haploid karyotype length (hkl) was statistically different between the studied species. the polyploid p. pubescens showed the largest hkl value, 93.10 µm. in general, karyotypes were symmetrical with predominance of metacentric chromosomes. chromosome size was small in most species (<4 µm), while s. diploconos, c. laevigatum, and c. mariquitense, species with high hkl values, exhibited larger chromosomes. genome size estimation were unpublished for ten studied species and were the first estimation for the genera acnistus, brunfelsia and physalis. were observed about eight-fold differences between species with averages varying from 2c=2.57 pg to 2c=20.27 pg. as both hkl and gs showed a continuous variation. we observed partial similarity in the species ordered according to hkl and gs. the solanaceae genera showed a constant chromosome number and a tendency to posse symmetrical karyotypes. the genome size also showed differences, which suggests that chromosome evolution in the group could be driven by alterations in the repetitive fractions of the genome. keywords. acnistus, brunfelsia, cestrum, physalis, solanum, karyotype evolution. introduction the solanaceae family comprises about 2,500 species and 100 genera and have cosmopolitan distribution. the greatest diversity of the family is found in neotropical regions (d’arcy 1991; hunziker 2001). members of solanaceae have great ecological and morphological diversity, characteristics which favoured the occupation of diverse habitats, such as desert regions, tropi106 amanda teixeira mesquita et al. cal rainforests, and even disturbed areas (d’arcy 1991; knapp 2002). the family includes several species of important global food crops with high economic value, such as tomatoes (solanum lycopersicum), potatoes (solanum tuberosum), eggplants (solanum melongena), and chilli peppers (capsicum spp.), widely used drug plants, such as tobacco (nicotiana tabacum), “datura” (datura stramonium), and “angel’s tears” brugmansia suaveolens, as well as many ornamental plants, such as species of the genus brunfelsia, cestrum and petunia. many solanaceae species, including tomatoes, potatoes, and tobacco, are model organisms for various biological studies, and their genomes are some of the most well studied among angiosperms (knapp et al. 2004). karyotype information about species and groups are important for taxonomic and evolutionary studies, whereas karyological changes accompany speciation and, consequently, the diversification of the groups (guerra et al. 2008, 2012, chiarini et al. 2018). the chromosome number, nuclear dna content, total length of the chromosome complement, asymmetry indices, and number and location of the rdna sites and heterochromatic bands are the main data used in cytotaxonomic studies. chromosome number data is the most available information and is not influenced by external agents, such as age of individuals, environmental conditions, and gene expression, providing accurate data about species evolution (dobginy et al. 2004, guerra et al. 2008, 2012). cytogenetic characterization, accompanied by a genome size (gs) study, can offer important information about genome organization, phylogenetic relationships, and evolutionary trends. this approach has been successful used in some solanaceae (mishiba et al. 2000, moscone 2003, chiarini et al. 2014). chromosome data is available for some genera of solanaceae, while for other genera there is not enough data or information about their chromosomes. lycium and solanum present constant chromosome number (2n=24 and polyploids) (bernadello and anderson 1990; bernadello et al. 1994; chiarini and bernadello 2006; rego et al. 2009; stiefkens et al. 2010; melo et al. 2011; chiarini et al. 2014), while capsicum shows 2n=24 and 2n=26 (moscone 1993; moscone et al. 2007; aguilera et al. 2014; grabiele et al. 2014; romero-da cruz and forni-martins 2015; romero-da cruz et al. 2017). for the cestreae tribe, composed of cestrum, sessea, and vestia, the only chromosome number reported to date is 2n=16 (fregonezi et al. 2006; las peñas et al. 2006; fernandes et al. 2009; urdampilleta et al. 2015). the greatest range in chromosome number is found in nicotiana (n=12 to n=32, and polyploids, chase et al. 2003). only about 8% of solanaceae taxa have available gs data. this character has more variability than chromosome number (soltis et al. 2003). in solanum, the gs ranges of from forty-fold in species with 2n=24 chromosomes. the smallest reported c-value is in s. chacoense, 1c=0.63 pg (bennett and smith 1976), while the largest value is 1c= 24.80 pg, found in s. hartwegii (pringle and murray 1991). nevertheless, there are still many gaps in karyotypic knowledge for the solanaceae family and such information (i.e. genome size, chromosome number, and karyotype variables) is important to complete current data and to better understand the systematic relationships and chromosome evolution of the family. therefore, the objectives of this study were: (1) to report original chromosome numbers and describe the karyotype variables in distinct genera of the solanaceae family, (2) to determine the genome size (gs) using flow cytometry for the first time for many species. material and methods plant material sixteen species from the genera acnistus, brunfelsia, cestrum, solanum, and physalis were collected in southeastern brazil. voucher specimens were deposited into the herbarium at the university of campinas (uec). data collection is detailed in table 1. chromosome preparations seeds of at least three individuals per species were germinated in petri dishes. in some cases, 1 ml gibberellic acid (ga3) was applied to break seed dormancy (ellis et al. 1985). according to previous tests, root meristems were pre-treated with different solutions to block the cell cycle to obtain good chromosome spread and condensation (table 2). the root apices were fixed in 3:1 ethanol: acetic acid (v:v) mixture that was stirred for a minimum of 12 h at room temperature (rt) and stored at -6ºc until slide preparation. slides were made using root meristems that were previously digested in a solution of 1% macerozima, 2% cellulase, and 20% pectinase for 10-15 minutes at 37ºc and squashed in a drop of 45% acetic acid. coverslips were removed after freezing in liquid nitrogen for 15 minutes. the cells were photographed under a microscope olympus bx51 with a dp72 camera attached and images were captured using olympus dp2 bsw program (olympus corporation). 107chromosome number and genome size diversity in five solanaceae genera karyotype analysis five metaphases of each species, with the same degree of chromosome condensation, were used to determine the chromosome number. the measurements were taken using the micromeasure© software (3.3). ideograms were made using measurements of the following means for each chromosome pair: s (short arm length), l (long arm length) and c (total chromosome length) using the formula c= s+l. in addition, haploid karyotype length (hkl) was calculated by the sum of the haploid chromosome lengths. the arm ratio (r) was calculated using the formula r= l/s and was used to classify chromosomes according to levan et al. (1964). for ideograms, chromosomes were first grouped by morphology (r=1.00-1.69 metacentric-m; r=1.70-2.99 submetacentricsm; r=3.00-6.99 subtelocentric-sm) and then by decreasing size order within each group. the karyotype symmetry was described using the indices a1= 1-[(σbi/bi)/n] (bi = mean of the short arm table 1. cytogenetics data of solanaceae species: species and voucher specimen; provenance of materials; chromosome number, haploid karyotype formula (hkf), median haploid karyotype length (hkl), variation in chromosome length (vcl); symmetry indices a1 and a2; median dna content (2c values). species (voucher specimen) provenance 2n hkf hkl – µm (ci) vcl – µm a1 a2 2c values – pg (ci) acnistus arborescens schltdl. (monge 2787) brazil: rio grande do sul; aratinga 24 12m 45.93 (2.78) 3.17-4.38 0.17 0.10 6.56 (0.06) brunfelsia. uniflora d. don (mesquita 15) brazil; são paulo; campinas 22 7m+4sm 50.51 (0.50) 3.89-5.37 0.32 0.10 6.58 (0.13) cestrum laevigatum schltdl. (mesquita 12) brazil; são paulo; campinas 16 6m+2sm 78.72 (2.96) 7.92-10.88 0.23 0.10 20.27 (0.43) c. mariquitense kunth (mesquita 14) brazil; são paulo; campinas 16 7m+1sm 73.91 (6.38) 7.35-11.39 0.21 0.12 physalis pubescens l. (vasconcellos neto 00-068) brazil; são paulo; jundiaí 48 19m+5sm 93.10 (2.87) 1.43-2.80 0.32 0.18 12.98 (0.09) solanum cyphomandra clade solanum diploconos (mart.) bohs (mesquita 23) brazil; são paulo; jundiaí 24 8m+4sm 74.72 (1.86) 4.63-7.49 0.32 0.14 19.22 (0.43) dulcamaroid clade s. flaccidum vell. (mesquita 07) brazil; são paulo; campinas 24 9m+2sm+1st 26.73 (1.14) 1.83-2.50 0.27 0.10 2.57 (0.25) s. inodorum vell. (vasconcellos neto 20401) brazil; são paulo; jundiaí 24 5m+7sm 38.33 (3.90) 2.83-3.86 0.39 0.09 4.63 (0.06) geminata clade s. pseudocapsicum l. (mesquita 24) brazil; são paulo; jundiaí 24 9m+3sm 28.61 (7.70) 1.76-2.72 0.28 0.13 2.94 (0.11) leptostemonum clade acanthophora section s. acerifolium sendt. (mesquita 02) brazil; são paulo; campinas 24 10m+2sm 36.17 (1.09) 1.71-3.87 0.26 0.23 5.69 (0.15) s. palinacanthum dunal (mesquita 20) brazil; são paulo; ubatuba 24 5m+7sm 37.86 (0.72) 2.51-3.86 0.41 0.13 5.00 (0.10) torva section s. adspersum witasek (monge 2748 c 240) brazil; rio de janeiro; arraial do cabo 24 9m+3sm 25.09 (1.62) 1.77-2.44 0.25 0.09 3.19 (0.04) s. scuticum m. nee (vasconcellos neto 8503) brazil; são paulo; jundiaí 24 9m+3sm 27.66 (2.10) 1.95-2.79 0.31 0.04 3.42 (0.06) s. variabile mart (monge 2324) brazil; são paulo; itacaré 24 9m+3sm 33.45 (0.51) 2.15-3.26 0.24 0.11 3.54 (0.09) uncertain position s. concinnum schott ex sendtn. (mesquita 08) brazil; são paulo; campinas 24 6m+6sm 31.82 (0.37) 2.26-2.86 0.39 0.09 3.65 (0.25) s. sanctae-catharinae dunal (vasconcellos neto 20873) brazil; são paulo; jundiaí 24 10m+2sm 23.15(2.56) 1.68-2.35 0.26 0.09 3.79 (0.07) ci – confidence interval at 95% of semi range. 108 amanda teixeira mesquita et al. of each chromosome pair, bi = average of the long arm of each chromosome pair, n = number of chromosome pairs) and a2=x/s (s = standard deviation; x = average chromosome complement length) (zarco 1986). a1 index measures intrachromosomal asymmetry which indicates differences in the size of chromosome arms. a2 index measures the interchromosomal asymmetry and indicates the variation in chromosome lengths. in terms of length, chromosomes were classified according to lima de faria (1980) as very small (≤1 µm), small (>1 µm and ≤4 µm), intermediate (>4 and ≤12) and big (>12 and ≤60). flow cytometry the same species that were cytogenetically analysed (except for cestrum mariquitense) were cultivated in a greenhouse and used for gs measurements. for each species, three individuals were measured in three repetitions, for a total of nine samples. approximately 1 cm2 of young leaf tissue was used to prepare the nuclear suspensions, according to dolezel et al. (2007). the material of each species of interest and a piece of internal leaf standard (pisum sativum “ctirad” 2c=9.09 pg ( dolezel et al. 1998) were sliced with a razor blade and placed into a petri dish on ice. about 1 ml of lb01 buffer (dolezel et al. 1989) was used to extract the nuclei. a nylon mesh with 40 microns was used to filter the sample (celltrics, partec), then, 25 µl 1 mg/ml propidium iodide and 25 µl 1 mg/ml rnase were added to the nuclear suspension. the measurement was performed on a bd facs calibur flow cytometer, for each sample an average of 10,000 nuclei were analysed. the 2c value was calculated using the linear relationship between fluorescence signals from stained nuclei of the unknown sample and the reference standard. the nomenclature for genome size classification followed leitch et al. (1998) with modification by soltis et al. (2003): values <1.4 pg and between 1.4 to 3.5 pg correspond to “very small” and “small” genomes, respectively. on the other hand, values between 3.5113.99 pg, >14 pg and >35 pg are considered “intermediate,” “large”, and “very large” genomes, respectively. statistical analyses the hkl values, as well of gs values of each species, were compared using past 3.18 ® (øyvind hammer, natural history museum, university of oslo). the kruskalwallis nonparametric test was performed to compare the averages among the species and dunn’s post-hoc test (dunn 1954) was carried out after significant kruskalwallis test. results karyotype analysis the somatic chromosome numbers were 2n=2x=24 (acnistus and solanum), 2n=2x=22 (brunfelsia), 2n=2x=16 (cestrum) and 2n=4x=48 (physalis) (table 1; fig. 1). although the differences in hkl between some species were significant (p<0.05) according to statistical analysis (table 1; fig. 2), this variation was gradual, and no groups were formed. solanum sanctae-catharinae showed the lowest median value (23.15 µm) with a variation of 1.68-2.35 µm from the smallest to largest chromosome pair. other solanum species also presented low hkl (except for s. diploconos), with values reaching 38.33 µm in s. inodorum (2.83 to 3.86 µm). species with intermediate hkl values were a. arborescens (45.93 µm, 3.17 to 4.38 µm) and b. uniflora (50.51 µm, 3.89 to 5.38 µm). high hkl values were found in c. mariquitense with 73.91 µm (7.35 to 11.39 µm), s. diploconos with 74.72 µm (4.63 to 7.49 µm), and c. laevigatum with 78.72 µm (7.92 to 10.88 µm). physalis pubescens showed fig. 1. somatic metaphases of five genera of solanaceae. a solanum flaccidum. b s. adspersum. c s. sanctae-catharinae. d s. inodorum. e physalis pubescens. f acnistus arborescens. g brunfelsia uniflora. h s. diploconos. i cestrum laevigatum. bar=10 µm. 109chromosome number and genome size diversity in five solanaceae genera the highest hkl value (93.10 µm), even though it is a polyploid species with chromosomes ranging from 1.43 to 2.8 µm. karyotypes are symmetrical with a1 and a2 values for each species ranging from 0.17 to 0.41 and from 0.04 to 0.23, respectively. most species presented a predominance of metacentric chromosomes (table 1, fig. 3) that characterized most intrachromosomal symmetry shown in the a1 index. acnistus arborescens had the most symmetrical karyotype, composed of only metacentric chromosomes and a1=0.17. three species had less symmetrical karyotypes: solanum inodorum and s. palinacanthum showed predominance of submetacentric chromosomes (5m+7sm) and a1 value of 0.39 and 0.41, respectively. solanum concinnum also presented a1=0.39, but karyotype formulae 6m+6sm. interchromosomal index a2 showed that all species have few variations in chromosome size of the karyotypes. solanum scuticum showed the small a2 value (0.04) and solanum acerifolium presented the highest a2 value (0.23), characterizing the most interchromosomal asymmetry among studied species (table 1). c-value genome size estimates of all the studied species are shown in table 1 and histograms for selected species are shown in fig. 4. according to statistical analysis, gs showed significant differences among some of the studied species (fig. 5). a variation of about eight-fold was observed, ranging from 2c=2.57 pg (s. flaccidum, fig. 4a) to 2c=20.27 pg (c. laevigatum, fig. 4d). the gs presented continuous variation, so distinct groups were not characterized (fig. 5). most species had small (2c=2.57 pg in s. flacidum to 2n=3.79 pg in s. sanctaecatharinae) and intermediate genomes (2c=4.63 pg in s. inodorum to 6.56 pg in a. arborescens and 6.58 pg in b. uniflora). the species with larger genomes were p. pubescens (2n=12.98 pg), s. diploconos (2c=19.22 pg), and c. laevigatum (2c=20.27 pg). discussion chromosome number the chromosome number data found here are new for s. adspersum, s. inodorum, s. flaccidum, and s. sanctae-catharinae, with 2n=24 chromosomes, as well as for b. uniflora, with 2n=22. for the remaining species, the chromosome number obtained corroborated with data found in the literature for acnistus (2n=24), cestrum (2n=16), solanum (2n=24), and physalis (2n=48) (heiser 1963; pedrosa et al. 1999; fernandes et al. 2009; rego et al. 2009; urdampilleta et al. 2015). all the species in this study, except for p. pubescens, which is a tetraploid, are diploid. although diploid is the most frequent ploidy level (including other species of physalis), polyploidization has played an important role in the evolution of some solanaceae genera (e.g., nicotiana, chase et al. 2003; s. elaeagnifolium, scaldaferro et al. 2012). the chromosome number most frequent in the family is 2n=24, found in more than 85% of the previously studied solanaceae species (olmstead et al. 2008) though a diploid series from 2n=14 to 2n=26 is present in some genera (eg. petunia and calibrachoa, mishiba et al. 2000, cestrum, sessea and vestia, las peñas et al. 2006, capsicum, moscone et al. 2007). many authors have postulated hypotheses for the ancestral chromosome base number in the family. raven (1975) proposed x=7 and 12 for the order solanales and solanaceae family, respectively, while badr et al. (1997) suggested the hypothesis of x=7 or x=8. moscone (1992) corroborate with the proposition of badr et al. (1997), suggested x=7 as the basic chromosome number for solanaceae. olmstead and palmer (1992) and olmstead et al (2008) based in phylogenetic studies, suggests an ancestral position of subfam. cestroideae (x=8), and x=12 as a derivate basic chromosome number the family. fig. 2. boxplots illustrating the continuous variability of hkl (haploid karyotype length), as inferred from de kruskal wallis analysis. the numbers on the x axis represent the species ordered by crescent hkl values (in µm): s. sanctae-catharinae (1), s. adspersum (2), s. flaccidum (3), s. scuticum (4), s. pseudocapsicum (5), s. concinnum (6), s. variabile (7) s. acerifoium (8) s. palinacanthum (9) s. inodorum (10), a. arborescens (11) b. uniflora (12) c. mariquitense (13) s. diploconos (14) c. laevigatum (15) p. pubescens (16). the central box represents 50% of the data from de upper to lower quartile. the horizontal bar expresses the median position. the extremity of the vertical lines indicates minimum and maximum values of hkl, if they are no outliners. when outliners are present, they are represented by circles. 110 amanda teixeira mesquita et al. fig. 3. ideograms of the investigated solanaceae species based on median chromosome values. bar=5 µm. 111chromosome number and genome size diversity in five solanaceae genera the lack of chromosomal data for several genera and for the solanaceae sister group, the family convolvulaceae, as well as the presence of distinct basic numbers in other solanales families, as in hydroleaceae, x=8 and 10 (constance 1963) and montiniaceae, x=12 (goldblatt 1979), has hampered to establish a consensus about a basic chromosome number and understand the direction of chromosome number evolution for the family. karyotype structure differences in chromosome size were seen between the solanaceae species here investigated. the relatively small chromosome size and hkl observed in the species here of solanum, except for s. diploconos (statistically distinct, and previously considered a species of the distinct genus cyphomandra), and p. pubescens, have been reported in some studies for solanum (bernardello and anderson 1990; acosta et al. 2005; chiarini et al. 2006; rego et al. 2009; melo et al. 2011; moyetta et al. 2013), and another solanaceae genera, as lycianthes and vassobia (rego et al. 2009) and lycium, (stiefkens and bernadello 2002,) acnistus arborescens and b. uniflora shows chromosomes and consequently, hkl values, with intermediate size, when compared to solanum and physalis. these karyotype characteristics are also present in capsicum (moscone 1996), sclerophylax and nolana (lujea and chiarini 2017), genera also belonging to solanaceae. although the intermediate size of the chromosomes, a constant chromosome number, karyotype symmetry and chromosomes majority metacentric appear to maintain in these groups. the tribe cestreae (subfam. cestroideae) embraces the genera cestrum, sessea and vestia, presents the largest chromosomal sizes of the family (fregonezi et al. 2006, peñas et al. 2006). cestrum laevigatum and c. mariquitense, investigated here, showed the largest chromosome size high hkl values, confirming the trend for the tr. cestreae (fregonezi et al. 2006). such increase in chromosome size for the tribe can be due to the absence of arabidopsis-type telomeres (tttaggg)n for short interstitial telomeric sequences (sits), leading to the lack of control of the telomerase-dependent replication. these sequences may associate with other dna sequencfig. 4. flow cytometry histograms (iodide propidium fluorescence intensity of nuclei) showing dna amounts from leaf tissues of some solanaceae species. 1 pisum sativum “ctirad” (standard). 2 s. flaccidum. 3 p. pubescens. 4 a. arborescens. 5 c. laevigatum. fig. 5. boxplots illustrating the continuous variability of gs (genome size), as inferred from de kruskal wallis analysis. the numbers on the x axis represent the species ordered by crescent c-values (in pg): s. flaccidum (1), s. pseudocapsicum (2), s. adspersum (3), s. scuticum (4), s. variabile (5), s. concinnum (6), s. sanctaecatharinae (7) s. inodorum (8) s. palinacanthum (9) s. acerifolium (10), a. arborescens (11) b. uniflora (12) p. pubescens (13) s. diploconos (14) c. laevigatum (15). the central box represents 50% of the data from de upper to lower quartile. the horizontal bar expresses the median position. the extremity of the vertical lines indicates minimum and maximum values of hkl, if they are no outliners. when outliners are present, they are represented by circles. table 2. pretreatments used for each genus of solanaceae studied. genus pretreatments acnistus and physalis 8-hydroxyquinoline 0.002m + cycloheximide 25mg/l (1:1), 8 h, 4ºc brunfelsia 8-hydroxyquinoline 0.002m, 6 h, 14ºc cestrum colchicine 0.1% 6 h, rt* solanum saturated solution of r-dichlorobenzene 2 h, rt* solanum (s. diploconos) saturated solution of r-dichlorobenzene 5 h, rt* *rt=room temperature. 112 amanda teixeira mesquita et al. es and assist in their dispersion leading to an increase in genome size (sykorova et al. 2003a, b). besides chromosome number, other widely conserved karyotype characters in solanaceae genera, are chromosome morphology and karyotype symmetry. symmetrical karyotypes with a predominance of metacentric chromosome pairs are found in the five genera studied here. acnistus arborescens was the unique specie that had only metacentric chromosomes, thus, had the most symmetrical karyotype and only s. flaccidum showed a subtelocentric chromosome pair. other genera of the family solanaceae in which it is possible to observe these characteristics arecapsicum (pozzobon et al. 2006; moscone et al. 1993, 2007), lycium (stiefkens and bernadello 2012), lycianthes, and vassobia (rego et al. 2009). among the angiosperms, karyotype asymmetry can be associated with derivate taxa (stebbins 1971) in some groups of the solanaceae family, intermediate asymmetry values, can be observed, such as the tr. cestreae (las peñas et al. 2006), solanum sect. acanthophora (chiarini et al. 2014) however, for species, the karytotype asymmetry was not associate with basal or derived position of the taxa in the phylogeny. regarding karyotype asymmetry, no evolutive trend was found for the analysed genera or among the representatives of solanum. overall, karyotype asymmetry seems to occur randomly within some groups of the family. our study analysed five species from other sections (acantophora and torva) of the leptostemonum clade. in sect. acantophora (s. acerifolium and s. palinacanthum), we observed greater hkl and karyotype asymmetry than in sect. torva (s. adspersum, s. scuticum and s. variabile). karyotype asymmetry was previously reported for the lepstotemomun clade (chiarini et al. 2014). in other cases, asymmetry was random within a group, as in solanum morelloid and dulcamaroid clades (moyetta et al. 2013). both species belonging to clade dulcamaroid that were studied corroborated this data, while s. inodorum (hkl=38.33 µm) presented a more asymmetrical karyotype (5m + 7sm and a1=0.39, a2=0.09) than s. flaccidum (hkl=26.73 µm, 9m + 2sm + 1st and a1=0.27, a2=0.10). the karyotype characteristics described for the studied species and genera as well as in other solanaceae groups, a constancy in the chromosome number, karyotype symmetry and chromosome morphology, indicates karyotype orthoselection, which preserves similar chromosomal complements, regardless of chromosome size (acosta et al. 2005; moscone et al. 2003). according to wu and tanksley (2010), inversions have occurred at a much higher rate than translocations throughout the evolutionary history of solanaceae, thereby preserving chromosome morphology favouring chromosomal uniformity c-value/dna content despite the great number of representatives in solanaceae, the gs estimation is available for a small proportion of species and genera. only 12 solanaceae genera have data about gs, representing approximately 10% and 186 species that corresponding to 7% of solanaceae representatives. genome size data for a. arborescens, b. uniflora and p. pubescens are the first estimation for the relative genera. some species of cestrum and solanum have their gs measured but the data here obtained are unpublished for c. laevigatum, s. flaccidum, s. inodorum, s, adspersum, s, scuticum, s. variabile, s. concinnum and s. sanctae-catharinae. the gs variation observed in the species studied partially coincides with the variation observed in hkl. in general, species with small, intermediary, or high hkl presented the same gs classification. among the five species with small hkl, four presented small values of dna content (s. adspersum, s. flaccidum, s. pseudocapsicum and s. scuticum). similarly, of the six species with high hkl, five showed high values of dna content (a. arborescens, b. uniflora, s. diploconos, c. laevigatum and p. pubescens). the estimation of nuclear dna using flow cytometry is more accurate than measuring chromosomes. this accuracy is supported by statistical tests and by species boxplots, where the dispersion of hkl data (figure 2) was greater than gs data (figure 5). calculating hkl is more subject to external effects (stace 2000). methodological standardization, especially the degree of chromosomal condensation in the mitotic metaphase, is important for obtaining chromosomal sizes and comparing the results obtained between species (stace 2000). although angiosperms have high diversity in their dna content, the predominance of a small genome size causes a tendency with modal values equal to 1c = 0.7 pg (leitch et al. 1998). this distribution, strongly skewed towards small genomes, is associated with the ancestral condition of the group and large genomes could have arose more than once during angiosperm evolution (leitch et al. 1998; soltis et al. 2003). among the species studied, and in the solanaceae family in general, we observed a predominance of small genomes (see bennett and leitch 2012). the cosmopolitan distribution of the family and occurrence in a wide variety of habitats (d’arcy 1991; hunziker 2001; knapp 2002) are related to some phenotypic characteristics that 113chromosome number and genome size diversity in five solanaceae genera are correlated to the low dna content. species with low dna content tend to be found in varying habitats and those with very large genomes appear to be excluded from extreme habitats (knight and ackerly 2002). despite the predominance of small genomes in solanaceae, there are some groups with intermediary or large genome sizes, such as species of the genera nicotiana, cestrum, capsicum, and the cyphomandra clade of solanum (see bennett and leitch 2012). there are two main factors associated with increased genome size in plants, polyploidy events or whole genome duplication (soltis et al. 2003; leitch and leitch 2013; wendel et al. 2015) and an increase in the repetitive elements of dna (mainly transposable elements) (leitch and leitch 2013; bennetzen and wang 2014). in solanaceae, it is likely that the changes in genome size of these groups is related to repetitive elements, since there are few groups with polyploidy relatives causing an increase in dna content. conclusions we conclude that some karyotype characters are well conserved in the solanaceae family at the generic level. chromosome numbers are very constant, with few reports of polyploidy and aneuploidy and the predominance of chromosome morphology and karyotype symmetry. the family represents a model for karyotypic orthoselection and the karyotype evolution in solanaceae may have been driven by repetitive dna reorganization that led to gs diversification, but did not affect chromosome number and morphology. acknowledgments: the authors thanks the comissão de aperfeiçoamento de pessoal do nível superior (capes), conselho nacional de desenvolvimento científico e tecnológico (cnpq), and fundação de amparo à pesquisa do estado de são paulo (fapesp) (grant number 2016/17096-9) for the financial support. we thank the empresa brasileira de pesquisas agropecuária (embrapa), unidade gado de leite for the infrastructure granted to execute part of the study. we thank espaço da escrita – coordenadoria geral da universidade – universidade estadual de campinas (unicamp) for the language services provided. funding details this work was supported by the fundação de amparo à pesquisa do estado de são paulo (fapesp) under the grant 2016/17096-9. references acosta mc, bernardello g, guerra m, moscone ea. 2005. karyotype analysis in several south american species of solanum and lycianthes rantonnei (solanaceae). taxon 54: 713–723. aguilera pm, debat hj, garcía ys, martí da, grabiele m. 2014. capsicum rhomboideum. in: marhold k, breitwieser i (eds.), iapt/iopb chromosome data 18. taxon 63: 6, e1. bennett md. 1972. nuclear dna content and minimum generation time in herbaceous plants. proc. roy. soc. london, ser. b, biol. sci. 181: 109–135. bennett md. 1998. plant genome values: how much do we know? proc natl acad sci usa 95: 2011–2016. bennett md, leitch ij. 1995. nuclear dna amounts in angiosperms. ann. bot. 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naik, a.k. patel, s.k. mishra, a. nag, j. panigrahi (2019) characterization of intraspecific hybrid in clitoria ternatea (l.) using morphophysiological, cytogenetic, metabolic and molecular markers. caryologia 72(3): 11-22. doi: 10.13128/caryologia-754 published: december 13, 2019 copyright: © 2019 a. naik, a.k. patel, s.k. mishra, a. nag, j. panigrahi. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. characterization of intraspecific hybrid in clitoria ternatea (l.) using morphophysiological, cytogenetic, metabolic and molecular markers aparupa naik1, amiya k. patel1, sujit k. mishra1, atul nag2, jogeswar panigrahi1,2,* 1 plant biotechnology laboratory, department of biotechnology and bionformatics, sambalpur university, jyoti vihar-768019, sambalpur, odisha, india 2 department of biotechnology, central university of rajasthan, nh-8, bandarsindri, kishangarh, rajasthan *corresponding author: drjpanigrahi@gmail.com abstract. clitoria ternatea (l.) is a medicinal plant possessed with bioactive molecules such as taraxerol, delphinidin, kaempoferol and quercetin etc. for the development genotype with higher content of these bioactive molecules, marker-assisted breeding is one of the best strategies and it initiates with the development of f1 hybrids. thus, an intraspecific f1 hybrid was raised involving two contrasting genotypes of c. ternatea acc. ctb3-sl1 (blue flowered) and c. ternatea acc. ctw2-bl1 (white flowered). the hybridity of the f1 plant was confirmed by assessing the phenotypic traits, such as colour of the petal, pod shape and seed coat colour, 100 seed weight, and the content of taraxerol and delphinidin. the pollen mother cells in the f1 hybrid showed eight bivalents with preponderance of ring bivalents and 8i:8i segregation at metaphase-i and anaphase-i, respectively. sds-page seed albumin and globulin detected three pollen parent-specific polypeptides (mw 31.62, 22.38 and 18.81kda), and were inherited to f1 hybrids, which evidenced the hybridity of putative f1 plants. further, dna marker analysis also showed the inheritance of 11 rapd, six scot and one issr markers to putative f1 plant, which affirmed the hybrid nature of the f1 plant. this study also evidenced that combined use of morphophysiological, cytogenetic, protein and dna marker analyses could be effective for precise characterization of intra-specific hybrids in c. ternatea. these f1 hybrid and its derived future progenies could also be used for mapping of qtls or genes contributing higher accumulation of taraxerol and delphinidin in different plant parts. keywords.hybridity, meiosis, seed protein profile, dna marker, taraxerol, delphinidin. introduction clitoria ternatea (l.) belongs to the family-leguminosae (fabaceae) with somatic chromosome count 2n=2x=16 and was distributed worldwide 12 aparupa naik et al. (joson and ramirez 1991; npgs2008). the c. ternatea is also known as shankhpushpi, butterfly pea, aparajita, gokarni, girikarnika (bishoyi et al. 2014). the plant is elongated, slender, climbing herbaceous vine with five leaflets, white to purple flowers, and has deep roots. butterfly pea is predominantly a self-pollinated species, but sometimes considered as often cross-pollinated due to the appearance of segregating genotypes in some populations (cook et al. 2005). this plant has been considered as important medicinal plant, and the phyto-chemical studies explored several bioactive metabolites such as alkaloids, triterpenoids, flavonoids, glycosides, anthocyanins and lactones etc. in this species (mukherjee et al. 2008; sethiya et al. 2009). thus the plant parts based extracts have been prepared and used as a memory enhancer, nootropic, anti-stress, anxiolytic, anticonvulsant, tranquilizing and sedative agent (parrotta 2001; prajapati et al. 2003; khare 2004; kapoor 2005; margret et al. 2015). among the bioactive metabolites, content of kaempferol, delphinidin and taraxerol having anti-cancer and anti-tumour properties have also been documented in this species (braig et al. 2005; chen and kong 2005; niering et al. 2005; singletary et al. 2007; swain et al. 2012). the white-flowered genotypes of c. ternatea were found to be medicinally rich in taraxerol and kaempferol, whereas the blue flowered genotypes were affluent in delphinidin, quercetin and isoquercetin etc. for the development of improved genotype with higher content of these bioactive metabolites marker assisted breeding (mab) is one of the finest strategy, where f1 hybrid serves as the starting material for all future breeding efforts for the genetic improvement. thus intra-specific f1 hybrid involving blue f lowered and white flowered genotype can be used as the starting material for widening genetic base of this medicinal species in term of bioactive metabolites composition and production of advanced breeding lines containing desired metabolites of pharmaceutical importance. however, interaction between both the genome in either of the parental cytoplasmic background often led to genetic variation due to genetic recombination, differential gene action, penetrance and expressivity. f1 hybrid being the starting material for all breeding efforts, its precise identification at early stage is mandatory. thus morphological, cytological, biochemical and molecular markers have been effectively used to ascertain hybridity of f1 plants in many species. but the reproducibility of morphological, cytological and biochemical markers in consonance to environmental variation and developmental regulations limits their applicability. therefore seed protein and dna sequence based marker analysis could also be utilized to screen and identify f1 hybrids at an early stage because of their stability, uniformity, reliability and reproducibility across the environment and are also free from penetrance and expressivity. the seed protein markers have been effectively employed for cultivar characterization, genetic diversity assessment, and verification of hybridity in many species (mohanty et al. 2001; panigrahi et al. 2007; jisha et al. 2011; mishra et al. 2012). similarly dna markers, such rapd, issr and ssr have been used for characterization of hybrids in different species (lima-brito et al. 2006; muthusamy et al. 2008; goldmann et al. 2008; hemalatha et al. 2010; bianco et al. 2011; mishra et al. 2012; mishra et al. 2017). in case of c. ternatea, few of these dna markers have only been used for genetic diversity studies (chandra 2011; swati et al. 2011; ganie et al. 2012; ali et al. 2013: bishoyi et al. 2014). however, no report has been made so far on the development of intra-specific hybrid in c. ternatea and its characterization. in the present study, an intra-specific f1 hybrid of c. ternatea, involving blue flowered [c. ternatea acc. ctb3sl1] and white flowered [c. ternatea acc. ctw2-bl1] genotype, was raised and its hybridity was affirmed by simultaneous use of morpho-physiological and cytogenetic analyses, estimation of bioactive metabolite, profiling of seed protein and dna based markers. materials and methods plant materials genotypes of c. ternatea (including of five white genotypes, six blue genotypes, and four bipetaloid blue genotypes) were collected from sambalpur and bargarh districts of odisha and maintained at the experimental garden, school of life sciences, sambalpur university, odisha, india (table 1). among these genotypes, two genotypes of c. ternatea acc. ctb3-sl1 and c. ternatea acc. ctw2-bl1 were identified on the basis of their bioactive metabolites (delphinidin and taraxerol) content, and used as seed parent and pollen parent, respectively for the development of f1 hybrid. morpho-physiological traits characterization different morpho-physiological traits like colour of standard petal, flower length, flower breadth, floral bud size, anther size, style length, stigma length, seed coat colour and 100 seeds weight were studied for three f1 plants along with their parents. the morpho-physiological traits unique to pollen parent were used as visual dus marker for the characterization of f1 hybrid. 13characterization of intraspecific hybrid in clitoria ternatea cyto-genetic characterization to study the chromosome homology, mitotic and meiotic analysis of f1hybrids and their parents were carried out following behera et al. (2010). well-developed roots (1-3 cm) obtained from the seedlings at 8.00-8.30 a.m. and incubated with pre-chilled p-dichlorobenzene (pdb) solution for two hours at 20ºc, followed by fixation in 1:3 aceto-alcohol and kept overnight at room temperature. subsequently, the root tips were transferred to 70% ethanol and stored at 40c. hydrolysis of the root tips was carried out in preheated 1n hcl at 600c for 10 min followed by staining with the help of 1.5% acetoorcein for one hour and squashed with 45% propionic acid. for meiotic analysis, the flower buds of appropriate size were fixed in 1:3 aceto-alcohol and kept overnight at 25 ± 2oc and then it was transferred to 70% ethanol and stored at 4oc. the anthers of suitable size were squashed in a drop of 1.5% acetocarmine, and the meiotic behaviour of chromosomes at diakinesis, metaphase-i and anaphase-i were observed. suitable stages of mitosis and meiosis were observed under a compound microscope (unilab, india) and were documented using nikon coolpix-4500 camera. bioactive metabolites characterization estimation of taraxerol in root tissues: roots of c. ternatea genotypes and their f1 hybrid were collected after 30 days of initiation of flowering, air dried under shade and ground to f ine powder. powder of each sample (appx. 20gm) was subjected to extraction with 70% alcohol for 5h at 60ºc, filtered (using whatmann no.1 filter paper) and dried under vacuum in a rotary evaporator (rv-10, ika, germany). the hydroalcoholic extract (appx. 8.4 g) was suspended in water and sequentially extracted using hexane, chloroform, ethyl acetate and n-butanol as described by kumar et al. (2008). the hexane and chloroform fractions were subjected to chromatography using chlorofom: methanol (1:1, v/v) as eluent and the eluted fractions were further chromtographed using hexane: ethyl acetate (80:20, v/v) as eluent and yielded the delphinidin as described earlier (kumar et al. 2008). this compound was dissolved in ethanol (1mg.ml-1) and 10 µl aliquot of each sample was used for hptlc assay along with standard taraxerol solution (10-100µg.ml-1) as described by kumar et al. (2008). thin layer chromatography was carried out using aluminum backed hptlc plates (100cm2; 0.2 mm thickness) of silica gel 60 f254 (merck, germany) in a hptlc system (camag, switzerland) consisting of linomat-iv sampler, twin plate development chamber and camag tlc scanner 3 with wincats software. the derivatized plates were scanned under visible light and the content of taraxerol was estimated densitometrically by measuring absorption at 420 nm by tlc scanner-3 integrated with wincats v 1.4.2 software (slit dimension6 mm x 0.45 mm; scanning speed20 mm.s-1). estimation of delphinidin in f lowers: the f lowers were collected, air dried and extracts of petals were prepared following fukui et al. (2003). the petal table 1. fourteen accessions of c. ternatea with their flower colour, petal configuration and the geographical coordinates of collection sites located in odisha, india. sl. no. accession (acc._) collection site latitude longitude mean sea level(m) flower colour & petal structure 1 ctw1-bg1 bargarh 21⁰22’50”n 83⁰44’48”e 186 white unipetaloid 2 ctw2-bl1 sriram vihar (burla) 21⁰28’46”n 83⁰53’5”e 172 white unipetaloid 3 ctw3-bl2 burla town 21⁰28’46”n 83⁰53’5”e 172 white unipetaloid 4 ctw4-bgk1 kandahata (bargarh) 21⁰15’57”n 83⁰39’54”e 186 white unipetaloid 5 ctw5-bg2 bargarh 21⁰22’50”n 21⁰22’50”e 186 white unipetaloid 6 ctw6-bg3 bargarh 21⁰22’50”n 21⁰22’50”e 186 white unipetaloid 7 ctb1-kl1 kuchinda 21⁰37’34”n 83⁰19’0”e 254 blue unipetaloid 8 ctb2-bgk2 kandahata (bargarh) 21⁰15’57”n 83⁰39’48”e 186 blue unipetaloid 9 ctb3-sl1 sambalpur 21⁰46’81”n 83⁰97’54”e 151 blue unipetaloid 10 ctb4-pl1 padampur (bargarh) 21⁰0’0”n 83⁰3’46”e 205 blue unipetaloid 11 ctb5-pl2 padampur (bargarh) 21⁰0’0”n 83⁰3’46”e 205 blue unipetaloid 12 ctbb1-pl3 padampur (bargarh) 21⁰0’0”n 83⁰3’46”e 205 blue bi-petaloid 13 ctbb-pl4 padampur (bargarh) 21⁰0’0”n 83⁰3’46”e 205 blue bi-petaloid 14 ctbb-pl5 padampur (bargarh) 21⁰0’0”n 83⁰3’46”e 205 blue bi-petaloid 14 aparupa naik et al. extracts were dissolved in 0.2 ml of 6 n hcl and kept at 100 ºc for 20 min. the hydrolyzed anthocyanidins were extracted with 0.2 ml of 1-pentanol. hplc was performed using an ods-a312 column as described by katsumoto et al. (2007) using acetic acid : methanol : water (15 : 20 : 65) as solvent with flow rate 1ml per min, and the delphinidin content was estimated by measuring absorbance from 400-600 nm on photodiode array detector (spd-m10a; shimadzu co., ltd). under these hplc conditions, the λmax of delphinidin and retention time were 540 nm and 4 min, respectively which were validated with those of delphinidin chloride (sigma-aldrich). proteomic characterization using seed protein profiling the albumin and globulin fraction of the seeds were extracted and denatured as described by panigrahi et al. (2007). protein samples (appx. 25.0 μg) were separated under discontinuous sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page; laemmli et al. 1970) using 10 % resolving gel (0.375 m tris–hcl, ph 8.8) and 4% stacking gel (0.125 m tris–hcl, ph 6.8). tris-glycine (0.1% sds, 25 mm tris-glycine, ph 8.3) was used as running buffer, and electrophoresis was carried out at 1.5 ma per well constant current until tracking dye reaches the separating gel, and then current supply was increased to 2 ma per well till tracking dye reach bottom of the gel. the molecular weight marker-pmwm (genei pvt. ltd.) was used as a standard, and the size of polypeptides was estimated by standard curve method. genomic characterization using dna markers the genomic dna from f1 hybrid and its parents were isolated using the modified ctab method (sivaramakrishnan et al. 1997) and purified (mishra et al. 2012). dna was dissolved in 2.0 ml te (trisedta) buffer (10 mm tris, 1 mm edta, ph 8.0) and stored at -20°c. purity and concentration of the dna sample were measured using a uv-vis spectrophotometer (uv 1601, shimadzu, kyoto, japan) by taking te buffer as the blank. the quantification of dna was validated by analyzing the purified dna on 0.8 % (w/v) agarose gel by taking diluted, uncut phage lambda dna as standard. the dna samples were equilibrated to 10 ngμl−1 in te buffer. for rapd, scot and issr marker analysis, the pcr amplification of 25ng of genomic dna was carried out using 30 random decamer oligonucleotide primers (opa-01-20 and opb-01-10; operon technologies, alameda, ca, usa), 36 scot primers (scot -1 to scot -36; collard and mackill 2009) and seven issr primers from the set 100/9 (ubc-861, ubc-865, ubc-868, ubc-873, ubc-872, ubc-808, ubc-807; university of british columbia, vancouver, canada), respectively. the pcr amplification reaction (25μl) contained 25 ng template dna, 2.5μl of 10x assay buffer [100 mm tris-cl, ph 8.3; 0.5 m kcl; 0.1 % (w/v) gelatin], 1.5 mm mgcl2, 200 μm of each dntp, 0.25μm primer, 1.0 units taq dna polymerase (bangalore genei pvt. ltd., bangalore, india). for r apd, issr and scot analysis, the amplification were carried out using a thermal cycler (geneamp-9700; applied biosystems, foster city, usa), and conditions are as below: amplification conditions rapd issr scot initial denaturation 94 °c for 5 min pcr cycles 45 cycles 40 cycles 35 cycles cyclic denaturation 94 °c for 60 s 94 °c for 30 s 94 °c for 30 s annealing of primers 37 °c for 60 s 40-60 °c for 60 s 50 °c for 60 s elongation 72 °c for 2 min 72 °c for 2 min 72 °c for 2 min final elongation 72 °c for 5 min 72 °c for 5 min 72 °c for 5 min storage of sample 4 °c for ∞ min the amplified products were mixed with gel loading buffer [20 % (w/v) sucrose; 0.1 m edta, 1.0 % (w/v) sds; 0.25 % (w/v) bromo-phenol blue; 0.25 % (w/ v) xylene cyanol] and separated in 1.4 % (w/v) agarose gel containing 0.5μgml−1 ethidium bromide in tae buffer (40 mm tris acetate, ph 8.0; 2 mm edta) at 50v constantly. the separated dna fragments were documented using gel documentation system (gel doc xr system, biorad, usa), size of amplified fragments was estimated using tl-120 software (non-linear dynamics, total lab ltd., newcastle upon tyne, uk) and 250 bp step-up ladder (bangalore genei pvt. ltd.) as standard. results in this study, the putative intra-specific f1 hybrids were raised by convetional hybridization and were characterized by using morpho-physiological and cytogenetic analysis, estimation of bioactive metabolites, seed protein (albumin and globulin) profiling and dna marker analysis. 15characterization of intraspecific hybrid in clitoria ternatea morpho-physiological and metabolite characterization of the intraspecific f1 hybrid morpho-physiological traits including flower colour, pod beak, and seed coat colour distinguish the blue flowered parental genotype c. ternatea (acc. ctb3-sl1) from white flowered one (c. ternatea acc. ctw2-bl1). the size of flower and 100 seed weight were also distinguishes both the parents. in many such morpho-physiological traits, the f1 hybrid was intermediate between the parents with predominance of the characters of c. ternatea, acc. ctw2bl1, such as seed coat colour, petal colour being the pollen parent, and their appearance in the intermediate form was also very vivid for the identification of the f1 hybrid (table 2; fig. 1a, b). in most of the quantitative traits, such as leaf size, flower size, and 100 seed weight, the f1 plant resided well around the mid-parental value (table 2). the raised f1 hybrids were assessed along with their parents for two important bioactive metabolites (taraxerol and delphinidin). taraxerol was obtained mainly from root tissues whereas delphinidin was obtained from the petals of the f lowers. the f1 hybrid contains 0.856±0.031 mg.g-1 taraxerol in its root tissue and 0.372±0.019 mg.g-1 delphinidin in its flowers. on comparison with its parents taraxerol content in root tissue of f1 hybrid was almost at par with the donor parent (c. ternatea acc. ctw2-bl1) whereas delphinidin content was intermediate between both the parents (table 2; fig. 1c). cyto-genetical characterization of the intraspecific f1 hybrid appropriate stages like metaphase, anaphase, diakinesis, metaphase-i, anaphase-i were observed in the table 2. morpho-physiological and metabolite characterization of intra-specific f1 hybrid of c. ternatea. morphological traits c. ternatea acc. ctw2-bl1 c. ternatea acc. ctb3-sl1 f1 hybrid shape of the leaflets lanceolate lanceolate ovate-lanceolate base of the leaflets cuneate oblique oblique flower length (cm) 5.23±0.15 5.03±0.15 5.08±0.1 flower breadth (cm) 3.2±0.1 3.04±0.06 3.07±0.06 average days to flowering 56 days 64 days 64 days colour of the petal white blue intermediate nature of the ovary & style pubescent glabrous glabrescent seed colour black brown blakish brown number of seeds per pod 4-5 5-6 4-5 100 seed weight (g) 5.82 ± 0.19 5.16 ± 0.2 5.26 ± 0.18 taraxerol content (mg.g-1) 0.821 ± 0.026 0.377 ± 0.014 0.856 ± 0.031 delphinidin content (mg.g-1) 0.104 ± 0.02 0.514 ± 0.019 0.372 ± 0.019 fig. 1. characterization of intra-specific f1 hybrid along with its parents (c. ternatea acc. ctb3-sl1 and c. ternatea acc. ctw2-bl1). floral attributes including petal colour (a), mature seeds showing colour of the seed coat and aril (b); graphical representation of bioactive metabolites (taraxerol and delphinidin) content. 16 aparupa naik et al. pmcs of both the parents and f1 hybrid. chromosome analysis of both the parents revealed 16 chromosomes at metaphase in each of them, and the separation at anaphase occurs in a normal fashion. the mitotic metaphase of f1 hybrid showed 16 distinct chromosomes similar to its parents (fig. 2a). as expected, the pmcs of the f1 hybrid showed formation of eight bivalents (ii) at diakinesis and metaphase-i (fig. 2b, c) and 8ii: 8ii separation at anaphase-i (fig. 2d). the pollen fertility in the f1 hybrid was almost 86% and was equivalent to its parents. proteomic and genomic characterization of the intraspecific f1 hybrid sds-page of seed albumins of two parental genotypes, including c. ternatea acc. ctb3-sl1 and acc. ctw2-bl1, and their f1 hybrids led to the detection of 33 polypeptide bands with molecular weight 12.59 to 84.14 kda. out of which 30 polypeptides were monomorphic, and rest three were varied for their expression (table 3; fig. 3). the putative f1 hybrid possessed with three polymorphic polypeptides (mw 31.62, 22.38 and 18.81 kda) specific to pollen parent c. ternatea acc. ctw2-bl1 along with the monomorphic polypeptides (table 3; fig. 3). since c. ternatea acc. ctw2-bl1 was used as pollen parent, the appearance of these unique albumin polypeptides in the f1 hybrid can potentially be used as markers for identification of hybrids involving at least c. ternatea acc. ctw2-bl1 as pollen parent. in the present study, all three kinds of dna markers showed polymorphism at par. thirty rapd primers have amplified 127 rapd fragments ranging from 130 to 2389 bp, and among them, 22 amplified fragments (17.32%) showed parental polymorphism (table 4). contrasting to this amplification with nine issr and 36 scot primers generated 39 and 224 fragments ranging from 437 to 3154 bp and 116 to 3916 bp, respectively (table 4, 5). both issr and scot analysis showed lower polymorphism (2.56% and 5.80%) in comparison to rapd markers. among the parental polymorphic markers 22 rapd, one issr and 13 scot markers were inherited to the putative f1 hybrid, and among them 11 rapd and six scot markers, unique to pollen parent (ctw2-bl1), were very vivid in its appearance for the identification of f1 hybrid (fig. 4). the total number of fragments amplified, percentage of polymorphism, inheritance of polymorphic fragments to the f1 hybrid were shown in table 4 and 5. fig. 2. cytogenetic characterization of f1 hybrid showing 2n=2x=16 chromosome configuration at mitotic metaphase (a), association of eight bivalents at diakinesis (b) and metaphase-i (c), and segregation of chromosome at anaphase-i (d). fig. 3. inheritance of seed albumin markers to intra-specific f1 hybrid (c. ternatea acc. ctb3-sl1 x c. ternatea acc. ctw2-bl1) intra-specific f1 hybrid; lane ‘m’ represents mol. weight marker (pmw-m, genei, india), and arrow indicates on right hand side indicate the polypeptides inherited to the f1 hybrid (mw in kda). 17characterization of intraspecific hybrid in clitoria ternatea discussion the f1 hybrids have been considered as starting material for all breeding endeavours including the development of mapping population, mapping, and tagging of traits, marker-aided selection and generation of advanced breeding lines before the release of cultivars. thus, identification and characterization of f1 hybrids at an early stage during hybridization programme is quite essential (lima-brito et al. 2006; mishra et al. 2012). in this study, the putative intra-specific f1 hybrid was characterized by using morpho-physiological and cytogenetic analyses, estimation of bioactive metabolites (taraxerol and delphinidin), seed protein (albumin and globulin) profiling, and dna marker analysis. the f1 hybrid was intermediate between the parents (c. ternatea acc. ctb3-sl1 and acc. ctw2-bl1),in term of morpho-physiological traits distinguish the parents, such as flower colour, pod beak and seed coat color, size of flower and 100 seed weight. although no pervious report is available in c. ternatea, in several species the character(s) distinguish the parents were appeared in its intermediate form in f1 hybrid (mishra et al. 2012; mishra et al. 2017) and in some cases the pollen parent specific traits were also predominated as observed in the present study. either the appearance of pollen parent specific traits in the f1 or appearance of traits in intermediate form could be used vividly for the identification of hybridity. the consistency of metabolites in the f1 hybrid of any medicinal plant in consonance to their parents is very vital in the perspective of their use as source material either to harvest therapeutics compounds or to generate breeding lines. c. ternatea plant parts are source of many important bioactive metabolites, and also have wide range of biological and pharmacological activities (mukherjee et al. 2008; sethiya et al. 2009). in view of this, the raised f1 plants were assessed along with their parents for two important bioactive metabolites, taraxerol and delphinidin, mostly used for the treatment of various kind cancer and tumours (braig et al. 2005; chen and kong 2005; niering et al. 2005; singletary et al. 2007; swain et al. 2012). taraxerol was obtained mainly from root tissues whereas delphinidin was obtained from the petals of the flowers. the f1 hybrid contains 0.856±0.031 mg/g taraxerol in its root tissue and 0.372±0.019 mg/g delphinidin in its flowers. on comparison with its parents taraxerol content in root tissue of f1 hybrid was almost at par with the pollen parent (0.821±0.026) whereas delphinidin content was intermediate between both the parents (c. ternatea acc. fig. 4. inheritance of pollen parent specific fragments (→) to c. ternatea acc. ctb3-sl1 x c. ternatea acc. ctw2-bl1 intra-specific f1 hybrid, generated by rapd primers (a), issr primers (b) and scot primers (c). (m: 250 bp step-up ladder, b: c. ternatea acc. ctw2-bl1, f: f1 hybrid and s: c. ternatea acc. ctb3-sl1). table 3. details of seed albumin and seed globulin markers inherited to the intraspecific f1 hybrid. marker total no of bands range of molecular weight (kda) no. of polymorphic polypeptides no. of polymorphic bands inherited to f1 from parents specific bands in f1 from (kda) ctw2-bl1 ctb3-sl1 ctw2-bl1 ctb3-sl1 seed albumin 20 14.13-66.83 03 (15.0%) 03 -31.6, 22.4 & 18.8 -seed globulin 13 12.59-84.14 -----total 33 13.34-112.2 03 (9.09%) 03 -31.6, 22.4 & 18.8 18 aparupa naik et al. table 4. details of rapd and issr markers used for characterization of intraspecific f1 hybrid showing the inheritance of parent specific markers to the intraspecific f1 hybrid. primer sequence (5 ’ → 3’) no. of fragments amplified range of amplified fragments (bp) polymorphic bands percentage of polymor-phism (%) no. of nonparental fragments* in f1 parent specific polymorphic bands (bp) in f1 from ctw2-bl1 ctb3-sl1 rapd marker opa-01 caggcccttc 7 176-1500 ---opa11500 ----opa-02 tgccgagctg 3 432-1233 opa-021233 33.33 ----opa-021233 opa-03 agtcagccac 4 273-968 --0 ------opa-04 aatcgggctg 9 170-1500 opa-04170 opa-04314 22.22 --opa-04170 opa-04314 opa-05 aggggtcttg 6 506-1233 opa-05 506 16.66 ----opa-05 506 opa-06 ggtccctgac 8 276-2045 opa-061516 opa-06612 25 ----opa-061516 opa-06612 opa-07 gaaacgggtg 5 530-1937 ----------opa-08 gtgacgtagg 4 130-1019 opa-07895 25 --opa-07895 --opa-09 gggtaacgcc 5 461-1401 ----------opa-10 gtgatcgcag 2 277-911 ----------opa-11 caatcgccgt 6 139-2389 opa-111079 16.66 --opa-111079 --opa-12 tcggcgatag 5 330-2333 opa-122333 20 --opa-122333 --opa-13 cagcacccac 3 758-1144 ----------opa-14 tctgtgctgg 2 599-717 ----------opa-15 ttccgaaccc 9 284-2250 opa-152250 opa-15622 22.22 --opa-152250 opa-15622 --opa-16 agccagcgaa 9 299-2187 opa-162167 opa-16622 22.22 --opa-162167 opa-16622 opa-17 gaccgcttgt 1 437 --0 ------opa-18 aggtgaccgt 2 569-974 opa-18974 50 --opa-18974 --opa-19 caaacgtcgg 1 1193 opa-191193 100 ----opa-191193 opa-20 gttgcgatcc 1 1000 --0 ------opb-01 gtttcgctcc 3 777-1417 opb-011417 opb-01976 66.66 --opb-011417 opb-01976 --opb-02 tgatccctgg 2 759-898 ----------opb-03 catccccctg 2 637-1689 ----------opb-04 ggactggagt 2 942-2187 ----------opb-05 tgcgcccttc 6 539-1653 ----opb-05539 ----opb-06 tgctctgccc 6 750-1575 --0 -----opb-07 ggtgacacgg 2 741-1377 opb-07741 50 --opb-07741 --opb-08 gtccacacgg 8 520-1520 opb-081386 opb-081000 opb-08870 opb-08520 50 opb-091530 opb-091164 --opb-081386 opb-081000 opb-08870 opb-08520 opb-09 tgggggactc 0 ----0 ------opb-10 ctgctgggac 4 956-2156 --0 ------total 127 130-2389 22 17.32 4 11 11 issr marker ubc-861 (acc)6 4 ------------ubc-865 (ccg)6 6 445-1000 ----------ubc-868 (gaa)6 10 539-1889 ubc-8681486 10.0 ----ubc-8681486 ubc-873 (gaca)4 6 505-1541 ----------ubc-872 (gata)4 2 1250-3038 ----------ubc-808 (ag)8c 12 429-2036 ---ubc8081058 ubc808981 ---- ubc-807 (ag)8t 3 592-924 ----------39 437-3154 2 2.56 2 0 1 19characterization of intraspecific hybrid in clitoria ternatea ctb3-sl1: 0.104±0.02; acc. ctw2-bl1: 0.514±0.019). this variation might be attributed to the genetic recombination favouring conglomeration of suitable alleles, expression of genes producing key enzymes of metabolic pathways and growth environment which probably necessitated the production and accumulation of more taraxerol as reported for different bioactive metabolites in several medicinal species (amoo and van staden 2013). mitotic analysis of f1 hybrid revealed its chromosome count as 2n=16 similar to its parents. sixteen distable. 5. details of scot markers used for characterization of intraspecific f1 hybrid showing the inheritance of parent specific markers to the intraspecific f1 hybrid. primer sequence (5 ’ → 3’) no. of fragments amplified range of amplified fragments (bp) polymorphic bands percentage of polymorphism (%) no. of nonparental fragments* in f1 parent specific polymorphic bands (bp) in f1 from ctw2-bl1 ctb3-sl1 scot -01 caacaatggctaccacca 5 389-1077 ----------scot -02 caacaatggctaccaccc 7 293-1218 ----------scot-03 caacaatggctaccaccg 12 341-1593 ----scot-03341 ----scot -04 caacaatggctaccacct 4 684-2437 ----------scot -05 caacaatggctaccacga 3 500-1250 ----------scot -06 caacaatggctaccacgc 8 151-1706 ----------scot -07 caacaatggctaccacgg 6 967-2096 ----scot-07539 ----scot-09 caacaatggctaccagca 5 394-2260 ----------scot-10 caacaatggctaccagcc 5 1666-3916 ----------scot-11 aagcaatggctaccacca 4 509-1255 ----------scot-12 acgacatggcgaccaacg 3 366-676 ----------scot-13 acgacatggcgaccatcg 2 310-607 ----------scot-14 acgacatggcgaccacgc 6 313-1351 ----------scot-15 acgacatggcgaccgcga 2 257-358 ----------scot-16 accatggctaccaccgac 5 550-1658 ----------scot-17 accatggctaccaccgag 3 590-1546 ----------scot-18 accatggctaccaccgcc 8 316-1805 ----------scot-19 accatggctaccaccggc 14 341-2231 scot-19583 7.14 scot-19548 scot-19480 --583 scot-20 accatggctaccaccgcg 5 500-2210 ----------scot-21 acgacatggcgacccaca 5 231-1023 ----------scot-22 aaccatggctaccaccac 7 269-1132 ----------scot-23 caccatggctaccaccag 4 275-977 ----------scot-24 caccatggctaccaccat 6 480-1669 scot-24562 16.66 ----scot-24562 scot-25 accatggctaccaccggg 8 369-2654 scot-251308 12.5 --scot-251308 --scot-26 accatggctaccaccgtc 7 294-963 scot-26566 14.28 ----scot-26566 scot-27 accatggctaccaccgtg 8 527-2386 scot-27647 12.5 --scot-27647 --scot-28 ccatggctaccaccgcca 10 474-1552 scot-281431 scot-28981 20.0 ----scot-281431 scot-28981 scot-29 ccatggctaccaccggcc 14 335-2523 --0 ------scot-30 ccatggctaccaccggcg 8 390-2954 --12.5 scot-30438 ----scot-31 ccatggctaccaccgcct 11 326-2477 scot-31774 9.09 scot-31326 scot-31774 --scot-32 ccatggctaccaccgcag 9 116-2000 scot-321030 scot-32830 22.22 --scot-321030 scot-32830 --scot-33 ccatggctaccaccgcag 5 210-1000 --0 ------scot-34 accatggctaccaccgca 4 339-1176 --0 ------scot-35 gcaacaatggctaccacc 6 210-2555 scot-35 1430 scot-35210 33.33 --scot-351430 scot-35210 --scot-36 gcaacaatggctaccacc 5 449-899 scot-36899 20.0 ----scot-36899 total 224 13 5.80 6 7 6 20 aparupa naik et al. tinguished chromosomes were also observed in the mitotic metaphase and they were separated in normal fashion during anaphase. meiotic analysis revealed formation of eight bivalents at diakinesis and metaphase-i and 8ii: 8ii separation at anaphase-i, which might be due to homology between the parents. as result the pollen fertility of f1 hybrid is almost equivalent to its parents. these cytological observations along with morphophysiological traits could be helpful for the characterization of the f1 hybrids of c. ternatea as reported in many species. the homologous multigene families control the expression seed protein profile across the species, thus the seed protein marker exhibits monogenic segregation where the presence of polypeptide being completely dominant over absence, and in some cases, codominance for molecular weight variants also noticed (osborn 1988). mutations or deletions of structural genes coding for these polypeptides or their regulatory loci might lead to lack of expression of the concerned polypeptides (panigrahi et al. 2007). this kind of variations in seed protein marker profiling led the use this as as reliable markers for verification of hybridity of inter-varietal crosses (bennet et al. 1991), and inter-specific (panigrahi et al. 2001, jisha et al. 2011, mishra et al. 2012). in the present study sds-page of seed albumins revealed inheritance of three polymorphic polypeptides (mw 31.62, 22.38 and 18.81 kda) specific to pollen parent c. ternatea acc. ctw2-bl1 in the f1 hybrid. since c. ternatea acc. ctw2-bl1 was used as pollen parent, the appearance of these unique albumin polypeptides in the f1 hybrid can potentially be used as markers for identification of hybrids involving at least c. ternatea acc. ctw2-bl1 as pollen parent as reported in cajanus cajan (panigrahi et al. 2007) rapd, scot and issr marker analysis relies on differential enzymatic amplification of targeted dna fragments on the basis of primer annealing sequence of the genome. rapd is being random in nature, this kind of dna markers were ubiquitously distributed throughout the genome, and capable of detecting a high level of polymorphism. whereas issr is simple sequence repeat specific and scot is the specific to the conserved sequence around the initiating codon of the gene. these markers have also been successfully utilized in several crop species for diverse breeding efforts including identification and characterization of the hybrids. in the present study, rapd, issr and scot markers showed 17.32, 2.56 and 5.80% polymorphism among the parents. both issr and scot analysis showed lower polymorphism (2.56% and 5.80%) in comparison to rapd markers in the present study. there are some contradictory reports on detection of polymorphism by rapd and issr markers, issr markers showed more polymorphism than rapd markers (godwin et al. 1997; limabrito et al. 2006; nagaoka et al. 1997; zietkiewicz et al. 1994) and vice versa (muthusamy et al. 2008). this contradiction might be due to the use of different decamer oligonucleotides or ssr motifs as primers, and varied primer-annealing site in the genomes. again, issr and scot polymorphism depends on the frequency of ssr motifs and conserved sequence around the initiating codon, respectively (depeiges et al.1995; collard and mackill 2009) which vary within a species or even varieties targeted. identification of inter and intra-specific hybrids has been carried out in several species using either rapd or issr markers individually, or in combination (goldmann et al. 2008; jisha et al.2011; bianco et al. 2011, mishra et al. 2012). in this study 22 rapd, one issr and 13 scot markers were found to be inherited to the putative f1 hybrid, and among them 11 rapd and six scot markers, unique to pollen parent (c. ternatea, acc. ctw2-bl1), were very vivid in its appearance for the identification of f1 hybrid. in the present study, several non-parental fragments have also been amplified in the f1 hybrid, and this might be due to either dna recombination followed by minor genomic reorganization during the hybridization (huchett et al. 1995), or loss of priming sites due to chromosomal crossing over during meiosis (smith et al.1996). as the objectives is to identify the hybrids and to confirm the hybrid nature of putative seedlings at the juvenile stage, screening of the putative f1 hybrids using pollen parent-specific rapd, issr and scot markers contribute economic significance to this medicinal plant. the findings from the present study, it has been assorted that use of seed protein profiling and dna marker analysis complements the characterization of intra-specific f1 hybrid along with morpho-physiological traits and cytogenetic analyses more precisely. further, these inherited seed albumin and dna markers could also be used for further studies in gene mapping, 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72(3): 53-62, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-761 citation: s.i.r. conceição, a.s. róis, a.d. caperta (2019) nonreduction via meiotic restitution and pollen heterogeneity may explain residual male fertility in triploid marine halophyte limonium algarvense (plumbaginaceae). caryologia 72(3): 53-62. doi: 10.13128/caryologia-761 published: december 13, 2019 copyright: © 2019 s.i.r. conceição, a.s. róis, a.d. caperta. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. nonreduction via meiotic restitution and pollen heterogeneity may explain residual male fertility in triploid marine halophyte limonium algarvense (plumbaginaceae) sofia i. r. conceição1, ana sofia róis1,2, ana d. caperta1,* 1linking landscape, environment, agriculture and food (leaf), instituto superior de agronomia (isa), universidade de lisboa, tapada da ajuda, 1349-017 lisboa, portugal 2school of psychology and life sciences, universidade lusófona de humanidades e tecnologias (ulht), campo grande, 376, 1749-024 lisboa, portugal * corresponding author; e-mail address: anadelaunay@isa.ulisboa.pt abstract. the cosmopolitan halophylic genus limonium (plumbaginaceae) presents high cytogenetic interest because of the natural occurrence of diploid and polyploid variants. natural triploids are very rare in nature but common in this genus, including the widespread triploid limonium algarvense found in the iberian peninsula and in morocco. this study describes male sporogenesis and gametogenesis, pollen formation and germination, and seed production in triploid l. algarvense and diploid limonium ovalifolium using various cytological approaches. the diploid species presented regular meiosis. the triploid species was defective in male meiosis due to unpaired chromosomes, trivalent and tetravalent pairing, unbalanced chromosome segregation in meiosis i, and meiotic restitution in both meiosis i and ii. these results may be explained by indeterminate and broad first meiotic restitution. dyads and restitution nuclei at meiosis i were the most frequent meiotic products in the triploid species. cytomixis was observed in both species, and callose deposition did not differ among them. in the diploid species, regular, tricolpate pollen grains, which germinated in vitro were found. contrastingly, the triploid species produced heterogeneous pollen in morphology and size, with moderate to no viability that poorly germinated in vitro. we conclude that even if most triploids male gametes are non-functional, they seem to generate small numbers of viable gametes via nonreduction of chromosomes. flow cytometric seed screening demonstrated that the diploid species presented a diploid progeny whereas triploids only showed triploid progenies. in the triploids low pollen fertility coupled with viable seed production may assure their persistence in natural populations. keywords. apomixis, in vitro pollen germination, limonium, male sporogenesis and gametogenesis, meiotic restitution, polyploidy. introduction in flowering plants, polyploidy (i.e. the condition of having three or more copies of the basic set of chromosomes) has been considered to be one of the 54 sofia i. r. conceição, ana sofia róis, ana d. caperta main drivers of plant speciation (ramsey and schemske, 1998; adams and wendel, 2005). a major route of polyploidization rely on alterations of the meiotic cell cycle involving meiotic nuclear restitution during micro and megasporogenesis, originating unreduced gametes (bretagnolle and thompson, 1995; de storme and mason, 2014). several processes can lead to these gametes like cytokinetic defects, omission of a meiotic division (de storme and geelen, 2013) or alterations in spindle biogenesis and polarity. these processes can lead to spindle absence or malformation during metaphase i or ii (mi or mii), spindle co-orientation (parallel spindles) in mi (bretagnolle and thompson, 1995), and tripolar and fused spindles in mii (rim and beuselinck, 1996). meiotic restitution nuclei can be associated with first division restitution (fdr) or second division restitution (sdr) due to the omission of the first or second meiotic division, respectively (ramanna and jacobsen, 2003). the cosmopolitan species-rich genus limonium mill. (plumbaginaceae) contains complex aggregates of diploids and polyploids (róis et al., 2016; caperta et al., 2017; róis et al., 2018). these species are of interest because of the occurrence of diploid (2n = 2x = 16, 18 chromosomes), triploid (2n = 3x = 24, 25, 27), tetraploid (2n = 4x = 32, 35, 36), pentaploid (2n = 5x = 43), and hexaploid (2n = 6x = 51, 54, 56) variants (e.g., erben 1978, 1993; brullo and pavone 1981; arrigoni and diana 1993; castro and rosselló 2007). meiotic studies in the genus are scarce (d’amato, 1940a, 1940b, 1949; róis et al. 2012), although essential to assess fully the nature of cy tological variation of polyploid species. studies on male and female sporogenesis and gametogenesis revealed that diploid limonium ovalifolium (poir.) kuntze (2n = 2x =16) presented regular meiosis, whereas tetraploid limonium multiflorum erben (2n = 4x =35, 36) showed unbalanced and irregular meiosis (róis et al., 2012; róis et al., 2016). triploid cytotypes appear to be the predominant limonium cytotypes in the iberian peninsula and in the balearic islands (erben, 1978, 1979; cowan et al., 1998; castro and rosselló, 2007). however, meiotic studies in triploids are limited to a few works on female development in statice oleaefolia scop. var. confusa godr. (synonym limonium virgatum (willd.) fourr.; 2n = 27 chromosomes; erben, 1993) (d’amato, 1940a, 1940b, 1949). in the present study, our goal was to compare male sporogenesis and gametogenesis in triploid limonium algarvense erben with diploid l. ovalifolium (poir.) kuntze. both the diploid and triploid species are perennial and capable of vegetative reproduction, and have a widespread occurrence in the iberian peninsula and in morocco (erben, 1993; fennane et al., 2014; caperta et al., 2017). flowers of the triploid species have potential agrofood industry applications due to antioxidant and antiinflammatory properties (rodrigues et al., 2015, 2016). material and methods cytological analysis of microsporogenesis and gametogenesis five l. algarvense (2n = 25 chromosomes, 5.69 ± 0.15 pg/2c; caperta et al., 2017) and one l. ovalifolium (2n = 16, 3.58 ± 0.04 pg/2c; róis et al., 2012) genotypes from ex situ collections established in a greenhouse at instituto superior de agronomia (lisbon, portugal) were used. microsporogenesis and gametogenesis were analysed in floral buds in distinct developmental stages, as described in róis et al. (2012). buds selection was based on size: bud with 0.1 – 0.3 cm (from pre-meiotic interphase to metaphase i stage i); and bud > 0.3 – 0.5 cm (from anaphase i to pollen grain -stage ii). in brief, staged buds were fixed in a fresh absolute ethanol : glacial acetic acid (3:1) solution overnight and stored in 70 % ethanol solution at –20 º c until used. then, buds were digested in a pectolytic enzyme mixture [2 % cellulase (sigma), 2 % cellulase ‘‘onozuka r-10’’ (serva), and 2 % pectinase enzyme (sigma)] in 1xeb in a humid chamber for 2 h at 37 °c. meiocytes, chromosomes and pollen grains spreads were prepared from anthers. preparations were stained with 4’, 6-diamino-2-phenylindole hydrochloride (dapi) (1 mg ml –1) in vectashield (vector laboratories). the percentage (%) of meiotic products from cell fusion in the first division was calculated as the number of fused dyads/telophase i cells; and the percentage of meiotic products from cell fusion in second division was calculated as the number of fused triads or tetrads/ telophase ii cells, respectively. for tetrad analysis, staged flower buds previously fixed in ethanol : glacial acetic acid (3:1) solution were used. the buds were further placed in an aceto-carmine solution for 1 h, and dissected in a drop of aceto-carmine solution. for histochemical callose staining, f lower buds at stages i and ii were collected and stained through a modified procedure by musiał et al. (2015). buds were kept in 80 % ethanol for 30 min in agitation and transferred to 1 m naoh solution for 3 h at 37 ºc. then, the buds were washed twice in distilled water for 2 min with agitation, and placed in 0.1 m kpo4 for 2 min at room 55residual male fertility in triploid marine halophyte limonium algarvense temperature (rt), and subsequently in 0.1 % aniline blue (merck) in 0.1m kpo4 for 48 h, at rt. finally, anthers were dissected in multiwall-slides in a drop of 0.1m kpo4: glycerol (1:1). pollen size, viability and germination anthers from mature flowers stored in 70 % ethanol were stained using alexander’s stain (alexander, 1969) under a coverslip, and observed under light microscopy. total pollen viability estimates were performed by one person using three to five flowers per plant and counted with a 63x objective. about 300 pollen grains per flower were recorded. pollen grains dimensions were estimated as described in róis et al. (2012) by calculating mean, standard deviation, and standard error. for pollen tube growth analysis, five flowers (five anthers each f lower) per plant were used following the procedure described in róis et al. (2012). the pollen grains were collected from plants soon after anther dehiscence and cultured in a media containing 20 mm boric acid, 6 mm calcium nitrate, 0.1 % casein hydrolysate and 7 % sucrose (zhang et al., 1997). a dialysis tubing and filter paper support combined with 23 % polyethylene glycol –20,000 as an osmoticum in the medium, provided appropriate physical conditions for pollen germination. pollen grains were incubated at 37 ºc during 48 h or 72 h in the dark. the grains were considered germinated when they present a tube length that was equal or greater than the diameter of the pollen grain. for measurement of pollen tube length, 10 pollen tubes were selected randomly from each treatment, and measured on micrographs recorded with a 63x objective using axiovision 4.0 (zeiss) software. optical microscopy analysis and imaging slides of cell preparations, meiocytes, pollen grains, and pollen tubes were observed using a zeiss axioskop 2 fluorescence microscope and photographed with an axiocam mrc5 digital camera (zeiss). flow cytometric screening of seeds flow cytometric seed screening (matzk et al., 2000) was used to estimate the genome size of seeds derived from each plant progenies in at least 40 seeds, which were analyzed in pooled groups of 20 seeds. nuclei were isolated following the procedure of galbraith et al. (1983), in which 0.5 cm2 of fresh leaf tissue of each sample was chopped with a razor blade, simultaneously with 0.5 cm2 of fresh leaf tissue of the internal reference standard, in a petri dish containing 1 ml of wpb buffer (loureiro et al., 2007). as internal standard secale cereale ’dankovske’ (2c = 16.19 pg) (doležel et al., 1998) was utilized. the suspension was filtered through a 50 µm mesh nylon filter, and propidium iodide (50 µg/ml) was added to stain the dna. a partec cyflow space flow cytometer (partec gmbh, görlitz, germany) equipped with a green solid state laser (cobolt samba 532 nm, operating at 30 mw; cobolt, stockholm, sweden) was used to measure the relative f luorescence of stained nuclei. results were obtained using partec flomax software (v. 2.9). about 1300 nuclei per sample were analyzed. the dna-ploidy level was inferred as a relative position of the sample g1 peak to that of the internal standard. the exact chromosome numbers and dna ploidy level of the progenitor plants were determined by chromosome counting (please see caperta et al., 2017). the value of genome size in mass units (2c in pg; sensu greilhuber et al., 2005) was obtained for each individual analysed using the following equation: limonium 2c nuclear dna content (pg) = (limonium g1 peak mean / reference standard g1 peak mean) * genome size of the reference standard. statistics analysis an analysis of variance (anova) was applied to assess the significance of differences among the studied individuals in relation to meiotic products and pollen types (p < 0.05 and p < 0.001) (khan and rayner, 2003). percentages were logit-transformed before the statistical analysis to ensure homogeneity of variance. results the diploid l. ovalifolium presented a regular meiosis whereas triploid l. algarvense showed several meiotic abnormalities (fig.1). at pre-meiotic interphase, the triploid species exhibited the maximum of three nucleoli while the diploid species showed two nucleoli (data not shown). in the beginning of first meiotic division at the pachytene stage, abnormal meiocytes were observed in l. algarvense, with unpaired chromosomes (fig.1b), although l. ovalifolium showed full pairing of all chromosomes (fig. 1a). in l. ovalifolium eight bivalents were found at diakinesis (fig. 1c) whereas in l. algarvense, chromosome abnormalities proceeded as meiosis advanced to the next prophase stages. in this lat56 sofia i. r. conceição, ana sofia róis, ana d. caperta ter species, at the diplotene stage trivalents were detected besides bivalent formation (fig. 1d), and univalents were also visible in metaphase i (fig. 1f ). conversely, in l. ovalifolium metaphase i cells were regular (fig. 1e). at anaphase i, the majority of l. ovalifolium meiocytes were generally normal, although occasionally lagged chromosomes were found (fig. 1g). by contrast, in l. algarvense most anaphase i cells presented chromosome laggards (data not shown) and chromosome bridges (fig. 1h). at the end of telophase i, some dyads and fused dyads fig. 1. chromosome pairing and segregation in dapi-stained male sporocytes in diploid limonium ovalifolium and triploid limonium algarvense. a full pairing of chromosomes at pachytene in l. ovalifolium; b pachytene with unpaired (unp, arrowed) chromosomes in l. algarvense; c diakinesis showing eight bivalents in l. ovalifolium; d diplotene with trivalents (tri) and different chromosome associations in l. algarvense; e metaphase i in l. ovalifolium; f l. algarvense metaphase i showing an univalent (uni, arrowed); g anaphase i in l. ovalifolium; h abnormal anaphase i with chromosome bridges in l. algarvense (arrowed); i dyad and fused dyads in l. algarvense (j); k chromosome arrangement after a tripolar spindle in l. algarvense showing three groups of chromosomes (10, 6 and 9, respectively totalizing 2n = 25 chromosomes); l prophase ii in l. ovalifolium; m metaphase ii and abnormal anaphase ii (n) showing chromosome bridges in l. algarvense (arrowed); o triad fusion in l. algarvense; p co-existence of a fused dyad and a fused tetrad in l. algarvense. bars = 5 µm. 57residual male fertility in triploid marine halophyte limonium algarvense resulting in first division nuclei in the triploid species were found (fig. 1i-j). however, nuclei fusion was seldom seen in the diploid species. after telophase i in l. algarvense, tripolar spindles originate a particular chromosome arrangement where it was possible to detect 2n = 25 chromosomes arranged in three groups, respectively ten, six and nine chromosomes (fig. 1k). in this chromosome arrangement, associations of chromosomes with different sizes and shapes was moreover detected. in meiosis ii, regular prophase ii cells were observed in l. ovalifolium (fig. 1l). in l. algarvense chromosome bridges were still visible in metaphase ii and in anaphase ii (fig. 1m, n). at the end of meiosis, the diploid species showed mostly tetrads, while dyads were the more common meiotic product in the triploid species (table 1). in addition to monads and dyads, the triploid species showed triads and tetrads. the coexistence of fused dyads (fig. 1p) and tetrads (fig. 1p) as well as fused triads (fig. 1o) (second division restitution nuclei) and dyads with micronuclei (fig.2). although, the frequency of polyads formation is low in both the diploid and triploid species, its occurrence was rarer in triploid (0.2 %) than in diploid (5.5 %) species (table 1; fig. 2d). in both species, the presence of cy toplasmatic bridges with passage of nuclear content from one cell to another (cytomixis, fig. 2a) was observed, although with very rare incidence in diploid l. ovalifolium. no significant differences were detected between l. algarvense individuals in relation to the frequency of the meiotic products types for dyads (p =0.36), triads (p = 0.0692) and tetrads (p = 0.29). no significant differences were found between fused meiotic products for dyads (p =0.49), triads (p = 0.869) and tetrads (p = 0.29). to substantiate possible causes of nuclei fusion, callose deposition was verified using aniline blue labelling during different stages of microsporogenesis. at tetrad stage both in diploid and triploid plants, bright fluorescence callose labelling was visible around the meiotic products (fig. 3), without differences between species. the diploid species formed regular tricellular pollen grains with one vegetative nucleus and two sperm nuclei (fig. 4a) whereas in the triploid species bicellular pollen grains having only one or two vegetative nuclei and one sperm nucleus were observed (fig. 4b). in diploid (100%) and triploid species (l. algarvense ~ 70%) most pollen grains showed three colpi (fig. 5c, table 2). nevertheless, in the triploid species pollen grains with one colpus, two, four and five colpi were also found (fig. 5, table 2). in general, l. ovalifolium pollen grains measured 53.52 ± 5.6 µm (n = 41). by contrast, pollen grain size fig. 2. tetrad analysis of l. algarvense. a cytoplasmatic bridges with chromosome passing (cytomixis); b unbalanced triad (a micronucleus is arrowed); c tetrad; d polyad (a micronucleus is arrowed). bars = 5 µm. table 1. percentage of meiotic products from ex-situ collection limonium plants used. species accession number meiotic products 1st division restitution nuclei 2nd division restitution nuclei total of cells analysedmonads dyads triads tetrads polyads l. ovalifolium 2009i4sr 0 10.9 0 83.6 5.5 0 0 65 l. algarvense 2009i1al 0 23.3 18.5 7.2 0 45.8 5.2 249 2009i2al 0 59.6 6.1 0.5 0 28.8 5.1 198 2009i7al 10 69.5 5 2.1 0.2 9.4 4.0 479 2009i18al 21.9 20.6 1.3 0 0 57.6 2.6 155 2010i15pa 0 36.9 0 0.2 0.2 57.6 5.2 465 58 sofia i. r. conceição, ana sofia róis, ana d. caperta differences were detected in the triploid species: one colpus (17.5 ± 2.0 µm, n = 2), two (37.8 ± 4.4 µm, n = 1), three (56.2 ± 6.3 µm, n = 65), four (68.8 ± 3.0 µm, n = 51) and five (78.4 ± 1.3 µm, n = 9) colpi. the pollen grain types did not have significant differences among the studied triploid individuals for 1 colpus (p = 0.177), 3 colpi (p = 0.836), 4 colpi (p = 0.224), 5 colpi (p = 0.587), and for a significance level between 0.05 and 0.1 for 2 colpi (p = 0.0545). still, pollen grains viabilit y and germination revealed marked differences between species. comparing to the diploid species, which had 84.8 % (n = 1006) of viable grains, the triploid species ranged from low (genotype 2009i1al, 14.8 %; n = 824) to moderate (genotype 2010i15pa, 41.2 %; n = 900) viable pollen grains. the diploid species showed the highest frequency (60.5 %, n = 885) of germinated grains, while in the triploid species pollen germination frequencies varied among accessions, from 0.8 % (n = 900, in 2009i1al) to 8.2 % (n = 883, in 20109i15pa). the triploid species was able to produce seeds (c. 150/per scape) with a moderate seed germination frequency 65 % (n = 587). the estimation of embryo and residual endosperm nuclear dna contents by f low cytometry showed that in l. ovalifolium only histograms with a single 2c dna peak was found representing diploid seeds whereas in l. algarvense histograms with an unique 3c dna peak was detected (fig. 6). discussion polyploid plants can arise by the fusion of unreduced gametes or through a mechanism that employs an intermediate step generating triploids (triploid bridge hypothesis) (ramsey and schemske, 1998). triploids are considered to be meiotically unstable, resulting in frequent chromosome loss and fragmentation (mcclintock fig. 3. callose deposition in limonium ovalifolium and l. algarvense. a anther from a flower bud at stage ii with callose labelling in l. ovalifolium (bar = 50 µm); b anther from a flower bud at stage ii exhibiting bright fluorescence in l. algarvense (details shown in the inset – b’) (bar = 50 µm); tetrads with strong labelling in l. ovalifolium (c) and in l. algarvense (d) (bar = 5 µm). fig. 4. dapi staining of pollen grains. a regular tricellular pollen grain in l. ovalifolium with one vegetative nucleus and two sperms nuclei; b bicellular polar grain showing one vegetative nucleus (vn) and one sperm nucleus (sn). bars = 5 µm. fig. 5. pollen grains types in l. algarvense. pollen grains with one colpus (a), two (b), three (c), four (d) and five (e) colpi. bars = 5 µm. 59residual male fertility in triploid marine halophyte limonium algarvense 1929). these plants can produce diploid, triploid and tetraploid progeny as well as populations of aneuploid individuals with diverse karyotypes (henry et al., 2005). during microspore formation, triploid l. algarvense showed diverse division anomalies related to chromosome pairing and segregation. in prophase i at pachytene, l. algarvense presented unpaired regions, probably as a result of a lack of chromosome homology in some of these regions and reduced recombination. at diplotene and diakinesis the presence of triand tetravalents involving non-homologous chromosomes reinforces the hypothesis of intergenomic recombination. moreover, the tendency for chromosome nondisjunction and a high association of certain groups of chromosomes (with different size and morphology) revealed a high homology between some chromosome regions, which difficult their table 2. percentage of pollen grains morphotypes in l. algarvense. the percentage of each of the different pollen types and standard deviation is indicated. accession pollen grains 1 colpus 2 colpi 3 colpi 4 colpi 5 colpi 2009i1al 0.24 ± 0.2 (2) 1.09 ± 0.5 (9) 74.15 ± 1.8 (611) 23.18 ± 1.8 (191) 1.33 ± 0.7 (11) 2009i7al 0.61 ±0..3(5) 3.03 ± 1.5 (25) 75.30 ± 3.7 (622) 20.10 ± 2.4 (166) 0.97 ± 0.7(8) 2010i15pa 1.11 ± 0.5 (10) 4.33 ± 2.2(39) 74.11 ±1.5 (667) 19.78 ±3.3(178) 0.67 ± 0.6 (6) 2010i16pa 1.11 ± 1.0 (10) 4.89 ± 1.6 (44) 78.89 ± 5.3 (710) 14.56 ± 7.1 (131) 0.56 ± 0.7 (5) 2009i18al 0.35 ± 0.3 (3) 1.76 ±1.5 (15) 69.65 ± 6.3 (592) 25.65 ± 3.3 (218) 2.59 ± 2.7 (22) figure 6. flow cytometric histograms of seeds of l. ovalifolium (a) and l. algarvense (b-f). 60 sofia i. r. conceição, ana sofia róis, ana d. caperta normal dissociation during meiosis. furthermore, the chromosome organization presented in fig. 1k can be a clue to triploid l. algarvense hybrid origin. in this late anaphase i derived from a tripolar fuse, three distinct chromosome groups of six, nine and ten chromosomes were clearly visible, perhaps pointing to three genomes involved in this species formation. this meiotic behaviour strongly suggest parental genome differences for triploid l. algarvense, which may imply allopolyploid origin (i.e., attained by hybridization). distinct genomes usually have several differences at chromosome level as well as modifications in sequence, structure, and/ or gene order that difficult or inhibit homologous pairing (ramsey and schemske, 2002). these chromosome irregularities during the first meiotic division can be better explained by a broad fdr-type of meiotic restitution (de storme and geelen, 2013) or an indeterminatetype meiotic restitution (imr-type) (lim et al., 2001). in this latter case, meiotic non-reduction involved a reductional division of bivalents together with an equational segregation of univalents (lim et al., 2001). compared to diploid l. ovalifolium, triploid l. algarvense frequently showed fused nuclei at first division and second division, and dyads, triads, tetrads and polyads. in our study, coexistence of fused dyads and fused tetrads in the same nuclei spread is a strong indication of the occurrence of meiotic restitution. fdrand/or sdr-type meiotic restitution were considered as important processes for polyploid formation in e.g., triticeae (jauhar, 2007; ressurreição et al., 2012), solanum (den nijs and peloquin, 1977), arachis (lavia et al., 2011), and taraxacum (van dijk et al., 1999). these processes lead to unreduced gametes formation (bretagnolle and thompson, 1995; brownfield and kӧhler, 2011; de storme and geelen, 2013). callose is an essential barrier between meiocytes and defects on its deposition could lead to an ectopic genome doubling and cell fusion (spielman et al 1997; yang et al., 2003; de storme and geelen, 2013). our results showed that nuclear fusion might occur before callose deposition, since throughout anther development its deposition was regular in both species. another parallel phenomenon that occurred in both species was cytomixis that consists in the movement of the nuclei content between cells (singhal et al., 2010; kaur and singhal, 2012; mandal et al., 2013), and may lead to unreduced gametes. although it was not obvious at which stage of meiosis cytomixis occurred, it probably took place at meiosis i before callose deposition. this phenomenon can be one of the precursors of chromatin bridges, micronuclei, triads and polyads, as found in spergularia diandra (kaur and singhal, 2012). compared to the diploid species that produced regularly sized pollen (róis et al., 2012), a great diversity of pollen morphology and size was revealed in the triploid species. as previously observed for diploid (l. ovalifolium) and tetraploid (l. multiflorum) limonium species (róis et al., 2012), our study supports that pollen size and ploidy are not correlated in the limonium system. moreover, a direct correlation seems to exist between pollen grain morphology, viability and pollen tube germination, since such processes were only observed in grains predominantly with three colpi. in limonium, pollen viability appeared to be high in diploids whereas in polyploids, low to high fertility was reported (erben, 1978). a high pollen viability was observed in triploid turnera sidoides, which had irregular meiotic behaviour (kovalsky et al., 2018). in both diploid and triploid species studied here spontaneous seed production occurred, as insect pollinations were not frequent in our greenhouse. both species showed a high percentage of seeds per scape, with moderate to high germination. the exact chromosome numbers and dna ploidy level of the progenitor plants were determined in a previous study by combined flow cytometry and chromosome counting (caperta et al., 2017). in the present study, flow cytometric seed screening investigations demonstrated that in both species only one dna peak was found, which corresponds to the embryo peak, since mature seeds were characterized by one embryo and a well-developed starchy endosperm without nuclei (róis et al., 2012). no quantitative variation in seed ploidy was found in the progeny of diploid or triploid plants and thus l. ovalifolium only produced a diploid progeny whereas l. algarvense originated a triploid progeny. conclusions triploid l. algarvense plants displayed extremely unbalanced meiotic cell division, probably originating non-functional aneuploid gametes. however, as found in other natural triploids (ramsey and schemske, 1998), these plants may also generate small numbers of euploid (x, 2x) gametes and 3x gametes via non-reduction. even if the importance of these triploids as pollen donors is limited, they spontaneously produce viable seeds. although, the reproductive mode of triploid l. algarvense is not yet determined, this species show stable populations widespread in the iberian peninsula and in morocco (caperta et al., 2017), probably originated by apomixis (asexual seed production). 61residual male fertility in triploid marine halophyte limonium algarvense acknowledgements the authors gratefully acknowledge ana paula paes (leaf/isa) for help in plant maintenance and wanda viegas for useful comments and suggestions (leaf/ isa). funding this work was funded by portuguese national funds through fundação para a ciência e a tecnologia (fct, www.fct.pt/) projects ptdc/agrpro/4285/2014 and uid/agr/04129/2013, grants ptdc/agrpro/4285/ bm/2014, bpd/uid/agr/04129/2013 attributed to sirc and adc. references adams kl, wendel jf. 2005. polyploidy and genome evolution in plants. curr opin plant biol 8(2): 135–141. alexander mp. 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substantia an international journal of the history of chemistry vol. 2, n. 1 march 2018 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 72(3): 87-95, 2019 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-770 citation: s. chatterjee, s. ray (2019) clastogenic and cytotoxic effects of aerial parts’ aqueous extract of synedrella nodiflora (l.) gaertn. on wistar rat bone marrow cells. caryologia 72(3): 87-95. doi: 10.13128/caryologia-770 published: december 13, 2019 copyright: © 2019 s. chatterjee, s. ray. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. clastogenic and cytotoxic effects of aerial parts’ aqueous extract of synedrella nodiflora (l.) gaertn. on wistar rat bone marrow cells saumabha chatterjee1,2, sanjib ray2* 1 department of zoology, durgapur govt. college, durgapur-713214 2 molecular biology and genetics unit, department of zoology, the university of burdwan, golapbag, burdwan-713104, west bengal, india *corresponding author: ray.sanjibray@gmail.com abstract. synedrella nodiflora is a traditionally used medicinal plant. the aim of the present study was to analyze the clastogenic and cytotoxic effects (cces) of aerial parts’ aqueous extract of s. nodiflora (aaesn) on wistar rat bone marrow cells (wrbmcs). the cces of aaesn were analyzed with light and fluorescence microscopes respectively. the data indicate a dose-dependent (100-500 mg/kg body weight [bw]) increase in the aberrant cell frequencies, chromosomal structural aberrations and a significant (p<0.001) increase in apoptosis in the aaesn treated wrbmcs. the chromosomal aberration per 100 cells (apoptosis %) were calculated as 2.01±0.241 (5.02±1.72), 4.76±0.05 (46.73±2.34), 5.37±0.32 (66.92±2.92) and 6.58±0.14 (76.79±0.73) respectively for the aaesn doses of 0, 100, 300 and 500 mg/kg bw. in conclusion, the aaesn may contain phytochemicals with clastogenic and cytotoxic efficacy on wrbmcs, indicating, having the anticancer as well as carcinogenic potentials. therefore, it demands further an elaborate study to explore the active principle(s) and a proper care should be taken while it is prescribed in traditional medicine. keywords. apoptosis, clastogenic, cytotoxic, synedrella nodiflora, genotoxic, anticancer, secondary metabolites. 1. introduction cancer is an important worldwide health problem and the antiproliferative pharmacological activities of plant-derived secondary metabolites appear to explain the anticancer effects (figueroa-hernandez et al. 2005). a variety of bioactive components were isolated from the different medicinal herbs (cragg et al. 1996). alkaloids, fixed oils and fats, polyphenols, flavonoids, saponins, glycosides, terpenoids etc., having medicinal value, were extracted from a wide variety of plant species (tamilarashi et al. 2000). many plantbased active compounds act as antitumor and apoptotic cell death inducer in tumors (sato et al. 1994). acetogenins like uvaribonin, 22-epicalmistrin, and chalcone showed significant antiproliferative activity against a panel of cancer cell lines (pettit et al. 2008). generally, the cell cycle components are the 88 saumabha chatterjee, sanjib ray prime targets of most of the efficient anticancer agents (li et al., 2002). the discovery of competent anticancer drugs like vincristine and vinblastine were isolated from catharanthus roseous; paclitaxel (taxol®) extracted from taxus brevifolia represent trustworthy proof that plants are potential sources of novel anticancer chemotherapeutic drugs (cragg et al., 1996). the antiproliferative, clastogenic, and cytotoxic pharmacological activities of plant-derived secondary metabolites appear to elucidate the chemo-preventive or anticancer effects. therefore, searching for the phytochemicals having the clastogenic and cytotoxic effects (cces) are of renewed interest in drug discovery for cancer treatment. the cces of a medicine provide a glimpse into its mechanism of action. a clastogen can cause chromosomal structural alterations like chromatid break, deletion, sister chromatid exchanges, sister chromatid union, dicentric chromosome, acentric fragments, micronuclei etc. that subsequently may lead to the various cytotoxic effects including cell killing, apoptosis, and necrosis. moreover, the medicinal plants having these effects, one can also assume their toxicity risk factor for its indiscriminate use in the traditional therapeutic purpose (thybaud et al. 2007). cancer chemotherapeutic drugs are generally cytogenotoxic, hence subjected to non-target destruction but non-cancerous cells can revive better than the cancerous one (choudhury et al. 2000; palo et al. 2009). since these clastogenic chemotherapeutic drugs might enhance the chance of secondary cancer development, dose optimization, target specification, and combination chemotherapy with the other antioxidants are highly recommended (pandit and choudhury, 2011). many anti-cancer agents cause dna damage at a very high level leading to the cell cycle checkpoint activation and programmed cell death (helleday et al. 2008). administration of the many plant-derived anticancer agents, including paclitaxel, can affect spindle stability leading to abnormal mitosis and chromosomal aberration (dumontet and jordan, 2010). synedrella nodiflora (l.) gaertn. (family: asteraceae) is an ephemeral flowering weed. it is indigenous to tropical america and also distributed in india, malaysia, bangladesh, china, japan, and other indopacific countries (wiart 2006). in india, s. nodiflora leaves are traditionally applied for the remedy of rheumatism. in ghana, oral application of warm aqueous juice of this plant results in the remedy of epilepsy. in malaysia, it is used externally as a medicine for the treatment of inflammation, headache, and earache. the leaves are also used for the treatment of hiccup, stomachache, and threatened abortion cases (rathi and gopalkrishnan 2005; rahmatullah et al. 2010; bhogaonkar et al. 2011). the toxicological (olukunle and abatan 2008; dutta et al. 2012), insecticidal (rathi and gopalkrishnan, 2005), larvicidal (ghayal et al. 2010), antibacterial, antioxidant (wijaya et al. 2011), antidiarrhoeal, hypoglycaemic (zahan et al., 2012), anti-inflammatory properties (haque et al. 2012) of this plant have been reported. our previous study revealed the antiproliferative activity of the aerial parts’ aqueous extract of s. nodiflora (aaesn) on root apical meristem cells and wistar rat bone marrow cells (wrbmcs) as well as the presence of different phytochemicals like alkaloids, flavonoids, terpenoids, tannins, phlobatannins, and saponins in the aaesn (ray et al. 2013b). however, the cces of aaesn on the mammalian system has not been well-studied. thus, the present study is focused on the assessment of the clastogenic and cytotoxic effects of the aerial parts’ aqueous extract of s. nodiflora on wrbmcs in vivo condition. nabeel et al. (2008) described the cytogenetic effect of the aqueous extract of arum maculatum on the bone marrow cells of the swiss male mice. some of the parameters recorded by the scientists were also taken into consideration in this study. the novel aspects of this study are that it explored the cces of aaesn, a source of future anticancer chemotherapeutic drugs, and raised the question against its indiscriminate use in traditional medicine. 2. materials and methods 2.1. chemicals colchicine, glacial acetic acid, and methanol were obtained from bdh chemicals ltd., uk. edta was procured from gibco, grand island, n.y, usa. ethidium bromide and acridine orange were purchased from sigma, st. louis, m.o., usa and s.d. fine-chem. ltd., mumbai, india respectively. other chemicals used in this work were of analytical grade from reputed manufacturers. 2.2. plant products collection, storage, and extract preparation plant aerial parts collection, storage, and extract preparation procedures were described in detail in our earlier report (ray et al. 2013b) and briefly the fresh aerial parts’ of s. nodiflora were collected from golapbag campus of the university of burdwan, taxonomically authenticated by prof. ambarish mukherjee, and the voucher specimen (no.bugbsc013) is maintained in the department ( figure 1). the dried and pulverized plant product was boiled in double-distilled water 89clastogenic and cytotoxic effects of synedrella nodiflora (1:10, w/v) in a water bath for 30 min. the extract was allowed to cool to room temperature, filtered by whatman filter paper no. 1 (sigma-aldrich, inc., st. louis, mo, usa), and then refrigerated at -20°c for further use. for the measurement of extract value (17.64%w/w) and extract concentration (11.3 mg/ml), 10 ml of extract was kept for evaporation to complete dehydration in a hot air oven at 60 ºc. 2.3. experimental animals male wistar-albino rats (age 4-6 weeks; weight 40–60 g) were purchased from local vendors and maintained in the departmental animal house (in community cages) at room temperature (25±2oc), controlled illumination (12 h light and 12 h dark cycle), and with standard rat diet and water. the rules of the “institutional animal care and use committee” were strictly followed throughout the whole experiment and the required total 24 rats were euthanized with prior approval from the dissection monitoring committee (dmc) of the university of burdwan (no: r-s/n-1/646, dated 30-03-2016; under ref. no. budmc/2016/01/05(a) dated 13.07.2016). 2.4. treatment and clastogenicity analysis the aaesn (100, 300 and 500 mg/kg body weight [bw]) was injected into the peritoneal cavity of the male wistar rats (ray et al. 2013b). control rat groups were injected an equal volume of double distilled water. at each data point, six rats were used. after 12 h of aaesn injection, colchicine (10 mg/kg bw), a standard metaphase arresting agent, was injected into the peritoneal cavity of the rats irrespective of control and treatment groups for 3 h (ray et al 2013b). then the animals were euthanized by cervical dislocation just before the femur bones were dissected out (ray et al. 2013b) and the wrbmcs were fixed in aceto-methanol (1:3) after the required hypotonic (0.56% kcl) treatment. control group was considered for nullifying the sole toxic effect of colchicine. the detailed procedure of metaphase plate preparation and giemsa staining procedure were described earlier (ray et al. 2013b). briefly, the femur bones were dissected out, the bone marrow cells were collected in 15 ml centrifuge tubes by flushing with pre-warmed (37°c) 2.5 ml of 0.56% aqueous kcl solution with 5 ml hypodermic syringe, the cells were maintained in a hypotonic solution for 30 min at 37°c in a water bath and then the cells were fixed with aceto-methanol (methanol 3 parts and acetic acid 1 part). metaphase plate preparation was done through flame-drying technique and 2% giemsa (staining duration: 35 min) was used for staining. the giemsa stained slides were mounted with a coverslip in synthetic medium and the different types of chromosomal abnormalities like chromatid break, terminal deletion, fragmented chromosome, centric fusion, centromeric association, ring chromosome, chromatid gap, chromosomal association, end to end association etc. were scored (kumpawat et al. 2003). 2.5. fluorescence microscopic cytotoxicity analysis the acridine orange-ethidium bromide (ao-eb) double staining procedure (bustillo et al. 2009) was used to determine cytotoxic effects of aaesn in terms of early and late phases of apoptosis with the fluorescence microscope by observing changes in nuclear morphology and apoptotic blebbing. it is the established fact that acridine orange can infiltrate in both the live and dead cells while ethidium bromide enters only in dead cells. the ao-eb staining strategy causes color differentiation with blue-filter excitation; the living cells (stained only with acridine orange) give green fluorescence, the early apoptotic cells (permitting limited penetration of ethidium bromide) show green to yellowish nuclei with perinuclear chromatin condensation, the late apoptotic cells show a dark red color with fragmented or condensed chromatin, and the necrotic cells give red color with large nucleus having no condensed chromatin. here, the aaesn treatment and wrbmcs collection procedures were same as described for clastogenicity analysis, except, the harvested cells were washed in 1x pbs instead of hypotonic kcl solution. after cenfig. 1. showing the aerial parts (leaf, stem, and flower) of synedrella nodiflora (l.) gaertn. 90 saumabha chatterjee, sanjib ray trifugation at 1000 rpm for 5 minutes and discarding the supernatant, the precipitates were stained with acridine orange-ethidium bromide mix (conc. 100 µg/ml) in 1:1 ratio for 5 minutes in 2 ml eppendorf tubes. then, cells were washed thrice with 1x pbs repeating the centrifugation steps. the cells were resuspended in 100 µl 1x pbs and then 20 µl cell suspensions were taken on grease free slide, the percentages of dead cells (apoptotic vs necrotic) and live cells were scored under leica fluorescence microscope. 2.5. scoring and statistical analysis all the results were expressed as mean±sem. aberrant cell percentage were analyzed through one way anova (d.f.=11) followed by tukey-kramer tests. the differences between the untreated and treated groups for cellular viability and chromosomal abnormalities were analyzed with the 2x2 contingency χ2-test and were considered statistically significant at p<0.05, p<0.01 and p<0.001. 3. results 3.1. clastogenic effects of aaesn metaphase chromosomal abnormalities were scored in all the aaesn treated groups of rats and were compared with the untreated controls. the various clastogenic effects were observed in the aaesn treated rats. the number of cell abnormality percentage was calculated as 4.76±0.03, 5.37±0.32, and 6.58±0.14 after aaesn treatment respectively with 100, 300, and 500 mg/kg bw as compared with the control (2.01±0.24 %) (table 1). among the different clastogenic effects scored, chromatid break, terminal deletion, centric fusion, centromeric association, ring chromosome, chromosomal association, and an end to end association followed a dose-dependent increase in frequencies. here, the chromosomal association (0.98±0.01) percentage was found to be the highest frequency of chromosomal abnormality followed by centric fusion (0.89±0.03), fragmented chromosome (0.88±0.01), end to end association (0.86±0.02), centromeric association (0.83±0.01), chromatid break (0.54±0.03), ring chromosome (0.51±0.06), and terminal deletion (0.45±0.02) after the treatment with 500 mg/kg bw of aaesn (figure 2 and table s1). 3.2. fluorescence microscopic analysis for cytotoxicity apoptotic cells were examined under a f luorescence microscope after acridine orange and ethidium bromide (ao-eb) combined staining of wrbmcs. a dose-dependent increase in the apoptotic cells (%) was observed in aaesn treated samples. as compared with the untreated cells, a significantly (p<0.001) increased percentages of early and late apoptotic cells, and necrotic cells were observed in aaesn treated wrbmcs. the dose-dependent response was more prevalent in apoptotic cell death than that of necrosis. the maximum percentages (85.40±1.71) of viable cells were counted in the table 1. pooled data showing the aaesn induced aberrant cell percentage of wrbmcs. aaesn treatment dose (mg/kg bw) tm ab.cell(%)= (tac/tmc)x100 range mean±sem (% increase) 00 100 1.72-2.48 2.01±0.24#♯ᵟ 100 96 4.69-4.84 4.76±0.05*♯ᵟ (136.8) 300 94 4.81-5.93 5.37±0.32*#ᵟ (167.2) 500 109 6.42-6.86 6.58±0.14*#♯ (227.4) *significant at p < 0.05 as compared to the control, # at p < 0.05 as compared to the 100 mg/kg aaesn treatment, ♯ at p < 0.05 as compared to the 300 mg/kg aaesn treatment, ᵟ at p < 0.05 as compared to the 500 mg/kg aaesn treatment by one way anova (d.f.=11) followed by tukey-kramer procedure. bw; body weight, tm; total no. of metaphases counted for studying chromosomal abnormality, ab.cell %; aberrant cell per percentage; tac; total number of aberration count, tmc; total metaphase count. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 * * * * *** * * * * ** *** ** * * ** * * * ** ** 00 100 300 500 d .a b/ 10 0 c el ls doses of aaesn (mg/kg bw) cb td fc cf cta rc ca ea fig. 2. influence of aaesn on wrbmcs in terms of different chromosomal abnormalities. *significant at p<0.05, **at p<0.01 and ***at p<0.001 as compared to the control by 2x2 contingency χ2-test (d.f.=1). the data were represented as mean±sem. d.ab./cell: different abnormalities per cell scored; cb: chromatid break; td: terminal deletion; fc: fragmented chromosome; cf: centric fusion; cta: centromeric association; rc: ring chromosome; ca: chromosomal association; ea: end to end association. 91clastogenic and cytotoxic effects of synedrella nodiflora untreated samples and the maximum percentages of early apoptotic (20.92±0.34), late apoptotic (55.87±0.69) and the total apoptotic cell frequency (76.79±0.73) were calculated in aaesn (500 mg/kg bw) treated samples. the maximum percentage (5.34±0.57) of necrotic cells were scored at a dose of 300 mg/kg bw of aaesn for 500 mg/ kg of aaesn treatment (figure 3 and table s2). 4. discussion the leaves of synedrella nodiflora are traditionally used for the remedy of rheumatism, epilepsy, inflammation, headache, stomachache, and earache (rathi and gopalkrishnan 2005; rahmatullah et al. 2010; bhogaonkar et al. 2011). in the present study, we have tested in vivo cces of aaesn on the wrbmcs. the rapidly dividing bone marrow cells are the ideal model to demonstrate antiproliferative activity of herbal extracts (ray et al., 2013 a, b). the wrbmcs are also considered as a basic model for clastogenicity testing and many authors have used it to study the side-effects of anti-cancer/anti-inf lammatory medicines (pearse et al. 2009; pandit and choudhury 2011; haque et al. 2012; ray et al. 2013a; ray et al. 2013b; gomaa 2018). generally, the bone-marrow mito-depressive drugs show anticancer activity (prasanthi, 2016). the previous studies indicated the mutagenic activity of pyrethroids on murine bone marrow cells, human peripheral blood lymphocytes, and in aquatic animals (barrueco et al. 1992; oraby 1997). the general non-steroidal antiinflammatory drugs (nsaids) may also inhibit the proliferation of bone marrow cells without any intervention of hormonal activities in murine models (chang et al., 2007). the study of the bone marrow suppression is designated as a common toxicity assessment of cytotoxic agents and this toxicity assay had been included in the preclinical study of the four-stage trial system (heidelberg and fox, 1990). our earlier study indicated a dose-dependent decrease in metaphase frequency (ray et al. 2013b) and in the present study a dose-dependent increase in chromosomal abnormalities (both total and differential counts) and apoptotic cells percentage in wrbmcs. several anticancer agents put forth their influence through cell cycle events (salmon et al., 1984). the antiproliferative activities of the several herbal extracts are related to their ability to obstruct dna synthesis (akinboro and bakare 2007; mercykutty and stephen 1980). toona sinensis leaf aqueous extract has been reported to have an anti-proliferative influence on human lung cancer cells (laosinwattana et al. 2007, 2009). in the present study, clastogenicity of aaesn on the wrbmcs revealed that several types of chromosomal abnormalities including chromosomal fragmentation, chromatid break, ring chromosome formation, centromeric association etc. were induced by aaesn. kumpawat et al. (2003) showed that raw betel-nut extract introduced clastogenicity on mouse bone marrow cells and human peripheral blood lymphocytes. there are similar kinds of study reports where they explored the genotoxic activity of plant extracts on wrbmcs and human peripheral blood lymphocytes (pandit and choudhury 2011; sakamoto-hojo et.al. 2017). we previously reported the cytogenotoxic alteration of onion apical meristem cells that were exerted by the aerial parts’ aqueous extract of ampelocissus latifolia (chaudhuri and ray 2014). nefic (2008) described the effect of ascorbic acid on human peripheral blood lymphocytes, where vitamin-c at a higher concentration (1,000μg/ml) could induce mitotic arrest and chromosomal abnormalities. the similar kinds of dose-optimization studies were performed using the methanolic extracts of artemisia annua and pyracantha coccinea on allium cepa root apical meristem cells (karaismailoglu mc 2014, 2017). pandit and choudhury (2011) narrated the clastogenic effect of a chemotherapeutic drug on mouse bone marrow cells. gewirtz (1999) revealed cytogenotoxic effect of anthracyclin antibiotics due to suppression of topoisomerase-ii and thus hindering topoisomerase-ii mediated dna cleavage and re-ligation. moreover, it also triggers ros generation (dorosho, 1983). 0 20 40 60 80 100 ** ** * ** * ** * ** * ** * ** * ** * ** * ** * * ** * ** * * ** * 50030010000 c el l ( % ) doses of aaesn (mg/kg) nvc eac lac tac nc fig. 3. shows aaesn induced cytotoxicity observed on wrbmcs under a fluorescent microscope. nvc: normal viable cells; eac: early apoptotic cells; lac: late apoptotic cells, tac: total apoptotic cells. nc: necrotic cells. experiments were done in three sets and data were represented as mean±sem. *significant at p<0.05, **at p<0.01 and ***at p<0.001 as compared to the control by 2x2 contingency χ2-test (d.f.=1). 92 saumabha chatterjee, sanjib ray many anti-cancer agents cause dna damage at a high level leading to checkpoint activation and programmed cell death (helleday et al. 2008). administration of anticancer agents like paclitaxel can affect spindle stability leading to abnormal mitosis and chromosomal aberration (dumontet and jordan, 2010). one noticeable thing is that colchicine, a mitostatic drug, was also used here to arrest the cells at metaphase but it was equally administered to both control and treatment groups. here, the treatment group showed a significantly higher level of clastogenic alterations of chromosomes in comparison to the control group in a dose-dependent manner. so, apart from colchicine, there must be extra clastogenic effects of aaesn. we previously reported cell cycle retardation effect as well as the increased prophase-metaphase frequency on allium cepa root apical meristem cells treated by the same extract. recently, bonciu et al. (2018) used allium cepa root apical meristem cells as a genotoxicity test system. the mito-retarding effect was also noticed in case of wrbmcs in that study in a dose-dependent manner, apart from the influence of colchicine in both control and treatment groups (ray et al., 2013b). hence, the strong probability of interaction of the phytochemical(s) present in aaesn with mitotic spindle could not be ignored. however, a detailed mechanism of clastogenic action of aaesn is subjected to further detailed study. the ao-eb combined staining assay data indicate a dose-dependent increase in the apoptotic cell frequency to a much greater extent than that of necrotic cells. treatment with aaesn caused the characteristic changes related to apoptotic morphologies in wrbmcs indicating that s. nodiflora is an extensive source of natural bioactive substances with apoptotic cell death-inducing activity on wrbmcs. another important observation was that the necrotic cells’ increased frequency did not follow a dose-response relationship and the data, in turn, suggest an apoptotic potential of aaesn. the maximum percentage (5.34±0.57) of necrotic cells were scored at a dose of 300 mg/kg bw of aaesn, indicating, unlike apoptosis, the necrotic cell frequencies did not follow a dose-dependent response pattern. there are similar reports showing the apoptotic cell death-inducing effects of some of the anticancer agents like isodeoxyelephantopin (farha et al., 2013) and farnesiferol c (hasanzadeh et al., 2017). previously, we also described the cytotoxic effect of aerial parts’ aqueous extract of a. latifolia on apical meristem cells using ao-eb staining method (chaudhuri and ray 2014). chu et al. (2014) showed the antiproliferative and cytotoxic effect of camptothecin-20(s)-o-(2-pyrazolyl-1) acetic ester (cpt6) on breast tumor mcf-7 cells by increased sub g1 cell population and apoptosis induction among the treatment groups. farha et al. (2013) reported isodeoxyelephantopin (idoe) mediated apoptosis on nasopharyngeal carcinoma (kb) cells by obtaining more apoptotic morphologies through ao-eb combined staining assay in idoe treated kb cells. our results are in agreement with those of ichikawa who reported the apoptosis-inducing effects of isodeoxyelephantopin in various cells (ichikawa et al., 2006). our previous study revealed the presence of alkaloids, tannins, terpenoids, f lavonoids, phlobatannins, and saponins as active ingredients in this extract (ray et al. 2013b). these active ingredients might have interacted with the cell cycle machinery and/or with dna thus inducing cell cycle delay, clastogenicity, and apoptosis. the aaesn-induced abnormal mitosis in wrbmcs might lead to the mitotic catastrophe through apoptosis and necrosis. mitotic catastrophe is an intrinsic mechanism that senses mitotic failure / abnormal mitosis and responds by driving a cell to an irreparable antiproliferative fate of death or senescence (margaret 2015; vakifahmetoglu et al. 2008). here, the mitotic catastrophe induced by aaesn was more prone to result in apoptosis rather than necrosis which is more desired outcome in cancer chemotherapy. our results related to toxicity of this plant are in agreement with the histopathological toxicity and brine shrimp lethality of s. nodiflora (olukunle et al. 2008; dutta et al. 2012). 5. conclusion the phytochemicals present in aerial parts’ aqueous extract of synedrella nodifolia could induce cytotoxicit y, clastogenicit y and mitotic catastrophe in wrbmcs and thus indicate its potential use in cancer chemotherapy in the near future. there is ample scope to explore the active principle(s) of aaesn and the detailed molecular mechanism of the cces. moreover, awareness should be raised among the concerned tribals regarding its probable side-effects due to indiscriminate use and overdose. funding financial support was received from the ugc (jrf), india and infrastructural 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95clastogenic and cytotoxic effects of synedrella nodiflora wijaya s. nee t k, jin k, hon tlk, san lh, wiart c. 2011. antibacterial and antioxidant activities of synedrella nodiflora (l.) gaertn. (asteraceae). j. complement. integr. med. 8(1): 1-13. zahan r, nahar l,. haque a, mosaddik a, faza, a, alam z, and haque me. 2012. antidiarrhoeal and hypoglycemic effects of synedrella nodiflora. phytopharm. 2(2): 257-264. table s1. supplementary pooled data of fig.2 showing the aaesn induced different categories of aberrant cell percentage of wrbmcs. doses (mg/ kg bw) of aaesn tm d.ab/cell (%) cb td fc cf cta rc ca ea 00 100 0.14±0.04 0.05±0.04 0.48±0.05 0.15±0.03 0.31±0.04 0.1±0.03 0.27±0.05 0.11±0.03 100 96 0.43±0.11b 0.09±0.00 0.93±0.01b 0.60±0.05c 0.49±0.03 0.37±0.04c 0.87±0.03c 0.5±0.03c 300 94 0.46±0.11c 0.27±0.05c 0.87±0.05a 0.65±0.05c 0.59±0.02a 0.46±0.08c 0.96±0.04c 0.63±0.08c 500 109 0.54±0.04c 0.45±0.03c 0.88±0.01b 0.89±0.04c 0.83±0.02c 0.51±0.08c 0.98±0.01c 0.86±0.03c asignificant at p<0.001, bat p<0.01 and cat p<0.05 as compared to the control by 2x2 contingency χ2-test (d.f.=1). tm; total no. of metaphases counted for studying chromosomal abnormality, d.ab./cell: different abnormalities per cell scored; cb: chromatid break; td: terminal deletion; fc: fragmented chromosome; cf: centric fusion; cta: centromeric association; rc: ring chromosome; ca: chromosomal association; ea: end to end association. table s2. supplementary pooled data of fig. 3 showing aaesn induced apoptosis and necrosis in wrbmcs in vivo. doses (mg/ kg bw) of aaesn tc nvc eac lac tac nc tc mean±sem tc mean±sem tc mean±sem tc mean±sem tc mean±sem 00 1728 1476 85.40±1.71 102 6.26±0.78 150 8.75±1.23 252 15.02±1.72 0 100 2015 1035 51.44±2.00a 240 11.93±0.89c 700 34.80±1.54a 940 46.73±2.34a 40 2.00±0.33c 300 1677 468 27.92±1.16a 258 15.43±0.86a 861 51.49±2.12a 1119 66.92±2.92a 90 5.34±0.57a 500 2066 404 19.57±0.45a 432 20.92±0.34a 1154 55.87±0.69a 1586 76.79±0.73a 76 3.68±0.24b conc.: concentration; tc: total cells; nvc: normal viable cells; eac: early apoptotic cells; lac: late apoptotic cells, nc: necrotic cells, tac: total apoptotic cells asignificant at p<0.001 as compared to the control by 2x2 contingency χ2-test (d.f.=1). experiments were done in triplicate and data were represented as mean±sem. substantia an international journal of the history of chemistry vol. 2, n. 1 march 2018 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 73(3): 45-54, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-867 caryologia international journal of cytology, cytosystematics and cytogenetics citation: w.m. amer, r.a. hassan, a.s. abdo (2020) cytogenetic and molecular studies of the egyptian capsella bursa-pastoris (brassicaceae). caryologia 73(3): 45-54. doi: 10.13128/caryologia-867 received: february 23, 2020 accepted: april 19, 2020 published: december 31, 2020 copyright: © 2020 w.m. amer, r.a. hassan, a.s. abdo. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. cytogenetic and molecular studies of the egyptian capsella bursa-pastoris (brassicaceae) wafaa m. amer, rania a. hassan*, amany s. abdo botany and microbiology department, science faculty, cairo university, egypt * corresponding authors. e-mail: raniaali@sci.cu.edu.eg; raniaaly2006@yahoo.com abstract. capsella bursa-pastoris  (brassicaceae) is one of the most successful tetraploid species in the world. it showed high morphological diversity within egyptian populations. morphological investigations of herbarium specimens and fresh collected populations grouped them under three distinguished morphotypes (lobed “l”; simple “s” and lobed-simple “ls”) depending mainly on the basal leaves structure. this high degree of phenotypic variation has received our critical attention. until recently, the previous studies on c. bursa-pastoris  attributed its phenotypic variation to environmental factors. but in egypt, these three morphotypes were traced in mixed populations along with the species geographical range, so the environmental factors have no influence on their distribution or phenotypic variation. accordingly, our primary concern in this study was to  determine the factors controlling this variation. the cytogenetic studies revealed that the three identified morphotypes are three distinct genotypes with three different chromosome numbers: 2n=2x=16 (diploid) for “l”; 2n=3x=24 (triploid) for “s”; 2n=4x=32 (tetraploid) for “ls”. the triploid genotype “s” showed rare occurrence among the studied populations and is postulated to be a new record of a hybrid in egypt. karyotyping of the three genotypes showed significant differences in the genome and chromosomes relative lengths. molecular study using cpssr technique supported the cytogenetic results and differentiated the three studied genotypes. the retrieved results revealed that the phenotypic diversity within the egyptian c. bursa-pastoris populations is genetically controlled. keywords: capsella bursa-pastoris, cytogenetic studies, genotypes, karyotyping, molecular study, phenoplasticity. introduction brassicaceae (cruciferae) or mustard family is one of the largest angiosperm families, it comprises 3977 species and 341 genera and 52 tribes (kiefer et al., 2014). one of the most important genera in the brassicaceae is genus capsella medik. molecular systematic studies confirmed that genus capsella belongs to the tribe camelineae (neuffer et al., 2014). this genus is an excellent model for molecular evolutionary studies due to its phylogenetic relations within the brassicaceae. studying its genetics, speciation and sympatric distribution is important for agricultural matters. genus capsella is represented worldwide by five species: the two selfcompatible tetraploid c. bursa-pastoris (l.) medik and c. thracica velen 46 wafaa m. amer, rania a. hassan, amany s. abdo (2n=4x=32); the self-incompatible diploid c. grandiflora (fauche & chaub) boiss; the two self-compatible diploid c. rubella rent. and c. orientalis klokov (hurka et al., 2012; neuffer et al., 2014). these species differ greatly in their geographical distribution, where, c. grandiflora is limited to northwestern greece and albania; c. rubella has a broad mediterranean / central european distribution; c. orientalis is found from eastern europe to central asia (hurka et al., 2012); c. thracica is endemic to bulgaria (neuffer et al., 2014). capsella bursa-pastoris (shepherd’s purse) is an annual to biennial species, extremely variable in size and leaf form, distinguished by terminal and axillary raceme inflorescences. its silicula fruit is obcordate-obtriangular in shape (amer et al., 2019). this species is the second most common flowering plant in the world (zhou et al., 2001), grows as a common weed of agriculture in almost all countries of the world from tropical to subarctic habitats (holm et al., 1979), and shows a high phenotypic plasticity (korsmo, 1954; holzner and numata, 1982). accordingly, the evolution of polyploidy and weediness in c. bursa pastoris is interesting to agricultural research (st onge, 2010). many taxonomic studies were carried out on this species depending on morphological characters and resulted into many species, subspecies, varieties, microspecies, biotypes, and segregates (aksoy et al., 1998; aksoy et al., 1999; neuffer, 2011). a considerable amount of literature has attributed the phenotypic variation in c. bursa-pastoris to environmental or geographical factors like seasonality, temperature, shade, rainfall, latitudinal and altitudinal gradients (almquist, 1929; neuffer, 1989; neuffer and bartelheim, 1989; stace, 1989; neuffer, 1990; aksoy, 1996; aksoy et al., 1999; neuffer and hoffrogge 2000). in egypt, capsella is a monospecific genus represented by c. bursa-pastoris (boulos, 1999). the field study and morphological investigations of this species – based on the leaves, inflorescence and fruit characters – showed the presence of high degree of phenotypic variation and revealed the presence of three morphotypes namely: lobed “l”, simple “s” and lobed-simple “ls” (amer et al., 2019). these three morphotypes were traced in mixed populations along with the species geographical range, so the environmental factors have no influence on their distribution or phenotypic variation. therefore, this study aims to determine the factors controlling the phenotypic variation of c. bursa-pastoris morphotypes through cytogenetic and molecular studies to clarify the impact of genetic diversity on their phenoplasticity. materials and methods morphological study the morphological investigations of c. bursa-pastoris were carried on 36 old populations deposited as herbarium specimens in cairo university herbarium (cai) and assiut university herbarium (astu). in addition to 66 fresh populations collected from menoufia (abu sleem village), faiyum (sinnuris district, el siliene) and el saff regions during our field work conducted in 20162018. the studied specimens (old & fresh) from different distribution localities are shown in table 1. from 66 fresh populations, 25 specimens/ population were undergone morphological investigations using different morphological criteria of leaves, inflorescence and fruit. acronyms of herbaria follow thiers (2019). the morphological investigations distinguished three morphotypes of c. bursa-pastoris in all the studied populations namely: (l) lobed, (s) simple and (ls) lobed-simple (amer et al., 2019). cytogenetic studies sample preparation for cytogenetic studies, the specimens collected from faiyum region (marked with * in table 1) were selected to nullify the environmental factors. seeds of 30 specimens representing the three morphotypes (10/ morphotype) were collected, soaked in distilled water for 2 hours, and germinated at room temperature. root tips of about 1 cm length were treated with colchicine (c22h25no6, 0.025 %) for 2 hours at room temperature, and washed thoroughly with distilled water. fixation was done using ethyl alcohol: glacial acetic acid (3:1, v/v). samples were washed thoroughly with water and hydrolyzed using 1 n hcl at 64° c for 5 minutes. the slides were prepared by squashing the root tips using 45% acetic acid and stained with aceto orcein solution. chromosome count and karyotyping chromosome count was performed on mitotic metaphase cells. for each morphotype, ten clearly observable metaphase cells from ten individuals were selected and photographed using standard and high resolution automated karyotyping software processing (leica cw4000). metaphase chromosomes of each morphotype were placed in pairs, arranged and numbered in order of size, with keeping in view the centromere position to consti47cytogenetic and molecular studies of the egyptian capsella bursa-pastoris (brassicaceae) tute a karyotype. the length of the short arm (p) and the long arm (q) was measured for each chromosome, and the total length (tl=p+q) was calculated. the relative length (rl) of the chromosomes (tl / sum tl × 100) and the mean relative length (mrl) of each chromosome pair were calculated. the centromeric index (ci) was estimated by (p / tl x 100), the mean centromeric index (mci) was calculated to represent the centromeric index value of a particular chromosome pair, then the chromosomes were classified according to levan et al. (1964). molecular study from t he sa me 30 specimens col lected f rom faiyum region (table 1) for cy togenetic studies, 14 specimens representing the three morphotypes were chosen for molecular study (4 lobed, 4 simple and 6 lobed-simple). dna extraction a total genomic dna was extracted from 1 g young leaves using ctab (cetyl-trimethyl ammonium bromide) extraction buffer procedure described by doyle and doyle (1990) and modified by allen et al. (2006). pcr reactions and data analysis for each 25 μl pcr reaction, add 12.5 μl dream taq green pcr master mix (2x), 1 μl forward primer (5 -́ gcc tac cgc atc gaa ata ga-3´), 1 μl reverse table 1. collected specimens of capsella bursa-pastoris (l.) medik. in egypt with their geographical distribution (arranged from north to south). collection & herbarium date longitude latitude locality amer 8312 (cai) 18.1.1987 30°26’33” 31°25’15” beheira province, rosetta fahmy 963 (cai) 2.5.1988 27°14’55” 31°21’23” mersa matruh, el sallum road fayed & el naggar s.n. (astu) 15.3.1984 30°00’44” 31°17’00” alexandria, el montazha amer 16225 (cai) 6.3.1988 30°32’58” 31°12’17” beheira province, mahmudiya abdel fattah & abdel aziz s.n. (cai). 19.3.1974 31°23’00” 31°01’53” el mansoura gun romée 443 (cai) 12.3.1968 30°55’33” 30°47’13” tanta amer 1515 (cai) 18.3.1982 31°48’59” 30°43’19” sharkiya, faqus g. täckholm s.n. (cai) 7.1.1927 31°12’46” 30°41’40” barrage (zifta) el naggar s.n. (astu) 30.1.1985 31°11’11” 30°27’19” banha, kafor mousa el bakry 2708 (cai) 29.4.1981 31°33’43” 30°24’57” bilbeis chrtek, kosinova & slavikova s.n. (cai) 4.4.1977 31°17’00” 30°12’01” bahtim el batanony s.n. (cai) 7.2.1957 31°14’14” 30°07’26” el menoufia amer et al. s.n. (cai) 27.1.2017 31°12’54” 30°06’45” el menoufia, abu sleem village el hadidy s.n. (cai) 17.1.1952 31°11’58” 30°04’52” imbaba el hadidy s.n. (cai) 12.1.1956 31°12’27” 30°01’39” giza, faculty of science farm taher el sayed s.n. (cai) 19.11.1926 31°11’45” 30°01’12” giza, in clover fields chrtek & kosinova s.n. (cai) 13.4.1971 31°12’29” 30°01’05” giza, faculty of agriculture farm chrtek & kosinova s.n. (cai) 1.4.1971 31°13’12” 30°00’35” giza, el harraniya village chrtek, kosinova & imam s.n. (cai) 27.4.1967 31°15’48” 29°35’21” el saff, fields along the road amer et al. s.n. (cai) 23.4.2018 31°15’17” 29°34’57” el saff abd el ghani 5820 (cai) 13.3.1983 30°51’27” 29°24’48” faiyum, sinnuris district, el siliene amer et al. s.n. (cai) 27.1.2017 30°51’27” 29°24’48” *faiyum, sinnuris district, el siliene abd el ghani 5234 (cai) 8.3.1983 30°48’56” 29°21’20” faiyum district, beni saleh abd el ghani 5320 (cai) 8.3.1983 30°27’11” 29°19’16” faiyum district, in clover fields fayed et al. s.n. (astu) 2.5.2010 34°18’25” 27°56’48” southern sinai, farsh elias zareh & fayeds.n. (astu) 5.12.1990 31°12’05” 27°10’20” assiut, sohag east road zareh s.n. (astu) 5.12.1990 31°20’18” 27°02’44” assiut, elmatmar zareh & fayed s.n. (astu) 30.1.1991 31°22’02” 26°57’05” assiut, sedfa zareh s.n. (astu) 28.2.1962 32°00’10” 26°14’08” el-balliana, sohag *specimens subjected to cytogenetic and molecular studies. 48 wafaa m. amer, rania a. hassan, amany s. abdo primer (5 -́ caa gaa agt cgg cca gaa tc-3´), 2 μl template dna, and complete to 25 μl by water (nuclease-free). the pcr was performed using the recommended thermal cycling conditions: one cycle of initial denaturation at 95°c for 5 minutes, 35 cycles of denaturation at 95°c for 45 seconds followed by annealing at 57°c for 45 seconds then extension at 72°c for 60 seconds, and one cycle of final extension at 72°c for 10 minutes. the reaction products were separated by electrophoresis on 1.6 % agarose gel in 1x tbe buffer and run in the same buffer at 100 v for 1 hour, and visualized by staining with 0.5 µg/ml of ethidium bromide and photographed under uv light. the cpssr locus atcp31017 was sequenced and dna was amplified using the previously mentioned primers (castro et al., 2014). the amplified fragments were sequenced in abi377 dna sequencer (abi, usa). then blast programs were used for searching dna databases for sequence similarities. mega software was used to carry out multiple sequence alignment and calculate genetic distances among studied taxa. neighbourjoining dendrogram was constructed showing the genetic relationships among 14 specimens of the three studied genotypes. results morphological diversity the morphological investigations and taxonomic revision of 36 old herbarium specimens and 66 recently collected populations of c. bursa-pastoris based on 25 morphological characters including plant height, basal and cauline leaves features, as well as inflorescence and fruit characters (amer et al., 2019). the most differential characters were that of the basal leaves. the results of that study (amer et al., 2019) revealed the presence of three morphotypes in egypt namely: lobed “l” with all basal leaves are lobed, simple “s” in which all basal leaves are simple, and lobed-simple “ls” in which basal leaves are mixed lobed with simple (figure 1). the three identified morphotypes were co-distributed and traced in the field as mixed populations along with the species geographical range. where, the “ls” morphotype was the most common type and showed the highest phenotypic variation, while the “s” morphotype showed rare occurrence in all studied localities. the environmental factors such as shading, temperature, soil type and rainfall showed no influence on the distribution of these morphotypes (amer et al., 2019). figure 1. morphological diversity within the egyptian c. bursa pastoris genotypes; l: lobed, s: simple, ls: lobed-simple. 49cytogenetic and molecular studies of the egyptian capsella bursa-pastoris (brassicaceae) cytogenetic analysis chromosome number chromosome count for each morphotype of c. bursa-pastoris was done in the mitotic metaphase (figure 2). the three studied morphotypes recorded three different chromosome numbers. accordingly, they are treated as three distinct genotypes. the lobed “l” genotype has diploid chromosome set of 2n=2x=16. the simple “s” genotype has triploid chromosome set of 2n=3x=24 with minor aneuploidy in chromosome no. 6 and 8 (2n=3x2=22). this triploid genotype is recorded for the first time in egypt. while the lobed-simple “ls” genotype recorded the presence of tetraploid chromosome set of 2n=4x=32 (figure 2). karyotype analysis the karyotyping data of these three genotypes are provided in figure 3 and table 2. the retrieved results showed that the chromosomes are small in size, the total genomic length ranges from 43.48 µm in the lobed “l” genotype to 67.76 µm in lobed-simple “ls” genotype, while the simple “s” genotype has an intermediate value of 61.46 µm. furthermore, the chromosomes are highly variable referring to their mean relative length (mrl), as shown in figure 4. the chromosome pair 1 is the longest in the three genotypes, its length ranges from 3.21 µm in lobed-simple “ls” genotype to 4.48 µm in simple “s” one, and its mean relative length (mrl) ranges from 4.74% in lobed-simple “ls” genotype to 7.99% in lobed “l” one. the length of the shortest chromosome pair 8 ranges from 1.54 µm in simple “s” to 1.74 µm in lobed “l, and its mean relative length (mrl) ranges from 2.35% in simple “s” genotype to 3.99% in lobed “l” with an intermediate value of 2.52% in lobed-simple “ls” genotype. the eight chromosome pairs were grouped based on the centromere position into four types: acrocentric, metacentric, submetacentric and subtelocentric (table 2). the chromosome pairs from 1 to 4 are metacentric in all the studied genotypes. the chromosome pair no. 5 is metacentric in genotypes “l” and “s”, while it is acrocentric in “ls” genotype. the chromosome pair 6 is acrocentric in “l” genotype, while it is submetacentric in “s” and “ls” genotypes. the chromosome pair 7 appeared acrocentric in “l” genotype, submetacentric in “s” genotype, and metacentric in “ls” genotype. the chromosome pair 8 is metacentric in “l” and “s” genotypes, while subtelocentric in “ls” genotype. molecular analysis gel electrophoresis of the pcr amplification products of 14 studied specimens (4 lobed, 4 simple and 6 lobed-simple) produced 14 bands of good quality. the statistical results of cpssr locus atcp31017 sequences developed a neighbour-joining dendrogram (figure 5) that separated the genotypes “l”, “s”, and “ls” into three genetic clusters. the first cluster included four specimens (l1-4) that represented the lobed “l” genotype (2n=16). in this cluster, specimens l1 and l3 showed high genetic similarity to each other. the second cluster included also four specimens (s5-8) that represented the simple “s” genotype (2n=24). in this cluster, specimens s5 and s7, and specimens s6 and s8 showed high genetic similarity to each other. the third cluster included six specimens (ls9-14) that represented the lobed-simple “ls” genotypes (2n=32). in this cluster, specimen ls9 showed the lowest genetic similarity figure 2. photomicrographs of well spread mitotic metaphase in the three genotypes of c. bursa pastoris: l: lobed diploid with 2n=2x=16; s: simple triploid with 2n=3x=24; ls: lobed-simple tetraploid with 2n=4x=32. 50 wafaa m. amer, rania a. hassan, amany s. abdo with other specimens. while specimens ls10 and ls12, also ls13 and ls14 showed high genetic similarity to each other. the dna sequences of the studied 14 specimens of c. bursa-pastoris were registered on the national center for biotechnology information (ncbi) under the following accession numbers mn602606, mn602607, m n6 026 08, m n6 026 09, m n6 02610, m n6 02611, m n6 02612 , m n6 02613, m n614131, m n614132 , mn614133, mn614134, mn614135 and mn614136. table 2. karyotyping data for the studied c. bursa pastoris genotytpes, l: lobed, s: simple, ls:lobed-simple, a: acrocentric, m: metacentric, sm: submetacentric, st: subtelocentric. l s ls long arm (q) µm 1 1.74±0.05 2.74±0.63 1.63±0.05 2 1.58±0.00 1.97±0.12 1.29±0.16 3 1.58±0.10 1.37±0.10 1.10±0.26 4 1.37±0.10 1.23±0.04 1.05±0.10 5 1.37±0.00 1.19±0.09 1.63±.38 6 2.32±0.00 1.51±0.25 1.34±0.24 7 2.21±0.00 1.23±0.23 0.95±0.15 8 1.08±0.50 0.84±0.49 1.35±0.22 short arm (p) µm 1 1.74±0.05 2.10±0.32 1.58±0.11 2 1.58±0.00 1.86±0.16 1.21±0.16 3 1.53±0.05 1.33±0.11 0.95±0.11 4 1.32±0.05 1.19±0.04 1.00±0.11 5 1.21±0.05 1.16±0.10 0.22±0.10 6 0.26±0.15 0.70±0.39 0.45±0.44 7 0.21±0.00 0.74±0.35 0.84±0.06 8 0.66±0.34 0.70±0.39 0.36±0.15 total length (p+q) µm 1 3.48±0.10 4.84±0.95 3.21±0.16 2 3.16±0.00 3.83±0.28 2.5±0.32 3 3.11±0.15 2.70±0.20 2.05±0.37 4 2.69±0.15 2.42±0.08 2.05±0.21 5 2.58±0.05 2.35±0.19 1.84±0.48 6 2.58±0.15 2.21±0.64 1.79±0.58 7 2.42±0.00 1.97±0.55 1.79±0.21 8 1.74±0.54 1.54±0.79 1.71±0.37 mean relative length (mrl) 1 7.99 7.38 4.74 2 7.27 5.83 3.69 3 7.14 4.12 3.03 4 6.18 3.69 3.03 5 5.94 3.59 2.72 6 5.94 3.37 2.64 7 5.57 2.99 2.64 8 3.99 2.35 2.52 mean centromeric index (mci) 1 50 44.41 49.13 2 50 48.55 48.41 3 49.23 49.18 45.67 4 49.02 49.17 48.82 5 46.88 49.25 12.3 6 9.94 30.69 25.01 7 8.68 36.63 47.16 8 40.11 47.53 21.93 type 1 m m m 2 m m m 3 m m m 4 m m m 5 m m a 6 a sm sm 7 a sm m 8 m m st fig. 3. karyotypes of c. bursa-pastoris genotytpes. l: lobed diploid (2n=2x=16); s: simple triploid (2n=3x=24); ls: lobed-simple tetraploid (2n=4x=32). fig. 4. mean relative length (mrl) of each chromosome pair in the three studied genotypes of c. bursa-pastoris. 51cytogenetic and molecular studies of the egyptian capsella bursa-pastoris (brassicaceae) discussion the taxonomic revision of capsella bursa-pastoris in egypt revealed the presence of a high degree of phenoplasticity in all the studied populations (old and fresh) and resulted in three distinctive morphotypes (“l” lobed, “s” simple and “ls” lobed-simple) based mainly on the basal leaves structure (amer et al., 2019). these morphotypes were traced as mixed populations in all the studied localities (table 1), so the environmental factors have no influence on their distribution or phenoplasticity (amer et al., 2019). however, positive correlation between the degree of genetic heterogeneity and environmental variability was reported by aksoy et al. (1998), neuffer et al. (1999), neuffer and hurka (1999) and castro et al. (2014). in addition to han et al. (2015) who reported that c. bursa-pastoris populations from different regions are polymorphologically different. the study of genetic diversity of c. bursa-pastoris in egypt has received little attention so far. in this study, we tried to elucidate the role of genetic diversity within the egyptian morphotypes in their phenoplasticity. the chromosome counting of the identified morphotypes recorded three different chromosome numbers (2n=16 for lobed “l”, 3n=24 for simple “s”, 4n=32 for lobed-simple “ls”), so they are treated here as genotypes. shull (1909) was the first to distinguish between four biotypes with different leaf types (simplex, rhomboidea, tenuis, and heteris) and cleared the correlation between leaf shape and chromosome number. he recorded the tetraploid number for simplex and rhomboidea biotypes, while heteris and tenuis were diploid. nonetheless, the egyptian morphotypes are not equivalent to shull’s biotypes (amer et al., 2019). worldw ide, t he tet raploid c . bursa-pa stor i s (2n=4n=32) is one of the most successful plants that have high polymorphic level (han et al., 2015). similarly in egypt, the tetraploid “ls” genotype is the most common and diverse type with a wide range of leaf forms on the same plant (amer et al., 2019). this is supported by shull (1929), löve and löve (1956), davis (1965), raj (1965), hsu (1968), svensson (1983) and hurka (1984). the diploid chromosome number (2n=2x=16) recorded in lobed “l” genotype, was early recorded in europe by bosbach and hurka (1981), in greece by svensson (1983), and in kashmir by jeelani et al. (2013). although c. bursa-pastoris is a self-compatible species, different percentages of outcrossing were recorded by many authors: aksoy et al. (1998) recorded 1-2%, hurka et al. (1989) recorded 3-12%, and hurka and neuffer (1997) recorded up to 20%. in reviewing the literature, no data were found on the triploid “s” genotype (2n=3x=24) which is recorded for the first time in egypt. its rare presence within c. bursa-pastoris populations comparing with the other two genotypes (amer et al., 2019) may support its hybrid origin. the lack of chromosome no. 6 and 8 (aneuploidy, 2n=3x-2=22) in some individuals of this genotype supports our postulation. bretagnolle and thompson (1995) cleared that the triploid offspring are typically sterile due to problems in chromosomal pairing and segregation during meiosis, which may cause aneuploid gametes and result in sterility. karyotype analysis showed that the chromosomes are small in size, this result agrees with schmidt and bancroft (2011) who reported that mitotic chromosomes of crucifer species are generally very small in size. the karyotyping of the three genotypes (fig. 3 & table 2) showed distinctive variations within genotypes in the fig. 5. diagonal matrix of the genetic distances and neighbour-joining dendogram showing the genetic relationship among 14 specimens of the three studied genotypes (l, s and ls). 52 wafaa m. amer, rania a. hassan, amany s. abdo genome and chromosomes mean relative lengths (mrl) in addition to the centromere position. our results are supported by guerra (2008) who claimed that the karyological data in taxonomy contribute to evaluate the genetic relationships among species or populations and lead to better understanding of the way they diverged from each other. as reported by amer et al. (2019), the “ls” populations were large in size, up to 80 cm length with inflorescence up to 75 cm, while “l” and “s” populations were small in size (up to 50 cm long with inflorescence up to 40 cm). these results indicate that the growth rate of tetraploid populations is greater than that of other genotypes, and agree with neuffer’s finding (1989) that the rate of growth was greater for the tetraploid groups. on the other hand, the small size of the diploid “l” and the triploid “s” genotypes can be explained by deletion of redundant genes which can result in downsizing of the genome, as reporteded early by devos  et al. (2002), blanc and wolfe (2004), vitte and bennetzen (2006). the molecular results achieved by sequencing the cpssr locus atcp31017 of 14 specimens of c. bursa-pastoris genotypes support the cytogenetic results, where the neighbour-joining dendrogram (shown in figure 5) separated the studied genotypes “l”, “s”, and “ls” into three distinctive genetic clusters. the first cluster included four specimens for the diploid “l” genotype. in this cluster, specimens l1 and l3 showed high genetic similarity with each other, this result may be reflected from high morphological similarity (both specimens had plant length up to 50 cm and inflorescence up to 40 cm and similar cauline leaves). the second cluster included four specimens for the triploid “s” genotype. in this cluster, specimens s5 and s7 showed high genetic similarity reflected from morphological similarities with each other (plant length up to 50 cm, its inflorescence up to 40 cm). moreover, specimens s6 and s8 showed high genetic similarity as both samples had very small plant size (stem length was up to 20 cm, its inflorescence was up to 15 cm). the third cluster included six specimens for the tetraploid “ls” genotype. in this cluster, specimen ls9 had the lowest genetic similarity with the other specimens. this may be due to its very large size (stem length was up to 80 cm, its inflorescence was up to 75 cm) and presence of both simple and lobed cauline leaves on the same plant. on the other hand, specimens ls10 and ls12 were genetically similar (both specimens had large plant size and simple cauline leaves). also, specimens ls13 and ls14 were genetically similar, as both specimens had small plant size and simple cauline leaves. the lobed-simple “ls” genotype with the highest phenotypic plasticity (as shown in fig. 1), also showed high genetic diversity (fig. 5). the position of the triploid “s” genotype in the obtained dendrogram is supporting our postulation that it is a hybrid between “ls” and “l” genotypes. together these results provide important insights about the correlation of the phenotypic diversity within the egyptian c. bursa-pastoris and the genetic diversity. conclusion the phenotypic diversity within the egyptian c. bursa-pastoris populations is genetically controlled. the three studied morphotypes represent distinct genotypes. the environmental factors have no effect on their phenoplasticity. the postulated hybrid (triploid genotype) may suffer from sterility and disappear in the near future. references aksoy a (1996). autecology of capsella bursa-pastoris (l.) medik. ph.d thesis, university of bradford, bradford, england. aksoy a, dixon jm, hale wh (1998). biological flora of the british isles. capsella bursapastoris (l.) medikus (thlaspi bursa pastoris l., bursa bursa-pastoris (l.) shull, bursa pastoris (l.) weber). j ecol 86: 171–186. doi: 10.1046/j.1365-2745.1998.00260.x aksoy a, hale wh, dixon jm (1999). towards a simplified taxonomy of capsella bursapastoris (l.) medik. 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international journal of cytology, cytosystematics and cytogenetics citation: s. gateva, g. jovtchev, c. chanev, a. georgieva, a. stankov, a. dobreva, m. mileva (2020) assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of bulgarian rosa alba l. essential oil. caryologia 73(3): 71-88. doi: 10.13128/caryologia-260 received: may 13, 2019 accepted: june 02, 2020 published: december 31, 2020 copyright: © 2020 s. gateva, g. jovtchev, c. chanev, a. georgieva, a. stankov, a. dobreva, m. mileva. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of bulgarian rosa alba l. essential oil svetla gateva1,*, gabriele jovtchev1, christo chanev2, almira georgieva3, alexander stankov1, anna dobreva4, milka mileva5,* 1 institute of biodiversity and ecosystem research, bulgarian academy of sciences, 2 gagarin str., bulgarian academy of sciences, sofia 1113, bulgaria 2 university of sofia, department of chemistry, 1 j. bourchier str., sofia 1164, bulgaria 3 institute of neurobiology, bulgarian academy of sciences, 23 acad. g. bonchev str., sofia 1113, bulgaria 4 institute for roses and aromatic plants, 49 osvobojdenie blvd., kazanlak 6100, bulgaria 5 the stephan angeloff institute of microbiology, bulgarian academy of sciences, 26 acad. g. bonchev str., sofia 1113, bulgaria *corresponding author. e-mail: spetkova2002@yahoo.co.uk; milkamileva@gmail.com abstract. bulgarian rosa alba l. essential oil is widely used in perfumery, cosmetics and pharmacy. the scarce data about its cytotoxic/genotoxic effect and anti-cytotoxic/anti-genotoxic potential gave us a reason to set our aim: i) to study its cytotoxic/ genotoxic activities, iii) to explore its cytoprotective/genoprotective potential against the experimental mutagen n-methyl-n’-nitro-n-nitrosoguanidine (mnng) in two experimental test-systems barley and human lymphocytes using appropriate endpoints and iii) to assess its antioxidant properties. findings about chemical composition of rose essential oil would help us to explain its activities. chromatogaphic profile of rose essential oil was obtained by gas chromatography-mass spectrometry and quantificaton of particular constituents was done with a gas chromatographyfid system. superoxide anion radical scavenging, dpph inhibition and iron ion chelating activity were used to study a possible antioxidant potential of the rose oil. its defense potential was investigated by induction of chromosome aberrations and micronuclei in both test-systems. cytogenetic analysis showed a low cytotoxic effect in both test-systems and no high genotoxic effect in human lymphocytes in vitro in a dose-dependent manner. rose oil possessed a well-expressed anti-cytotoxic/antigenotoxic potential against mnng manifested by decreasing both of chromosome aberrations and micronuclei regardless of the experimental schemes used. a wellexpressed concentration-depended free radical scavenging activity of the essential oil was obtained. current data suggest a promising ethnopharmacological potential of bulgarian white rose essential oil. keywords: rosa alba l. essential oil, rose oil components, genotoxicity, anti-cytotoxicity, anti-genotoxicity, antioxidant effect. 72 svetla gateva et al. introduction аs a general rule, living organisms exist in conditions of continuous attack by various environmental pollutants such as alkylating agents, oxidative stress inducers, etc. as a result serious alteration in the main hereditary molecule dna could be induced. there is a constant need to obtain products, which could decrease or eliminate the harmful effects of the genotoxins. plants are known as a rich source of various bioactive natural compounds, which are widely used in various areas of human life. a cursory look at the literature cited in relation to plants’ essential oils in recent years indicates that there is a growing interest in evaluation of the biological activities of various extracts of essential plants, their antimutagenic and antigenotoxic potential (blasiak et al. 2002; mezzoug et al. 2007; bakkali et al. 2008; vicuña et al. 2010; siddique et al. 2010; arumugam et al. 2010; leffa et al. 2012; gokbulut et al. 2013; madrigal-santillán et al. 2013; oyeyemia and bakare 2013; reddy and devi 2014; shohayeb et al. 2014; laribi et al. 2015), and presuming their role in the prevention of degenerative diseases and other human ailments including cancer (hajhashemi et al. 2002; raut and karuppayil 2014; horváth and ács 2015). the genus rosa is one of the largest and most important aromatic and medicinal genera of the rosaceae family. numerous rose phytocomplexes, including essential oils isolated from rosa damascena mill., rosa x centifolia l., rosa gallica l., rosa alba l. and rosa rugosa thunb. have been identified and used for therapeutic purposes as well (rangaha 2001; degraf 2003; moein et al. 2010). the rose extracts help in the reduction of thirst, healing of old cough, special complaints of women, abdominal and chest pain, digestive problems and show skin health effects (tabrizi et al. 2003; boskabady et al. 2006; ema/hmpc 2013). the rose essential oils and by-products of rosa damascena, from shafaa, taif, saudi arabia (shohayeb et al. 2014) and from amman and ajloun areas, jordan (talib and mahasneh 2010) have antimicrobial activity against various gram-positive, gram-negative, acid-fast bacteria and fungi. absolute oil of rosa damascena trigintipetala dieck has an antimutagenic activity against mitomycin c in normal human blood lymphocytes (hagag et al. 2014). methanolic and aqueous extracts of rosa damascena white variety from iran show better anti-radical activity than some synthetic antioxidants (kashani et al. 2011). unfortunately, little is known about singleand repeat-dose cytotoxicity, genotoxicity, carcinogenicity, reproductive and developmental toxicity, local tolerance or other special studies of preparations from rosae flos in animals and humans according to current state-of-the-art standards (hagag et al. 2014). rosa alba l. is the second most important plant for bulgarian rose production. kovatcheva et al. (2011) and dobreva (2010) demonstrated that bulgarian rosa alba l. has a similar oil composition but some of compounds are with lower content to that of rosa damascena mill. collected from bulgaria. rosa alba l. essential oil, known as bulgarian rose oil of white rose, has been defined as “original, exclusively fine, only suitable for the highest perfumery” (degraf 2003). fukada et al. (2011) found that in an experimental model of acute stress in rats, inhalation with rosa alba l. essential oil (supplied by kanebo cosmetics) lowered corticosterone levels almost twice. water extract of calyces of rosa alba from india might be a useful memory restorative agent in the treatment of cognitive disorders (naikwade et al. 2009). our previous study indicated well-expressed antimicrobial activities of bulgarian rosa alba l. essential oil (mileva et al. 2014). insufficient data exist about cytotoxic/genotoxic effect and anti-cytotoxic/anti-genotoxic potential of essential oil from bulgarian rosa alba l. this gave us a reason to set our aim in the present paper: i) to study cytotoxic and genotoxic activities of bulgarian rosa alba l. essential oil, ii) to explore its cytoprotective and genoprotective potential against well known experimental mutagen – alkylating agent n-methyln’-nitro-n-nitrosoguanidine (mnng) in two types of widely used experimental test-systems (higher plant and human lymphocytes in vitro) using appropriate for this purpose endpoints, and iii) to assess its antioxidant properties. phytochemical analysis of rose essential oil would help us to explain its cytotoxic/genotoxic and anti-cytotoxic/anti-genotoxic effects. material and methods chemicals used all chemicals, standards and solvents used for analysis of rose essential oil (gc-ms, gc-fid), methods for testing of the antioxidant activity and cytogenetic analysis were of high purity (>99%). tetracosane [64631-1] ga14075, ec 2114745, free puriss. p.a. > 99.5 % (gc) used as a reference compound in predicted relative response factors calculations was purchased from fluka, usa. nitroblue tetrazolium (nbt), xanthine, α-tocopherol, butylated hydroxytoluene (bht), ascorbic acid, ferrous chloride, ferrozine, edta, dimetylsulfoxyde (dmso) used for examination of antioxidant properties and the chemicals used for cell cultivation and cytogenetical analysis (rpmi 1640, bovine serum, phytohaemagglutinin pha, giemsa) were purchased from sigma-aldrich, germany. α-bromonaphthaline, 73assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of bulgarian rosa alba l. essential oil colchicine, kcl were purchased from merck, germany, n-methyl-n’-nitro-n-nitrosoguanidine (mnng) from fluka – ag, switzerland and schiff ’s reagent from riedel-de haen ag, germany. plant material and distillation of rosa alba l. essential oil fresh flowers of rosa alba l., from the experimental field of the institute of rose and essential oil plants (ireop), in kazanlak, bulgaria were used as raw material in the study. the flowers were picked up in the mornings in may/june 2009, from 8 to 10 am, in a phase of semiblooming – blooming. roses were distilled immediately by water-steam distillation using ireop’s semi-industrial processing line. process parameters of the distillation were as follows: a raw material for charge – 10 kg; hydro module 1:4, rate of 8-10% and duration – 150 min. the aromatic water was re-distilled in the same apparatus. the essential oil, obtained of each charge is the sum of primary and secondary oil in their natural ratio. total oil is a mixture of distillates collected over 15 days – the time of the collection campaign of rosa alba l. for 2009. finally, it was dried with sodium sulfate (merck, germany), filtered and stored at 4 °c for further use. the concentration of the main compounds as well as the physicochemical parameters and characteristics of the rose essential oil are controlled through implementation of national (bds iso9842:2006) and international standards (iso 9842:2003, www.iso.org) only for oil obtained from rosa damascena mill. so, in our study we used the essential oil from rosa damascena mill. cultivated in bulgaria, kazanlak as one of the controls in the tests for antioxidant activities. gas chromatography-mass spectrometry (gc-ms) chromatogaphic profile of rose essential oil was obtained by gas chromatography-mass spectrometry. gc-ms analysis was carried out on a hp 6890 “plus” gas chromatograph interfaced with a 5975 mass selective detector. separations were performed using a hp-5ms silica-fused capillary column – 30 m × 0.25 mm coated with 0.25 μm film of (5%-phenyl)methylpolysiloxane as the stationary phase (agilent technologies, usa). the flow rate of carrier gas (helium) was maintained at 0.8 ml/min. the injector and the transfer line temperature were kept at 250 and 300 °c respectively. the oven temperature program used was 60 °c for 2 min then 3 °c/ min to 300 °c for 8 min, total run time 90 min. the injections were carried out in a split mode with a split ratio of 25:1. the mass spectrometer was scanned from 30 to 550 m/z. the injection volume was 1 µl. the quantitative analysis was carried out on a hp 5890 “series ii” gas chromatograph equipped with a fid detection system. we analysed the same sample and the separation was performed at the same chromatographic conditions, column, carrier gas and temperature program. gc-fid – eluted constituents were identified on the basis of a kovats retention index (ri), determined with reference to a homologous series of n-alkanes (c10-c28), under identical experimental conditions. gc-ms – eluted constituents were identified using ms library search (nist version 2.1), as well as by comparison with the fragmentation pattern of the mass spectra with data published in the literature (adams 2007). for each identified constituent, from gc-ms analysis was obtained its kovats ri. every particular value for these indices was confirmed by the literature. the differences between the measured and published kovats ri exceeding 10 units were not reported, except these for n-alcanes and a few unsaturated hydrocarbons. the percentage compositions of rosa alba l. essential oil were determined from the gc-fid peak ’s areas corrected with predicted relative response factors for the constituents calcutated by formula given by tissot et al. (2012). examination of antioxidant properties superoxide scavenging properties the generation of superoxide anion radical o2•in the model system xanthine-xanthine oxidase (xo) and the changes occurring upon the rosa alba l. oil effect were investigated by the nitroblue tetrazolium (nbt) test. the detailed procedure was described elsewhere (mileva et al. 2000). briefly, the spectrophotometric registration of superoxide was carried out measuring the amount of formazan generated by o2•induced reduction of nbt. the investigated samples of a volume 1 ml pbs contained: 1 mmol/l xanthine, 2.10-3 iu xo, 0.04 mmol/1 nbt, as well as oil at concentrations from 0 to 100 μg/ ml. samples were incubated at 37 oc and the amount of the formed formazan was measured by absorption at 560 nm. the time of incubation was selected so that the absorption for the controls was 0.2. the decrease of absorbance in the presence of oil indicated the consumption of superoxide anion in the reaction mixture. data were calculated in percentage as spectrophotometric scavenger index (spsi) the ratio of the absorption at 560 nm for the sample with oil, and the same absorption for the controls (without oil). the scavenger activity of oil was compared with that of α-tocopherol – commonly used as superoxide radical scavenger. 74 svetla gateva et al. dpph test hydrogen atoms and electron-donating potential of essential oils were measured from the bleaching of the purple-colored ethanol solution of dpph (sigmaaldrich, germany). all compounds were dissolved in ethanol to a concentration of 100 mg/ml stock solutions for the follow dilutions. dpph assay was performed as follows: freshly prepared ethanolic solution of dpph (100 mm) was incubated with tested substances in the concentration of 10 to 100 μg/ml, and the absorbance (a) monitored spectrophotometrically at 517 nm after 30 min incubation in dark at room temperature. inhibition of dpph in percentage (dpph inhibition, %) was calculated as given below: dpph inhibition (%) = [(a control a sample)/(a control)] x 100 bht and ascorbic acid served as positive controls. each experiment was carried out in triplicate and data were presented as a mean of the three values (singh et al. 2008). iron binding capacity of essential oil metal chelating activity of rosa alba l. essential oil on ferrous ions was determined according to the method of decker and welch (1990). the percentage of inhibition of ferrozine fe2+ complex formation was calculated using the formula: chelating activity (%) = [(a control – a sample)/a control] × 100 where a control is the absorbance of the ferrozine fe2+ complex, and a sample is the absorbance of essential oil. as a positive control edta was used. all results for antioxidant properties are presented as mean ± sd, and compared against routinely used reference positive controls. the data were collected from three independent experiments with three parallel measurements for each experiment. analysis of cytotoxic/anti-cytotoxic and genotoxic/anti-genotoxic effects of rosa alba l. essential oil rose essential oil and chemicals preparation rose essential oil was dissolved in 1% dimetylsulfoxyde (dmso). a standard well-known experimental mutagen n-methyl-n’-nitro-n-nitrosoguanidine (mnng) 50 μg/ml was used as dna damage agent in the chromosome aberrations and micronucleus assays. mnng was dissolved in bidistilled water. test-systems two types of experimental test-systems were used – hordeum vulgare (barley) and human lymphocytes in vitro. barley seed meristems and human lymphocyte cultures preparation as well as the schemes of treatment are given below. hordeum vulgare (barley) meristem cells as a testsystem. seeds of reconstructed karyotype of hordeum vulgare mk14/2034, 2n = 14 (künzel and nicoloff 1979) presoaked for 1 hour in tap water were germinated for 18h in petri dishes on moist filter paper at 24oc. wellsynchronized seeds with a root meristem size of 1-2 mm were selected for further treatment. experimental designs used for cytogenetic analysis. three types of experimental schemes were applied. first to assess cytotoxic/genotoxic effects of rose essential oil, whole germinated seeds of barley with root meristems were treated with essential oil in concentrations from 250 to 1000 μg/ml. to assess the protective potential of rose oil germinated seeds were conditioning treated with 250 and/or 500 μg/ml for 60 min followed by 4h intertreatment time and subsequent challenge treatment with 50 μg/ml mnng (60 min). third part of germinated seeds was pretreated with 250 and/or 500 μg/ml of rose oil (60 min), followed immediately by 50 μg/ml mnng (60 min) without any inter-treatment time. for chromosome aberrations evaluation the germinated seeds were treated at 24 oc for 2 hours with 0.025% colchicine in a saturated solution of α-bromonaphthaline after the treatment and recovery times for 18, 21, 24, 27 and 30 hours. the extracted embryos were fixed in a mixture of ethanol and acetic acid (3:1), hydrolyzed in 1n hcl at 60oc for 9 min and stained with schiff ’s reagent at room temperature for 1h. the root tips were macerated in a 4% pectinase solution for 12 min and squashed onto slides for scoring of metaphases with chromatid aberrations. for scoring of micronuclei, the root tips were fixed after 30h recovery time without colchicine treatment. human lymphocytes in vitro as a test-system. lymphocy te cultures (1x106 mol/l) were prepared from venous blood of three healthy nonsmoking/nondrinking donors (men and women) aged between 33 to 50 years according to the standard method of evans (1984). each 75assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of bulgarian rosa alba l. essential oil culture contained 3.5 ml rpmi 1640 medium, heatinactivated fetal bovine serum (20%), phytohemagglutinin pha (0.1%) and 40 mg/ml gentamycin (pharmacia, bulgaria). the study complies with the declaration of helsinki. voluntary written informed consent was taken from all study participants. experimental designs used for cytogenetic analysis. lymphocyte cultures were treated with rose oil in a range of concentrations from 50 to 500 μg/ml to assess cytotoxic and genotoxic effect of rosa alba l. essential oil. to study its protective potential, non/or low cytotoxic concentrations of rose essential oil 50 and/ or 200 μg/ml were applied as a conditioning treatment (60 min) followed by 4 hours inter-treatment time and a challenge treatment (60 min) with 50 μg/ml mnng. another part of the lymphocyte cultures was pretreated with 50 and/or 200 μg/ml of rose oil (60 min), followed immediately by mnng 50 μg/ml (60 min) without any inter-treatment time. after each treatment the lymphocyte cells were washed with a fresh rpmi medium. untreated cells were used as a negative control. at the 72nd hour of cultivation to each culture was added 0.02% colchicine, then the cells were hypotonized in 0.56 % kcl; afterwards fixed in a mixture of methanol: glacial acetic acid (3:1, v/v) and stained in 2% giemsa for assessment of chromosome aberrations. lymphocytes were directly fixed without colchicine for assessment of micronuclei. cytogenetic analysis. cytotoxicity of bulgarian rosa alba l. essential oil for both test-systems mentioned above was assessed by mitotic index (mi) using a formula: mi‰=a/1000, where a is a number of dividing cells. genotoxic effect was evaluated by chromosome aberrations (ca) and micronuclei (mn) induction. percentage of metaphases with chromosome aberrations (mwa % ± sd) was calculated. 1000 well spread metaphases (in m1 mitosis) of each treatment variant in both test-systems were assessed. chromatid breaks (b’), isochromatid breaks (b”), translocations (t), intercalary deletions (d), duplication-deletions (dd) and dicentrics (dc) were determined. “aberration hot spots” in the plant chromosomes were determined to obtain information about the dna segments with higher susceptibility to dna damage. for analyzing the locus specificity of aberration induction the metaphase chromosomes of h. vulgare were subdivided into 48 segments of approximately equal sizes. the segments are numbered with respect to their position in the standard karyotype as described earlier by künzel and nicoloff (1979). percentage of micronuclei was calculated (mn % ± sd) where 4000 nuclei per experiment and treatment variant were assessed. data analysis the results were calculated statistically by student’s t-test and chi-square method. all experiments were repeated three times. an adapted formula was used for comparison of the upper limit of the confidence interval of the expected and observed chromatid aberrations in individual loci and evaluation of aberration „hot spots” in barley (rieger et al. 1975; künzel and nicoloff 1979; jovtchev et al. 2010). for multiple comparisons, a oneway analysis of variance (anova) was employed, followed by bonferroni s̀ correction. results chromatogaphic profile of rosa alba l. essential oil the chromatographic composition of the essential oil of bulgarian rosa alba l. is presented in table 1. the values were compared with literature data. the main groups are aliphatic hydrocarbons (ah) – 40.92% with high molecular weight (c15 – c27) as heneicosane (12.75%), tricosane (2.69%), eicosane (1.46%), pentacosane (1.0%), heptacosane – 0.86%, (z)-3-heneicosene-0.64%, etc., followed by oxygenated monoterpenes (om) – 40.35% as citronellol – 17.69 %, geraniol –16.64 %, trans-citral – 2.12%, linalool – 1.37%, β-citral – 0.68%, etc., sesquiterpenes hydrocarbons (sh) – 5.68% as caryophyllene – 1.52%, α-muurolene – 0.41%, b-cubebene – 0.14%, etc., and oxygenated sesquiterpenes (os) – 1.72%. the remaining 11.27% of the constituents were not reported due to their low content, less than 0.05% by mass, and/or not sufficiently reliably identification. after squalene some constituents leaving the column were in fact overlay with the column bleed and were also discarded from the final results. superoxide scavenging analysis of rosa alba l. essential oil the decrease of absorbance at 560 nm in semples with antioxidant supplements indicates the consumption of superoxide anion radical in the reaction mixture. as can be seen from figure 1, rosa alba l. essential oil exhibits moderate activity in superoxide scavenging assay in concentration range of 0–500 μg/ml. it is sig76 svetla gateva et al. table 1. volatile oil constituents of bulgarian rosa alba l. essential oil № name class r. alba oil, relative percetage kovats ri (measured) kovats ri (literature) reference 1 linalool om 1.37 1092 1099 babushok et al. 2011 2 cis rose oxide hm 0.06 1124 1128 babushok et al. 2011 3 a-terpineol om 0.27 1186 1190 babushok et al. 2011 4 citronellol om 17.69 1227 1228 babushok et al. 2011 5 β-citral om 0.68 1244 1249 jalali-heravi et al. 2006 6 geraniol om 16.64 1256 1255 babushok et al. 2011 7 trans-citral om 2.12 1269 1276 bertuzzi et al. 2013 8 citronellyl format om 0.23 1277 1277 babushok et al. 2011 9 geranyl formate om 0.31 1289 1303 babushok et al. 2011 10 citronellol acetate om 0.12 1346 1352 babushok et al. 2011 11 3,7-dimethyl-2,6-octadien-1-ol acetate om 0.92 1380 1385 wannes et al. 2009 12 tetradecane ah 0.05 1400 1400 13 caryophyllene sh 1.51 1419 1420 babushok et al. 2011 14 b-cubebene sh 0.14 1430 1434 facey et al. 2005 15 humulene sh 0.16 1453 1453 babushok et al. 2011 16 naphthalene, 1,2,3,4,4a,5,6,8a-oct sh 0.33 1479 1480 marongiu et al. 2006 17 α-muurolene sh 0.41 1504 1498 babushok et al. 2011 18 g-cadinene sh 0.19 1528 1523 babushok et al. 2011 19 salvial-4(14)-en-1-one sh 0.19 1557 1563 pavlovic et al. 2006 20 caryophyllene oxide sh 2.75 1580 1580 babushok et al. 2011 21 3-octadecine ah 0.15 1615   22 heptadecane ah 0.54 1700 1700 23 (2z,6e) farnesol os 1.72 1722 1722 babushok et al. 2011 24 z-5-nonadecene ah 5.20 1880 1885 tigrine-kordiani et al. 2006 25 nonadecane ah 12.18 1900   26 3-nonadecyne ah 0.57 1911   27 3-eicosene, (e)ah 0.28 1953   28 eicosane ah 1.46 2000   29 10-heneicosene (c,t) ah 0.34 2070   30 (z)-3-heneicosene ah 0.64 2085   31 1-heneicosene ah 0.55 2091   32 heneicosane ah 12.75 2100   33 docosene ah 0.41 2200   34 9-tricosene, (z)ah 0.74 2290   35 tricosane ah 2.69 2300 2300 36 1-pentacosene ah 0.19 2490   37 pentacosane ah 1.00 2500 2500 38 hexacosane ah 0.09 2600 2600 39 1-heptacosene ah 0.08 2697   40 heptacosane ah 0.86 2700 2700 41 octacosane ah 0.15 2800 2800 heterocyclic monoterpenes (hm) – 0.06 % oxygenated monoterpenes (om) – 40.35% sesquiterpenes hydrocarbons (sh) – 5.68% oxygenated sesquiterpenes (os) – 1.72% aliphatic hydrocarbons (ah) – 40.92% total identified 88.73% 77assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of bulgarian rosa alba l. essential oil nificantly lower than that of alpha tocopherol, but close to that of the rosa damascena mill. оur previous works have shown that essential oils of rose species from different origins in principal does not have high potential as a scavenger of superoxide anion radical (mileva et al. 2014). dpph – radical scavenging assay rosa alba l. oil, as well as citronellol and geraniol as main aromatic components of the essential oil were tested in terms of their dpph – radical scavenging activity. the dpph assay usually involves a hydrogen atom transfer reaction (li et al. 2009). dpph radical scavenging test is a sensitive antioxidant assay and depends on substrate polarity. as can be seen from the chromatographic composition of the essential oil, it is rich of components which possess ideal structural chemistry for dpph radical scavenging activity (table 1). the presence of multiple hydroxyl functions could be considered as an option for hydrogen donation and/or radical scavenging activity. as can be seen in figure 2, we found that bulgarian rosa alba l. essential oil exhibits higher potency in scavenging dpph radicals than geraniol and citronellol in the applied system, but significantly lower than the benchmark substances (bht and ascorbic acid). in the concentrations in range of up to 50 μg/ml, rosa alba l. and rosa damascena mill. rose oils have close activity, but in higher concentracions, over than 50 μg/ml, rosa damascena mill. oil exhibits about 10% higher activity. iron binding capacity of rosa alba l. essential oil one of the possible mechanisms of the antioxidant activity of essential oils is the chelation of transition metals. among the transition metals, iron is known as the most active pro-oxidant, due to its high reactivity. the ferrous form of iron accelerates lipid peroxidation by breaking down hydrogen peroxide and lipid peroxides to reactive free radicals by fenton’s reaction (li et al. 2010). the products of these reactions are able to oxidise cell lipid membranes, modify proteins, as well as to damage dna. chelating agents may inactivate metal ions and potentially inhibit the metal-dependent processes (li et al. 2010). ferrous ion chelating activities of the oil, citronellol and edta are shown in figure 3. the chelating test provided that rosa alba l. essential oil inhibits lipid peroxidation up to 75%. the antioxidant activity obtained in this test is significantly lower than the activity of edta but similar to this of citronellol. in the concentration in range of up to 50 μg/ml the two rose oils have close activity, but in higher than 50 μg/ml concentrations rosa alba l. exhibits increasing chelating activity. figure 1. superoxide scavenging activity of rosa alba l. essential oil. data were calculated in percentage as spectrophotometric scavenger index (spsi) – the ratio of the absorption at 560 nm for the sample with oil, and the same absorption for the controls (without oil). the essential oil from rosa damascena mill. was used as a control. data are expressed as mean ± sd of three independent experiments. ***p<0.001 compared with alpha -tocopherol. figure 2. dpph scavenging activity (%) at various concentrations (μg/ml) of bulgarian rosa alba l. essential oil and its main ingredients geraniol and citronellol. the essential oil from rosa damascena mill. was used as a control. data are expressed as mean ± sd of three independent experiments. ***p<0.001 compared with citronellol (a), compared with bht and ascorbic acid (b). 78 svetla gateva et al. cytotoxic and genotoxic effects of rosa alba l. essential oil rosa alba l. essential oil didn’t show any cytotoxic effect in barley (figure 4a). here, human lymphocytes were found to be more susceptible than barley to rose oil in a concentration range (50-500 μg/ml) used in the present study. the rose oil decreased in a low extent the value of mitotic activity (figure 4b) compared with the negative control in the lymphocyte cells (p < 0.01), whereas after treatment with mnng (50 μg/ml) a wellexpressed cy totoxic effect in both test-systems was observed (p < 0.001) (figure 4a, 4b). rosa alba l. essential oil treatment enhanced the induction of chromosome aberrations (mwa) compared to non-treated cells in both test – systems. these results showed its genotoxic effect (p < 0.05; p < 0.01, p < 0.001). the effect is clearly depended on the concentration applied (figure 5a, 5b). human lymphocyte cultures were more sensitive to rosa alba l. oil than hordeum vulgare. genotoxic effect of rose oil was much lower (p < 0.001) than the alkylating agent mnng (50 μg/ml) in both test-systems (figure 5a, 5b). analysis of the chromosome aberrations distribution showed that in barley, rose essential oil induced only isochromatide breaks (data not shown). in human lymphocytes the observed chromosome aberrations were preferably b” 92.0 %, followed by b’ 8.0 %. in comparison with the rose oil, mnng treatment (50 μg/ml) induced more diverse spectrum of chromosome disturbances in both test-systems. in hordeum vulgare were obtained predominantly b”97.0 %, followed by b’ 3.0%, whereas in lymphocyte cultures b” were 88.6%, b’10.4%, dc-1%, respectively (data not shown) (figure 6a, 6b). treatment with rose oil enhanced the frequency of micronuclei (mn) clearly depending on the test-system and the concentration applied (figure 6a, 6b; figure 7a, 7b). no increase of the frequencies of this endpoint were observed in barley meristem cells (figure 7a). the formation of micronuclei increased (p < 0.001) above two-fold (1.1%±0.3 for 50 μg/ml to 1.7%±0.2 for 500 μg/ ml) compared to the negative control (0.5%±0.2) in lymphocyte cultures. the genotoxic effect of rose oil assessed as micronuclei is much lower than mnng in the concentration applied in our study (p < 0.001) (figure 7b). anti-cytotoxic and anti-genotoxic potentials of rosa alba l. essential oil anti-cytotoxic potential two types of experimental schemes were applied: i) conditioning treatment with nontoxic or low toxic figure 3. chelating activity of bulgarian rosa alba l. essential oil and its main constituent citronellol. the essential oil from rosa damascena mill. was used as a control. data are expressed as mean ± sd of three independent experiments. ***p<0.001 compared with edta. figure 4. value of mitotic activity (mi) observed after rosa alba l. essential oil treatment in hordeum vulgare (a) and in human lymphocyte cultures (b). mitotic activity is calculated as a percent of the control. data are expressed as mean ± sd of three independent experiments. **p<0.01; ***p<0.001 compared with the negative control. figure 5. frequency of chromosome aberrations (mwa) calculated after rosa alba l. essential oil treatment in hordeum vulgare (a) and human lymphocyte cultures (b). data are expressed as mean ± sd of three independent experiments.*p<0.05; **p<0.01 and ***p<0.001 compared with the negative control. 79assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of bulgarian rosa alba l. essential oil concentrations of rose essential oil followed by challenge treatment with alkylating agent mnng (50 μg/ ml) and 4 hours inter-treatment time, ii) treatment without any inter-treatment time between treatments (figure 8a, 8b). mitotic activity (mi) obser ved after treatment showed clear dependence on the experimental design and test-systems (figure 8a, 8b). the value of mitotic index was significantly increased (p < 0.01) after treatment applying both schemes of experimental design, compared to those obtained after mnng (50 μg/ml) treatment alone in both test-systems (figure 8a, 8b). a lack of any difference was obtained between the values of mitotic activity after rose essential oil conditioning treatment (250, 500 μg/ml for barley and 50, 200 μg/ml for human lymphocytes, respectively) followed by challenge treatment with mnng (50 μg/ml) with 4 hours figure 6. types of chromosome aberrations (ca) and micronuclei (mn) observed after treatment with rosa alba l. essential oil in hordeum vulgare (a) and in human lymphocyte cultures (b). figure 7. induction of mn observed after rosa alba l. essential oil treatment in hordeum vulgare (a) and in human lymphocyte cultures (b). data are expressed as mean ± sd of three independent experiments. **p<0.01 and ***p<0.001 compared with the negative control. 80 svetla gateva et al. inter-treatment time and treatment without any intertreatment time. anti-genotoxic potential significantly (p < 0.05 till p < 0.001) lower frequency of chromosome aberrations was observed in hordeum vulgare after conditioning treatment with rose essential oil (250, 500 μg/ml) prior to mnng challenge (50 μg/ ml) with 4 hours inter-treatment time, compared to that induced after treatment with alkylating agent only (figure 9a). the reduction of mnng induced chromosome aberrations was nearly three times lower in samples after rose oil 500 μg/ml conditioning (7.5%±1.6) compared to mnng single treatment (20.7%±2.0). similar anti-genotoxic effect (p < 0.001) was observed in human lymphocytes after applying the same experimental scheme of treatment – conditioning with rose essential oil (50, 200 μg/ml) followed by challenge with mnng (50 μg/ml) with 4 hour intertreatment time (figure 9b). chromosome injuries were decreased approximately three times in samples conditioned with rose oil 50 μg/ml (5.6%±1.7) and with 200 μg/ml (6.0%±1.3) compared to mnng treatment alone (16.3%±1.9). lower structural chromosome disturbances were also calculated after treatment with rose essential oil and alkylating agent without any inter-treatment time (figure 9a). the frequencies of chromosome aberrations were decreased between 2.2-times for samples conditioned with 50 μg/ml rose oil to 1.8–times for samples conditioned with 200 μg/ml. reduction of the frequencies of chromosome aberrations was observed in barley cells but to a lower extent (p < 0.01) also after treatment following the scheme without any inter-treatment time compared to those after mnng treatment alone (figure 9a). the spectrum of chromosome disturbances induced after applying experimental schemes for assessing antigenotoxic potential of rose essential oil (250, 500 μg/ml) in h. vulgare showed predominantly b” 93%, followed by t 4% and b’ 3% in samples with 4 hours inter-treatment time, and b” 93%, t-5%, d-1%, b’-1% in samples without any inter-treatment time (data not shown). in human lymphocytes conditioning treated with rose oil (50, 200 μg/ml) followed by mnng and inter-treatment time of 4 hours were obtained b” 84.4%, b’-11.8%, t-1.9%, rc-1% respectively, whereas in samples treated with rose oil and mnng without any inter-treatment tine were observed mainly b” 85.5%, b’ 12.1% and t 2.4% (data not shown). by calculating the frequency of micronuclei as another endpoint for genotoxicity, it was observed that rose essential oil showed similar anti-genotoxic effect (p < 0.01, p < 0.001) in both test-systems (figure 10a, 10b). the effect was obtained applying both schemes of experimental design. in hordeum vulgare conditioning treatment with rose essential oil decreased mn approximately two-times (0.97%±0.12 for 250 μg/ml and 1.02%±0.10 for 500 μg/ml) compared to that induced after treatment with the alkylating agent (1.95%±0.18). similarly, lower mn frequency was obtained after treatment of barley cells without any inter-treatment time (figure 10a). frequency of micronuclei was decreased roughly 2 times – 1.0%±0.11 for 250 μg/ml and 1.04%±0.14 for 500 μg/ml. in human lymphocytes conditioning treatment with rose figure 8. anti-cytotoxic potential of rose essential oil assessed by mitotic index (mi) applying experimental schemes with: rosa alba l. essential oil conditioning treatment prior to mnng challenge (50 μg/ml) with 4 hours inter-treatment time and, without any inter-treatment time in hordeum vulgare (a) and in human lymphocyte cultures (b). mitotic activity is calculated as a percent of the control. data are expressed as mean ± sd of three independent experiments. **p<0.01 compared with mnng. figure 9. anti-genotoxic potential of rose essential oil assessed by induction of chromosome aberrations (mwa) applying experimental schemes with: rosa alba l. essential oil conditioning treatment prior to mnng challenge (50 μg/ml) with 4 hours inter-treatment time and, without any inter-treatment time in hordeum vulgare (a) and in human lymphocyte cultures (b). data are expressed as mean ± sd of three independent experiments. *p<0.05, **p<0.01 and ***p<0.001 compared with mnng. 81assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of bulgarian rosa alba l. essential oil oil decreased mn approximately four-times 1.0%±0.2 for 50 μg/ml and 1.6%±0.3 for 200 μg/ml compared with those induced by mnng alone (3.9%±0.4). the frequency of micronuclei was from 3-fold (for 50μg/ml) to 2.4fold (200 μg/ml) lower than that of mnng after treatment without any inter-treatment time (figure 10b). “aberration hot spots” in barley. “aberration hot spots” are a good expression tool to investigate genotoxic activity as well as anti-genotoxic potential of rosa alba l. essential oil. they give additional information about the effect of the essential oil on hordeum vulgare root meristem cells. the potential of mnng to induce “aberration hot spots” in plant chromosomes was analyzed in the reconstructed barley karyotype mk14/2034. seven out of the 48 inspected segments showed significant deviation from a random distribution of isochromatid breaks. aberration clustering of isochromatid breaks (table 2) was found in segment 10 and segment 14 of chromosome 2 (4.5% and 5.8%, respectively), in segment 17 of chromosome 34 (6.4%), in segment 21 of chromosome 43 (4.2%), in segment 30 chromosome 5 (9.1%), and in segments 44 and 48 of chromosome 71 (7.3%, resp. 6.7%). all of these segments are located directly adjacent to the centromeres in the heterochromatin rich regions and are not connected with the regions of chromosome reconstruction. after r. alba l. essential oil treatment alone concentration dependent aberration hot spots (2 or 3, resp.) were observed (see table 2), namely: after treatment with r. alba l. oil 250 µg/ml – segment 30 of chromosome 5 (8.2%) and segments 41 of chromosome 6 (14.2%); after treatment with r. alba oil 500 µg/ ml – segment 14 of chromosome 2 (6.2%), segment 21 of chromosome 43 (6.2%) and segment 30 of chromosome 5 (10.0%)and after treatment with r. alba l. essential oil 1000 µg/ml – segment 17 of chromosome 34 (7.2%), figure 10. anti-genotoxic potential of rose essential oil assessed by induction of micronuclei (mn) applying experimental schemes with: rosa alba l. essential oil conditioning treatment prior to mnng challenge (50 μg/ml) with 4 hours inter-treatment time and, without any inter-treatment time in hordeum vulgare (a) and in human lymphocyte cultures (b). data are expressed as mean ± sd of three independent experiments. **p<0.01; ***p<0.001 compared with mnng. table 2. observed aberration “hot spots” in chromosomes of barley root tip meristem cells of the reconstructed karyotype mk14/2034 after different treatment procedures treatment variants non-spot segments hot spot segments chr.17 chr.2 seg. 10 chr.2 seg. 14 chr.34 seg. 15 chr.34 seg. 17 chr.43 seg. 21 chr.5 seg. 30 chr.6 seg. 41 chr.71 seg. 1 chr.71 seg. 44 chr.71 seg. 48 1. control 100% 2. mnng (50 µg/ml) 56.0% 4.5% 5.8% 6.4% 4.2% 9.1% 7.3% 6.7% 3. rosa alba oil (250 µg/ml) 77.6% 8.2% 14.2% 4. rosa alba oil (500 µg/ml) 77.6% 6.2% 6.2% 10.0% 5. rosa alba oil (1000 µg/ml) 72.2% 7.2% 10.3% 10.3% 6. rosa alba oil (250 µg/ml) → mnng (50 µg/ml) 60.3% 5.6% 6.3% 6.3% 6.9% 14.6% 7. rosa alba oil (250 µg/ml) → 4h it → mnng (50 µg/ml) 60.6% 10.5% 11.4% 7.9% 9.6% 8. rosa alba oil (500 µg/ml) → mnng (50 µg/ml) 73.0% 8.5% 8.5% 10.0% 9. rosa alba oil (500 µg/ml) → 4h it → mnng (50 µg/ml) 81.5% 7.6% 10.9% 82 svetla gateva et al. segment 30 of chromosome 5 (10.3%) and segment 41 of chromosome 6 (10.3%). conditioning treatment with r. alba l. essential oil in concentrations of 250 µg/ml and 500 µg/ml, without any inter-treatment time prior to mnng challenge decreased the aberration “hot spots” to five or three out of the 48 inspected segments (table 2): r. alba l. essential oil 250 µg/ml – segment 15 chromosome 34 (5.6%), segment 21 of chromosome 43 (6.3%), segment 30 of chromosome 5 (6.3%), segments 1 and 48 of chromosome 71 (6.9% and 14.6%, resp.); r. alba l. oil 500 µg/ ml – segments 10 and 14 chromosome 2 (both 8.5%) and segment 48 of chromosome 71 (10.0%). after intertreatment time of 4 hours between conditioning and challenge treatment only 4 aberrations “hot spots” for conditioning with r. alba l. oil 250 µg/ml showed a significant deviation from a random distribution of isochromatid breaks – segment 14 of chromosome 2 (10.5%), segment 15 of chromosome 34 (11.4%), segment 41 of chromosome 6 (7.9%) and segment 48 of chromosome 71 (9.6%). two aberrations “hot spots” were found after conditioning with r. alba l. essential oil 500 µg/ml, namely segment 30 of chromosome 5 (7.7%) and segment 44 of chromosome 71 (10.9%) (table 2). “aberration hot spots” were found in all treated variants in barley chromosomes – karyotype mk 14/2034, with exception of chromosome 17 (table 2). in summary, applying both experimental schemes for assessing anti-genotoxic effect of r. alba l. essential oil the frequency of aberration “hot spots” was statistically significant decreased compared to mnng 50 µg/ ml (7 aberration “hot spots”) (see table 2). after consecutive treatment r. alba l. oil 500 µg/ml – 4 h intertreatment time – mnng the best result was achieved – reduction of aberration “hot spots” to 2 (table 2). discussion in the current study valuable information was obtained about cytoprotective/genoprotective and antioxidant potentials of rosa alba l. essential oil. the use of two types of test-systems – barley and human lymphocytes in vitro, which are widely applied in the genotoxic studies makes evaluation more representative. our results showed that rose essential oil possessed good expressed dpph radical scavenging activity; the effect increased with the increasing of the concentration. it confirmed the data of hatano et al. (1989) who proposed that rose’s extract radical scavenging ability is due to the polar-bounded hydrogen. the compounds, which are able to donate hydrogen, are able to break the chain reaction of lipid peroxidation at the first initiation step, and/or to produce redox-silent compounds. gordon (1990) reported that the chelating agents are effective as secondary antioxidants because they reduce the redox potential thereby stabilizing the oxidized form of the metal ion. the data shown in figure 3 reveal that the bulgarian rosa alba l. oil demonstrates an effective capacity as iron binding agent, depending on the concentration applied, so its behavior as liposomal membrane peroxidation protector could be due to its iron binding capacity (mileva et al. 2014). moreover, in concentrations higher than 400μg/ml rosa alba l. oil has a higher activity than rosa damascena mill. in the present study bulgarian rosa alba l. essential oil didn’t show significant cytotoxic effect in barley but it has relatively low cytotoxicity in human lymphocytes in vitro depending on the concentration. sinha et al. (2014) demonstrate that essential oils may be safe at low concentrations, but show toxicity to humans at high concentrations represented as lethal doses. as typical lipophiles, essential oils can pass through the cell membrane and cytoplasmic membrane. they disrupt the structure of the different layers of polysaccharides, fatty acids and phospholipids and permeabilize through them (bakkali et al. 2008). their mode of action affects several targets at the same time. the cytotoxic activity of some essential oils for example of ocotea quixos and others is mostly due to the presence of phenols, aldehydes and alcohols (bruni et al. 2003; sacchetti et al. 2005). in our study aliphatic hydrocarbons (ah) are the major compounds of rosa alba l. essential oil. here heneicosane (12.75%) and tricosane (2.69%) are representatives of the main components of this group (table 1). similar oil composition but in higher concentrations for some ingredients was detected for rosa damascena mill. by kovacheva et al. (2010; 2011) and mileva et al. (2014). the essential oils of plants such as ceratonia siliqua (hsouna et al. 2011), ailanthus altissima (albouchi et al. 2013) and viscum album leaves (cebovic et al. 2008), contain the same compounds in similar concentrations as in the white rose oil. they showed obvious cytotoxic effects, high antioxidant and phytotoxic activities. shokrzadeh et al. (2017) reported a sensitivity of cancer cell line (a549) to high concentrations of rose oil obtained from rosa damascene mill. from kashan. probably these substances play a role in the cytotoxicicity of rose essential oil that we obtained for human lymphocytes. the higher resistance of hordeum vulgare cells compared to the human lymphocytes is probably due to their different cell permeability and a cell wall existence compared to lymphocyte cells. the current investigation detects genotoxic activity of bulgarian rosa alba l. essential oil depending on 83assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of bulgarian rosa alba l. essential oil the concentrations in both test-systems applied. shokrzadeh et al. (2017) also reported that at concentrations of 50-200 μg/ml rosa damascena mill. oil significantly increased the frequency of micronuclei in human lymphocytes. according to bakkali et al. (2008) in the case of cytotoxicity and genotoxicity, essential oils can damage the cellular and organelle membranes and can act as pro-oxidants on proteins and dna via production of reactive oxygen species (ros). as it is known numerous plant extracts or phytochemicals have dual aspects, showing both genotoxicity and anti-genotoxicity against mutagens and carcinogens in vivo and in vitro test-systems (kopaskova et al. 2012; reddy et al. 2014; gateva et al. 2015). here we made an attempt to study the defense potential of bulgarian rosa alba l. essential oil and to determine the experimental conditions under which it can occur against alkylating agent mnng. this is a typical mutagenic agent, which damages dna. as a result it induced intra-strand, interstrand crosslinks and double-strand breaks as well as base methylations, respectively (kinzella and radman 1980; black et al. 1989). extracts and essential oils of various plants were used to decrease cytotoxicity and genotoxicity induced by numerous genotoxins including alkylating agents (vicuña et al. 2010; mezzoug et al. 2007; leffa et al. 2012; madrigal-santillán et al. 2013; agabeyli 2012; kuzilet et al. 2013; matić et al. 2015). the current results clearly show anti-genotoxic potential of bulgarian rosa alba l. essential oil manifested by decreasing of the frequencies of chromosome aberrations and micronuclei after conditioning treatment with rose oil before mnng challenge and 4 hours inter-treatment time in both experimental test-systems used by us. this is in agreement with our study (gateva et al. 2019) where the preventive effect of acyclic monoterpenoid geraniol (which is one of the major rose essential oil compounds) was obtained against mnng in human lymphocytes in vitro and hordeum vulgare. geraniol was found to be effective in limiting the genotoxic effect of mnng applied as conditioning treatment (4h inter-treatment time) prior challenge with mnng compared to the samples treated with mnng alone. our current study also showed an increased resistance to the damaging effect of mnng both in barley and in human lymphocytes after treatment with rose essential oil and mnng without any inter-treatment time. hence the defence potential of bulgarian rosa alba l. essential oil is manifested regardless of the experimental conditions. the results about anti-cy totoxic and anti-genotoxic effect of the rose oil corresponded with those for antioxidant activity of rose essential oil obtained by dpph test and iron chelating analysis. as a rule, the anti-cytotoxic, anti-genotoxic, and antioxidant properties of the plant extracts cannot be attributed of activities to single constituents. their biological activity could be explained with the combination of effects to one another. as can be seen on chromatographic profil of rosa alba l. oil (table 1), geraniol and citronellol are in major amounts in oil, so they could have an over additive participation in total antioxidant and iron chelating properties. ruberto and baratta (1999) demonstrated that most radical scavenging activities of essential oils are mainly due to the cumulative effect of ingredients nerol, citronellol and geraniol, within whose structure polar bounded hydrogen has been observed. undoubtedly, dpph radicals have little relevance as presence in biological systems, but the results are indicative of the capacity of the bulgarian white rose oil to scavenge free radicals which relate to hydrogen atom or electron donation ability. there are data indicating that not only geraniol and citronellol, but citral (which belongs to the bioactive compounds of the bulgarian rosa alba l. essential oil) also possess antioxidant activity (raut and karuppayil 2014). madankumar et al. (2013) showed that geraniol has a potent antioxidant effect by scavenging oxygen-free radicals and increasing the level of total glutathione content (gsh) in murine skin. it could modulate the activity of enzymatic and non-enzymatic antioxidants to exert its chemopreventive activity against 4-nitroquinoline 1-oxide induced oral cancer in rats. manoharan and selvan (2012) proposed that geraniol inhibits abnormal cell proliferation occurring in skin carcinogenesis by modulating the activities of phase ii detoxification agents and through free radical scavenging potential. geraniol and camphene were found to significantly decrease lipid peroxidation, inhibit nitric oxide release (83.84% and 64.61%) and ros generation in the pre-treated cells as compared to stressed cells (tiwari and kakkar 2009). kashani et al. (2011) described a significant correlation between the phenolic content and dpph scavenging capacity of white rose extracts. our results indicate good iron chelating and dpph radicalscavenging activities, medium superoxide scavenging ability of bulgarian r. alba l. essential oil, which is probably due to its chemical composition. some authors reported that α-terpineol (which is one of the white rose essential oil compounds obtained by us) has remarkable ferrous ions chelating agent and possees antimutagenic activity against 2-aminoanthracene in s. typhimurium ta100 (di sotto et al. 2013). according to bakkali et al. (2008) the mechanism of the decrease of mutagenicity did not depend only of the type of essential oil but on the type of mutagen, thus on the type of lesions and consequently on the dna repair 84 svetla gateva et al. or lesion avoidance system involved. the protective effect obtained by us for bulgarian rosa alba l. essential oil against alkylating agent mnng suggests an activation of repair pathways in addition to antioxidant and scavenging activity of rose essential oil. it is well known that n7-alkylg is responsible for about 90% of the total frequency of alkylation events among the alkylation damages induced by alkylating agents. quantity of o6-alkylg (dna adduct formed by the alkylating agent) is less, but if not repaired could led to dna damage such as cross-linking, strand breaks and modification of bases (kondo et al. 2009). however n7-alkylg is considered to be as mutagenic as o6-alkylg because it is efficiently repaired by base excision repair (ber) pathway (drablos et al. 2004). o6-methylg lesions are repaired by direct damage reversal repair carried out by the enzyme o6-methylguanine-dna methyltransferase (mgmt) also referred to as alkylguanine transferase (agt). mgmt efficiently repairs o6-methylg before replication, through direct transfer of the adducted methyl group from the oxygen in the guanine to a cysteine residue in the catalytic site of mgmt (ramos et al. 2011). niture et al. (2006; 2007) showed that both the ethanolic and aqueous extracts and compounds of some medical plants increased mgmt expression and its activity in lymphocytes and cancer cell lines. to understand the real mechanism of protective potential of rose essential oil when applied in combination with alkylating agents more studies are needed. conclusion bulgarian rosa alba l. essential oil has low cytotoxicity in barley and human lymphocytes in vitro in a dose-dependent manner, as well a good cytoprotective/genoprotective effect against dna damaging agent mnng when applied both with 4 hours between treatments and without any inter-treatment time. anti-genotoxic potential of rose essential oil was manifested by the decrease of the frequency of chromosome aberrations and the micronuclei in both test-systems. something more, white rose’s oil demonstrated well-pronounced anti-oxidant potential and very good metal chelating activity. the results show that rose oil contained protective compounds that can decrease dna damage. data suggest a promising ethnopharmacological potential of bulgarian white rose essential oil. it could serve in medical cosmet as a prophylactic agent, and as an adjuvant in cancer prevention and therapy. geolocation fresh flowers of rosa alba l., from the experimental field of the institute of rose and essential oil plants (ireop), in kazanlak, bulgaria were used. all studies were conducted in the laboratories of the republic of bulgaria. acknowledgments this work was supported by the project “environmental and genetic assessment of the state of the environment, management and strategies for overcoming the risk” – bulgarian academy of sciences, sofia as well as partially supported by the project of bulgarian national science fund № kp-06 n36/17 (granted to m. mileva). references adams rp. 2007. identification of essential oil components by gas chromatography quadropole mass spectrometry; allured publishing co. carol stream, il: usa,; pp. viii + 804 pp. agabeyli ra. 2012. antimutagenic activities extracts from leaves of the morus alba, morus nigra and their mixtures. int. j. biol. 4 (2): 166–172. doi:10.5539/ijb.v4n2p166 albouchi f, hassen i, casabianca h, hosni k. 2013. phytochemicals, antioxidant, antimicrobial and phytotoxic activities of ailanthus altissima (mill.) swingle leaves. s. af. j. bot. 87: 164–174. arumugam p, ramamurthy p, ramesh a. 2010. antioxidant and cytotoxic activities of lipophilic and hydrophilic fractions of mentha spicata l. 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cytotoxicity in plectranthus barbatus using the allium cepa test. caryologia 73(2): 145-153. doi: 10.13128/caryologia-947 received: march 23, 2020 accepted: may 24, 2020 published: july 31, 2020 copyright: © 2020 k.c. cauana trapp, c.a.l. hister, h.d. laughinghouse iv, a.a. boligon, s.b. tedesco. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 1 universidade federal de santa maria, avenida roraima, 1000, cidade universitária, cep 97105-900, santa maria/rs, brazil 2 agronomy department, fort lauderdale research and education center, university of florida – ifas, 3205 college avenue, davie, fl 33314 usa * corresponding author: e-mail: hlaughinghouse@ufl.edu abstract. plectranthus barbatus, popularly known as brazilian boldo or false boldo is mainly used for digestive problems. the aim of our study was to evaluate the proliferative and genotoxic potential of aqueous extracts obtained from fresh and dry leaves and stems of p. barbatus using the in vivo allium cepa test. for the treatments, 6 g of fresh material was first weighed. this was then dried in the microwave, resulting in 0.9 g stems and 0.64 g leaves. the same amount of material was dried naturally at room temperature. we prepared the aqueous extracts by infusion of the leaves and decoction of the stems. water was used as a negative control and glyphosate 1.5% as a positive control. the extracts were analyzed by high performance liquid chromatography (hplc). statistical analysis was performed using the chi-square and scott-knott tests (p <0.05). the results showed that the extracts increased the cellular division of roots. no treatment found genotoxicity. the hplc showed the predominance of isoquercitrin and kaempferol in the leaves, and isoquercitrin, kaempferol, and quercitrin in the stems. we concluded that the aqueous extracts from both leaves and stems of p. barbatus have proliferative and non-genotoxic activity on the cell division of a. cepa, which can be extrapolated to other eukaryotic cell types. keywords: brazilian boldo, proliferation, liquid chromatography, mitotic index, medicinal plants. introduction people have used medicinal plants for the treatment of ailments for thousands of years. though current medicine is well developed, the world health organization (who) has found that a large part of the population in developing countries (85%) still uses traditional folk medicine for their basic health care (brasil, 2006). however, the use of some plants to treat diseases is still based on empirical knowledge, thus without science-based data. the indiscriminate consumption of these plants as herbal teas or other products can 146 kássia cauana trapp et al. result in negative side effects, mainly due to the lack of knowledge on their chemical constituents. only plants whose effects are known should be consumed, thus understanding putative toxic effects (martins et al., 2003). plectranthus barbatus andrews is a species used in traditional medicine, known in brazil as boldo, fakeboldo, and malva-santa. this species has a characteristic bitter taste when using its leaves in herbal teas, which is absent in its stems, but the substance causing this taste has not yet been identified (lorenzi and matos, 2008). aqueous extracts of the leaves are used to treat heartburn, gastric discomfort, dyspepsia, hangovers, as a laxative (lorenzi and matos, 2008), and to prevent or control oral diseases (figueiredo et al., 2010). it has also been shown to have contraceptive activity in rats (almeida and lemonica, 2000), and potential use as an antimalarial drug (kiraithe et al., 2016). studies that validate putative genotoxic and proliferative effects of medicinal plants are necessary, and the use of cytogenetic bioassays which use plants for detecting chromosomal damage that may be caused by other plant extracts, complex mixtures, chemicals, etc. are highly recommended. the allium cepa l. test is one of the few direct methods to measure damage to cells exposed to mutagenic or potentially carcinogenic agents and to assess the effects of such damage by observing chromosomal changes (tedesco and laughinghouse iv, 2012). several studies have used the a. cepa test to evaluate plant extracts for cytotoxicity, such as tedesco et al. (2015), hister et al. (2017), and sousa et al. (2018). rank and nielsen (1994) found an 82% correlation between the a. cepa test and the mouse carcinogenicity test, concluding that a. cepa was even more sensitive than the ames test. teixeira et al. (2003) found the same results in tests comparing meristematic cells of onion roots, bone marrow cells of rats, and human lymphocytes as bioindicators, validating the safety of a. cepa for cytogenetic studies. extracts from medicinal plants are a complex mixture of various bioactive compounds. to better understand these mixtures, high performance liquid chromatography (hplc) can be used for phytochemical analyses (trapp et al., 2016). this technique is able to analyze the amount of chemical compounds in a sample in one single analysis, revealing the chromatographic or fingerprint profile of the extract (alaerts et al., 2007). our work aimed to evaluate the possible genotoxic and proliferative activities of fresh and dried leaves and stems of p. barbatus. this was undertaken using two different methods: 1) analyzing the cell cycle of root tips of a. cepa and 2) determining the phenolic compounds in the extracts. material and methods sampling of the material material was obtained from an adult plant grown in a natural garden and acclimatized for 4 years in a 10kg large pot. the plant was collected in santa maria, rio grande do sul, brazil (29°42’54.8”s and 53°43’12.0”w) and taxonomically identified by dr. ts canto-dorow. treatments and criteria for analysis the meristematic cells of roots of a. cepa (onion, 2n = 16) were used as a test system to evaluate morphological and structural changes in chromosomes and to determine mitotic indices. groups of four bulbs, each corresponding to a treatment, were placed in tap water for rooting. after rooting, one group remained in water (negative control), another group was placed in a 1.5% glyphosate solution (positive control), since this herbicide induces chromosomal alterations and inhibits cell division in meristematic cells (dimitrov et al., 2006; souza et al., 2010; rodrigues et al., 2017). the other bulbs were transferred to the treatment solutions for 24 hours with the different extracts of p. barbatus. we used 24 hours because this is the duration of the mitotic cycle in a. cepa (matagne, 1968). the experiment was divided into two steps, using both dried and fresh material: step 1: 6 g (brasil, 2011) of fresh material (leaves and stems) was weighed. a microwave was used to dry 6 g for comparison (5 minutes for leaves and 7 minutes for stems), which resulted in 0.64 g of leaves and 0.9 g of stems. the following treatments were then prepared: t1 water (negative control); t2aqueous extract of 6 g l-1 of fresh stems; t3aqueous extract of 0.9 g l-1 of microwave dried stems; t4aqueous extract of 6 g l-1 of fresh leaves; t5aqueous extract of 0.64 g l-1 of microwave dried leaves; t6glyphosate 1.5% (positive control). step 2: stems and leaves were dried naturally at room temperature, resulting in the same dry weight as those dried in the microwave. the following treatments were used: t1water (negative control); t2aqueous extract of 0.64 g l-1 of room temperature dried leaves; t3aqueous extract of 0.9 g l-1 of room temperature dried stems; t4glyphosate 1.5% (positive control). after treatment, a. cepa rootlets were collected, fixed in ethanol:acetic acid (3:1) and stored in 70% ethanol at 4oc for further analysis. for slide preparation, a modified squashing technique was used, and acetic acid was used to stain the genetic material (guerra and souza 2002). 147determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test slide analyses were performed using a leica icc50 hd light microscope, at 400x magnification and for each bulb two replicates were used (two slides). five hundred cells per slide were counted, totaling 1000 cells per bulb and 4000 cells per treatment. mitotic index (mi) values were calculated based on the percentage of dividing cells, according to the formula: mi = (number of cells in mitosis / total number of cells) x 100. in addition, we observed the phases of cell division to verify possible irregularities, such as chromosomal breakage, chromosome bridges, laggard chromosomes, and micronuclei. analysis of extracts by high performance liquid chromatography (hplc-dad) a sample of each extract of p. barbatus was run on an hplc-dad at the phytochemical laboratory of the department of industrial pharmacy at ufsm. general chemistry, apparatus and procedures all chemical reagents were of analy tical grade. methanol, formic acid, gallic acid, caffeic acid, ellagic acid, and boldine were purchased from merck (darmstadt, germany). quercetin, quercitrin, isoquercitrin, rutin, and kaempferol were purchased from sigma chemical co. (st. louis, mo, usa). hplc was performed with a shimadzu (kyoto, japan) and shimadzu self-injector system (sil-20a) equipped with alternative pumps (shimadzu lc-20at) attached to a degasser (20a5 dgu) with an integrator (cbm 20a), diode arrangement detector (spd-m20a) and software (lc solution sp1 1.22). quantification by hplc chromatographic ana lyses were performed in reverse phase under gradient conditions using a c18 column (4.6 mm x 150 mm) loaded with particles of 5 μm diameter; the mobile phase used water containing 1% formic acid (a) and methanol (b), and the gradient was: 13% b for 10 minutes, following 20%, 30%, 50%, 60 %, 70%, 20% and 10% b at 20, 30, 40, 50, 60, 70 and 80 minutes, respectively, following the method described by abbas et al. (2014) with minor modifications. the aqueous extracts of the leaves of p. barbatus (fresh and dry), stems of p. barbatus (fresh and dry), and the mobile phase were filtered with a 0.45 μm membrane filter (millipore) and then degassed with an ultrasonic bath before use. the extracts were analyzed at a concentration of fresh leaves at 6 g l-1, microwave dried leaves at 0.64 g l-1, fresh stems at 6 g l-1, microwave dried stems at 0.9 g l-1. the flow used was 0.7 ml min-1, injection volume of 40 μl, and the wavelength was 254 nm for gallic acid, 302 nm for boldine, 327 nm for caffeic and ellagic acids, and 366 nm for quercetin, quercitrin, isoquercitrin, kaempferol, and rutin. the reference solutions were prepared in the mobile phase for hplc at 0.025 0.300 mg ml-1 for quercetin, quercitrin, isoquercitrin, rutin, and kaempferol; 0.05 0.45 mg ml-1 for ellagic, gallic, and caffeic acids; and 0.006 0.250 mg ml-1 for boldine. chromatographic peaks were confirmed by comparison of their retention time to standards and by dad spectra (200 to 500 nm). calibration curve for gallic acid: y = 13174x + 1273.6 (r = 0.9999), boldine: y = 12583x + 1274.9 (r = 0.9999), caffeic acid: y = 11992x + 1367.1 (r = 0.9999), ellagic acid: y = 13286x + 1264.1 (r = 0.9999), quercetin: y = 12837x + 1364.5 (r = 0.9999), isoquercetin: y = 12769x + 1326.5 (r = 0.9999), rutin: y = 13158x + 1173.9 (r = 0.9998), quercetin: y = 13627x + 1292.5 (r = 0.9996), and kaempferol: y = 13271x + 1324.6 (r = 0.9999). the extracts of the naturally dried leaves and stems were also analyzed at a concentration of room temperature dried leaves at 0.64 g l-1 and room temperature dried stems at 0.9 g l-1. in this case, the flow used was 0.7 ml min-1, injection volume of 40 μl, and the wavelength was 254 nm for gallic acid, 302 nm for boldine, 327 nm for caffeic and ellagic acids, and 366 for quercetin, quercitrin, isoquercitrin, kaempferol, luteolin, and rutin. the reference solutions were prepared in the mobile phase for hplc at the concentrations of 0.045-0.300 mg ml-1 for quercetin, quercitrin, isoquercitrin, rutin, luteolin, and kaempferol; 0.02 0.35 mg ml-1 for ellagic, gallic, and caffeic acids; and 0.006 0.250 mg ml-1 for boldine. chromatographic peaks were confirmed by comparing their retention time to standards and by dad spectra (200 to 600 nm). calibration curve for gallic acid: y = 12683x + 1197.5 (r = 0.9998), boldine: y = 12481x + 1238.9 (r = 0.9999), caffeic acid: y = 11983x + 1267.1 (r = 0.9999), ellagic acid: y = 12670x + 1325.8 (r = 0.9992), quercetin: y = 13056x + 1264.5 (r = 0.9999), isoquercitrin: y = 11979x + 1286.5 (r = 0.9996), rutin: y = 12758x + 1345.3 (r = 0.9999), y = 12629x + 1198.6 (r = 0.9999), luteolin: y = 13540x + 1317.1 (r = 0.9993), and kaempferol: y = 11978x + 1257.6 (r = 0.9997). all chromatographic operations were performed at room temperature and in triplicate. the limit of detection (lod) and the limit of quantification (loq) were calculated based on the standard deviation of the responses and the slope, using three independent analytical curves. lod and loq were calculated as 3.3 and 10 σ.s-1, respectively, where σ is the standard deviation of 148 kássia cauana trapp et al. the response, and s is the slope of the calibration curve (boligon et al., 2013). statistical analysis the experimental design was completely randomized. mitotic index values were compared by the chisquare test (χ2) (p <0.05), using bioestat 5.o (ayres et al., 2007). the means of phenolic compounds from hplc were compared using the scott-knott’s test (p <0.05) using assistat®, beta version 7.7 (silva and azevedo, 2016). results and discussion the a. cepa test is an ideal bioindicator to identify potential cytotoxic or mutagenic effects from medicinal plants, which can be harmful to human health (bagatini et al. 2007). table 1 shows that the treatments with the aqueous extracts increased cell division, compared to the negative control. this means that both the leaves and stems from fresh and microwave dried material induced cell proliferation in meristematic cells of a. cepa rootlets. thus, the substances tested can be considered cytotoxic, since they modify normal mi, increasing or decreasing it (leme and marin-morales, 2009; vieira and silveira, 2018). in the case of p. barbatus, the aqueous extracts increased the cell division of a. cepa roots. this is similar to data by iganci et al. (2006) on aqueous extracts of fresh leaves of p. barbatus (30 g l-1) on seeds of a. cepa, where the authors found a significant increase in cell division after 6 days of treatment. the highest mitotic index was induced by the extracts of stems of p. barbatus, with t2 = 5.5% (extract from the decoction of fresh stems). for leaf extracts, the microwave-dried leaf extract (t5) differed the most from the other extracts, but did not differ (p <0.05) from the treatment with microwave-dried stems (t3). in addition, all the mitotic indices of the treatments differed (p <0.05) from the positive control, which had a high cytotoxic effect. in table 2, we see that extracts from stems of p. barbatus dried at room temperature decreased cell proliferation in relation to water (t1), except t2 (aqueous extract by infusion of 0.64 g l-1 dry leaves), which was not significantly different from the negative control. the mitotic indices of naturally dried stems and microwave-dried stems were different, suggesting that forced drying is able to intensify the cell division of a. cepa (table 1 and table 2). still, the extract from fresh stems had the highest overall mitotic index. although the extracts induced cell division in a. cepa, we found no cells with chromosomal alterations in any of the treatments. probably the secondary compounds in the extracts, which are complex mixtures with numerous bioactive secondary metabolites, induced the cell division. iganci et al. (2006) also observed an increase in mi with no genotoxic effects when testing aqueous extracts of p. barbatus on seeds of a. cepa. in contrast, a study by costa (2002) found that p. barbatus leaves were toxic to the liver and kidneys of mice treated over seven days. similarly, souza and maia (2000) reported chronic toxicity of hydroalcoholic extracts of p. barbatus in rats treated with doses 20x greater than those used in folk medicine (680 mg kg-1). therapeutic actions of medicinal plants are determined by their secondary metabolites (taiz and zeiguer, 2013), producing phenolic compounds with several functions, such as defense against pests and diseases, protection from ultraviolet radiation, and they can also attract pollinators (ignat et al., 2011). as a secondary function, the phenolic compounds can have anti-inflammatory (smolarek et al., 2009) and antimicrobial effects (medina table 1. number of analyzed allium cepa cells and mitotic indices (mi) of treatments with fresh and dried leaves and stems of plectranthus barbatus. treatments – step 1 tco cells in interphase p m a t mitotic index (%) t1 – negative control 4000 3893 66 19 8 14 2.68d* t2 – decoction of 6 g l-1of fresh stems 4000 3780 102 46 36 36 5.5a t3 – decoction of 0.9 g l-1of microwave-dried stems 4000 3840 77 22 30 31 4.0b t4 – infusion of 6 g l-1of fresh leaves 4000 3877 91 15 7 10 3.1c t5 – infusion of 0.64 g l-1of microwave-dried leaves 4000 3846 70 36 24 24 3.9b t6 – positive control 4000 3975 10 8 2 5 0.63e tco = total cells observed; p= prophase; m= metaphase; a= anaphase; t= telophase. *means followed by the same letter are not significantly different among themselves, using the chi-square test at 5% probability. 149determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test et al., 2011). in our study, hplc analyses revealed different phenolic compounds in the aqueous extracts of leaves and stems: gallic acid (peak 1), boldine (peak 2), caffeic acid (peak 3), ellagic acid (peak 4), rutin (peak 5), quercitrin (peak 6), quercetin (peak 7), isoquercitrin (peak 8), kaempferol (9), and luteolin (peak 10) (figure 1a, 1b, 1c, 1d, 1e and 1f). table 3 shows the mean amount of each of the phenolic compounds present in the aqueous extracts of p. barbatus leaves analyzed by hplc. we found that in aqueous extracts of fresh leaves, isoquercitrin was the most abundant compound, followed by quercitrin and ellagic acid. in extracts from the microwave-dried leaves, isoquercitrin was also the pretable 2. number of analyzed allium cepa cells and mitotic indices (mi) of treatments of dried leaves and stems of plectranthus barbatus at room temperature. treatments – step 1 tco cells in interphase p m a t mitotic index (%) t1 – negative control 4000 3847 84 28 15 26 3.83 a* t2 – infusion 0.64 g l-1 naturally dried leaves (room temperature) 4000 3860 75 19 18 28 3.5 a t3 – decoction de 0.9 g l-1 of naturally dried stems (room temperature) 4000 3918 50 12 6 14 2.05 b t4 – positive control 4000 3946 36 6 7 5 1.3 c tco = total cells observed; p= prophase; m= metaphase; a= anaphase; t= telophase. *means followed by the same letter are not significantly different among themselves, using the chi-square test at 5% probability. figure 1. representative hplc profile of aqueous extracts of leaves and stems of plectranthus barbatus: a) infusion of fresh leaves; b) infusion of dry leaves (microwave); c) infusion of dry leaves (room temperature); (d) decoction of fresh stems; e) decoction of dry stems (microwave); (f ) decoction of dry stems (room temperature). uv detection at 327 nm. gallic acid (peak 1), boldine (peak 2), caffeic acid (peak 3), ellagic acid (peak 4), rutin (peak 5), quercitrin (peak 6), quercetin (peak 7), isoquercitrin (peak 8), kaempferol (9) and luteolin (peak 10). 150 kássia cauana trapp et al. dominant compound (including more than in the fresh leaf extract), followed by kaempferol and quercetin. however, in the aqueous extracts of naturally dried leaves, both isoquercitrin and kaempferol were most abundant, followed by quercetin, caffeic acid, and luteolin. luteolin is a phenolic compound found only in the extract of naturally dried leaves. these results are similar to those by grayer et al. (2010), who analyzed the chemical composition of p. barbatus and observed kaempferol and quercetin, in addition to pires et al. (2016) who detected quercitrin, quercetin, and kaempferol. the extract of fresh leaves had the highest amount of total quantified phenolic compounds, followed by the extract of microwave-dried leaves and then the extract of naturally dried leaves. thus, slowly drying the leaves caused a greater loss of compounds compared to fresh leaves. studies by pereira et al. (2000) support this finding, where they identified that higher levels of bioactive compounds are linked with rapid drying. in table 4, we see that in fresh stems there were low amounts of phenolic compounds, which is a different pattern than seen in the fresh leaves. isoquercitrin was the table 3. phenolic compounds in aqueous extracts by infusion of plectranthus barbatus leaves using high performance liquid chromatography (hplc). compound extract of fresh leaves mg g-1 extract of microwavedried leaves µg ml-1 lod mg g-1 loq µg ml-1 extract of naturally dried leaves lod loq gallic acid 5.28 h* 2.02 h 0.023 0.075 1.98 f 0.020 0.067 boldine 7.21 f 5.64 f 0.008 0.026 3.76 e 0.012 0.039 caffeic acid 5.11 h 7.05 e 0.015 0.049 7.12 c 0.019 0.061 ellagic acid 15.29 c 3.89 g 0.027 0.093 5.70 d 0.027 0.093 rutin 12.82 d 3.86 g 0.019 0.062 1.87 g 0.015 0.049 quercitrin 16.44 b 14.92 d 0.013 0.042 2.02 f 0.034 0.113 quercetin 10.14 e 16.09 c 0.024 0.079 9.92 b 0.008 0.026 isoquercitrin 17.82 a 19.65 a 0.017 0.056 13.57 a 0.017 0.056 kaempferol 5.63 g 17.23 b 0.035 0.115 13.62 a 0.025 0.081 luteolin 7.03 c 0.013 0.042 total 95.74 90.35 66.59 results are expressed as mean of three determinations. lod is the limit of detection and loq is the limit of quantification. * means followed by different letters differ by the scott-knott test (p <0.01). table 4. phenolic compounds in aqueous extracts by decoction of plectranthus barbatus stems using high performance liquid chromatography (hplc). compound extract of fresh leaves mg g-1 extract of microwavedried leaves µg ml-1 lod mg g-1 loq µg ml-1 extract of naturally dried leaves lod loq gallic acid 1.95 g* 3.08 f 0.023 0.075 2.81 e 0.020 0.067 boldine 3.44 e 7.12 c 0.008 0.026 4.99 c 0.012 0.039 caffeic acid 1.10 i 6.50 d 0.015 0.049 4.08 d 0.019 0.061 ellagic acid 4.75 d 2.37 g 0.027 0.093 1.34 g 0.027 0.093 rutin 2.15 f 2.34 g 0.019 0.062 1.41 g 0.015 0.049 quercitrin 6.07 b 6.52 d 0.013 0.042 7.87 a 0.034 0.113 quercetin 1.35 h 3.97 e 0.024 0.079 2.22 f 0.008 0.026 isoquercitrin 8.25 a 8.32 b 0.017 0.056 7.88 a 0.017 0.056 kaempferol 5.98 c 9.06 a 0.035 0.115 7.34 b 0.025 0.081 luteolin 1.49 g 0.013 0.042 total 35.09 49.28 41.43 results are expressed as mean of three determinations. lod is the limit of detection and loq is the limit of quantification. * means followed by different letters differ by the scott-knott test (p <0.01). 151determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test most abundant compound in fresh stems, followed by quercitrin and kaempferol. the extracts of microwavedried stems contained the highest amount of compounds among the stems, with kaempferol being the major compound, followed by isoquercitrin and boldine. in the aqueous extract of the naturally dried stems, isoquercitrin and quercitrin were both the most abundant, followed by kaempferol and boldine. as in the leaf extracts, luteolin was also found in the extract of naturally dried stems. stem extracts had nearly 50% less phenolic compounds than the leaves, while inducing higher mitotic indices. phenolic compounds such as flavonoids (quercitrin and isoquercitrin) are able to sequester free radicals (decker, 1997) and their abundance has been positively correlated with antimicrobial action in salmonella enteritidis (medina et al., 2011). interestingly, a study by boligon et al. (2012) found that isoquercitrin isolated from scutia buxifolia reissek had a protective effect against human lymphocyte damage caused by hydrogen peroxide. the authors hypothesize that this was due to the reduction of oxidative stress due to its antioxidant nature. carvalho et al. (2007) found that phenols, such as ellagic acid and gallic acid, can inhibit seed germination, plant growth, and fungi. according to tomás-barberán and espín (2001), this group of secondary metabolites is related to the prevention of cardiovascular diseases and cancer. in summary, aqueous extracts prepared with naturally dried material at room temperature did not affect the mitotic index of a. cepa. on the other hand, fresh and microwave-dried extracts of leaves and stems increased cell division (mitotic index). no cells with chromosomal alterations were found in any of the treatments. phytochemical analyses found a high amount of isoquercitrin in the aqueous extracts of leaves, with the major compound in naturally dried leaves being kaempferol. the total amount of phenolic compounds was much lower in stems. isoquercitrin was the major compound in the extracts of fresh and dried stems at room temperature, and in the latter quercitrin was also predominant. the extracts of the microwave-dried stems were dominated by kaempferol. our results are preliminary, but we emphasize the importance to identify putative harmful compounds found in plants, to increase the safety for their use in folk medicine. acknowledgements we thank prof. m.l. athayde (in memorian) for her valuable collaboration. partial financial support was made through a pibic/cnpq ic fellowship. hdl would like to thank the usda nifa hatch project # flaftl-005697. references abbas sr, sabir sm, ahmad sd, boligon aa, athayde, ml. 2014, phenolic profile, antioxidant potential and dna damage protecting activity of sugarcane (saccharum officinarum). food chem 147:10-16. alaerts g, matthijs n, verbeke j, heyden y. 2007. chromatographic fingerprint development for herbal extract: a screening and optimization methodology on monolithic columns. j chromatogr a 1172:1-8. almeida fcg, lemonica ip. 2000. the toxic effects of coleus barbatus on the different periods of pregnancy in rats. j ethnopharmacol 73:53–60. ayres m, ayres jrm, ayres dl, santos as. 2007. bioestat: aplicações estatísticas nas áreas das ciências biomédicas [bioestat: statistical applications in biomedical sciences]. belém: ong mamiraua. bagatini md, silva acf, tedesco sb. 2007. uso do sistema teste de allium cepa como bioindicador de genotoxicidade de infusões de plantas medicinais [use of the test system allium cepa as a bioindicator of genotoxicity in medicinal plant infusions]. rev bras farmacogn 17:444-447. bianchi mlp, antunes lmg. 1999. radicais livres e os principais antioxidantes da dieta [free radicals and the main antioxidants in the diet]. rev nutr 12:123130. boligon aa, sagrillo mr, machado lf, souza filho o, machado mm, cruz, ibm, athayde ml. 2012. protective effects of extracts and flavonoids isolated from scutia buxifolia reissek against chromosome damage in human lymphocytes exposed to hydrogen peroxide. molecules 17(5):5757-5769. boligon aa, kubiça tf, mario dn, brum tf, piana m, weinblen r, lovato l, alves sh, santos rcv, alves cfs, athayde ml. 2013. antimicrobial and antiviral activity-guided fractionation from scutia buxifolia reissek extracts. acta physiol plant 35:2229-2239. brasil. agência nacional de vigilância sanitária. 2011. formulário de fitoterápicos da farmacopéia brasileira [form of phytotherapics of the brazilian pharmacopoeia]. brasília: anvisa. brasil. ministério da saúde. secretaria de ciência, tecnologia e insumos estratégicos. 2006. política nacional de plantas medicinais e fitoterápicos [national policy of medicinal plants and herbal medicines]. brasília: editora ms. 152 kássia cauana trapp et al. carvalho jct, gosmann g, schenkel ep. 2007. compostos fenólicos simples e heterosídicos [simple and heterosidic phenolic compounds]. in: simões, cmo (editor), farmacognosia: da planta ao medicamento [pharmacognosy: from plant to medicine]. 6th ed. porto alegre: editora da ufrgs; p. 13-28. costa mccd. 2002. aspectos farmacológicos de plectranthus barbatus andr. 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(plantaginaceae) saeed mohsenzadeh*, masoud sheidai, fahimeh koohdar a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding timir baran jha1,*, partha sarathi saha2, sumita jha2 chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india harminder singh2, jaswant singh1,*, puneet kumar2, vijay kumar singhal1, bhupendra singh kholia2, lalit mohan tewari3 population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies raheleh tabaripour1,*, masoud sheidai1, seyed mehdi talebi2, zahra noormohammadi3 induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions girjesh kumar, shefali singh* karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(1): 3-12, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-951 caryologia international journal of cytology, cytosystematics and cytogenetics citation: b. yuksel, o. aksoy, m. karatas (2021) the cytological and molecular investigation of the toxic effects of the herbicide roundup on cucumis sativus. caryologia 74(1): 3-12. doi: 10.36253/caryologia-951 received: may 25, 2020 accepted: october 16, 2020 published: july 20, 2021 copyright: © 2021 b. yuksel, o. aksoy, m. karatas. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. the cytological and molecular investigation of the toxic effects of the herbicide roundup on cucumis sativus burcu yuksel1,*, ozlem aksoy2, melis karatas2 1 vocational school of kocaeli health services, kocaeli university, kocaeli, turkey 2 department of biology, faculty of letters and sciences, kocaeli university, kocaeli, turkey *corresponding author. e-mail: burcu.yuksel@yahoo.com abstract. in the current study, it is aimed to investigate the toxic effects of a widely used herbicide roundup containing active ingredient glyphosate on cucumber (cucumis sativus) by cytological and molecular investigation. three different concentrations (0.6%, 1.2% and 2.4%) of roundup were applied to cucumber for 48 and 72 hours. at the end of the application procedure, the germination percentage, mean root length, mitotic frequency and mitotic abnormalities, rapd profiles and genomic template stability (gts) were determined in root apical meristematic cells. for rapd pcr analysis 10 rapd primers were used, 8 of them produced band patterns and it was found that 5 rapd primers among them produced unique polymorphic band patterns and subsequently were used to produce a total of 24 bands. observed percentage of polymorphism was 26%. the changes in rapd profiles after roundup treatment was included variations as gain and/or loss of bands compared with the control group. genomic template stability changed in rapd profiles at various roundup concentrations. keywords: cucumis sativus, rapd pcr , root growth, genotoxicity, glyphosate. introduction recent in vivo and in vitro experiments have been reported the impact of chemical groups of pesticides as they are regarded a significant set of environmental pollutants (khan 2016; lushchak et al. 2018; alvarez et al. 2017). pesticides are classified into different groups such as organophosphates, organochlorines, carbamates and pyrethroids (vakonaki et al. 2013). nevertheless, previous findings about the genotoxicity of the most of the pesticides are rare and the findings of the different studies are inconsistent (sarath et al. 2019). roundup includes the glyphosate [n(phosphonomethyl)glycine] as the active ingredient, and is a well-known and popular brand name of a universal, broad-spectrum herbicide manufactured in u.s. it is the top selling herbicide in the world at least for 40 years, as well (ho and cummins 2010). the main ingredient of the roundup, the glyphosate, destroys the organisms it targets by inhibiting the enzyme, 5-enolpyruvoyl-shikimate3-phosphate 4 burcu yuksel, ozlem aksoy, melis karatas synthetase (epsps), which is a vital source for the development of popular aromatic amino acids including phenylalanine, tyrosine and tryptophan (schaumburg et al. 2016). the chief beneficiaries and application areas of pesticides are plants, sometimes they themselves are the target organism as in the case of weeds and sometimes they carry hazardous targets on them such as pests, insects and pathogenic fungi, etc. sources of exposure include the direct application or via soil and water as well as atmospheric drift. pesticides enter reaction with various nucleophilic centers of cellular biomolecules, including dna because of their reactivity and electrophilic behaviours (benedetti et al. 2018; bolognesi 2003). they can also create other more volatile electrophilic products that can either transform cellular components or are digested to some other steadier products. control and treatment group design studies, qualitatively and quantitative, can elicit the effects of genotoxicity of the pesticides. in the studies that use rapd method, the previous research utilized diagnostic analysis by examining the change in band intensity or disappearance and/or appearance of rapd bands, and the phenetic numerical analysis that would give us ideas about the general genetic mixture of populations, which is also labelled as the genetic similarity analysis (lynch and milligan 1990; de wolf et al. 2004). glyphosate is usually used in two ways: it can be put on foliage or added to freshly cut stumps. it works by progressing through the plant to its actively growing areas and inhibiting protein synthesis. similar in chemical structure to an amino acid, glyphosate prevents plants from creating three amino acids required for growth (poletta et al. 2009). thus it is aimed to investigate the toxic effects of a widely used herbicide roundup containing active ingredient glyphosate on cucumber (cucumis sativus) by cytological and molecular studies, which has not been done before. the use of parameters such as germination and root growth to assess the toxicity of various substances is rapidly increasing, as data on germination can inform us about the lethal effects of the herbicides used. delay in germination or root growth can provide information about non-lethal but metabolic activity. material and methods determination of ec50 we used 6 different concentrations of the herbicide roundup including the suggested concentration to find out the ec50 (effective concentration that lower the root length 50% of the control). after 48th and 72th hour, for each concentration, the mean value of 100 roots was extracted as a percent of the control value. then, the obtained result was utilized to determine the ec50 value. cytological experiments cytological response was observed in the root apical meristem of. the root tips were placed in a solution of carnoy with the following concentration of 3:1, alcohol: acetic acid, and hydrolyzed in 1 n hcl at 60°c for approximately 5–10 minutes and then were crushed in a 2% orcein stain in 45% acetic acid. slides of the cucumber were stored in a freezer and scrutinized 30 days later (rank and nielsen 1994). mitotic analysis. mitotic index was calculated by counting at least 1000 cells from each of the paste preparations (equation 1). the percentage mitotic index was determined by dividing the number of cells divided by the total number of cells and multiplying by 100; mitotic index (%) =( number of divided cells)/ (total number of cells) x100 (1). detection of mitotic abnormality percentage and frequencies the chromosomal abnormalities and cellular anomalies determined at each division stage were determined and divided by the number of normal cells, and mitotic abnormality percentage and frequencies were calculated (equation 2).chromosomal abnormalities were evaluated separately in all phases of mitosis and photographic data was obtained using an olympus bx51 photomicroscope. three replicates were prepared for each concentration. mitotic abnormality percentage=(chromosomal abnormalities and cellular anomalies)/(number of divided cells total number of cells) x100 (2) germination percentage and root length values in our study, as a result of the treatment of c. sativus seeds treated for 48 and 72 hours with different concentrations of roundup (0.05%, 0.1%, 0.5%, 1%, 1.2%, 2%), the change in germination percentage and differ5the cytological and molecular investigation of the toxic effects of the herbicide roundup on cucumis sativus ences in root lengths were determined. all experiments were carried out in triplicate. rapd pcr analysis a qiagen dneasy plant mini kit was utilized to express genomic dna from 0.1-0.2 g powdered root tissue. the spectrophotometer shimadzu uv-mini 1240 was used to assess and calculate the quantity and quality of dna. a commercial set of 10 random 10-mer primers was obtained (thermo scientific). pcr amplifications were carried out in a 25 μl reaction mixture with 10 ng of template dna, 1x taq polymerase buffer and 1 u of taq polymerase, and 2.5 mm mgcl2, 1 μm dntp, 1 mm primer. amplifications were done in a tc-3000 thermal cycler. the cycle programmed was made up of a preliminary denaturation step at 94°c for 5 min, followed by 40 cycles of 94°c for 1 min, 30°c for 1 min, 72°c for 1 min, and a final elongation at 72°c for 5 min. the pcr products were kept on a 1 % agarose gel with ethidium bromide (0.5 μg/ml) and all digital photographs were taken by the uvp geldoc-it 310 imaging system. a 1 kb dna ladder was used as size marker (fermentas). 10-mer oligonucleotide primers of 60–70% gc content were benefitted from during the monitoring c. sativus genome for the modification. a negative control with no dna template was also performed in each pcr amplification to validate the absence of any contamination. estimation of genomic template stability the genomic template stability (gts) was determined as follows: gst% = (1a/n) x 100. this reads as where (a) rapd polymorphic profiles found in each treated sample and (n) the number of total bands in the control (atienzar et al. 1999; liu et al. 2007). polymorphism detected in rapd profiles comprised of disappearance of a normal band and appearance of a new band compared to the control rapd profiles (atienzar et al. 2002). the average was later computed for each experimental group that were treated with various roundup concentrations. data analysis because of the dominant characteristics of rapd markers, each band was acknowledged as a representative of the phenotype at a single biallelic locus. a binary matrix consisting of present (1) or absent (0) was created by marking each amplified fragment from each individual. in the marking, only pure and different bands were counted. bands with the same gel mobilities were taken as homologous. the matrix was used to create an input file and evaluated with the software program popgene 1.32 (nei 1978). results genotoxic characteristics of the pesticide roundup in c.sativus root tip cells were examined in this study. because of its popularity in everyday use in the field of agriculture, a commercial form of the pesticide was examined. c.sativus was utilized as the test system due to its usage on plants in agriculture and plants might yield exceptional genotoxic metabolites. the ec50 value of roundup was calculated as 1.2% ml/l and we treated the root tips were with the concentrations of 0.6% (ec50/2), 1.2% (ec50), and 2.4% (2xec50) ml/l as shown in figure 1. figure 2 shows that the root lengths are reduced by half with the ec50 concentration of 1.2% roundup treatment. table 1 shows as a result of the treatment of c.sativus seeds with roundup different concentrations, it was observed that the germination percentage, which was 100% after 72 hours in the control group, decreased figure 1. c.sativus seeds treated with roundup concentrations a) control, b) 0.6%, c) 1.2%, d) 2.4%. 6 burcu yuksel, ozlem aksoy, melis karatas in parallel with the roundup concentration increase, respectively. results of the study revealed that roundup modified the mitotic cycle and reduced the mitotic index in c.sativus root tip cells. a significant decrease was observed in all concentrations compared to the control (table 2). the mitotic index in the control group was 26.7, and 8.5, 5.2 and 1.7 at 0.6%, 1.2% and 2.4% roundup concentrations, respectively. it was also observed that the amount of the dose had effects on the reduction of the mitotic at index, which were all significant at different concentrations according to p<.005. these results demonstrated that concentrations of roundup were cytotoxic in cucumber. previous literature reported similar results in mitosis from the treatment of the herbicides racer (yuzbaşıoğlu et al. 2003), atrazine (bolle et al. 2004), and arsenal (grisolia et al. 2004). this might stem from some potential mechanisms for chemically reduced mitotic index in plant cells. firstly, the reduction in the mitotic index can be caused because of the blocking of g1 suppressing dna synthesis (shcneiderman et al. 1971). the second potential reason might be a hinderance of g2 which blocks the cell from ingoing mitosis. the reduction in the mitotic index could be a result of the inhibition of dna synthesis at the s-phase (sudhakar et al. 2001). we also examined the mitotic abnormality percentages and frequencies for prophase, metaphase, anaphase and telophase for different concentrations at different hours (table 3). we observed that mitotic abnormality increased for all measurements as the concentration amount increased. compared to the control group, the highest percentage of abnormal dividing cells was observed at 2.4% roundup concentration metaphase (300) and 1.2% roundup concentration in telophase (300) stage.later, 2.4% roundup concentration was determined in prophase (209), at 0.6% in metaphase (144) and at 1.2% in prophase (140). these data show that increasing coumarin concentrations increase the amount of abnormal dividing cells in each division phase. abnormal and normal cells were not observed figure 2. root length percentages in c.sativus treated with roundup showing ec50 value(72th h). 100 86 70 63 50 18 12 0 20 40 60 80 100 120 c ontrol 0.05 0.1 0.5 1.2 2 4 c o n c en tra tio n (% ) r o o t l en g th (% ) table 1. the mean of determined values of the germination percentages in 48th and 72th hour. roundup concentration (%) germination percentage means 48th hour 72th hour control 100 100 0.05 97.5 100 0.1 95 95 0.5 90 90 1 87.5 92.5 1.2 30 77.5 2 57.5 87.5 4 45 60 table 2. the effect of roundup concentrations on mitotic index. roundup concentration % the number of divided cells mitotic index (%) ± sd standard error p value control 267 26.7 ± 1.117 .22 0.6 85 8.5 ± .812 .11 .000 * 1.2 52 5.2 ± .652 .08 .000* 2.4 17 1.7 ± .213 .02 .000* *p<.005 table 3. the effect of roundup concentrations on the mitotic abnormality percentage and frequencies.(n: normal dividing divider, a; abnormal dividing cells,%; percentage data). concentrations prophase metaphase anaphase telophase n a % n a % n a % n a % control 66 18 27.2 19 5 26 11 3 27 7 1 14 0.6% 50 27 54 9 13 144 8 5 62 3 2 66 1.2% 30 42 140 10 13 130 8 6 75 1 3 300 2.4% 11 23 209 3 9 300 3 4 133 7th e cytological and molecular investigation of the toxic eff ects of the herbicide roundup on cucumis sativus at a concentration of 2.4%, possibly due to the failure of the telophase phase (table 3). roundup boosted t he percentage of abnorma l cel ls in c.sativus. this grow t h was signif icant in all concentrations in comparison to the control and the amount of the dose was also a contributing factor. the abnormalities that were commonly observed were stickiness in chromosomes, nuclei degeneration, micronuclei formation and vacuolization in cytoplasm (figure 3). aft er roundup treatment, genomic dna profi les and genomic dna quantities and purities were shown in figure 4. by evaluating the agarose gel images, it was decided that the dna belonging to the control group and roundup groups were sufficient and purity for rapd-pcr experiments. th e list of polymorphic and monomorphic rapd primers (table 4), the number of primers compared between 0.6%, 1.2% and 2.4% treatments of roundup and the percentage of polymorphism for all primers (fig. 5) were determined. 10-mer oligonucleotide primers of 60–70% gc content were benefitted from during the monitoring c. sativus genome for the modification, but among all, only eight primers produced precise and steady results. th e total number of bands was 26 for untreated control treatments and 70 for all treatments ranging from 258 to 1170 pb. one primer generated the same rapd profi les for the roots (figure 6). conversely, 8 rapd profi les demonstrated important alterations between untreated control and treated figure 3. c.sativus root tip cells treated with roundup concentrations a) control, b) stickiness, c) shift in the equatorial plane of metaphase, d) vacuolization, e) and f ) nuclei degeneration, g), h) and i) micronuclei formation. figure 4. genomic dna profi les of cucumber exposed to untreated control (c), 0.6% (0.6), 1.2% (1.2) and 2.4% treatments of roundup. table 4. th e list of polymorphic and monomorphic rapd primers compared to 0.6%,1.2% and 2.4% treatments of roundup. concentration(%) monomorphic primers polymorphic primers 0.6 opu-6 opc-5, opc-6, opu-7, opc-8, opc-9, opu-3 1.2 opu-6, opu-3 opc-5, opc-6, opu-7, opc-8, opc-9, opu-2 2.4 opu-6,opu-3 opc-5, opc-6, opu-7, opc-8, opc-9, opu-5 figure 5. the percentage of polymorphism for all primers in roundup treated cucumber. 8 burcu yuksel, ozlem aksoy, melis karatas roots (figure 7) with apparent alterations (disappearance and/or appearance) in the quantity and extent of amplified dna fragments for various primers. the modifications in rapd profiles were reported for treated c. sativus when compared with their controls (table 5). polymorphic bands were perceived at some of the treatments for 8 primers. polymorphisms were because of the appearance and disappearance of the amplified bands in the treated profiles when compared with control profiles. value of polymorphisms p (%) was observed at 26%. on the other hand, value of polymorphisms p (%) for roundup treatments: 0.6%, 1.2% and 2.4%; 41.%, 79% and 77%, respectively. the genomic template stability (gts, %) values, which is a qualitative tool that measures modifications in rapd profiles, was determined for each 8 primers and showed in table 6. gts values reduced at a significant amount in 0.6% roundup concentration. rapd profiles showed significant differences (loss of a normal band and / or formation of a new band) in the number and size of the replicated dna bands between the control and treated c. sativus roots. these changes, which were determined in the rapd profiles of the applied c. sativus roots, are given in table 4 with all the details. the maximum band increase was seen in 1.2% and 2.4% roundup applications of the opc 6 primer. the maximum band change in total was observed in 0.6% roundup application (table 6). figure 6. monomorphic rapd profile of cucumber exposed to untreated control (c), 0.6% (0.6), 1.2% (1.2) and 2.4% (2.4) treatments of roundup. rapd profiles were generated using primer opu-6. table 5. genomic template stability (gts, %) of c.sativus exposed to untreated control, 0.6%, 1.2% and 2.4% treatments of roundup. primers roundup concentration (%) control 0.6 1.2 2.4 opc-5 100 66 83 66 opc-6 100 0 0 0 opc-8 100 0 50 50 opc-9 100 66 100 100 opu-2 100 100 100 100 opu-3 100 0 100 100 opu-5 100 100 100 100 opu-7 100 0 100 100 average 100 41 79 77 table 6. the number of bands in control and molecular sizes (base pair, bp) of disappearance (-) and/or appearance (+) of dna bands for all primers in roundup treated cucumber (vision worksls image analyzer software). primers control roundup concentration (%) 0.6 1.2 2.4 opc-5 6 + 1223;694 1245 1245 596;314;254 0 0 opc-6 2 + 651;524 651;524;915;734 651;524;915;734 230 230 0 opc-8 2 + 618;430 625 600 477 477 477 opc-9 3 + 504 0 0 200 552;350;200 200 opu-2 3 + 0 0 0 0 237 0 opu-3 1 + 421;228 0 0 0 262 262 opu-5 4 + 0 0 0 0 537 537;795 opu-7 3 + 867;528;426 0 0 0 352 352 total 24 12(+) ; 6(-) 6(+) ; 9(-) 7(+) ; 6(-) 9the cytological and molecular investigation of the toxic effects of the herbicide roundup on cucumis sativus the distance values between the dendrogram and the treatment groups obtained by separate evaluation of the protein band profiles obtained by sds-page in the control group and roundup treated c. sativus roots were shown in figure 8. roots treated and untreated in dendrograms were composed of two main clusters. while 1.2% and 2.4% roundup applications were on the same branch, control and 0.6% roundup application were observed on the same branch. discussion the findings of this study demonstrated that the abnormalities were existent in stages of the mitosis in all treatments. the generation of mitotic abnormalities seems to be a usual impact of most chemicals (shehata et al. 2011). the stickiness and disturbed stages were the most commonly observed abnormalities. provided of chromosome loss are underdeveloped chromosome, stickness, multipolarity and c-mitosis. substances that cause fractures in chromosomes may cause chromosome figure 7. polymorphic rapd profiles of cucumber exposed to untreated control (c), 0.6% (0.6), 1.2% (1.2) and 2.4% (2.4) treatments of roundup. rapd profiles were generated using primer opc-5, opc-6, opc-8, opc-9, opu-2, opu-3, opu-5 and opu-7. figure 8. dendrogram obtained by separate evaluation of protein band profiles which were obtained by sds-page in control group and round up treated c. sativus roots. 10 burcu yuksel, ozlem aksoy, melis karatas bridge formation or changes in chromosome structure (yuksel 2017, radic et al. 2010) backward chromosomes; as a result of disturbances in the organization or functions of spindle yarns (turkoglu 2012; bonciu et al.2018). in our study, the first type of abnormalities was the stickiness discovered in most phases of mitosis following various roundup treatments. the amount of sticky cells rose up in all stages of mitotic division as the roundup concentration upsurged in the most of the treatments. moreover, this characteristic was augmented via the extending of interval time from 24 to 48h then reduced in the 10 days period interval in most treatments. the results of our study supported previous research, such as (aksoy et al. 2008; yuksel and aksoy 2017; bonciu 2018). previous studies stated that the chromosome stickiness might stem from breakage and swap between chromatin fibers over adjoining chromosomes. another form of abnormalities was the ill-formed, which was seen in metaphase and anaphase in the experiments, and the ratio of this characteristic did not depend on the roundup concentration or period interval. this abnormality was found in previous research, for example polit et al., 2003 , horak, et al., 2015 (soybean) following many chemical treatments they claimed that the chromosomes disturbed might stem from the impact of the chemical treatment on proteins forming the spindle apparatus. the difference in the ratio between the number of histone and other proteins can increase the adhesiveness of the nuclear chromatin, which ensures optimal organization, usually causing the development of atypical metaphases and anaphasis, chromosomal bridges in the anaphase and telophase, and finally, inhibition of cytokinesis and the formation of binuclear cells can be observed. (bonciu et al.2018). laggard chromosomes were also seen in some roundup treatments in metaphase anaphase and telophase (frescura et al. 2013;dimitrov et al. 2006). laggard at metaphase could be caused by the crash of the spindle apparatus to manage and operate in a standard way (haiba et al. 2011). lastly, the emergence of these chromosomal abnormalities could be attributed to the mutagenic potential of roundup. in another study, cytotoxic and genotoxic effects of cycloxidime and quizalofop-p-ethyl herbicides on allium cepa were investigated, and it was observed that decreased mitotic index and chromosomal abnormalities were increased. cycloxidime and quizalofop-p-ethyl concentrations increased, compared to the control group of cells with the chromosome stickiness, as the most common chromosomal aberration in the root tips of allium cepa, where herbicide was applied. (rosculete et al. 2018). previous studies also found that adhesive chromosomes reflect highly toxic effects and possibly lead to cell death (donghua et al. 1996). genotoxicity is among the major side effects of pesticide exposure (boumaza et al. 2016). with these results, we can conclude that roundup has a toxic effect and reveals a cell death process with increased chromosomal anomalies. long-term application of herbicides for control of harmful pathogens in agriculture can economically affect plants important to humans and endanger their genetic material. herbicides should be safe, healthy and effective. therefore, prior examination of the genotoxic, cytotoxic and biochemical impact of herbicides on plants and other systems is important for their application for agricultural uses. in this study, rapd was utilized to identify dna mutilation in the roots of c.sativus and the value of polymorphisms p (%) were increased with increasing roundup concentration. on the other hand, gts values decreased obviously in 0.6% roundup concentration. inhibition of shoot and root development and proliferation of hg, b, cr and zn elements in the roots and leaves of bean were detected following an upsurge in the concentration. the amount of polymorphisms p (%) was 50.4% and 28.0% for the roots and leaves, respectively following rapd analysis. to sum up, findings of this study reinforce the notion that the rapd analysis is a reliable technique for the discovery of dna damage caused by environmental pollutants such as toxic chemicals (cenkci et al. 2009). similarly, the study conducted by enan (2006) discovered that 22 novel fragments emerged and 43 disappeared due to utilizing 350 mg/l heavy metals to inundate phaseolus vulgaris. less band appearance/disappearance was used during the application of 150 mg/l. the disappearance of bands be related with to the existence of dna photoproducts (like pyrimidine dimmers, 6–4 photoproducts), which can be a facilitator of inhibition or reduction (bypass event)of the polymerization of dna in the pcr reactions (donahue et al. 1994; nelson et al. 1996). nonetheless, new fragments can be augmented since some sites open up to the primer following structural modifications in the dna (pietrasanata et al. 2000; enan, 2006). the reason of this process might be point mutations and/or large rearrangements of the dna. a single point mutation within the primer site can cause dramatic modifications in rapd patterns (williams et al. 1990). conclusion most of the cytotoxic and molecular focused studies examine the effects of environmental pollutants affecting plants. long-term use of pesticides in high amounts causes various problems in the cytological, biochemical 11the cytological and molecular investigation of the toxic effects of the herbicide roundup on cucumis sativus and genetic mechanisms of plants. 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2017. cytological effects of coumarin on the mitosis of lens culinaris medik.  fresenius environ. bull., 26:6400-6407. yuksel b. 2017. determınatıon of cytotoxıc, bıochemıcal, and genotoxıc effects of coumarın on lentıl (lens culınarıs medıc). doctoral desertation. kocaelı ünıversity. yuzbaşıoğlu d, unal f, sancak c. 2003.cytological effects of the herbicide racer “flurochloridone” on allium cepa. caryologia 56:97-105. caryologia. international journal of cytology, cytosystematics and cytogenetics 73(2): 51-61, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-891 citation: a. lima-de-faria (2020) comparison of the evolution of orchids with that of bats. caryologia 73(2): 51-61. doi: 10.13128/caryologia-891 received: february 12, 2020 accepted: april 16, 2020 published: july 31, 2020 copyright: © 2020 a. lima-de-faria. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. comparison of the evolution of orchids with that of bats antonio lima-de-faria professor emeritus of molecular cytogenetics, lund university, lund, sweden e-mail: johanessenmoller@icloud.com abstract. the evolution of orchids and bats is an example of dna’s own evolution which has resulted in structures and functions which are not necessarily related to any obvious advantage to the organism. the flowers of orchids resemble: humans, apes, lizards, frogs and even shoes. the faces of bats resemble plant leaves but also horseshoes. these similarities are not accidental because they emerge repeatedly in different genera and different families. this evolutionary situation bewildered botanists and zoologists for many years, but is now elucidated by the molecular unification of plants and animals derived from the following evidence: (1) contrary to expectation, plant and animal cells (including those of humans) could be fused and the human chromosomes were seen dividing in the plant cytoplasm. (2) orchids, bats and humans have about the same number of genes: orchids, 21,841; bats, 21,237 and humans circa 20,000. (3) these three groups contain the same homeotic genes which decide: flower formation (orchids), body segmentation (bats) and body segmentation (humans). the leaf pattern, is formed in plants by the leafy master gene, but this pattern even appears in minerals, which have no genes, an indication that pure atomic processes are responsible for its emergence at the organism level. keywords: orchids, bats, evolution, dna’s own evolution. evolution is a well established phenomenon but its mechanism remains to be elucidated evolution is one of the best established phenomena in biology. its firm basis rests mainly on the following data: (1) the comparison of structures and functions in invertebrates and vertebrates. (2) the documentation from the fossil record. (3) analysis of cells and chromosomes in most well known organisms. (4) sequencing of dna, in a long array of species, that has allowed to establish phylogenetic relationships at the molecular level. (5) other molecular studies that included the structures and functions of rna and proteins and their key interactions. however, this does not mean that the mechanism that is responsible for evolution is known. (1) a mechanism can only be physico-chemical, and we are only approaching this stage of investigation with the building of synchrotron radiation 52 antonio lima-de-faria accelerators and spallation sources as those built at lund university, sweden, and in other countries. (2) one is also far from understanding the source of the ramification into many branches of organisms which has led to the establishment of the different alleys that are called: phyla, orders, families, and other natural divisions. examples of this situation are: (a) the origin of vertebrates from invertebrates which remains far from being understood (daeschler and shubin 2011). (b) the emergence of birds from reptiles which is a source of permanent debate (zhou 2004). (c) the classification of flowering plants, with their recurring symmetries, which bewilders botanists (denffer et al. 1971). (d) the comparative work, based on the sequencing of dnas. this has led to the creation of databases but many species have not yet been included (fang et al. 2015). (3) the own evolution of dna, as well as that of proteins and rna, continue to be virgin land. as pointed out by branden and tooze (1991), as long as we do not know the rules of the interactions between these molecules at the atomic level, evolution of the chemistry of life will remain in a primitive stage. however, every important phenomenon in science, demands an explanation. the recourse, called the ”prevailing theory”, has been the use of random mutation and selection. geneticists know well that random mutation and selection occur in nature, but these are antiquated ”solutions” that have been superseded . selection is solely a system of choice and as such cannot substitute a physico-chemical mechanism. random mutations occur, but have been shown to be of little importance in evolution. directed mutations have now been well established as positive events in species transformations (zhang and saier 2009). similarity between plants and animals. — the impossible became possible 1) in the early days of genetics it became established that plant and animal chromosomes needed to have a centromere and telomeres if they were to survive during cell division. but plants were so different from animals that these basic similarities were not considered significant. 2) genes started to be located in great numbers in the chromosomes of drosophila, humans and maize. however, plants had no brain, and no blood circulation, as a consequence they had to have quite different genes. 3) when the first genes were isolated in the test tube, the ribosomal rna genes could be recognized in bacteria, plants and animals, not having changed appreciably for millions of years. haemoglobin, the carrier of oxygen in animal blood, was also present in plants. again this similarity of molecular organization was a curiosity. 4) the genes for 18s and 28s ribosomal rna were found in over 500 species to be located not at random, but tended to appear in plants, animals and humans, near telomeres. their position could be defined by an equation (lima-de-faria 1973). genes were considered to occur at random, as one still tends to think today, and the response was that this was a particular case. 5) suddenly, what was considered impossible, became possible. the fusion between plant cells and human cells was considered impossible. but it was achieved rapidly when the enzymes to remove the cell wall of plant cells became available. the experiments were controlled by the use of the radioisotope tritium and the human chromosomes were seen to divide in the plant cytoplasm. later the fusion of human sperm with plant cells could be observed occurring under the microscope (dudits et al. 1976, lima-de-faria et al. 1983). actually this work opened the way to present day biotechnology. 6) molecular analysis brought the crucial information. the genes that decided the segmentation of the body of insects, were the same that led to the formation of vertebra in the human column and those which decided the formation of floral parts (sepals, petals, stigma and anthers) in a plant. these are the homeotic or hox genes (lu et al. 1996). 7) this does not mean, however, that we are in possession of the molecular cascades that occur between the gene and the final formation of traits that shape the pattern of animals and plants. this is why the comparison of the evolution of the orchids with that of bats becomes relevant. the structures and functions of orchids exhibit a remarkable evolutionary variation the orchids (family orchidaceae) have confused botanists for three centuries due to the following features: the richness of orchid species the orchids display an extraordinary variation. they constitute approximately 10% of flowering plant spe53comparison of the evolution of orchids with that of bats cies (zhang et al. 2017) having about 28,000 currently accepted species, distributed in about 763 genera (christenhusz and byng 2016). the number of orchid species is nearly equal to the number of bony fishes, more than three times the number of bird species, and about four times the number of mammal species. the origin of orchids and the fossil record about 135 million years ago the plant kingdom began to develop vascular plants with enclosed seeds, the angiosperms, which spread rapidly (barth 1985). orchid fossils trapped in amber, in the baltic sea, are 15 to 20 million years old (poinar and rasmussen 2017). but genetic sequencing indicates that orchids may have arisen 76 to 84 million years ago or may go back to 100 million years ago (chase 2001). the fossil record from rocks is poor because orchids ”are herbaceous plants and therefore are not good subjects for fossilization”. as a result they are poorly documented in sedimentary deposits. besides, fossils are not considered reliable because of their resemblance to present-day orchids. this means that ”most extant groups are probably very young” (arditti 1992). the result is that: ”there is no general agreement regarding the time of the origin of the orchids” (arditti 1992). dressler (1993) asks: ”to what other group of plants are the orchids most closely related?” his answer is ”unfortunately, there is little agreement on the proper classification of these plants”. orchid flowers assume the most unexpected shapes resembling: humans, apes, bees, wasps and even shoes it is not only the great variation in flower shape that has confused researchers but, above all, is the display of patterns that have no immediate relationship to the environment or any obvious advantage to the organism (table 1, fig. 1). blamey et al. (2013) in their ”wild flowers of britain and ireland” give the common names of near 20 species of orchids. most of them have a resemblance to animals and to humans. these last are called ”manikins” (meaning a little man). they are: (1) manikin orchid, burnt-tip orchid (neotinia ustulata). (2) manikin orchid, lady orchid (orchis purpurea). (3) manikin orchid, military orchid (orchis militaris). (4) manikin orchid, monkey orchid (orchis simia). (5) manikin orchid, man orchid (orchis anthropophora). (6) lizard orchid (himantoglossum hircinum). (7) frog orchid (coeloglossum viride). (8) greater butterf ly orchid (platanthera chlorantha). (9) bee orchid (ophrys apifera). (10) wasp orchid (ophrys trollii). (11) fly orchid (ophrys insectifera). (12) late spider orchid (ophrys fucif lora). (13) ghost orchid (epipogium aphyllum). (14) lady’s slipper (cypripedium calceolus). (15) tongue orchid (serapias lingua) (fig. 4). several features are remarkable: (1) the patterns are not accidental because the same shape reappears in species which do not belong to the same genus (i.e. are not closely related). this is the case of the human figure in neotinia and orchis. (2) the resemblance displayed by the flowers is so perfect that it is included in the scientific name: monkey-face orchid, dracula simia (simia = monkey), orchis anthropophora (anthro = human), ophrys apifera (apis = bee), ophrys insectifera (fly), serapias lingua (lingua = tongue). (3) the pattern that exhibits these unexpected similarities, is not displayed by all the parts of the flower, but is usually restricted to the lip. this is the lower petal of the flower called also ”labellum”, another constraint in pattern development. (4) the common names, given to these species, were coined by leading botanists who, generation after generation, recognized the same similarities (table 1). the structures and functions of bats display also a remarkable evolutionary variation like systematists dealing with the classification of orchids, zoologists were confronted with great difficulties when analyzing the evolutionary features of bats. the large variation of bat species the bats build the order chiroptera which is divided into 21 families. these comprise not less than 1,400 species, an impressive number since it represents about 20% of the described mammalian species (fang et al. 2015). besides, they are present on every continent except antarctica (wilson and mittermeier 2019). according to hill and smith (1984) they constitute one of the largest and most widely distributed groups of mammals. the origin of bats and the fossil record “the origin and evolution of bats is poorly understood” (hill and smith 1984) and they add that ”any 54 antonio lima-de-faria table 1. orchid species in which the flowers are similar to animal structures and other unexpected shapes. common and scientific names according to blamey et al. 2013, “wild flowers of britain and ireland”. the words used and the statements made by the authors are in quotation marks. common name species name resemblance described by botanists common spotted orchid dactylorhiza fuchsii common orchid general pattern pyramidal orchid anacamptis pyramidalis foxy—smelling green-winged orchid anacamptis morio fragrant, purple dark green veins manikin orchid burnt-tip orchid neotinia ustulata ”manikin” is the name given to a little man. manikin lip manikin orchid lady orchid orchis purpurea manikin lip ”lip” is the lower petal of an orchid flower, also called ”labellum” manikin orchid military orchid orchis militaris sepals (the ”soldier’s” helmet) manikin orchid monkey orchid orchis simia manikin lip having narrow “limbs” as a human manikin orchid man orchid orchis anthropophora lip with very narrow ”limbs” lizard orchid himantoglossum hircinum ”fancifully lizard-like by taking the manikin theme to an extreme” frog orchid coeloglossum viride ”flowers supposedly like a jumping frog” greater butterfly orchid platanthera chlorantha two petals diverging at right angles bee orchid ophrys apifera ”look remarkably like the rear of a small bumblebee” wasp orchid ophrys trollii wasp looking flowers fly orchid ophrys insectifera manikin lip. ”petals antenna-like (hence the ”fly”)” late spider orchid ophrys fuciflora ”hieroglyphic on its lip” ghost orchid epipogium aphyllum excellent camouflage lip bent back lady’s slipper cypripedium calceolus billowing unspurred lip heart-flowered tongue orchid serapias cordigera ”middle lobe shaped like an ace-of spades (not hearts)” tongue orchid serapias lingua middle lobe intermediate between the other two species monkey-face orchid dracula simia central part of flower ”bears a striking resemblance to a monkey’s face” (thorogood 2018) figure 1. three different types of orchid flowers, which represent their great variation in pattern. the shape of the flower is not related to any obvious advantage to the organism. (a) orchis morio, green-winged orchid. an example of a flower with the general shape. (b) orchis militaris, manikin orchid or military orchid. in this species the flower’s ”lip” resembles the human body with: head, open arms and open legs. (c) cypripedilum acaule, lady’s slipper. another species in which the ”lip” resembles a shoe or a slipper. 55comparison of the evolution of orchids with that of bats scenario concerning the origin and early evolution of bats is clearly speculation”. the reasons are: 1) the fossil record is poorly represented. 2) the 30 fossil genera that have been identified are most similar to present living bats. 3) some of these fossils are recent, dating from the ice age. 4) the fossils are so well preserved that the stomach contents remain visible. 5) the fossil record extends to approximately 60 million years ago, but it is suspected that the bats may have had originated earlier 70-100 million years ago. the orchids are considered to have arisen at the same time. of special importance is that, as noted by hill and smith (1984) ”although primitive in some features, these bats possessed some characteristics that are as advanced as some of modern living species of microchiroptera” and ”all existing evidence suggests that bats changed relatively little compared to other mammals as a group”. teeling et al. (2018) add that ”the evolutionary history of bats has stimulated some of the most passionate debates in science”. the facial traits of bats are highly varied and resemble the most unexpected shapes including those of plants wilson and mittermeier (2019) give the common names of the over 20 families of bats. several names refer to the shape of the tail, others to their feeding habits but most deal with the facial pattern of bats. these are: (1) hog-nosed bats (nose like that of pigs). (2) trident bats (nose with the shape of a plant leaf with 3 projecting parts). (3) old world leaf-nosed bats (frontal part of face as a large leaf ). (4) horseshoe bats (face having a horseshoe-shaped plate). (5) bulldog bats (looking like table 2. bat families and their resemblance to plant and animal structures and functions. common and scientific names according to wilson and mittermeier (2019), “handbook of the mammals of the world” vol. 9. the words used and the statements made by the authors are in quotation marks. common name family name resemblance described by zoologists old world fruit bats pteropodidae standard bat face. lack of laryngeal echolocation mouse-tailed bats rhinopomatidae free long tail like in wild mice hog-nosed bats craseonycteridae nose as in pigs falsevampires megadermatidae canine teeth and large molars like other carnivore mammals. feed on mammals or reptiles. trident bats rhinonycteridae noseleaf with 3 prongs. a ”prong” is a pointed projected part old world leaf-nosed bats hipposideridae frontal part of face as a large leaf. like leaves found in many plant families horseshoe bats rhinolophidae ”ornate facial growths including horseshoe-shaped plate” sheathtailed bats emballonuridae refers to the juxtaposition of the tail with the membrane stretching between the legs. ”use territorial songs that include six different ”syllables”.” slit-faced bats nycteridae long narrow cut on face as a distinctive cleft running longitudinally along muzzle madagascar suckerfooted bats myzopodidae ”distinctive sucker-like structure on wrists and ankles” that stick to surface. like those found in tadpoles of frogs and some insect species.”ears with mushroom-like structure” new zealand short-tailed bats mystacinidae ”known as singing bats. echolocation calls are multiharmonical. can have up to four harmonics”. ”walk on the forest floor. the most terrestrial bats in the world” bulldog bats noctilionidae face like that of a race of dogs. ”distinct from that of any other species of bat” smoky and thumbless bat furipteridae muzzle with oval or triangular nostrils disk-winged bats thyropteridae have adhesive disks on their hindfeet ghostfaced bats mormoopidae frightening appearance. modified lips that form a funnel naked-backed bats mormoopidae like naked mole rats. heterocephalus mustached bats mormoopidae like ”mustached monkey”. cerco pithecus new world leaf-nosed bats phyllostomidae fleshy noseleaf above nostrils. plant leaf face like the situation found in the body of some insect species funnel-eared bats natalidae large ears like those of hares free-tailed bats molossidae tail separated from wings as in birds long-fingered bats miniopteridae finger mutations. like those found in humans wing-gland bats cistugidae unlike glands found in other mammals, but probably like sebaceous glands vesper bats vespertilionidae ”vesper”, means active in the evening. like other species of vertebrates such as vesper mouse and vesper finch 56 antonio lima-de-faria a race of dogs). (6) ghost-faced bats (with frightening appearance). (7) new world leaf-nosed bats (with fleshy noseleaf above nostrils, the leaf pattern being similar to that present on the body of some insect species). the leaf pattern has arisen in not less than three independent families: rhinonycteridae, hipposideridae and phyllostomidae. thus, it is not an accidental event. the nose takes not only the shape of different animals but even of a horseshoe (horseshoe bats). this is a most unexpected pattern, like that of an orchid which resembles a ladie’s slipper (table 2, fig. 2). significant is that the common names given to all species were not coined by the general public but by leading zoologists. besides, successive generations of scientists continued to use the same designation, a confirmation that the patterns displayed are so striking that their names were not modified. selection has been invoked and denied to explain orchid and bat evolution dressler (1993) uses several new types of selection, which are called r-selection and k-selection, to explain the evolution of the orchids. but he feels obliged to conclude that ”at first glance, the production of many tiny seeds would seem to fit the characteristics of r-selection, but in other respects, most orchids fit this pattern poorly” and he adds: ”the classification of the orchids has been difficult because of the great amount of parallelism”. by parallelism he means the repetition of the same pattern that is seen in: pollen structures, flower form, seed formation and pollination patterns (table 7). the great difficulty for evolutionists who follow the general interpretation is that for selection to have a positive effect it has to have an advantage for the individual. but such is far from being the case when a flower looks like a shoe or a bat has a face that resembles a horseshoe. “mimicry is bizarre” (dressler 1993). ”there are many cases of generalized food flower mimicry, that do not involve a clear and recognizable model”. ”in generalized food flower mimics, the pollinators soon learn that the flowers offer no reward”. ”orchids do not just deceive figure 2. three different types of facial structures of bats that represent their great variation in pattern. the shape of the nose is not related to any obvious advantage to the organism. (a) bat species (name not indicated). common facial pattern with protruding nose. (b) phyllostomus hastatus. face with shape of leaf. belongs to family phyllostomidae, new world leaf-nosed bats. this species is called spearnosed bat because the leaf has a sharp point on the upper part like the leaf of many deciduous trees (e.g. oaks, elms, mangolias and others). (c) rhinolophus ferrumequinum. called mediterranean horseshoe bat. the facial pattern which resembles a horseshoe, is so striking that is included in the scientific name (ferrum = iron, equinum = horse). table 3. number of protein-coding genes in animals and plants. organism species gene number reference animal pteropus alecto (bat) 21,237 fang, j. et al. 2015 homo sapiens 20,000 pennisi 2003 merchant et al. 2007 ascaris suum (worm) 18,500 jex et al. 2011 daphnia pulex (water flea) 30,907 colbourne et al. 2011 plant apostasia shenzhenica (orchid) 21,841 zhang et al. 2017 chlamydomonas reinhardtii (unicellular alga) 15,143 merchant et al. 2007 arabidopsis (flowering plant) 26,341 merchant et al. 2007 medicago truncatula (legume plant) 62,388 young et al. 2011 cajanus cajan (pigeon pea) 48,680 varshney et al. 2012 57comparison of the evolution of orchids with that of bats pollinators through sexual deception of animals, but also through mimicry of other plants” (stevens 2016) and adds: ”how this type of deception evolved is also unclear”. zoologists were led to a similar approach when analysing the value of selection in the evolution of bats. some invoked ”positive natural selection” and ”darwintable 4. evolutionary similarities between orchids and bats. property orchids bats origin eastern asia 40 to 80 million years ago. no general agreement regarding time and origin australasia 30 to 60 million years ago. no general agreement regarding time and origin fossil record fossils poorly documented in sedimentary rocks fossils found from various periods but limited fossil preservation leaves and seeds preserved but ”no positive or useful record” stomach contents well preserved as in extant species fossil appearance fossils are already very similar to living orchids. ”evolved fully formed” fossils are already very similar to modern living bats systematic location under debate, included in the order asparagales no intermediate forms to other mammalian orders. location most uncertain number of species 22,000 to 30,000 1,400 extreme variation tremendous radiation. flowers with most unexpected forms face with most different forms resemblance to particular structures assuming the shape of: ghost humans apes frogs lizards butterflies bees wasps flies spiders assuming the shape of: ghost mouse hog horse shoes bulldog leaves plant exhibiting animal pattern and animal exhibiting plant pattern resemblance of flowers to bees and wasps is so striking that insect males copulate with flowers face with leaf form which is characteristic of several tree families repeated occurence of plant-animal pattern similarity to insects occurs in: 3 species of ophrys; and similarity to humans occurs in: neotinia and 4 species of orchis similarity to leaves occurs in 3 distinct families: 1) old world leaf-nosed bats 2) new world leaf-nosed bats 3) trident bats table 5. occurrence of structures with leaf shape from minerals to bats. minerals flowering plants insects bats native copper native gold native bismuth the typical shape of leaves is most common in deciduous trees wings with leaf shape kallima (butterfly) phyllium pulchrifolium (grasshopper) frontal part of head with leaf shape. old world leaf-nosed bats 90 species. new world leaf-nosed bats 217 species. no genes present. atomic selfassembly homeotic genes deciding formation and position of flower parts. master gene leafy deciding leaf formation homeotic genes deciding body segmentation which affects body pattern homeotic genes deciding body segmentation, but effect on facial pattern not yet investigated 58 antonio lima-de-faria ian selection” (hawkins et al. 2019, dong et al. 2016), but others considered selection inappropriate to explain the evolution of bats (hill and smith 1984), teeling et al. 2018) (table 7). similarity of gene number, and of genes, between orchids and bats elucidate the emergence of identical patterns and the appearance of traits not advantageous to the organism from the beginning it was assumed that humans had to have at least 200,000 genes. as late as 2000 gilbert (2000) gave the figure 150,000 genes, based on the number of proteins present in the human body. this value sprang from the one gene — one protein relationship accepted in the 1970s. soon, it became evident, that a single gene could give rise to several different proteins and later genes turned out to be large complex structures consisting of coding and non-coding regions (exons and introns). the sequencing of the bases in dna led to a surprising answer. humans had about 32,000 genes coding for proteins (bork and copley 2001), but this figure has subsequently been reduced to circa 20,000 (table 3). as dnas continued to be sequenced, in many different organisms, it turned out that the number of genes is not a good indicator of evolutionary relationships and moreover it is not related to organism complexity (lima-de-faria 2014). the flowering plant arabidopsis has 26,341 genes. some plants have even more genes than humans. medicago is a legume plant with 62,388 and cajanus (a pea) 48,680 genes. their large numbers are due to genome duplications. even more relevant is that daphnia (a minute water flea) has 30,907 genes. it is thus not surprising that bats, orchids and humans have about the same gene numbers: 21, 237, 21,841 and circa 20,000 respectively (table 3). table 6. structures and functions with no obvious positive effect for the organism and those with a positive effect. orchids bats no obvious positive effect positive effect no obvious positive effect positive effect flower resembling: lady’s slippers monkeys humans frogs lizards movement of flower lips. enhancing of pollination by insect trapping. enhancing of pollination by resembling bees and wasps. face resembling: leaf horseshoe hog bulldog movement of larynx producing sounds. echolocation used in insect trapping table 7. present interpretations of orchid and bat evolution, evoking selection as well as denying it. the statements made by the various authors are in quotation marks. orchids bats interpretation reference interpretation reference selection deciding evolution. new kinds of selection: r-selection and k-selection related to habitat and environment stearns 1977 bat genes submitted to ”positive natural selection” hawkins et al. 2019 genomes submitted to ”darwinian selection” dong et al. 2016 ”selection pressure” as the motor of evolution arditti 1992 bat genes have undergone ”relaxed natural selection” dong et al. 2016 selection considered inappropriate to explain evolution of orchids. ”great deal of parallel evolution” dressler 1993 selection considered inappropriate to explain evolution of bats. ”evolution of bats is clearly speculation” hill and smith 1984 fossils are similar to modern orchids arditti 1992 ”fossils are already very similar to modern microbats” wikipedia fossil record is limited and reveals little about evolution arditti 1992 ”the evolutionary history of bats has stimulated some of the most passionate debates in science” teeling et al. 2018 59comparison of the evolution of orchids with that of bats in addition many basic genes are common to mammals and plants. but one could hardly conceive that the homeotic genes, which decide the segmentation of the vertebral column in humans, are the same that determine the sequence of the flower parts in plants (lu et al. 1996). the lip of orchids is one component in the process of flower formation (table 5). moreover, the leaf pattern found in orchids, like in other plants, is decided by a series of leaf genes that have been sequenced, the master gene being called leafy (glover 2007). hence, the similarity between the patterns of orchids and bats is not fortuitous, but has a genetic basis (tables 4 and 5). remarkable is that minerals, which have no genes, and whose pattern emerged before dna and the cell appeared in evolution, also build leaf patterns (fig. 3). one should not forget that dna consists of the same atoms that are found in minerals and that different atom combinations result in the same mineral pattern (lima-de-faria 2017) (table 5). in this connection it is relevant to recall that the basic function of proteins and other macromolecules resides, not on their amino acid sequences, but on their metal atoms. this is the case in: haemoglobin (iron), chlorophyll (magnesium), vitamin b12 (cobalt) and zinc proteins (zinc). it is the atoms that are exposed to other molecules that create the final pattern of the organism. figure 3. the leaf pattern which occurs in minerals, plants and insects. (1) mineral, pure bismuth in native state. (2) plant, leaf of poison ivy rhus toxicodendron. (3) the leaf-like butterfly kallima. (4) the leaf-insect chitoniscus feedjeanus showing leaf-like modifications of the fore-wings, including a midrib and lateral veins. figure 4. an insect copulates with a flower. orchid ophrys insectifera. (1) flowering plant. (2) male of the insect species gorytes mystaceous making copulatory movements over the flower. (3) female of the same species. (4) flower lip drawn separately to show similarity to insect. (5) the flower of the orchid ophrys bombyliflora covered by the copulating male of the insect eucera sp. 60 antonio lima-de-faria dna’s own evolution does not necessarily lead to the building of organs with advantage to the organism it is usually not realized that dna has its own evolution which results in the formation of traits that may be of advantage but may also be of no advantage to the organism. by manipulation of eye genes, in which dna sequences were moved within the genome, gehring (1998) was able to produce fruit flies with eyes located on: the head, legs and even wings. flies, which normally have only two wings were also produced with four wings. this work was further extended to birds leading to the creation of birds with four wings instead of two (cohn et al. 1997). in all cases the new organs were normal and functional, being constituted by the same body parts such as muscles, veins and articulations. hence, dna can produce, by alteration of its own sequences novel structures that the organism gets as a ”surprise”. the evolution of the orchids and of the bats is a valuable example of the production of structures without any special advantage to the organism. but this does not exclude that there are also structures and functions which led to a subsequent positive effect to the organism’s survival or reproduction (table 6). at present, botanists and zoologists, continue in vain to evoke, or deny, the role of selection in the evolution of orchids and bats. but the use of the large accelerators of electrons and neutrons is transforming molecular biology into atomic biology. consequently it will furnish a better picture of the basic evolutionary similarities that unite these organisms. source of figures fig. 1 (a) gola, g. et al. 1943. tratado de botanica. editorial labor, barcelona, spain (fig. 718, page 924). (b) strasburger, e. 1943. tratado de botanica. manuel marin editor, barcelona, spain (fig. 831, page 671). (c) lindman, c.a.m. 1926. bilder ur nordens flora (plate on page 419). fig. 2 (a) from lima-de-faria, a., 1995, ”biological periodicity”, fig. 3.5, page 18, source: perrins, c. 1976. bird life. an introduction to the world of birds. elsevier, phaidon, london, uk (fig. page 28). (b) cabrera, d.a. et al. 1935. historia natural, volume 1, zoologia. instituto gallach, barcelona, spain (fig. page 37). (c) cabrera, d.a. et al. 1935. historia natural, volume 1, zoologia. instituto gallach, barcelona, spain (fig. page 37). fig. 3 from lima-de-faria, a., 1988, ”evolution without selection”, fig. 3.8, page 27, sources: (1) medenbach, o. and sussieck-fornefeld, c. 1983. minerais (translation of: mineralien. mosaik verlag, munich 1982). ed. publica, lisbon, portugal. (2) feininger, a. 1956. the anatomy of nature. crown publishers, new york, usa. 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mona h. darwish gene flow patterns reinforce the ecological plasticity of tropidurus hispidus (squamata: tropiduridae) fernanda ito, danielle j. gama-maia, diego m. a. brito, rodrigo a. torres* the technique of plant dna barcoding: potential application in floriculture antonio giovino1,*, federico martinelli2,*, anna perrone3 cytogenetic of brachyura (decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species alessio iannucci1, stefano cannicci1,2,*, zhongyang lin3, karen wy yuen3, claudio ciofi1, roscoe stanyon1, sara fratini1 comparison of the evolution of orchids with that of bats antonio lima-de-faria identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection mehdi zahravi1,*, panthea vosough-mohebbi2, mehdi changizi3, shahab khaghani1, zahra-sadat shobbar4 populations genetic study of the medicinal species plantago afra l. (plantaginaceae) saeed mohsenzadeh*, masoud sheidai, fahimeh koohdar a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding timir baran jha1,*, partha sarathi saha2, sumita jha2 chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india harminder singh2, jaswant singh1,*, puneet kumar2, vijay kumar singhal1, bhupendra singh kholia2, lalit mohan tewari3 population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies raheleh tabaripour1,*, masoud sheidai1, seyed mehdi talebi2, zahra noormohammadi3 induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions girjesh kumar, shefali singh* karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(2): 3-13, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-750 citation: s.s. sobieh, m.h. darwish (2020) the first molecular identification of egyptian miocene petrified dicot woods (egyptians’ dream becomes a reality). caryologia 73(2): 3-13. doi: 10.13128/caryologia-750 received: december 1, 2019 accepted: march 13, 2020 published: july 31, 2020 copyright: © 2020 s.s. sobieh, m.h. darwish. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. the first molecular identification of egyptian miocene petrified dicot woods (egyptians’ dream becomes a reality) shaimaa s. sobieh*, mona h. darwish botany department, faculty of women for arts, science and education, ain shams university, cairo, egypt e-mail: shimaa.sobieh@women.asu.edu.eg; mona.darwish@women.asu.edu.eg *corresponding author abstract. this is the first work on egyptian ancient dna (adna) from plant fossil remains. two adna extracts from miocene petrified dicot woods were successfully obtained, amplified, sequenced and recorded for the first time in the world using a dna barcoding technique. internal transcribed spacers (its) barcoding is a technique for delimiting and identifying specimens using standardized dna regions. the two miocene dicot woods: bombacoxylon owenii (malvaceae/bombacoideae) and dalbergioxylon dicorynioides (leguminosae/papilionoideae) were collected from the wadi natrun area in egypt and were identified by palaeobotanists on the basis of wood anatomy. the molecular identification by its region of bombacoxylon owenii did not match the wood taxonomic assignation. the molecular identification of bombacoxylon owenii suggested that it is more related to the extant genus ceiba rather than to the extant genus bombax. in contrast, the molecular identification by its of dalbergioxylon dicorynioides matched the identification of the palaeobotanist (related to extant genus dalbergia). therefore, we suggest that this region should be used as a starting point to identify several plant fossil remains and this work will be helpful in solving problems related to the identification of plant fossils. keywords: egyptian petrified woods, adna, dna barcoding, its. introduction over the past twenty years, several ancient dna studies have been published, but none has targeted ancient egyptian dna. initial studies on ancient plant dna were published in the mid-eighties (golberg et al. 1991). rogers and bendich (1985) reported the extraction of nanogram amounts of dna from plant tissues ranging in age from 22000 to greater than 44600 years old. dna from fossils facilitates the calibration of mutation rates among related taxa (poinar et al. 1993). ancient dna (adna) is the most important and informative biological component that scientists can find in archaeological areas for identification purposes. ancient dna analysis is used synergistically with other identifi4 shaimaa s. sobieh, mona h. darwish cation methods, such as morphological and anatomical observations and microscopic analyses. dna barcoding complements the microscopic techniques used in archaeobotany. dna analysis can be solely used for the identification of specimens when the morphological and anatomical characteristics are absent (hamalton 2016). ancient dna may be used to reconstruct proximal histories of species and populations. studies involving the extraction, sequencing, and verification of fossil dna demonstrate the existence of material that can be useful to both palaeontologists and evolutionary geneticists. this opens the possibility for coordinated studies of macroand microevolutionary patterns that directly approach the relationship between morphological changes on the one hand and genetic changes on the other. in addition, molecular evolutionary studies attempt to reconstruct relationships between concurrent taxa by deducing ancestral states and the genetic distances between them (golenberg 1994). ancient wood is found in high abundance, and samples are usually large enough to be analysed. for that reason, wood is an ideal target for ancient plant dna studies (kim et al. 2004). however, three problems obstruct the isolation and amplification of dna from any adna specimens (nasab et al. 2010). the first is the presence of contamination. the second is the existence of inhibitors of taq dna polymerase in ancient samples, while the third is the small quantity and low quality of dna that is regained from dead wood (kaestle and horsburgh 2002) and this is due to degradation of dna into small fragments in dead tissue (deguilloux et al. 2002). nevertheless, there are several reports of molecular analyses of adna from plants. ancient dna was extracted from 1600 year-old millet (panicum miliaceum) by gyulai et al. (2006) and in 1993, adna was extracted from 600year-old maize cobs (goloubinoff et al. 1993). wagner et al. (2018) characterized the adna preserved in subfossil (nonpetrified) and archaeological waterlogged wood from the holocene age (550–9,800 years ago). dna barcoding is used to identify unknown samples, in terms of a pre-existing classification (tripathi et al. 2013) or to assess whether species should be combined or separated. it is also used to establish a shared community resource of dna sequences that can be used for organismal identification and taxonomic clarification (tripathi et al. 2013). the nuclear ribosomal internal transcribed spacer (its) region is indicated as a plant barcoding region (hollingsworth et al. 2011). miocene fossils are believed to be the best-preserved fossils of egypt (el-saadawi et al. 2014). these fossils are chemically well preserved because of the low oxygen content and cold temperatures of the water in which they were deposited (kim et al. 2004). dna sequences can be obtained from miocene-age plant remains and the success rate is increased through the use of improved methods of dna extraction and the amplification of small segments of the fossil dna (kim et al. 2004). el-saadawi et al. (2014) reported that egypt contains the second largest deposit of miocene dicot woods in africa (containing 23 taxa) after ethiopia that contains 55 taxa. seven petrified dicot woods were collected from the wadi natrun area in egypt by prof. wagih el-saadawi and prof. marwa kamal el-din (botany department, faculty of science, ain shams university). they identified only three of them, namely (bombacoxylon owenii (leguminosae/papilionoideae), dalbergioxylon dicorynioides (fabaceae/faboideae) and sapindoxylon stromeri (sapindaceae) based on the wood anatomy (elsaadawi et al. 2014). therefore, the main purpose of the present study was to extract and amplify adna from these egyptian miocene petrified dicot woods to provide a complete identification. dna was successfully isolated from the wood samples of bombacoxylon owenii and dalbergioxylon dicorynioides. we used molecular techniques to confirm the wood anatomy identification of the two egyptian wood fossils using dna barcoding method. in addition, we validated the relationship between the plant fossil woods and the nearest living relative (nlr) based on molecular data acquired from the its barcode. material and methods population sampling fossil samples seven of the good quality egyptian ancient miocene petrified dicot wood specimens (23.03  to  5.33  ma. years ago) were used to extract the adna. only two specimens (bombacoxylon owenii (bombacaceae) and dalbergioxylon dicorynioides (fabaceae) (fig. 1a, b) were successfully identified to the genus level by the analysis of the its of the nuclear ribosomal dna and the other five samples gave negative results. these miocene petrified dicot woods were found in the wadi natrun area in egypt and were previously identified by palaeobotanists (el-saadawi et al. 2014; kamal el-din et al. 2015) on the basis of the wood anatomy. the wood specimens were housed in the palaeobotanical collection of the botany department, faculty of science, ain shams university, cairo-egypt. 5the first molecular identification of egyptian miocene petrified dicot woods (egyptians’ dream becomes a reality) nearest living relative (nlr) samples liv ing wood tissue from bombax ceiba and dalbergia sissoo was used in the present study as the nlr samples of bombacoxylon owenii and dalbergioxylon dicorynioides, respectively. dna extraction, amplification, and sequencing dna extraction total genomic dna was extracted from the living woods and fossil wood using the cetyltrimethylammonium bromide method (ctab) described by doyle and doyle (1987). as the extraction of adna in fossils is more difficult than the extraction of dna from living wood several modifications were made. layers of fossil surfaces were scraped with a sterile scalpel and were discarded under sterile conditions in order to remove any contamination, and mechanical disruption was used during the dna extraction procedure. the original fossil samples were loose fragments scattered on the sand surface ranging between 10-50 cm in length and 5-20 cm in diameter (el-saadawi et al., 2014). they were very hard and difficult to break so they were cut by marble cutting machine into pieces and then those pieces were grinded mechanically into fine powder. the starting weight of the fossil sample was five times (5 g) higher than the living wood samples. three volumes more of extraction buffer than the protocol suggested were added. polyvinyl pyrrolidone was added to the lysis buffer. the quality of the dna was estimated by checking the absorbance ratio at 260/280 nm using a spectronic 21d spectrometer. the dna samples from both the living and fossil samples were stored at -20°c for amplification and sequencing. dna barcode the internal transcribed spacers its of the nuclear ribosomal dna was amplified using its4 and its5 primers with sequences of its4: tcc tcc gct tat tga tat gc and its5: gga agt aaa agt cgt aac aag g (white et al. 1990). this region consists of a portion of 18s rdna, its1, 5.8s rdna, its2, and a portion of 28s rdna (van nues et al. 1994). the pcr mixture was a 25 μl solution containing 0.5 μl of dntps (10 mm), 0.5 μl of mgcl2 (25 mm), 5 μl of 5× buffer, 1.25 μlof primer (10 pmol), 0.5 μl of template dna (50 ng μl–1), 0.1 μl of taq polymerase (5 u μl–1) and 17.15 μl of sterile ddh2o. the amplification was carried out in a techni tc-312 pcr, stafford, uk system. the pcr cycles were programmed for the denaturation process for 4 min at 95°c (one cycle), followed by 30 cycles as follows: 94°c for 1 min; 53°c for 40 s;72°c for 1 min and finally one cycles extension of 72°c for 10 min and 4°c(infinite). the pcr products were run on 1.5% agarose gels, which were stained with ethidium bromide, at 120 v for 1 h. successful pcr products were sent to lgc genomics sequencing (germany) to be sequenced on a 3730xl dna analyzer (applied biosystemstm/ thermo fisher scientific). data analysis the sequence identity was determined using the blastn algorithm available through the national center for biotechnology information (ncbi) https://www. ncbi.nlm.nih.gov. the consensus sequences that showed a significant match with the earlier identified data in the ncbi were submitted to the barcode of life data system (bold) v4 http://www.barcodinglife.org to identify each sequence sample to the genus and species level. the new fossil sequences were submitted to the ncbi to be listed and recorded in the genbank database. the g+c content of the four samples were calcufig. 1. sections of bombacoxylon owenii (a) and dalbergioxylon dicorynioides (b). (a) (b) 6 shaimaa s. sobieh, mona h. darwish lated online using the cg content calculator website https://www.biologicscorp.com/tools/gccontent#.wrsk5ohubiu. the multiple dna sequences alignments (msa) were performed using t he molecu lar evolutionary genetics analysis version 6 (mega 6) (tamura et al. 2013), while double sequence alignment using the clustal w algorithm was performed according thompson et al. (1994). the genetic distances were computed using mega 6.06 according to the kimura-2-parameter (k2p) model (kimura 1980). phylogenetic reconstruction the aligned dna sequences by the clustal w algorithm of mega 6 were trimmed online using the trimming website: http://users-birc.au.dk/biopv/ php/fabox/alignment_trimmer.php. the final aligned sequences were used to construct the phylogenetic trees. sixteen species with their accession numbers (table 1) were used to construct the phylogenetic tree for cf. ceiba sp., and 36 species with their accession numbers (table 2) were used to construct the phylogenetic tree for cf. dalbergia sp. moreover, the sequences of persea pseudocarolinensis (accession number. ay337335) and persea palustris (accession number. ay3377330) from genbank, were chosen as outgroup to root the trees. the maximum likelihood (ml) analysis was applied to construct the phylogenetic trees. the ml analysis was constructed in mega 6 using the k2p model, with 1,000 bootstrap replicates. the codon positions were combined as 1st+2nd+3rd+noncoding. all positions containing gaps and missing data were eliminated. the tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to assume the phylogenetic tree. table 1. the eighteen species used for constructing the phylogenetic tree for cf. ceiba sp. with their accession numbers. accession number corresponding species mg603734 cf. ceiba sp. km453172 ceiba ventricosa km453167 ceiba erianthos km453170 ceiba pubiflora hq658387 ceiba crispiflora km453171 ceiba rubriflora hq658388 ceiba speciosa km488629 ceiba insignis km453168 ceiba jasminodora dq284851 ceiba pentandra hq658389 ceiba schottii hq658384 ceiba aesculifolia hq658385 ceiba acuminata hq658376 bombax buonopozens km453163 bombax ceiba dq826447 bombax malabaricum ay337335 persea pseudocarolinensis ay3377330 persea palustris table 2. the thirty-eight species used for constructing the phylogenetic tree for cf. dalbergia sp. with their accession numbers. accession number corresponding species mg450751 cf. dalbergia sp. km521409 dalbergia sissoo kp092712 dalbergia balansae km521377 dalbergia odorifera ab828610 dalbergia assamica  km521378 dalbergia hupeana km521413 dalbergia stipulacea ab828616 dalbergia bintuluensis ab828639 dalbergia hostilis km521372 dalbergia dyeriana ab828619 dalbergia bracteolata af068140 dalbergia congestiflora ab828632 dalbergia frutescens ab828633 dalbergia glomerata ab828649 dalbergia melanocardium km276143 dalbergia melanoxylon km276125 dalbergia latifolia ab828614 dalbergia benthamii ab828622 dalbergia canescens ab828608 dalbergia arbutifolia ab828626 dalbergia cultrate ab828605 dalbergia acariiantha ab828618 dalbergia bojeri ab828613 dalbergia baronii ab828640 dalbergia humbertii ab828635 dalbergia greveana ab828604 dalbergia abrahamii km521415 dalbergia trichocarpa ab828648 dalbergia martini fr854138 dalbergia tonkinensis ab828653 dalbergia parviflora hg313773 dalbergia entadoides km521404 dalbergia rimosa hg004883 dalbergia cf. kingiana hg313775 dalbergia dialoides km521414 dalbergia subcymosa ay337335 persea pseudocarolinensis ay3377330 persea palustris 7the first molecular identification of egyptian miocene petrified dicot woods (egyptians’ dream becomes a reality) results and discussion dna isolation as far as is known, this is the first time that dna from ancient egyptian wood samples was extracted. the absorbance ratios (a260/280 nm) of the dna extracts ranged between 1.811.94 (table 3), indicating good quality of the dna from both fossil and living specimens. the concentrations of the dna extracts were 175,285, 375 and 470 ng/ μl for dalbergioxylon dicorynioides, bombacoxylon owenii, bombax ceiba and dalbergia sissoo, respectively, as given in table 3. at the present time, publications of adna from plant fossils are still relatively infrequent; however, there are many adna publications from animals and humans which make up most samples in this field (gugerli et al. 2005). helentjaris (1988) indicated that plant material from archaeological sites may also be amenable to dna analysis. many researchers have explored the possibility of isolating dna from ancient wood samples. dna has been extracted from samples of modern papyri (writing sheets made with strips from the stem of cyperus papyrus) varying in age from 0-100 years bp and from ancient specimens from egypt, with an age-span from 1,300-3,200 years bp. the results showed that the dna half-life in papyri is approximately 19-24 years. this means that the last dna fragments will vanish within no more than 532-672 years from the sheets being manufactured (marota et al. 2002). in the case of ancient wood, the risk of contamination during handling and analysis is lower than with human or microbial dna (gilbert et al. 2005). earlier works on fresh wood by asif and cannon (2005), deguilloux et al. (2006) and studies of adna from ancient wood from quercus and cryptomeria by deguilloux et al. (2002) suggested the possibility of dna survival in ancient wood remains, which was confirmed by the current work. liepelt et al. (2006) reported that, it was possible to isolate dna from wood as old as 1000 years. depending on the mode of conservation and the climate at the excavation site, as well older samples could be isolated and analysed successfully (deguilloux et al. 2006). dna barcoding by its the dna barcoding affords an important step for the molecular identification of adna from petrified woods. the amplification of genomic dna uses the universal primers for the its region. two of seven adna extracts from the dicot wood fossil samples (bombacoxylon owenii and dalbergioxylon dicorynioides) were successfully used to amplify the its region. the pcr and sequencing success rates for the fossil and living samples were 100% (table 4). the genus and species identity results of the query sequences were then determined using the blast and bold databases to estimate the reliability of the genus identification. the results of both databases showed that its was 100% correctly identified at the genus level, while the success rates for species identification were 50 and 25% for blast and bold respectively (table 5). many studies have compared the discriminatory power revealed by the its region in its entirety with table 3. optical densities and concentrations of the dna isolated from fossil and living specimens. plant name optical density ratio 260/280 nm dna concentration (ng/µl)260 nm 280 nm bombacoxylon owenii 0.057 0.032 1.84 285 bombax ceiba 0.075 0.041 1.82 375 dalbergioxylon dicorynioides 0.035 0.018 1.94 175 dalbergia sissoo 0.094 0.052 1.81 470 table 4. success rates of the amplification and sequencing. barcode locus number of tested samples (fossil and living samples) no of samples amplified and percentage of pcr success number and percentage of pcr failure number and percentage of sequencing success its 4 4 (100%) 0 (0%) 4 (100%) 8 shaimaa s. sobieh, mona h. darwish its2, proposing the use of its2 as an alternative barcode to the entire its region (han et al 2013). its2 was previously used as a standard dna barcode to identify medicinal plants by chen et al. (2010) and a barcode to identify animals (li et al 2010). the length of the its2 region is sufficiently short to allow for the easy amplification of even degraded dna, and the its2 region has enough variability to distinguish even closely related species and has conserved regions for designing universal primers (yao et al. 2010). therefore, it could be used as a dna barcode for plant fossils in further investigations. in addition, all 4 raw nucleotide sequences were verified with the other available sequences in genbank using the blastn algorithm. the sequences of the two living samples of bombax ceiba and dalbergia sissoo showed an identity ratio of 99% with bombax ceiba (accession no. km453163) and dalbergia sissoo (accession no. ab828659), respectively (table 6). the identification of the fossil samples: based on the author’s knowledge, thus far, there has been no published work on adna from petrified wood. therefore, this is considered the first molecular identification of egyptian plant fossil remains and of petrified wood (bombacoxylon owenii and dalbergioxylon dicorynioides) worldwide. meanwhile, the authors hope that many other fields (anatomy and morpholog y) besides the molecular field will contribute to determining the relationship between living plants and their fossil remains. bombacoxylon owenii (cf. ceiba sp. accession no.: mg603734) the its sequence from the fossil specimen was amplif ied and produced a 704 bp fragment. the sequence was uploaded to the ncbi database and was documented, for the first time with accession number mg603734. bombacoxylon owenii was listed in the ncbi database as cf. ceiba sp. because the genbank policy is not to add fossil taxa to the taxonomy database, since it is a database of living or recently extinct organisms. bombacoxylon is a fossil genus for woods with features characteristic of the bombacoideae, not a whole plant. moreover, the molecular identification revealed a close resemblance of the submitted sequence to ceiba pentandra (the commercial kapok tree) rather than bombax as was expected by kamal el-din et al. (2015). this identification is not surprising since the two living genera (bombax and ceiba) are grouped in the same subfamily bombacoideae and have very few differences between them. moreover, the wood anatomy of both genera reveals the high resemblance between them, and they can be only distinguished by a combination of macroscopic characteristics, which are the shape of the vessel-ray pit, the ray width, the sheath cells and mineral inclusion (nordahlia et al., 2016). the nlr of some fossil wood taxa might be wrong, bombacoxylon shares characters with sterculiaceae and bombacaceae rather than only with bombax, grewioxylon with other members of the malvaceae with tile cells, (e.g., craigia) instead of only grewia (skala 2007). in addition, wickens (2008) stated that it must table 5. identification efficiency of the barcode loci using blast and bold. barcode locus no. of samples identified family level using blast family level using bold genus level using blast genus level using bold species level using blast species level using bold its  4 100% 100% 100% 100% 50% 25% table 6. identification matches of the its sequences using the blast and bold databases. sample identification plant order plant family plant subfamily blast search match blast similarity (%) bold search match bold similarity (%) cf. ceiba sp. (bombacoxylon owenii) malvales malvaceae bombacoideae cf. ceiba sp. 100 ceiba pantandra 90.83 bombax ceiba malvales malvaceae bombacoideae bombax ceiba 99 bombax malabaricum 99.14 cf. dalbergia sp. (dalbergioxylon dicorynioides) fabales fabaceae papilionoideae cf. dalbergia sp. 100 dalbergia odorifera 87.94 dalbergia sissoo fabales fabaceae papilionoideae dalbergia sissoo 99 dalbergia sissoo 98.57 9the first molecular identification of egyptian miocene petrified dicot woods (egyptians’ dream becomes a reality) not be assumed that the names of fossil wood necessarily represent species close to modern genera. the sequence of cf. ceiba sp. was compared with other available sequences in genbank using the blastn algorithm. the results showed that the sequences belonged to the homologous sequences of the genus ceiba. the sequence of cf. ceiba sp. showed identities with several living ceiba species rather than bombax. the identity ratios among the ceiba species indicated that the ceiba pentandra its nucleotide sequence (accession no. dq284851) was the nearest related its sequence for bombacoxylon owenii (cf. ceiba sp.). the sequence of cf. ceiba sp. was aligned with both bombax ceiba (accession no. km453163) and ceiba pentandra (accession no. dq284851) using clustal w (thompson et al. 1994). the identity between the cf. ceiba sp. its sequence and that of ceiba pentandra was 625 (80.23%) (fig. 2a), while the identity between the cf. ceiba sp. its sequence and that of bombax ceiba was 548 (76.54%) (fig. 2b). the final aligned sequences obtained by sequence trimming revealed that g+c content was obviously higher than of a+t content (table 7). genetic distances were calculated by the kimura-2-parameter (k2p) model (kimura 1980). moreover, both bombacoxylon owenii (cf. ceiba sp.) and ceiba pentandra shared similarities in the wood anatomy characteristics, with the presence of diffuse to semiring porous wood in both of them. bombacoxylon owenii (cf. ceiba sp.) and ceiba pentandra contain solitary vessels and have radial multiples of 2 to 4 and medium to large vessels that are often filled with tyloses. the growth rings in both are distinct or absent and the vessel frequency is 5 to 20 per mm2. the perforation plates are simple, and the intervessel pits are alternate. the vessel-ray parenchyma pits are like the intervessel pits and the fibres are nonseptate with thick-walls and diffuse to diffuse-in-aggregate axial parenchyma (table 8) (inside wood 2013; kamal el-din et al. 2015; nordahlia et al. 2016). dalbergioxylon dicorynioides (cf. dalbergia sp.accession no.: mg450751) the its sequence (610 bp) was amplified and recorded in the ncbi database with genbank accession no. mg450751. dalbergioxylon dicorynioides was recorded as cf. dalbergia sp. in the ncbi, since it is  a database of living or recently extinct organisms. dalbergioxylon dicorynioides is a fossil genus for woods not a whole plant. fig. 2. (a) sequence alignment between cf. ceiba sp. and ceiba pentandra (accession no. dq284851) using clustal w, identity (*): 625 is 80.23 %. (b) sequence alignment between cf. ceiba sp. and bombax ceiba (accession no. km453163 ) using clustal w, identity (*): 548 is 76.54 %. (a) (b) 10 shaimaa s. sobieh, mona h. darwish the total sequence length of its in the dalbergia genus ranged from 600 to 800 bp as reported by several records in the ncbi database for its in the dalbergia genus. the sequence was tested with other available sequences in genbank using the blastn algorithm. the results showed that the sequences belonged to the homologous sequences of the genus dalbergia. the sequence of cf. dalbergia sp. showed identities with several living dalbergia species, but when we compared the identity ratios among them we found that the dalbergia sissoo its nucleotide sequences (accession no. ab828659.1) were the nearest its sequence for dalbergioxylon dicorynioides (cf. dalbergia sp.), with an identity ratio of 91%. the final aligned sequences obtained by sequence trimming revealed that the g+c content was obviously higher than the a+t content (table 7). genetic distances for dalbergia sequences alignment were calculated by the kimura-2-parameter (k2p) model (kimura 1980). the comparison of the wood anatomy characteristics of dalbergioxylon dicorynioides (cf. dalbergia sp.) with those of living dalbergia species revealed that dalbergia sissoo was most closely related to dalbergioxylon dicorynioides (table 9) because both contained diffuseporous wood, solitary vessels and radial multiples of 2 to 3, indistinct or absent growth rings, exclusively simple perforation plates, alternate and vestured intervessel pits, vessel-ray pits similar to intervessel pits in size and shape throughout the ray cell, combinations of aliform, confluent and irregular banded (1 to 4 cells wide) axial parenchyma, 1-3 seriate rays up to 20 cells high, and thick-walled non-septate fibers (inside wood 2013; elsaadawi et al. 2014). phylogenetic analysis the phylogenetic ana lyses were conducted in mega6 (thompson et al. 1994) and the phylogenetic trees were inferred with the ml based on the kimura model (kimura 1980). nowadays, several programs can be used to construct maximum likelihood phylogenetic tree. the fastest ml-based phylogenetic programs that differ in implementations of rearrangement algorithms are phyml (guindon et al. 2010) and raxml/examl (stamatakis 2014). the topologies of the phylogenetic trees were evaluated using the bootstrap resampling method of felsenstein (1985) with 1000 replicates. the analysis involved table 7. sequence length and gc and at content. sample name full length g+c g+c% a+t a+t% cf. ceiba sp. 704 221+219 62% 134+130 38% bombax ceiba 692 231+234 66% 113+114 34% cf. dalbergia sp. 610 185+194 61% 135+96 39% dalbergia sissoo 610 189+211 64% 127+83 36% table 8. comparison of anatomical features between bombacoxylon owenii & ceiba pentandra. species feature bombacoxylon owenii ceiba pentandra (l.) growth ring distinct distinct, indistinct or absent porosity diffuse to semiring-porous diffuse-porous perforation plates simple simple intervessel pits alternate alternate radial diameter 240 μm (220 to260) 350 to 800 µm vessels groupings solitary and in radial multiples of 2 to 4  restricted to marginal rows tyloses common  common vessel/mm2 5 to 15(8) 5 to 20 vessel element length μm 335 μm 350 to 800 µm axial parenchyma diffuse, diffuse-in-aggregates, scanty, narrow vasicentric paratracheal and in narrow bands or lines diffuse, diffuse-in-aggregates, scanty, narrow vasicentric paratracheal and in narrow bands or lines rays 1 to 3 cells, seriate larger rays commonly 4 to 10 seriate fibers nonseptate with very thick walls nonseptate with thinto thick-walled 11the first molecular identification of egyptian miocene petrified dicot woods (egyptians’ dream becomes a reality) 18 nucleotide sequences (cf. ceiba sp., 12 species of ceiba and 3 species of bombax which were downloaded from the ncbi database), and persea pseudocarolinensis and persea palustris  were used as outgroups. there was a total of 1374 positions in the final dataset, and the ambiguous positions were completely eliminated for each sequence pair. the ml tree was divided into two clades, namely a and b. clade a included bombax members, while clade b included the ceiba species in addition to cf. ceiba sp. (bombacoxylon owenii). both cf. ceiba sp. and ceiba pentandra were on the same branch. therefore, the phylogenetic tree showed that bombacoxylon owenii (cf. ceiba sp.) was very similar to the ceiba genus, which previously was thought to resemble the bombax genus (kamal el-din et al. 2015) (fig. 3). in the ml tree, all the dalbergia species were divided into two clades, namely clade a and clade b (fig. 4). clade a includes cf. dalbergia sp. and dalbergia sissoo. the second group (clade b) was subdivided into many subclades that contained the other species of dalbergia. therefore, the present work matches the palaeobotanist table 9. comparison of anatomical features between dalbergioxylon dicorynioides & dalbergia sissoo. species feature dalbergioxlon dicorynioides dalbergia sissoo growth ring absent. distinct, indistinct or absent porosity diffuseporous diffuseporous perforation plates simple simple intervessel pits alternate alternate tangential diameter μm 170 μm (range 100 to 210 μm) 100 to 200 vessels groupings solitary and in radial multiples 2 to 3 solitary or grouped in radial multiples of 2 to3 cells. vessel groupings /mm2 8/mm2 (range 5 to13/ mm2) 5 to 20  vessel element length μm 330 μm (range 280 to 410 μm) <= 350 axial parenchyma aliform, confluent and irregular banded (1 to 4 celled wide) aliform, confluent and irregular banded, 4 (3to 4) cells per parenchyma strand rays 1to 3 seriate 1 to 3 cells fibers thick-walled, nonseptate very thick-walled, nonseptate  fig. 3. maximlikelihood (ml) cladogram showing the relationships of the its gene from cf. ceiba sp. in relation to its relatives. all analyses were performed with 1000 bootstrap replicates (arrow: fossil specimens, acc. no.: accession number). fig. 4. maximlikelihood (ml) cladogram showing the relationships of the its gene from cf. dalbergia sp. in relation to its relatives. all analyses were performed with 1000 bootstrap replicates (arrow: fossil specimens, acc. no.: accession number). 12 shaimaa s. sobieh, mona h. darwish assumption that there is a close relationship between dalbergioxylon dicorynioides (cf. dalbergia sp.) and dalbergia sissoo. conclusion the dna barcoding dataset in the present study provides an important first step towards establishing an effective molecular tool for the identification of adna from petrified woods. we hope that these results will encourage reliable adna studies of other petrified woods. the further studies of ancient wood dna from the abundant store of fossil plant remains will rely on this study and by the intensive works of researchers from different fields, and these findings could provide a powerful tool to increase world knowledge about the history of forests, plant evolution and historical biogeography. author contributions both authors suggested the point of the work and dr. shaimaa s. sobieh planned the experimental design to achieve this point. both authors supplied the financial support for the work. prof. mona darwish shared other palaeobotanists in the identification of dicot woods (see el-saadawi et al. 2014). the experimental part was done by dr/shaimaa s. sobieh. the writing of the manuscript was done by both authors. acknowledgements the authors would like to thank prof. wagih elsaadawi and prof. marwa kamal el-din (profs. of palaeobotany, botany department, faculty of science, ain shams university) for supplying them the fossil specimens. in addition, they thank fatma abdel naby mursi and aya abdel gawad (msc. students, botany department, faculty of women for art, science and education, ain shams university) for helping them in the extraction of adna. finally, the authors would like to thank dr enas hamdy ghallab (lecturer of medical entomology, entomology department, faculty of science, ain shams university) and mohamed emad el-din elsaid (biotechnology bachelors, misr university for science and technology) for helping the authors in understand many points in the bioinformatics programs. references asif 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2010. use of irs2 region as the universal dna barcode for plants and animals. plos one 5: e13102. doi. org/10.1371/journal.pone.0013102 caryologia international journal of cytology, cytosystematics and cytogenetics volume 73, issue 2 2020 firenze university press the first molecular identification of egyptian miocene petrified dicot woods (egyptians’ dream becomes a reality) shaimaa s. sobieh*, mona h. darwish gene flow patterns reinforce the ecological plasticity of tropidurus hispidus (squamata: tropiduridae) fernanda ito, danielle j. gama-maia, diego m. a. brito, rodrigo a. torres* the technique of plant dna barcoding: potential application in floriculture antonio giovino1,*, federico martinelli2,*, anna perrone3 cytogenetic of brachyura (decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species alessio iannucci1, stefano cannicci1,2,*, zhongyang lin3, karen wy yuen3, claudio ciofi1, roscoe stanyon1, sara fratini1 comparison of the evolution of orchids with that of bats antonio lima-de-faria identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection mehdi zahravi1,*, panthea vosough-mohebbi2, mehdi changizi3, shahab khaghani1, zahra-sadat shobbar4 populations genetic study of the medicinal species plantago afra l. (plantaginaceae) saeed mohsenzadeh*, masoud sheidai, fahimeh koohdar a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding timir baran jha1,*, partha sarathi saha2, sumita jha2 chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india harminder singh2, jaswant singh1,*, puneet kumar2, vijay kumar singhal1, bhupendra singh kholia2, lalit mohan tewari3 population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies raheleh tabaripour1,*, masoud sheidai1, seyed mehdi talebi2, zahra noormohammadi3 induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions girjesh kumar, shefali singh* karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(2): 111-119, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-544 citation: g. kumar, s. singh (2020) induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions. caryologia 73(2): 111-119. doi: 10.13128/caryologia-544 received: july 14, 2019 accepted: april 13, 2020 published: july 31, 2020 copyright: © 2020 g. kumar, s. singh. this is an open access, peerreviewed article published by firenze university press (http://www.fupress. com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions girjesh kumar, shefali singh* plant genetics laboratory, department of botany, university of allahabad, india *corresponding author. e-mail: shefalisingh.910@gmail.com abstract. cytomictic behaviour of chromosomes among pollen mother cells was observed in mutagenic studies in cluster bean (cyamopsis tetragonoloba (l.) taub.). the study of pollen mother cells (pmc) revealed various chromosomal aberrations among which cytomixis was notified due to its obtrusive peculiarity and is therefore given description in this article. cytoplasmic and chromatin transmigration were discernible among contiguous or slightly distant pmcs through recreation of passage via direct cell-to-cell fusion or channel formation. this cytomictic phenomenon was invariably more pronounced at meiosis i as compared to meiosis ii. plasmodesmatal connections play a paramount role in aiding this behaviour by establishing intercellular crosstalks. the cellular intermingling resulted in syncyte cells which were identified due to their doubled size. syncyte or unreduced pmc formation leading to unreduced fertile gametes is speculated to act as a possible way out for infraspecific polyploidization of species. pollen fertility was computed, alongwith this heterosized pollens of varying diameter were segregated. large sized pollens were 2n pollens; where size difference is a consequence of cytomixis. cytoplasmic connections among pollens were also observed sporadically. it is opined that syncyte formation and 2n pollen production have evolutionary significance. keywords: cyamopsis tetragonoloba, cytomixis, heterosized pollens, infraspecific polypoidy, pmcs, syncyte. introduction cytomixis is promiscuous intercellular interaction for exchange of nuclear material, dividing chromosomal bodies and other integral cytoplasmic organelles. the credit for first description of this phenomenon was conferred to arnoldy (1900) in gymnosperms. later on, the behaviour was also explored in pmcs of crocus sativus by koernicke (1901); however, the term ‘cytomixis’ was christened by gates (1911) during his findings in oenothera gigas and oenothera biennis. besides reproductive cells, cytomixis has also been witnessed in root meristematic cells (jacob 1941), tapetal cells (cooper 1952), shoot apex 112 girjesh kumar, shefali singh (guzicka and wozny 2004) and other diverse somatic cell systems. in angiosperms, it is invariably more frequent in family poaceae (reported in 82 species) and fabaceae (reported in 48 species) (mursalimov et al. 2013a) occurrence of cytomixis is speculated to be of pathological nature and is frequently documented in species with unbalanced genomes such as haploids, aneuploids, hybrids (de nettancourt and grant 1964), mutants (gottschalk 1970), triploids (salesses 1970). there are also few instances where cytomixis was more profound among polyploids than their diploid counterparts (semyarkhina and kuptsou 1974); where it is perceived to allow elimination of extra dna in order to stabilize the genome and produce balanced and/or reduced pollen grains (zhou 2003). the phenomenon has also been documented in pmcs of transgenic tobacco plants (sidorchuk et al. 2007). a unique pattern of b-chromosome pioneered cytomixis was observed in b-carrier plant poppy, where b-chromosome was the first entrants in the recipient cell and a-chromosomes followed them (patra et al. 1988), for which it was argued that heterochroatin blocks of b-chromosomes played a facilitating role for cytomixis. cautious contemplation has revealed that cytomixis is a sort of cell selection, which selects and preserves fitting variants but eliminates unbalanced and irreparable pmcs (kravets 2013). there is a difference of opinion regarding its significance; however general consensus by authors configures an evolutionary trail (boldrini et al. 2006; li et al. 2009). according to cheng et al. (1980), cytomixis acts as an additional facilitator in phylogenetic evolution of karyotypes by reducing or increasing the basic series. however guan et al. (2012) opined contrary views by asserting its deleterious effects on fertility while veilleux (1985) accredited cytomixis to be a potential means to conserve genetic heterozygosity of gametes. plethora of study recruited on cytomictic behaviour suggests that the phenomenon is a resulting event regulated by genetic and environmental factors rather than being due to fortuitous causes such as artifact produced by fixation, mechanical injuries or pathological anomaly (gottschalk 1970; song and li 2009). factors such as partial or total inhibition of cytokinesis during microsporogenesis (risuen˜o et al. 1969), effect of gamma radiation (kumar and yadav 2012; dwivedi and kumar 2018), action of chemical agents such as colchicine (gautam and kumar 2013), are reported to repercuss into cytomixis. several environmental constraints such as thermal stress (sidorchuk et al. 2016), cold harsh conditions also intrigue inter-meiocyte fusion and hence syncyte formation (singhal et al. 2011). depending upon the intensity and severity, cytomixis is categorized into three main types: weak (local), intensive, and destructive or pathological (kravchenko 1977). the study is significant because cytomixis is linked to evolution since it may lead to change in ploidy as well as often leads to unreduced gamete. furthermore, the study is of great relevance in assessing reasons and process of its occurence, and the complex process of microsporgenesis which is substantially affected by cytomixis. role of plasmodesmatal connections and callose insulation needs more detailed scrutiny. ionizing radiation i.e. gamma rays was used in the present study for exploiting its mutagenic role for improvement genetic characteristics of the plant system. role of gamma rays has also been anticipated for its role in inducing polyploids and aneuploids via cytomixis in several reports. gamma ray is ascribed to be most efficient factor that results in imbalanced genetic system (saraswathy et al. 1990). the pla nt materia l cluster bea n [cyamopsis tetragonoloba (l.) taub.] is an important legumes, thriving well in semi arid zones of indian and pakistan. the plant is highly valued for its guar gum that is extracted form the seed endosperm that add ons its economical value. besides this, cluster bean occupies a decent position in traditional folklore medicines and is nutraceutically also very important. cytomictic behaviour in cyamopsis tetragonoloba has been previously described spontaneously (sarbhoy 1980), but the present article is envisioned to reach new vistas by exploring multitude facets of gamma rays induced cytomixis. salient features and repercussions entailed in relation to meiotic behaviour and reproductive success will be ambit of this work. materials and methods plant material seeds of cluster bean [cyamopsis tetragonoloba (l.) taub.] were procured from central arid zone research institute (cazri) jodhpur, rajasthan, india. after preliminary screening, accession number rgc-1038 was selected for cytogenetical work. agroclimatic conditions of the experimental site present study was conducted in an experimental cage in roxburgh botanical garden, department of botany, university of allahabad, prayagraj, up, india during kharif season in july to november. the geographical location is 25o27’43.01’’n, 81o51’10.42’’e. prayagraj lies in sub-tropical climatic zone and receives an annual rainfall of 958mm where relative humidity is 59%. 113induced cytomictic crosstalk behaviour among micro-meiocytes of cluster bean treatment and sowing fresh seeds of cyamopsis tetragonoloba were arranged into different packets that were irradiated with gamma rays at increasing dose (viz. 100 gy, 200 gy and 300 gy) from a co-62 source radioisotope inside gamma chamber at national botanical research institute (nbri), lucknow, india at radiation speed of 2gy per second. these irradiated seeds were sown in respective pots in replicates in complete randomized block design (crbd) to raise the generation alongwith a control set that was maintained as a standard. bud fixation floral buds were fixed in carnoy’s fixative (solution constituting 3 parts of 90% alcohol: 1 part glacial acetic acid) for duration of 24 hours. buds were preserved in 70% alcohol at 4°c in refrigerator for future use. meiotic study flower buds of appropriate size were teased in a drop of 70% alcohol, followed by staining and mounting in 2% acetocarmine. squash of the bud was prepared using a tapper. after squash preparation, slides were observed under olympus light microscope whereas important stages were captured using nikon phase contrast research photomicroscope (nikon eclipse, e200, japan) at 40x resolution. pollen fertility was also computed on the basis of glycerine-acetocarmine stainability test using temporary mounts (marks 1954). adequately stained, globose, nucleated pollens were marked as fertile whereas sparsely stained, shrivelled and enucleated pollens were regarded as sterile. variation in pollen diameter was recorded. statistical calibration the data obtained were analysed using statistical software spss 16 and means were compared using duncan’s multiple range test (dmrt) (p≤0.05). all the results were expressed in form of mean ± standard error. results cytogenetical screening of microsporogenic cells is a reliable test for in-depth view of in cluster bean [cyamopsis tetragonoloba (l.) taub.]. cytogenetical studies revealed that chromosome complement set of the plant is n=7 (fig. 2b showing metaphase i), confirming the somatic chromosomal configuration to be 2n=14. meiocytes, in control, were perfectly normal and bivalents morphology was canonical with no considerable indication of aberrations; also there was no sign of cytomictic connections amongst pmcs. however, mutagenic treatment of gamma rays had impacted into a wide range of chromosomal anomalies alongwith cytomictic behaviour fig. 1. cytomixis via direct cell fusion (a-f) where a: direct cell fusion at diplotene; b: cell fusion between prophase 1 and metaphase i; c: chromatin transfer between two pmcs; d: horizontal transfer of chromosomes where one pmc is chromatin deficient; e: a chromosomal fragment in the transition phase; f: migrating chromatin pushed towards periphery as sticky chromatin band. cytomictic transmigration via channel formation (g–i) where g: single channel bridging two meiocytes; h: simultaneous transfer of chromatin from 1 pmc to 2 pmcs; multiple channel formation. group formation (j – l) where j and k: transitory micronuclei pushed at ends of meiocytes; l: association between cells at anaphase ii stage. scale bar: 10.45 µm. 114 girjesh kumar, shefali singh amidst dividing meiocytes. pmcs exhibiting cytomictic events the frequency of cytomixis was computed to be 7.29±0.33 % at 100 gy dose which increased from this value to 10.84±0.46% at 200 gy and 14.67±0.60% at 300 gy. it was witnessed to manifest either through direct cell to cell fusion or via cytoplasmic channels; where frequency of direct fusion was higher in comparison at all the three doses. table 1 represents data on cytomictic frequency at various stages of meiosis. traversing of cytoplasmic contents, chromatin material, cellular organelles and other vital intrinsic trophic factors between proximate pmcs was witnessed. onset of transitory events was witnessed by acquisition of cell polarization where nucleus was positioned towards the cell periphery i.e in between the communicating meiocytes unlike the non-cytomictic cells where nucleus was in the central space of pmc. direct fusion was recorded at diverse stages of division with different degree of cytomictic intensities. for categorising the intensity, three levels of cytomixis were identified according to kravchenko (1977). cells at lower doses had loose wall connexion; these formed pairs and led to cause local cytomixis since no indication of chromatin transmigration was observed. several pmcs deciphered rather intensive cytomictic phenomena where the migrating chromatin and micronuclei were encountered in between the associating pmcs or were pushed towards periphery of the parent pmc. transferring content was seen to pass via cytoplasmic channels as sticky chromatin bands. cytoplasmic channels (cc) were also of distinct morphology. it was, either, in the form of single channels (fig. 1g) or multiple bridging (fig. 1i) architectures through which nuclear transaction occurred. fig.1h shows simultaneous transfer of chromatin from one pmc to two pmcs through channel formation. at some instances, cytomixis occurred via group formation where multiple pmcs participated in the confluence (fig. 1j to l). distinguishing feature of such grouping was the attainment of chain transfer. chain transfer was peculiar where one cell donates a nucleus to the recipient cell and this recipient, in turn, transacts its nucleus to the succeeding one and so on. besides cytomixis, several other abnormalities were notifiable among which stickiness, univalents, disturbed polarity, unequal separation and laggards were more common alongwith less frequents anomalies such as bridges and micronuclei formation. an increasing trend for other chromosomal anomalies was recorded with respect to gamma irradiation i.e. from 9.80±0.29 at 100 gy to 16.72±0.40 at 300 gy gamma dose (table 1). syncyte manifestation a remunerative phenomenon of syncyte was witnessed at all the three doses of gamma irradiation viz. 100 gy (0.25±0.25%), 200 gy (0.55±0.28%) and 300 gy (0.66 ±0.33%). syncytes are recreated by complete confluence of two pmcs, where whole chromatin material is transferred to the recipient pmc. therefore the recipient pmc is complemented with doubled chromatin complement. fig. 2c is a syncytic cell representing 14 bivalents in place of 7 bivalents. conversely, hypoploid cells were also recorded with lesser number of bivalents (fig. 2d is a hypoploid cell). binucleate pmcs with supernumerfig. 2. consequence of cytomixis on late meiotic phases and pollen morphology. a: supernumerary nucleoli; b: normal pmc with seven bivalents; c: syncyte with 14 bivalents; d: hypoploid meiocyte; e: normal tetrad; f: polyad; g: normal fertile pollen; h: two nucleated pollen; i: two pollens fusing through wall dissolution; j: heterosized pollens; k: pollens with differential size and diameter; l: fertile and sterile pollens. scale bar: 10.45 µm. 115induced cytomictic crosstalk behaviour among micro-meiocytes of cluster bean ary nucleolus were seen that might have been engineered due to nucleolar transfer via common meiocytic links. abnormal sporads, pollen fertility and size variation besides meiocytic fusion, tetrad and pollen grain fusion (fig. 2i) were also recorded which is a rather uncommon and interesting phenomena to be reported in this study, since cytomictic behaviour is documented to occur more frequently at meiosis i. abnormal microspores including monads, dyad and polyads (fig. 2f) were also encountered. variation in pollen size (diameter) was also recorded which were accounted to be heterosized pollens. in case of control, pollen diameter was not variable but in treated sets, alongwith medium normal sized pollens, differential frequency of small and large sized ‘2n pollens’ were recorded. fig. 2j and 2k are heterosized pollens. smaller pollens were generally non-viable whereas larger ones had a differential degree of fertility. table 2 is a representation of pollen fertility in control and treated sets; it also depicts mean diameter and relative frequency of large, medium and small sized pollen grains. pollen fertility was also calculated and a sharp reduction was discernible from 97±0.57% at 100gy to 74.33±0.88% at 300 gy dose. discussion meiosis is a beautifully manoeuvred cellular event that has been established to ensure efficient chromosome partitioning, recombination and trait assortment. furthermore, its proper disposition is essential in restoring gametic viability. henceforth, it becomes seemingly important to follow and magnify knowledge on the meiotic cell cycle. a typical promiscuous cytomictic behaviour among pmcs of cluster bean (cyamopsis tetragonoloba (l.) taub.) was reported in response to mutagenic incidence of gamma rays. significance for recruiting studies on this phenomenon is vested in the fact that cytomictic events provide a larger view onto the mechanism of intercellular activities and cellular conveyance. cytomixis was more profuse at meiosis i than at meiosis ii and it appears to be active energy dependent process since kcn solution or decline in temperature checks it (zhang et al. 1985). the perplexity surrounding the mechanism through which chromatin transfer occurs was resolved after identification of the role of cytoplasmic connections (gates 1911). these connections form an important avenue for cytopasmic crosstalks among proximate pmcs. these are engineered ta bl e 1. e ffe ct o f g am m a ir ra di at io n on th e in ci de nc e of c yt om ix is a nd s yn cy te fo rm at io n in c lu st er b ea n [c ya m op si s te tr ag on ol o (l .) ta ub .]. tr ea tm en t c yt om ix is sy nc yt es (% ) fr eq ue nc y of c el ls s ho w in g cy to m ix is a t v ar io us s ta ge s of m ei os is , % (m ea n ± se ) o th er ab no rm al iti es (m ea n ± se ) n n o. o f pm c s (( m ea n) fr eq ue nc y of p m c s in vo lv ed in c yt om ix is ty pe o f c yt om ix is m ei os is i m ei os is i i d f c c pi m i a i t i m ii a ii t ii pt d p d k c on tr ol 33 15 10 0 g y 22 97 7. 29 ±0 .3 3 61 .6 6± 1. 66 38 .3 3± 1. 66 0. 25 ±0 .2 5 1. 91 ±0 .1 6 1. 70 ±0 .2 7 1. 00 ±0 .1 7 0. 77 ±0 .0 8 0. 67 ±0 .0 4 0. 99 ±0 .1 5 0. 45 ±0 .2 3 9. 80 ±0 .2 9 20 0 g y 33 00 10 .8 4± 0. 46 71 .0 2± 2. 41 28 .9 6± 2. 41 0. 55 ±0 .2 8 2. 02 ±0 .1 7 1. 65 ±0 .0 6 1. 68 ±0 .1 4 1. 34 ±0 .1 1 1. 24 ±0 .2 0 0. 77 ±0 .0 7 0. 97 ±0 .1 1 0. 68 ±0 .2 0 0. 42 ±0 .2 5 12 .0 2± 0. 31 30 0 g y 22 93 14 .6 7± 0. 60 75 .1 1± 1. 78 26 .4 5± 1. 45 0. 66 ±0 .3 3 2. 61 ±0 .1 8 2. 15 ±0 .0 5 2. 04 ±0 .0 6 1. 59 ±0 .1 5 1. 47 ±0 .1 3 1. 59 ±0 .2 4 1. 46 ±0 .0 7 0. 89 ±0 .2 8 0. 79 ±0 .1 1 16 .7 2± 0. 40 a bb re vi at io ns : p m c spo lle n m ot he r c el ls , c c -c yt om ic tic c ha nn el , d fd ir ec t f us io n, p ipr op ha se i , m im et ap ha se i , a ia na ph as e i, t ite lo ph as e i, m ii -m et ap ha se i i, a ii -a na ph as e ii , t ii -t el op ha se i i, pt -p ac hy te ne , d pd ip lo te ne , d kd ia ki ne si s. 116 girjesh kumar, shefali singh at prophase i of meiosis and are recognised as primary cc. persisting plasmodesmata expands its extremities, it forms passage of large interconnecting cells, which is termed as cytoplasmic channels. cell wall dissolution between the adjacent cells may also lead to cytoplasmic connections (falistocco et al. 1995). hydrolytic enzymes released by endoplasmic reticulum and golgi bodies are involved in cc formation (yu et al. 2004). these primary cc may form via fusion of several plasmodesmata or through enlargement of single plasmodesmata or de novo in the region where no plasmodesmata occurs (wang et al. 1998; mursalimov et al. 2013a). however the cellulose-pectin wall is gradually replaced by callose layer at subsequent stages, as explained by kravets (2013). the callose deposition insulates the cellular crosstalks and ceases the primary cc. it is for this reason that cytomixis is more profusely recorded at mieois i rather than meiosis ii. however cytomictic behaviour may still persits by the genesis of secondary cc which is formed by action of enzymes callase that acts on callose wall. specific organelles-spherosome like vesicles secreate callase and points at which callose catalyzes destruction of callose, secondary cc originates (mursalimov et al. 2013b). these secondary cc remain available for cytomixis at the later stages of meiosis. local cytomixis represents association of meiocytes into groups via cytomictic channels in the early prophase of meiosis without any participation of migrating chromatin. severe cytomictic channels such as fig. 1h was also seen where cytoplasmic content of one pmc emanates in two pmcs. this has also been reported in vicia faba (bhat et al. 2017). cytopathological symptoms are evident in intensive cytomixis, where transaction of chromatin; migration of the cytoplasmic content, nuclei etc are witnessed whereas destructive cytomixis involves complete destruction of the donor cells and severe pathological signs the filling of the anther cavity with agglutinated chromatin, and the impairment of remaining microsporocytes during meiosis. actually, destructive cytomixis represents rather the way of the msc autolysis than the way of communication between microsporogenic cells (kravets 2013). actin filaments play a key role in cytomixis since migration of cell contents through cytomictic channels is stopped due to cytochalasin b, a chemical that prevents the growth of actin filaments (zhang et al. 1985). several pertinent questions regarding functional state of the transferring chromatin were answered by conducive histone modification experiments using immunostaining technique mursalimov et al. (2015). migrating chromatin had no signs of selective heterochromatinization and was decrypted to be in transcriptionally active state. ultrastructural studies indicate that neither nucleus nor chromatin is damaged while traversing through cytomictic channel (mursalimov and deineko 2011). these arguments implicits ample evidence that cytomixis is a genetically controlled enigmatic phenomenon occurring due to environmental or physiological factors (bellucci et al. 2003); which has been installed in cells to facilitate inter-cellular transmigration of vital cellular components. several reports elucidates that cytomictic behaviour is linked to meiotic segregation and aberrant gene functioning at preceding meiotic or mitotic stages subverts to both chromosomal aberration as well as cytomixis. thus, cytomixis regulation may be controlled by genes responsible for the chromosome segregation such as the dif1 gene in arabidopsis thaliana (bhatt et al. 1999). an intriguing aspect revealed was presence of more than one nucleus in pmcs which displayed coenocytic behaviour. this behaviour is persistently encountered in intergeneric hybrids for example in meconopsis aculeate (singhal and kumar 2008). consequently, one cell gets an extra nucleus, leaving behind the other nucleus deficient cell. such coenocytes lead in formation of abnormal-sized pollen grains as suggested earlier by mendesbonato et al. (2001). furthermore, fusion of two pmcs led to syncyte formation, also documented in chrysanthemum (kim et al. 2009), mertensia echioides (malik et al. 2014). frequency of syncytes is quite low but it is easily detectible due to its invariably larger size compared single meiocyte. the product of such meiocytes resulted into the formation of ‘2n’ or large-sized pollen grains. jones and reed (2007) approved that presence of table 2. impact of gamma rays on pollen fertility and relative pollen size frequency in cluster bean [cyamopsis tetragonoloba (l.) taub.]. treatment pollen fertility (%) diameter(mm) relative frequency of different pollen size (%) small medium large small medium large control 97.00±0.57 19.17±0.61 100 100 gy 91.33±1.20 15.24±0.36 19.48±0.36 31.72±0.64 7.33±0.33 83.00±1.15 9.66±0.88 200 gy 82.66±0.66 15.94±0.50 19.98±0.40 32.83±0.57 11.66±0.33 71.00±0.57 17.33±0.88 300 gy 74.33±0.88 15.48±0.57 19.66±0.70 33.02±0.29 12.66±0.88 64.66±0.66 22.66±0.33 117induced cytomictic crosstalk behaviour among micro-meiocytes of cluster bean ‘giant’ pollen to be associated with 2n status. unreduced (diploid) gametes such as 2n pollen are good source for inducing polyploids (ghaffari 2006; latoo et al. 2006). syncytes are concluded to oblige with imperative significance since it results into aneuploids which are assets for cytogeneticists. it may have serve as an exemplary model for intergeneric polyploids production. it is witnessed that this additional supernumerary chromatin mass do not pair with main chromatin material of the recipient cell, instead it remains as a separate identity, which may later on from micronuclei or micropollen (bhat et al. 2006). however, its synthesis is of great future prospects since there induction is remunerative of infraspecifc polyploidization. it that may serve novel in the field of genetic variation and crop improvement. hypoploid cells are also quite prodigious tool from one viewpoint since they might become deficient in certain intrinsic genetic factors and their scrutiny is thus imperative. cytoplasmic connection among pollens, although a rare phenomenon, was also witnessed here. such connections among pollen grains had already been noticed in the intergeneric hybrids of roegneria tsukushiensis x psathyrostachys huashanica (sun et al. 1994) and in meconopsis aculeata (singhal and kumar 2008). heterosized pollens of varying diameter were also recorded. genesis of heterosized pollens stems from the aneuploid pmcs post cytomixis. pollen fertility was documented to decline with increasing gamma rays. the descending fertility is apparently an outcome of all the cumulative factors that led to cytogenetical aberrations which eventually affected the reproductive success of microsporogenesis. this study is succesfull documentation on gamma rays induced cytomixis in cyamopsis tetragonoloba (l.) taub. it also validates the efficacy of the ionizing radiation for inducing useful cytological variants such as aneuploids and infraspecific polyploids. gamma rays, plausibly has a substantial role in maintaining genetic heterogeneity (kravets 2013) or restoring and balancing the unbalanced genomes within the developing male gametophyte, as highlighted by (falistocco et al. 1995; ghaffari 2006; song and li 2009). if cytomixis is a means for synthesising infraspecific polyploids, it is also characterized as genome stabilizing, cell sorting checkpoint. for clearing all the mysteries and to expand our level of knowledge, we hope arrival of more concrete techniques, which might help in furthering our vision. acknowledgements authors express gratitude to cazri, india for providing seeds of clusterbean. authors extend sincere thanks to nbri, india for providing facilities for gamma irradiation on seeds. thanks to members of plant genetics laboratory for their suggestions and reviews. funding corresponding author was financially supported by university grant commission. references arnoldy w. 1900. beiträge zur morphologie der gymnospermen. iv. was sind die “keimbläschen” oder “hofmeisters-körperchen” in der eizelle der abietineen? 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the upland cotton aneuploidy. hereditas.138:65–72. caryologia international journal of cytology, cytosystematics and cytogenetics volume 73, issue 2 2020 firenze university press the first molecular identification of egyptian miocene petrified dicot woods (egyptians’ dream becomes a reality) shaimaa s. sobieh*, mona h. darwish gene flow patterns reinforce the ecological plasticity of tropidurus hispidus (squamata: tropiduridae) fernanda ito, danielle j. gama-maia, diego m. a. brito, rodrigo a. torres* the technique of plant dna barcoding: potential application in floriculture antonio giovino1,*, federico martinelli2,*, anna perrone3 cytogenetic of brachyura (decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species alessio iannucci1, stefano cannicci1,2,*, zhongyang lin3, karen wy yuen3, claudio ciofi1, roscoe stanyon1, sara fratini1 comparison of the evolution of orchids with that of bats antonio lima-de-faria identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection mehdi zahravi1,*, panthea vosough-mohebbi2, mehdi changizi3, shahab khaghani1, zahra-sadat shobbar4 populations genetic study of the medicinal species plantago afra l. (plantaginaceae) saeed mohsenzadeh*, masoud sheidai, fahimeh koohdar a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding timir baran jha1,*, partha sarathi saha2, sumita jha2 chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india harminder singh2, jaswant singh1,*, puneet kumar2, vijay kumar singhal1, bhupendra singh kholia2, lalit mohan tewari3 population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies raheleh tabaripour1,*, masoud sheidai1, seyed mehdi talebi2, zahra noormohammadi3 induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions girjesh kumar, shefali singh* karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(1): 43-51, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-839 caryologia international journal of cytology, cytosystematics and cytogenetics citation: a. lemos costa, c. furlan lopes, m. santos de souza, s. alves barcellos, p. giordani vielmo, r. josé gunski, a. del valle garnero (2021) comparative cytogenetics in three species of wood-warblers (aves: passeriformes: parulidae) reveal divergent banding patterns and chromatic heterogeneity for the w chromosome. caryologia 74(1): 43-51. doi: 10.36253/caryologia-839 received: january 22, 2020 accepted: april 26, 2021 published: july 20, 2021 copyright: © 2021 a. lemos costa, c. furlan lopes, m. santos de souza, s. alves barcellos, p. giordani vielmo, r. josé gunski, a. del valle garnero. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid alc: 0000-0003-4620-2989 cfl: 0000-0002-4783-4315 mss: 0000-0002-2130-6100 sab: 0000-0003-2863-9976 pgv: 0000-0003-3491-2115 rjg: 0000-0002-7315-0590 advg: 0000-0003-4252-8228 comparative cytogenetics in three species of wood-warblers (aves: passeriformes: parulidae) reveal divergent banding patterns and chromatic heterogeneity for the w chromosome alice lemos costa1,*, cassiane furlan lopes1, marcelo santos de souza1, suziane alves barcellos1, pâmela giordani vielmo2, ricardo josé gunski1, analía del valle garnero1 1 laboratório de diversidade genética animal, programa de pós-graduação em ciências biológicas, universidade federal do pampa, são gabriel, rio grande do sul, brazil 2 laboratório de diversidade genética animal, curso de graduação em ciências biológicas, universidade federal do pampa, são gabriel, rio grande do sul, brazil *corresponding author. e-mail: alicelemoscosta14@hotmail.com absract. chromosomal rearrangements are an important process in the evolution of species. it is assumed that these rearrangements occur near repetitive sequences and heterochromatic regions. avian karyotypes have diverse chromosomal band patterns and have been used as the parameters for phylogenetic studies. although the group has a high diversity of species, no more than 12% has been analyzed cytogenetically, and the parulidae family are extremely underrepresented in these studies. the aim of this study was to detect independent or simultaneous chromosomal rearrangements, and also to analyze chromosomal banding convergences and divergences of three woodwarblers species (myiothlypis leucoblephara, basileuterus culicivorus, and setophaga pitiayumi). our cbg-band results reveal an unusual w sex chromosome in the three studied species, containing a telomeric euchromatic region. the gtg and rbg bands identify specific regions in the macrochromosomes involved in the rearrangements. cytogenetic data confirm the identification of speciation processes at the karyotypic of this group. keywords: chromosomal evolution, karyotype, diploid number, chromosomal banding, constitutive heterochromatin. introduction the avian class is characterized by a bimodal karyotype, composed of many pairs of microchromosomes and just a few macrochromosomes (christidis 1990). the class presents several patterns of chromosomal bands. in cbg-banding, species of passeriformes usually reveal the w chromosome 44 alice lemos costa et al. heterochromatic (kretschmer et al. 2018a). in contrast, some struthioniformes species show a completely euchromatic chromosome (nishida-umehara et al. 2007). in other orders such as tinamiformes, this chromosome exhibits an intermediate cbg-banding pattern, containing euchromatic and heterochromatic blocks (garnero et al. 2006). some classical cytogenetic techniques provide patterns of positive and negative bands, exposing points of reference on the full length of the chromosome and enabling the creation of ideograms (ladjali et al. 1999). changes in these patterns suggest the possible types of rearrangements caused by chromosomal differences that may have occurred during the evolution of the genome (griffin et al. 2007). examples of this are the chromosomal rearrangements already reported by gtg and rbg bands in gallus gallus (galliformes), which identified a paracentric inversion in the long arm of chromosome 2 (nanda et al. 1994). chromosomal polymorphisms were identified by gtg bands in synallaxis frontalis (passeriformes), where pericentric inversion involving the first and third pairs was observed (de souza et al. 2019), and in treron phoenicoptera (columbiformes) in the first and second pairs (gupta and kaul 2014). c h r om o s om a l r e a r r a n g e m e nt s o c c u r du ring the evolutionar y process at the specimen level (kretschmer et al. 2018b). among these chromosoma l changes are commonly obser ved translocations, duplications, inversions, deletions, fusion, and fissions (stock and bunch 1982; nascimento et al. 1994; nanda et al. 2011). this occurs in regions involving repetitive sequences and in the proximity of heterochromatic regions (farre et al. 2016). less than 12% of the species of the aves class have been characterized by cytogenetic studies, where passeriformes order contains most of the species described (griffin et al. 2007; degrandi et al. 2020). parulidae (passeriformes) is strictly underrepresented in these studies, the family contains 119 species divided into 21 genera, but only 8% of all species have been investigated cytogenetically by giemsa staining, shown diploid variation from 76 to 80 chromosomes (carvalho 1989; hobart 1991). this study aimed to detect independent or simultaneous chromosomal rearrangements, and it also analyzes chromosomal banding convergences and divergences of three species of the parulidae family – myiothlypis leucoblephara, basileuterus culicivorus, and setophaga pitiayumi – using techniques of classical cytogenetics such as cbg, gtg, and rbg bands. material and methods sampling and collecting five specimens of wood-warblers were analyzed in the present study: myiothlypis leucoblephara (1 male and 1 female), basileuterus culicivorus (1 male and 1 female), and setophaga pitiayumi (1 female). all specimens were collected using a mist net in são gabriel, rio grande do sul state, brazil (latitude 30°20’38’’s and longitude -54°20’31’’w), under license sisbio nº 61047-3, and ceua/unipampa nº 010/2018. cell culture and chromosome preparation mitotic cells were obtained using a short-term bone marrow extraction technique (garnero and gunski 2000). initially, biological material was extracted from femurs in 10ml of rpmi 1640 medium and incubated with 0.01 ml of colchicine solution (0.05%) at 37°c for 1 h. cells were subsequently centrifugated and incubated for 20 min in hypotonic solution (0,075 m kcl) at 37°c. finally, the cells were fixed with methanol and acetic acid (3:1). we analyzed approximately 40 metaphases per specimen to determine the diploid number in an optical microscope (olympus dp53). for composing karyotype figures, it was used program corel draw12®, and the chromosomes were classified in decrescent order according to the long arm (p), short arm (q), arm radio (r) and centromeric index (i) (guerra 1986). cbg, gtg, and rbg banding regions of heterochromatic blocks were analyzed by cbg-banding (ledesma et al. 2006). after treatment in 0.2n hcl for 15 min, the slides were incubated in barium hydroxide (50%) for 17 min at 37°c. structural investigations of the gtg-banding were done according to schnedl (1971), with modifications to the immersion period in saline solution, which occurred for 1 min. to obtain the rbg-banding, the protocol by popescu (2000) was replicated with a modification of the incubation period in earl buffer (ph 5.1) saturated with na2hpo4, which occurred for 30 min at 87°c. subsequently, a wash step with distilled water was performed followed by immersion for 30 min in earl buffer (ph 6.4), without the addition of nahco3, at 87°c. in all banding protocols, metaphases were stained with giemsa (5% in 0.07 m phosphate buffer, ph 6.8). the gtg and rbg bands position were classified according to the international system of standardized 45comparative cytogenetics in three species of wood-warblers avian karyotypes (issak). band patterns were interpreted by comparison among the three species of this study, and the types of rearrangements were detected with the inferences by homology in model species gallus gallus (ladjali et al. 1999). results wood-warblers analyzed in this study showed differences in karyotypes. we identified a chromosome number of 2n=76 for myiothlypis leucoblephara, concomitant with the described by carvalho (1989) in a male specimen. basileuterus culicivorus presented a diploid number of 2n=78, and setophaga pitiayumi 2n=80 (figure 1). in the three species, the karyotypes exhibited 14 pairs of autosomal macrochromosomes and 1 pair of sex chromosomes zz or zw. the remaining pairs were composed of microchromosomes. autosomal macrochromosomes and sex chromosomes were morphometrically described, presenting only morphological divergences occurring among chromosomes 5, 6, and 7 in the three species (table 1). cbg-banding analysis identified constitutive heterochromatin in the centromeric regions of the macrochromosomes and revealed the w chromosome. this chromosome was positioned between the 6th and 7th pair and showed chromatic heterogeneity in cbg-banding in the three species. it was formed by a block of heterochromatin in the short arm and partial in the long arm, containing a telomeric euchromatic region in the long arm (figure 2). in all analyzed species, the z chromosome was euchromatic, with positive staining observed near the centromere and morphometrically positioned between the 4th and 5th pair of macrochromosomes. in this study, we describe by gtg-banding the first 10 autosomes macrochromosomes, and zw sex chromosomes (figure 3). m. leucoblephara presented 137 gtgbands distributed along the chromosomes, where the negatives integrated into terminal regions of the short and long arms of chromosomes 2, 4, 5, 6 and 7. other chromosomes contained positive bands in their terminal regions. b. culicivorus had a total set of 139 gtg-bands, of which the negatives were also distributed in the terminal regions of the short and long arms of chromosomes 1, 2, 4 and 5, and in the terminal region of the long arm of chromosomes 3, 6 and 7. other terminal regions of chromosomes contained positive bands. s. pitiayumi showed 137 gtg-bands along their chromosomes, with negatives forming the terminal regions of the short and long arms of the chromosomes 1, 3 and 4, and the terminal region of the long arm of chromosomes 2, 5, 6, 7 and 8. the terminal regions of other chromosomes consisted of positive bands. the reverse pattern was identified by rbg-banding, performed with the first 10 pairs of autosomal chromosomes and zw. this data was shown to be compatible with the results obtained by gtg-banding (figure figure 1. species complete karyotype. chromosomes arranged in descending order with giemsa staining, followed by sexual chromosomes z and w. myiothlypis leucoblephara (a), basileuterus culicivorus (b), and setophaga pitiayumi (c). 46 alice lemos costa et al. 3). homologous and non-homologous regions among the three species were identified and compared with the homologous regions of model species gallus gallus (ladjali et al. 1999). in chromosome 1, a fission in region 2 of the short arm of b. culicivorus, and a paracentric inversion in region 1 of this same arm in s. pitiayumi were detected. in the long arm of this same chromosome, in region 4, a paracentric inversion was found in b. culicivorus. for chromosome 3, an inversion followed by deletion in region 1 of the long arm was detected in b. culicivorus. in the 5th pair, b. culicivorus also presented a fusion in region 1 of the short arm. a break followed by pericentric inversion was found in the 6 pair of the species m. leucoblephara and s. pitiayumi. in chromosome 7, s. pitiayumi also presented a fission in region 1 of the short arm. m. leucoblephara and s. pitiayumi showed a fusion in region 1 of the long arm in chromosome 8 (figure 4). table 1. measurements and morphology of autosomal macrochromosomes and sex chromosomes of the species studied. chromosome myiothlypis leucoblephara   basileuterus culicivorus   setophaga pitiayumi short arma long arma rb cic morpho logyd short arma long arma rb cic morphologyd short arma long arma rb cic morphologyd 1 6.3 10.6 1.68 37.28 sm 6.1 11.1 1.82 35.47 sm 6.2 10.9 1.76 36.26 sm 2 4.1 9.3 2.27 30.60 sm 4.3 9.5 2.21 31.16 sm 3.9 9.6 2.46 28.89 sm 3 2.3 8.5 3.70 21.30 a 2.2 9.8 4.45 18.33 a 2.8 9.6 3.43 22.58 a 4 2.1 8.2 3.90 20.39 a 2.1 8.9 4.24 19.09 a 2.1 8.7 4.14 19.44 a 5 1.9 7.3 3.84 20.65 a 1.7 7.4 4.35 18.68 a 3.1 6.3 2.03 32.98 sm 6 1.5 6.7 9.70 23.15 a 0 9.8 9.80 9.80 t 2.1 6.5 3.10 24.42 a 7 2.1 4.7 2.24 30.88 sm 2.1 6.2 2.95 25.30 sm 1.2 5.4 4.50 18.18 a 8 0 6.3 6.30 6.30 t 0 7.2 7.20 7.20 t 0 6.3 6.30 6,30 t 9 0 5.8 5.80 5.80 t 0 6.3 6.30 6.30 t 0 6.1 6.10 6.10 t 10 0 5.3 5.30 5.30 t 0 5.9 5.90 5.90 t 0 5.7 5.70 5.70 t 11 0 4.9 4.90 4.90 t 0 5.1 5.10 5.10 t 0 5.2 5.20 5.20 t 12 0 4.1 4.10 4.10 t 0 4.5 4.50 4.50 t 0 4.8 4.80 4.80 t 13 0 3.7 3.70 3.70 t 0 3.9 3.90 3.90 t 0 4.1 4.10 4.10 t 14 0 3.5 3.50 3.50 t 0 3.6 3.60 3.60 t 0 3.6 3.60 3.60 t z 3.2 7.1 2.22 31.07 sm 3.3 7.4 2.24 30.84 sm 3.1 7.2 2.32 30.10 sm w 1.9 4.2 2.21 31.15 sm   1.8 4.3 2.39 29.51 sm   1.5 3.9 2.60 27.78 sm alength in micrometer (µm) q-long arm, p-short arm. brelationship between p/q. ccentromeric index. dchromosomal morphology: t-telocentric, a-acrocentric, sm-submetacentric. figure 2. cbg-banding metaphases with emphasis on the patterns of banding of sex chromosomes. myiothlypis leucoblephara (a), basileuterus culicivorus (b), and setophaga pitiayumi (c). 47comparative cytogenetics in three species of wood-warblers discussion the karyotypic structure of the three analyzed species in this study is similar to the typical avian karyotype (figure 1 and table 1), containing few pairs of macrochromosomes, many microchromosomes, a zw heterogametic sexual system for females and zz homogametic for males (christidis 1990). in the species of the family that has been previously studied, the frequency of the diploid number was within the standard, ranging from 76 to 80 chromosomes (carvalho 1989; hobart 1991). karyotypically, the three species presented the first pair of submetacentric chromosomes, supporting the theory that passeriformes retain this morphology among its oscines birds (guttenbach et al. 2003). during the evolutionary changes of this chromosome, a break followed by fusion with a microchromosome forming this biarmed chromosome has been historically suggested in galliformes (stock and bunch 1982). in passeriformes, it was shown by fluorescent in situ hybridization (fish) results that all species studied shared a fission of gga1 (kretschmer et al. 2018b). cbg-banding identified a preferential accumulation of constitutive heterochromatin in the centromeric regions (figure 2). the w chromosome showed a distinct banding pattern identified in passeriformes, which is generally heterochromatic (kretschmer et al. 2018a). in all three species, this chromosome has an euchromatic telomeric region in the long arm. we can infer that this chromosome has an intermediate cbg-banding pattern, it was seen in other orders such as tinamiformes in the crypturellus tataupa species, where euchromatic and heterochromatic blocks occur simultaneously (garnero et al. 2006). a similar pattern occurred in charadriiformes in the burhinus oedicnemus species, where a euchromatic band was found in the long arm of w chromosome (nie et al. 2009). neognathae birds tend to have a reduction in the size of the w chromosome. suggesting that this occurs due to loss of accumulated repetitive sequences and non-recombining regions. however, there are significant morphological differences in this chromosome, referring to loss and gain, followed by the accumulation of these sequences (furo et al. 2017). in some species such as neochmia faeton (passeriformes), ardeola grayii (pelecaniformes), gallinula melanops (gruiformes), amazona aestiva (psittaciformes), and crotophaga ani (cuculiformes), this chromosome is considered the largest or one of the largest among chromosomal complement (christidis 1989; mohanty and bhunya 1990; furo et al. 2017; gunski et al. 2019; kretschmer et al. 2021). the number of gtg and rbg bands obtained for the species was distinct (figure 3), collaborating with the observed diploid number. it is possible to suggest the occurrence of interchromosomal and intrachromofigure 3. description of gtg and rbg banding patterns and their respective ideograms. light bands: negative gtg and rbg positive. dark bands: positive gtg and negative rbg. 48 alice lemos costa et al. somal rearrangements for these species, since fission, fusion, inversion, and deletion processes can be detected by banding patterns, which could be used as a reference point of the genomic organization (ladjali et al. 1999; nanda et al. 2011). however, we suggest that results should be analyzed in future studies by fluorescent in situ hybridization (fish), giving additional information about this issue. comparisons of gtg and rbg band patterns among the three species showed distinct convergences and divergences (figure 4). the macrochromosome pairs 1, 2, and 3 have similar morphology, but the banding patterns were not the same in these chromosomes for the parulids. in this context, takagi (1974) found the same pattern in nine other orders of the aves class, for example in strigiformes, columbiformes, and gruiformes. some studies have shown that chromosomes 1, 2, and 3 are actively linked to intrachromosomal rearrangement processes in the passeriformes (nanda et al. 1994). our results indicate a sharing of number of chromosomal bands in m. leucoblephara and b. culicivorus species, in region 1 of the first pair in short arm compared to the g. gallus (ladjali et al. 1999). s. pitiayumi has a divergent pattern in this region, where possibly a paracentric inversion has caused this differentiation. b. culicivorus has rearrangement in region 2 of the short arm, where fission occurred in the telomeric region. also, in region 4 of the long arm, there is a higher number of bands compared the other parulids, where a break followed by paracentric inversion could have caused this pattern. b. culicivorus showed a reduction in the number of bands in region 2 of the long arm in the third pair compared to the other two parulids, which have similarities with g. gallus (ladjali et al. 1999) in this region. for this differentiation, a possible paracentric inversion and deletion may have occurred. in synallaxis frontalis species, there is a pericentric inversion involving the first and third pairs (de souza et al. 2019). nevertheless, this diversity of rearrangements involving chromosomes 1, 2, and 3 is not restricted to passeriformes. in columbidae, the treron phoenicoptera species has a chromosomic figure 4. ideograms of parulidae with compiled data obtained by gtg and rbg bands, demonstrating the type of chromosomal rearrangements with divergences and convergences among bands pattern. region of bands compared with gallus gallus available in ladjali et al. (1999). chr-chromosome. 49comparative cytogenetics in three species of wood-warblers rearrangement of inversion in first and second pairs (gupta and kaul 2014). in parulids, chromosomes 4 and 5 showed the same number of regions, containing differences only in morphology the 5 pair and number of bands. in region 1 of the short arm of chromosome 5, species b. culicivorus has three bands, while m. leucoblephara and s. pitiayumi contain two bands. the corresponding region of the g. gallus (ladjali et al. 1999) is similar, inferring that a possible fusion is related to the increase in bands in b. culicivorus. passeriformes have a unique evolutionary history for the 5th chromosome pair, where it is assumed to have occurred by fission of the short arm of chromosome 1 in the putative ancestral karyotype (pak) (kretschmer et al. 2018b). using g. gallus (gga) probes in passeriformes, gga1 usually hybridize two distinct chromosome pairs, for example, in saltator aurantiirostris (thraupidae) the second and fifth pairs (dos santos et al. 2015). the b. culicivorus chromosome 6 shows numerical conservation of positive and negative bands compared to the corresponding chromosome in g. gallus (ladjali et al. 1999), which contains 7 bands in region 1 of the long arm. m. leucoblepharus and s. pitiayumi showed 5 bands for the same chromosome in this region. a possible break followed by pericentric inversion may have occurred, resulting in the changes found in biarmed chromosome, which has 2 bands in region 1 of the short arm in the two species. this is a rearrangement type that has been previously found in treron phoenicoptera and synallaxis frontalis by gtg-bands (gupta and kaul 2014; de souza et al. 2019). morphology and number of bands in chromosome 7 found were similar in m. leucoblephara and b. culicivorus, which contained the same number of bands in the corresponding chromosome of g. gallus (ladjali et al. 1999). however, s. pitiayumi shown a reduction in the number of bands in region 1 and morphological difference in this chromosome. possibly, a fission in the terminal region of the short arm might have caused this reduction of bands and morphological differentiation in s. pitiayumi. in this perspective, multiple fragments of sites interstitial were found in non-telomeric regions in turdus merula (passeriformes), implying how active these regions are in relation to chromosomal rearrangements (nanda et al. 2002). in chromosome 8, region 1 of long arm, b. culicivorus showed similar patterns of bands number the g. gallus (ladjali et al. 1999). m. leucoblephara and s. pitiayumi had an increase in bands, thus inferring the occurrence of a fusion in the telomeric region. nevertheless, chromosomes 9 and 10 of the three species maintained morphological and numerical band similarities. the difference between m. leucoblephara and s. pitiayumi in chromosome 8 is a chromosomal rearrangement caused by fusion in the terminal region of the long arm, considering that b. culicivorus species has a similar pattern found in g. gallus (ladjali et al. 1999) in the correspondent chromosome. the telomeric region is an area rich in repetitive sequences which have been reported as hotspots of chromosomal fusion and fission (nanda et al. 2011). the similarities of chromosomes 9 and 10 of the three species suggest conservation. in the three species, z chromosome presented high evolutionary stability in terms of morphology and band patterns. in many passeriformes, the z chromosome has the same submetacentric morphology (kretschmer et al. 2018b). furthermore, some studies have shown that there is a high syntenic degree of this chromosome among several families of this group (griffin et al. 2007). it is important to emphasize that gtg and rbg bands analyses have already identified a paracentric inversion in the terminal region of the long arm of the z chromosome in alectoris chukar (galliformes) (ouchia and ladjali 2018). signals of hybridization in this chromosome also demonstrated that the accumulation of the repetitive sequences are responsible for the main cause of its enlargement, as in myiopsitta monachus (psittaciformes) (furo et al. 2017) and nyctibius griseus (caprimulgiformes) (de souza et al. 2020). in conclusion, the cytogenetic analyses performed in this study in the three parulids species provided an accurate description of the karyotypic structuring. through cbg, gtg, and rbg bands, the information was obtained on chromatic patterns and chromosomal rearrangements which should be analyzed by molecular cytogenetic techniques in the future. our results support the identification of speciation processes at the karyotypic of this group. acknowledgements the authors would like to thank all colleagues from the grupo de pesquisa diversidade genética animal from the universidade federal do pampa and dr. rafael kretschmer for comments on the manuscript. statement of ethics the protocols used in t his experiment were approved by the ethics 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www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-788 caryologia international journal of cytology, cytosystematics and cytogenetics citation: v. neves, w. viegas, a. d. caperta (2020) effects of high temperature on mitotic index, microtubule and chromatin organization in rye (secale cereale l.) root-tip cells. caryologia 73(4): 55-63. doi: 10.13128/caryologia-788 received: december 20, 2019 accepted: july 27, 2020 published: may 19, 2021 copyright: © 2020 v. neves, w. viegas, a. d. caperta. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. funding statement: this work was funded by portuguese national funds through fundação para a ciência e a tecnologia (www.fct.pt/) project ptdc/agrpro/4285/2014, ui/ agr/04129/2013, and grant leafagr/04129/bpd/2015 to adc. effects of high temperature on mitotic index, microtubule and chromatin organization in rye (secale cereale l.) root-tip cells vânia neves, wanda viegas, ana d. caperta* linking landscape, environment, agriculture and food (leaf), instituto superior de agronomia (isa), universidade de lisboa, tapada da ajuda, 1349-017 lisboa, portugal *corresponding author. e-mail: anadelaunay@isa.ulisboa.pt abstract. stressful high temperatures on plants can limit whole-plant function and decrease crop productivity. however, little is known regarding heat stress effects on microtubule cytoskeleton and chromatin in roots from intact plants. here we studied high temperature effects on cell division, microtubule and chromatin organization patterns in rye root tips from intact plants subjected to 40ºc for 4 h and after different recovery periods (0rt, 7rt, 24 rt). we showed that heat stress induced changes in nuclear morphology as detected by the unusual presence of interphase cells with irregularly shaped nuclei, probably associated with changes in chromosome segregation at anaphase, leading to micronuclei formation as well as changes in the mitotic index. these alterations were associated to differential effects in microtubules organization in both heat-stressed interphase and mitotic cells at 0rt and 7rt. although no changes in the distribution of h3 phosphorylation of ser 10 residues on chromatin were found in cells from heat-stressed plants, marked alterations in chromatin dna methylation patterns were detected. these effects included higher agglutination of 5-methylcytosine domains in both interphase and metaphase cells compared to controls. taken together these results seem to suggest that alterations in microtubule conformation upon heat stress influences nuclear chromatin organization and cell cycle progression. however, when seedlings recovered from stress (24rt), root tip cells presented microtubule configurations and chromatin organization patterns similar to controls. we conclude that in spite of heat stress markedly altered cell cycle progression and distribution of epigenetic marks, these responses are transient to cope with such stress conditions in the roots. keywords: dna methylation, heat stress, histone h3 ser 10 phosphorylation, microtubules, root. introduction high temperature is one of major environmental factors limiting crop growth and yield worldwide causing many physiological changes that affect crop yield and quality (suzuki et al. 2014). most studies on these effects focus on the above-ground tissues such as shoot and reproductive organs, 56 vânia neves, wanda viegas, ana d. caperta although roots can also be subjected to high temperature stress which can limit whole plant function and decrease productivity (heckathorn et al. 2013). a high degree of complexity in plant responses at the molecular, physiological and biochemical levels were described, largely controlled by different, and sometimes opposing, signaling pathways that may interact and inhibit each other (suzuki et al. 2014). however, knowledge concerning combined microtubule (mt) cytoskeleton organization and chromatin nuclear topology upon heat stress in intact plants is scarce, particularly in roots. in the plant cell cycle, the mt cytoskeleton composed of heteropolymers of αand β-tubulin undergo dynamic conformational changes in a process known as dynamic instability in response to the needs of the cell (horio and murata 2014). particularly, during cell division in somatic cells, mts are arranged into characteristic structures like the interphase cortical mts (cmt), pre-prophase band, mitotic spindle and phragmoplast (baluska et al. 1998). moreover, mts can undergo a number of posttranslational modifications that act to control specific mts-based functions in plants, including tyrosination, detyrosination, acetylation (smertenko et al. 1997a), phosphorylation (blume et al. 2008), polyglutamylation (wang et al. 2004), and transamidation (del duca et al. 2009). in plant cells disruption of mts occurs in response to various environmental factors namely to extreme temperatures such as heat stress (nicotiana tabacum, smertenko et al. 1997a; smertenko et al. 1997b; arabidopsis thaliana, müller et al. 2007), low temperature and abscisic acid treatments (triticum aestivum, khokhlova et al. 2003), hyperosmostic stress (triticum turgidum, komis et al. 2002), and affects post-translational modifications of tubulin as in cadmium stress (glycine max, gzyl et al. 2015). furthermore, plant mts in addition to their role in cell division and axial cell expansion, also have a thermosensory function that is of agronomical relevance in osmotic or cold stress conditions (triticum aestivum, abdrakhamanova et al. 2003; nick 2012). moreover, plants response to drought, cold and high salinity stress involve several epigenetic regulatory mechanisms like both dna and histone methylation and generation of small rnas implicated in genome regulation and structure (mirouze and paszkowski 2011; asensi-fabado et al. 2017). recent research has shown that environmental cues and abiotic stresses activate a stress memory that is mediated by epigenetic and chromatin-based mechanisms including chromatin modifications, such as cytosine methylation of dna, histone methylation and nucleosome occupancy (lämke and bäurle 2017). for example, exposure of arabidopsis plants to stresses, including salt, uvc, cold, heat and flood, resulted in a higher homologous recombination frequency, increased global genome methylation, and higher tolerance to stress in the untreated progeny. however, this transgenerational effect did not persist in successive generations (boyko et al. 2010)..by contrast, prolonged heat stress induces transcriptional activation of several repetitive elements of arabidopsis thaliana that requires minor changes in histone modifications but does not involve dna demethylation (pecinka et al. 2010). patterns of dna and histone modification can be altered in root tip cells of soybean seedlings grown at different temperatures (stępiński 2012). during male meiosis in secale cereale plants with b and without b chromosomes, heat exposure causes differential anomalies in chromatin structure in pachytene cells (pereira et al. 2017). heat-damaged pachytene cells displayed easily recognizable paired chromosome fibres and a single amorphous and heterochromatic mass closely associated with the nuclear periphery as well as disruption of the organization of sub-telomeric chromosome regions. however, no changes in dna methylation patterns were detected in untreated and treated plants (pereira et al. 2017). in this work we investigated the effects of heat stress (40ºc 4 h) on the organization of mt arrays and chromatin in rye root tips from intact seedlings, immediately after stress (0rt) and at different recovery periods (7rt, 24rt). in-depth cytological analyses of chromatin and microtubular organization were performed in root tip cells with dapi, and immuno-labelling with antibodies against tubulin, 5-methylcytosine (5-mc) and histone h3 phosphorylated at serine 10 residue (h3s10ph). materials and methods plant material and heat stress conditions rye (secale cereale l., 2n = 14 chromosomes) seeds were kindly supplied by neil jones (aberystwyth, uk). seeds were placed in petri dishes with moistened filter paper in the dark at 4°c for 3 days. seedlings were transferred to a growth chamber with controlled lighttemperature (rumed), with a photoperiod of 18 h light and 6 h dark, at 25°c ± 2ºc for 2 days. then, seedlings were subdivided in two sets of experiments: (i) kept at 25°c ± 2ºc with a photoperiod of 18 h light and 6 h dark (controls); and (ii) exposed to a ramp of increasing temperature from 25ºc to 40ºc (temperature increases 2°c / h), remaining 4 h at 40°c, after which the temperature fell gradually to 25°c (temperature decreases 2°c / h). the heat stress temperature was chosen based 57effects of high temperature on mitotic index, microtubule and chromatin organization in rye root-tip cells on agronomical relevant temperatures shown to have a significant effect in cool season grasses such as rye (xu, zhan et al. 2011). all seedlings were kept moist during heat stress. root tips were collected from plants in control conditions and from treated plants immediately after exposure to heat stress 40°c (0rt), and 7 h (7rt) and 24 h (24rt) during stress recovery. immunolabelling for immunostaining of α-tubulin root tips were prepared as described in caperta et al. (2006). briefly, roots were fixed in freshly prepared 4% paraformaldehyde solution (pfa) containing mt stabilizing buffer (1xmtsb) for 45 min at room temperature, and then rinsed twice in 1xmtsb for 5 min. for h3s10ph immunodetection, root tips were fixed in freshly prepared 4% pfa solution containing phosphate-buffered saline (1xpbs, ph 7.3) for 30 min, and then washed three times for 5 min in 1x pbs according to caperta et al. (2008). immunostaining of 5-mc was performed in root tips fixed in ethanol:acetic acid (3:1) as described in carvalho et al. (2010). for both h3s10ph and 5-mc immunolabelling, root tips were digested by treating with a pectolytic enzyme mixture [2% cellulase (sigma), 2% cellulase ‘‘onozuka r-10’’ (serva), and 2% pectinase enzyme (sigma) solution in 1xeb at 37º] until the material became soft. the macerated material was squashed in 1xpbs or 1xmtsb on a slide. slides were incubated for 1 h at 37ºc in a moisture chamber with a blocking solution (3% bovine serum albumin (bsa) in 1x pbs/1xmtsb, 0.1% tween 20), followed by an incubation at 10ºc overnight with the primary antibody diluted in 1xpbs/mtsb supplemented with 1% bsa. after three washes in 1xpbs/ mtsb for 5 min, the secondary antibodies diluted in 1xpbs/mtsb supplemented with 1% bsa were applied for 45 min at 37ºc. after final washes with 1xpbs, slides were counterstained with 4’6-diamino-2-phenylindole (dapi) and mounted in 1mg/ml citifluor antifade medium (af1, agar scientific). for mts immunolocalization a mouse monoclonal antibody to α-tubulin (clone dm1a, sigma, 1:100) was used, and the tyrosinated form of α-tubulin was detected with a rat antibody (yol 1/2, serotec, 1:100). an antimouse alexa 488 antibody (molecular probes) and an anti-rat antibody conjugated with biotin (serotec, 1:200) were used as secondary antibodies. this latter antibody was further detected with a streptavidin-cy3 conjugate antibody (sigma, 1:700). for detection of h3s10ph, a rabbit antibody (upstate, 1:200) was utilized and revealed using anti-rabbit rhoda mine-conjugated antibody (dianova, 1: 100). for revealing 5-mc a primary mouse antibody (abcam, 1:200) and a secondary antibody antimouse-cy3 (sigma, 1:100) were used. after three final washes, the slides were counterstained with dapi and mounted in 1mg/ml citifluor antifade medium (af1, agar scientific). all samples were examined using a zeiss axioskop 2 epifluorescence microscope, images were obtained using a zeiss axiocam digital camera, and the digital images were processed with photoshop (adobe systems). quantitative analysis of cell cycle progression and mt organization the nuclear morphology of well-preserved cells from control and in distinct recovery periods (0, 7 and 24 rt) after treatment was determined and the percentage of dapi stained cells with either regular or irregular shaped nuclei, and cells with micronuclei were calculated. cell cycle was evaluated by calculating the mitotic index as the percentage of mitotic cells identified in at least n = 200 cells for each treatment. the number of mitotic cells at different phases was moreover evaluated through tubulin immunolocalization of particular mts configurations (e.g. preprophase band, mitotic spindle or phragmoplast) in both control and heat-stressed cells after distinct periods of stress recovery. effects of heat stress on cmts organization were also evaluated in 200 cells from each treatment through the quantification of cells with normal or new mts arrangements in distinct recovery periods. the chi-square test (χ2, p< 0. 05) was utilized for statistical analysis. results and discussion changes in nuclear morphology and in mitotic index after heat stress are associated with new, transient mt arrangements interphase cells were classified as normal, when nuclei present regular shape and well-defined contour; abnormal, those showing nuclei with irregular shape; and cells with micronuclei. most control cells (n = 200) showed normal interphase nuclei (94%, table 1; fig. 1a,b) and the mitotic index was 6%, but decreased immediately after heat stress (0rt – 3%, n=302). in 0rt cells a significant difference in nuclei types was detected in comparison with controls (χ2 = 35.31, p< 0. 05), with an increase in the frequency of cells with abnormal nuclei (21%) and cells with micronuclei (3%). at 7rt cells (n = 300) significant differences between 58 vânia neves, wanda viegas, ana d. caperta heat-stressed cells and controls were found (χ2 = 92.36, p< 0.05) with a frequency of abnormal nuclei more than doubled (41%), and a small increase in cells with micronuclei (6%). the observed increase of micronuclei frequency attributable to root tips heat exposure is moreover associated with heat stress effects detected on mitotic cell cycle progression. the mitotic index was three times higher at 7rt (18%, table 1) than in controls. contrastingly, 24rt cells (n = 275) presented a high frequency of normal nuclei (90%) and a decrease in mitotic index (9%). the frequency of abnormal nuclei (7%) and micronuclei markedly decreased (3%). these findings support the hypothesis of mitotic arrest after 7rt of exposure to heat stress. root tips exposure to diverse chemical substances including fertilizers, heav y metals, herbicides, pesticides and radioactivity also affect the mitotic index in varying frequencies in allium cepa (bonciu et al. 2018). nonetheless, contrasting effects in the mitotic index were also observed in secale cereale plants exposed to chemical stresses like the mts-depolymerizing agent colchicine where mitotic arrest occurred in the lowconcentration treatment, whereas c-metaphase cells were able to progress into the cell cycle in the high-concentration treatment (caperta et al. 2006). heat stress effects in nuclear morphology can also result from perturbations of cmts organization as previously described for colchicine treatments (caperta et al. 2006). mts configurations were analyzed in untreated and heat-treated root tips through tubulin immunolocalization using antibodies that recognize α-tubulin (dm1a) and tyrosinated tubulin (yol 1/2). our results show that both antibodies presented coincident and similar immuno-signal distributions (fig. 2). control cell mts exhibited both α-tubulin and tyrosinated tubulin arrays table 1. percentage (%) of interphase cells with nuclear normal morphology (dapi), tubulin immunolabeled cells in interphase (cmts) and mitosis, and mitotic index. control and heat-stressed cells analyzed after 0 (0 rt), 7 (7 rt), and 24 (24 rt) h of recovery from the stress. the presence of cortical microtubules (cmt), preprophase band (ppb), mitotic spindle (sp) and phragmoplast (p) was scored. seedlings treatments frequencies (%) of dapi stained interphase cells with normal nuclear topology mitotic index frequencies (%) of interphase cells with typical organized cmts arrays frequencies of mitotic cells (%) at distinct phases number of mitotic cells analysedppb sp p control 94a 6a 98a 64a 24a 12 33 0 rt 76b 3b 16b 78b 11b 11 37 7 rt 53c 18c 37c 48c 27a 25 48 24 rt 90a 9d 95a 33d 48c 19 60 figure 1. dapi-stained secale cereale meristematic interphase root cells. 1a – control cells with a well-defined contour; and 1b. heat-stressed cells with irregular nuclei and/or with micronuclei (arrowed). bar: 5 μm. figure 2. indirect immunodetection of α-tubulin (mt) in control cells. nuclei, chromatin and chromosomes are stained with dapi (blue). tubulin containing arrays are detected with dm1a α-tubulin (green) and yol1/2 tyrosinated α-tubulin (red) antibodies. aa’’ interphase cell with cortical microtubules; b-b’’ prophase cell with the preprophase band; c -c’’ meta/anaphase cell with spindle; dd’’ ana/telophase cell with the phragmoplast. bar: 5 μm. 59effects of high temperature on mitotic index, microtubule and chromatin organization in rye root-tip cells configurations characteristic of higher plant cells: cmts at interphase, pre-prophase band, mitotic spindle and phragmoplast at the end of telophase (fig. 1). control cells presented organized cmts (98%, table 1), whereas at 0rt significant differences occurred (χ2 = 687. 57, p< 0.05), in which the majority of cells exhibited branched and wavy cmts with a disorganized orientation (84%) (fig. 3a-a’). previous studies showed that heat stress caused dissemblance of mts in mitotic tobacco suspension cultured cells after 30 min at 42ºc (smertenko et al. 1997b). in the present study we showed higher resilience of rye mts to heat stress since exposure of 2 days seedlings to 4 h at 40ºc only induced cmts disorganization at 0rt without total disruption. however, knowledge is inexistent with regard to the heat response of the rye variety used in this study (heat-resistant or heat-sensitive). compared to controls, at 7rt there was still a significant difference in the frequency of interphase cells with a disorganized and branched wavy-like mts configuration (63%, table 1, fig. 3d-d’) (χ2 = 424. 97, p< 0.05), although the frequency of interphase cells with normal mts arrangements doubled. at 24rt 95% of interphase cells present typical organized cmt arrays (table 1). disorganized wavy mts arrangements were also observed in root cells exposed to high colchicine concentration conditions (lazareva et al. 2003; caperta et al. 2006). therefore, it is tempting to suggest that new mts re-orientations result from perturbations induced by heat stress on cmts associations with plasma membrane, although the nature of mt attachments to the membrane is not yet totally clear both in animal (wolff 2009) and in plant cells despite all efforts to understand it (liu et al. 2015). it was demonstrated that cmts can change their orientation in response to a broad range of abiotic signals (nick 2013) by controlling the direction of cellulose deposition and reinforcing axial cell expansion (geitmann and ortega 2009). in the current work, the high frequency of interphase cells with abnormal nuclear morphology observed at 7rt probably reflects changes in cmts organization by inducing differential cytosol compartmentalization. earlier studies moreover reported that different mts arrays presented distinct sensitivities to temperature stress (smertenko et al. 1997b; abdrakhamanova et al. 2003; müller et al. 2007). the most heat-sensitive mt arrays are those of the mitotic spindle and the phragmoplast in tobacco cultured cells (smertenko et al. 1997b). in triticum chilling sensitive species, cmts are extremely cold-sensitive, whereas they persist at low temperatures in chilling-tolerant species (abdrakhamanova et al. 2003). in this study, mts from the pre-prophase band (ppb) appeared to be very sensitive to heat stress since most prophase cells at 0rt (94%) presented slightly disorganized pre-prophase bands (fig. 3b-b’). such marked changes in frequencies of prophase cells with abnormal ppb at 0rt were however not associated with perturbations in other mitotic phases since all metaphase (fig. 3c-3c’) and anaphase cells exhibited well-formed spindles, and telophase cells showed phragmoplast mts orthogonally disposed to the division plane. after 7rt, most prophase cells presented a reduction of abnormal pre-prophase bands (65%), although in some cells altered spindle and phragmoplast configurations were revealed (fig. 3f-f ’), which were absent at 24rt. compared to controls, the frequency of cells with ppbs presented the highest value at 0rt (78%) and the figure 3. indirect immunodetection of α-tubulin (mt) in heatstressed cells after 0 (0 rt), 7 (7 rt), and 24 (24 rt) h of recovery from the stress. nuclei, chromatin and chromosomes are stained with dapi (blue). tubulin containing arrays are detected with yol1/2 tyrosinated α-tubulin (red) antibody. a-a’ and d-d’ interphase cells showing branched, stringy cmts with disorganized orientation; b-b’ prophase cell with a slightly disorganized pre-prophase band with mts without the usual parallel orientation; metaphase cell (c-c’) and anaphase cell (e-e’) with normal spindles; f-f ’ telophase cell with thick cmts arrays and with remnants of phragmoplast. bar: 5 μm. 60 vânia neves, wanda viegas, ana d. caperta lowest one at 24rt (33%). these findings contrasted with frequencies of cells with spindles and phragmoplasts, which have low frequencies at 0rt (22%) and high frequencies at 7rt (52%), with a maximum of at 24rt (67%). taken together, the drastic dropping of mitotic index values at 0rt seems to be associated with perturbations on cmts organization as well as disturbances on ppb allowing however the progression of subsequent mitotic phases as only cells with normal spindles and phragmoplasts were detected. on the other hand, at 7rt the observed beginning of normal cmt organization reestablishment seemed to allow cells transition to mitosis associated with the marked increase in the mitotic index. the high frequencies of cells in metaphase, anaphase and cytokinesis at 7rt, associated with some perturbations in spindle and phragmoplast organizations appear to indicate why heat-stressed cells stay longer in mitosis. after 24 rt the mt cytoskeleton configurations in interphase and prophase cells were like the ones observed in controls, although the high frequencies of cells at metaphase, anaphase and cytokinesis revealed a delayed reestablishment reflected in the high mitotic index yet observed. nonetheless, it is not yet clear which microtubule structures, cortical mts or mitotic mts, are susceptible to tubulin modification induced by heat stress. heat stress induces changes in dna methylation patterns but no alterations in h3s10ph distribution patterns in both control and heat-stressed cells h3s10ph marks were absent during interphase. however, they were present in prophase cells in the pericentromeric heterochromatin and restricted to one nuclear hemisphere revealing rabl configuration (fig. 4). the rabl configuration, characteristic of rye genome (caperta et al. 2002) is maintained after heat stress as centromeres are all aligned in one nuclear pole. in interphase cells no labelling was found (fig. 4a-a’), which contrasted with prophase (fig. 4b-b’), metaphase (fig. 4a-a’ and c-c’) and anaphase (fig. 4a-a’) cells. in mitotic cells, a marked labelling was also found in chromosomes pericentromeric regions but no detectable changes were observed in signal dimensions or intensities in both control and heat-stressed cells (figs. 4d-d’, 4e-e’, 4f-f ’). instead, cold treatment of plant root meristems resulted in additional chromosomal sites of h3s10ph, besides the usual ones in pericentromeric regions (manzanero et al. 2000). also, up-regulation of the stress-inducible genes in arabidopsis t87 and tobacco by-2 cell lines is associated with increased phosphorylation of histone h3 at serine 10 residue at high salinity, cold and abscisic acid treatments (sokol et al. 2007). control cells showed interphase nuclei with disperse, dotted and intense 5-mc immunosignal all over the nucleus both in highly condensed (heterochromatin) and decondensed (euchromatin) chromatin (fig. 5a-a’’). instead, in heat-stressed cells at 0rt and 7rt a distinct, heterogeneous dna methylation distribution pattern with aggregated immunosignal was preferentially found more concentrated in the nuclear periphery (fig. 5c-c’’ and 5d-d’’). in metaphase cells, at both 0rt and 7rt a heterogeneous and discontinuous 5-mc labeling was also detected along chromosome arms (fig. 5e-e’’). after 24 rt, the distribution patterns of 5-mc were like those observed in control interphase and metaphase cells (fig. 5f-f ’’’). the results presented here in rye somatic cells from control plants are in accordance with earlier studies in rye metaphase chromosome spreads, which displayed a punctuated and uniform pattern of methylated dna residues along both the as and bs chromosomes, without any particular sites of accumulation (carchilan et al. 2007). however, no differences were found in the nuclear distribution of methylated cytosines between meiocytes of heat-stressed and control rye plants with figure 4. indirect immunodetection of histone h3 phosphorylated at serine 10 residue in secale cereale meristematic root cells. nuclei, chromatin and chromosomes are stained with dapi (blue). chromatin and chromosomes are detected with histone h3 phosphorylated at serine 10 residue (h3s10ph, green) antibody in control (a-a’c-c’) and heat-stressed cells (d-d’f-f ’). a-a’ interphase cell without h3s10ph labelling and an anaphase cell with h3s10ph marks; b-b’ prophase cells showing h3s10ph marks in the pericentromeric heterochromatin and restricted to one nuclear hemisphere revealing rabl configuration; c-c’ metaphase cell presenting h3s10ph dots only in a discrete region in pericentromeric chromatin; d-d’ cell at interphase showing no h3s10ph labelling and a metaphase cell with pericentromeric h3s10ph immnosignals; e-e’ cell at prophase with h3s10ph marks in the pericentromeric heterochromatin; and f-f ’ early anaphase cell revealing h3s10ph dots only in pericentromeric chromatin. bar: 5 μm. 61effects of high temperature on mitotic index, microtubule and chromatin organization in rye root-tip cells 0b or 2b chromosomes (pereira et al. 2017). a study on the effects of heat stress in the repetitive sequence genome fraction (coding and non-coding sequences) using leaves of intact rye plants suggests marked differences between these sequences that most likely reflect their distinct roles in the plant pathways involved in the stress response (tomás et al. 2013). hence, it appears that there are different temperature chromatin sensitivities accordingly with chromatin fractions, cell types and tissue origins. for instance, in soybean at normal temperature, root hairs were more hypermethylated than were stripped roots whereas in response to heat stress, both root hairs and stripped roots showed hypomethylation (hossain et al. 2017). these changes in dna methylation were directly or indirectly associated with expression of genes and transposons within the context of either specific tissues/ cells or heat stress (hossain et al. 2017). other studies in which both roots and above-ground tissues were heat-stressed indicate that roots are often more sensitive to heat stress than shoots in terms of cell division and physiological responses (heckathorn al. 2013). we conclude that heat stress induces transient changes in chromatin and mt organization rye root seedlings, which might indicate cell adjustments in stressful 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biomembranes 1788(7):1415-1433. xu y, zhan c, huang b. 2011. heat shock proteins in association with heat tolerance in grasses. int j proteomics. 2011:2011. caryologia. international journal of cytology, cytosystematics and cytogenetics 75(1): 155-164, 2022 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-956 caryologia international journal of cytology, cytosystematics and cytogenetics citation: masoomeh hasanbarani, fariba sharifnia, mostafa assadi (2022) molecular insights on some iranian species of delphinium l. and aconitum l. (ranunculaceae). caryologia 75(1): 155-164. doi: 10.36253/caryologia-956 received: may 28, 2020 accepted: november 27, 2021 published: july 6, 2022 copyright: © 2022 masoomeh hasanbarani, fariba sharifnia, mostafa assadi. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. molecular insights on some iranian species of delphinium l. and aconitum l. (ranunculaceae) masoomeh hasanbarani1,*, fariba sharifnia2, mostafa assadi3 1 department of biology, science and research branch, islamic azad university, tehran, iran 2 department of biology, north tehran branch, islamic azad university, tehran, iran 3 department of botany, agricultural research education and extension organization (areeo), research institute of forest and rangelands, iran *corresponding author. e-mail: mh_plantbiology@yahoo.com abstract. to be precise, 29 taxa of delphinium and 2 species of aconitum belonging to iran have been documented in flora iranica. in this research, with regard to both mentioned genera, a total of 25 species for the chloroplast trnl-f region and 11 species for the internal transcribed spacer (its) were investigated. after genome extraction, pcr and the sequencing of samples, the sequences were edited, and phylogenetic trees were prepared using bayesian methods. the phylogenetic study of this genera led us to the monophyletic aspect of them despite the segregation of aconitum and delphinium in their related classic taxonomy. it has been observed that there are some complicated species in the genus delphinium. the results of molecular analysis confirmed the separation of delphinium elbursense, delphinium speciosum, delphinium crispulum and delphinium dasycarpum (the complicated species of northern and northwestern iran). furthermore, based on the molecular results, it is suggested for d. elbursense var. gymnobotrys to have a higher taxonomic level as a distinct species. meanwhile, delphinium tuberosum, delphinium cyphoplectrum, delphinium quercetorum, delphinium pallidiflorum, and delphinium laxiusculum (western and northwestern species of iran), which are regarded as complex species, were placed in a distinct molecular tree. at the end, delphinium dolichostachyum was reported as a new record for iran, and the species has been compared to the related species delphinium carduchorum. keywords: pcr, bayesian, monophyletic, its, new record. introduction it has been reported that the family ranunculaceae contains five subfamilies, 43 genera and 2346 species at the present time (christenhusz & byng, 2016). the tribe delphinieae (aconitum l., delphinium l., consolida (dc.) gray, aconitella spach ) comprises 650-700 species, which amounts to some 25% of all ranunculaceae (jabbur & renner, 2012), and is distributed in the temperate regions of the northern hemisphere (tamura 1990; stevens 2001). the key feature of this tribe is the nectar placed in inner 156 masoomeh hasanbarani, fariba sharifnia, mostafa assadi tepal (jabbour & renner 2012; ilarslan et al. 1997; espinosa et al. 2017). based on flora iranica (iranshahr 1992), 29 species of delphinium and 2 species of aconitum are reported from iran. iranian species of delphinium are divided into two subgenera (differences between subgenera are in the form of seed and vegetative period): olighophyllon dimitrova and delphinium, which are perennial and annual species, respectively (iranshahr 1992; beltran et al., 2021; cabusora et al., 2020; fikirie et al., 2020). iranian aconitum species are also divided into two subgenera, which are aconitum and lycoctonum dc. (the difference between subgenera is the shape of galea) (iranshahr 1992). mobayen (1985) reported for the flora of iran; d. dasycarpum stev. ex dc., d. venulosum boiss. and d. micranthum boiss. & hohen. sharifnia et al. (2013) recorded d. kurdicum boiss. & hohen. for the first time for the flora of iran. recently, d. khorasanicum sharifnia & hasanbarani was reported as a new species from khorasan province (hasanbarani et al. 2017). in general, several studies have been carried out on the delphinieae in the world; for instance, seed morphology of 28 delphinium l. species has been studied (ilarslan et al. 1997; mieso & befa 2020; mustafa 2020; varamesh et al., 2014; rajaei et al., 2020; fataei et al., 2013). ozpelic & uztiirk (2000) worked on the morphology and ecolog y of 12 populations of d. cyphoplectrum boiss. in turkey. palynology study of 21 taxa from delphinium has also been performed (bursali & dogan 2005). the molecular analysis of nuclear and chloroplast sequences of delphinieae were studied in the geographical range of asia, the mediteraneaen, north america and the mountains of east africa; the monophyly of the genus consolida dc, aconitum l. and delphinium l. was proved (jabbour & renner 2011, 2012). wang et al. (2013) reported that based on molecular markers gymnoaconitum (stapf ) wei wang & z.d chen differs from the other species of aconitum and other genera of the tribe delphinieae. xiang et al. (2017) conducted a broad phylogenetic analysis within ranunculaceae using matk sequence and performed a series of analysis using four molecular markers focused on the tribe. micromorphological characters of the genus delphinium l. (sensu lato) seeds and fruits were studied using microscopic techniques (hadidchi et al. 2019). in china based on observations on living plants in the field, together with examination of herbarium specimens, demonstrated that delphinium iliense (ranunculaceae) is highly variable in the indumentum of peduncles, pedicels, bracteoles, sepals and also in the shape of bracteoles and their position on pedicels (li et al. 2019). during a taxonomic study on delphinium species in 2013-2018 based on herbarium specimens (tari) and also taking into account the descriptions and images of types, 31 species of delphinium were detected (table 1); among them, the subgen. delphinium includes the annual species: d. venulosum boiss. and d. peregrinum l., whereas the subgen. oligophyllon comprises perennial species, which have either tuberiformis or nontuberiformis roots (root form is one of the characters that is used in flora iranica delphinium key). d. speciosum m.b., d. lanigerum boiss. & hohen., d. elbursense rech.f., d. crispulum rupr. and d. dasycarpum stev. ex dc. are characterized by a non-tuberiformis root. these species have a similar distribution, and they are morphologically very closely related. the other species in the table 1. delphinium species in iran (following taxonomic studies of this genus in 2013-2017, endemic species are bold). species root form d. aquilegifolium (boiss.) bornm. tuberiformis d. biternatum huth. tuberiformis d. carduchorum chowdhuri & davis tuberiformis d. cyphoplectrum boiss. tuberiformis d. crispulum rupr. non-tuberiformis d. dasycarpum stev. ex dc. non-tuberiformis d. dasystachyum boiss. & bal. tuberiformis d. dolichostachyum chowdhuri & davis tuberiformis d. elbursense var. elbursense rech.f. non-tuberiformis d. elbursense var. gymnobotrys rech.f non-tuberiformis d. ilgazense p.h. davis tuberiformis d. jacobsii iranshahr tuberiformis d. khorasanicum sharifnia & hasanbarani tuberiformis d. kurdicum boiss. & hohen. tuberiformis delphinium lanigerum boiss. & hohen tuberiformis d. laxiusculum (boiss.) rouy tuberiformis d. macropogon prokhanov tuberiformis d. micranthum boiss. & hohen. tuberiformis d.ochrolecum stev. ex dc. tuberiformis d. pallidiflorum freyn tuberiformis d. peregrinum l. tuberiformis d. quercetorum boiss. & hausskn tuberiformis d. szowitsianum boiss. tuberiformis d. speciosum m.b. tuberiformis d. semibarbatum bienert ex boiss. tuberiformis d. saniculifolium boiss. tuberiformis d. schmalhausenii alboff tuberiformis d. tuberosum auch. ex boiss. tuberiformis d. turkmenum lipsky tuberiformis d. venulosum boiss. tuberiformis d. zalil aitch. & hemsl. tuberiformis 157molecular insights on some iranian species of delphinium l. and aconitum l. (ranunculaceae) genus delphinium (d. cyphoplectrum boiss., d. tuberosum auch. ex boiss, d. laxiusculum (boiss.) rouy, d. pallidiflorum freyn, and d. quercetorum boiss. & hausskn) have a tuberiformis root and non-yellow flower; they form a complex morphologically related species in this genus (iranshahr 1992). due to the large number of species distributed in iran and the controversies in taxonomical ideas among researchers, a taxonomic review of these species is required. moreover, we reported in our previous research that for the biosystematic study of delphinium species in iran, there is a strict necessity to have the help of molecular analysis methods to more confidently classify this genus. materials and methods plant materials in this research, in order to conduct molecular study, the plant materials were taken from central herbarium of iran (tari), and the samples were collected from the field dried on silica gel (this species is available in iaunt herbarium). it must also be mentioned that 25 species for the chloroplast marker (two species of aconitum) and 11 species for the its marker (one species of aconitum; aconitum iranshahrii endemic of iran and the sequences available in genbank) were investigated (table 2). dna extraction and pcr amplification total dna was extracted using the mbst kit (shayan et al. 2007). the amplification of dna fragments was carried out for its sequence and trnl-f region. the entire ribosomal its region was amplified using primers pairs ab101 (forward, 5 -acg aat tca tgg tcc ggt gaa gtg ttc g-3) and ab 102 (reverse, 5-tag aat tcc ccg gtt cgc tcg ccg tta c-3) (douzery et al. 1999), and the pcr reaction for nuclear marker was executed using a denaturation step of 5 min at 95c followed by 35 cycles of 30 s denaturation at 95c, 30 s of annealing at 56c, and 90 s extension at 72c, followed by a final extension step of 7 min at 72c. the trnl-f region was amplified using primers c (forward, 5-tac gac gat cty tct aaa caa gc-3) and f (reverse, 5gga aag att gct caa ata cca g-3) (taberlet & gielly 1991). the pcr reaction for chloroplast marker was carried out with a denaturation step of 5 min at 95c, followed by 35 cycles of 30 s denaturation at 95c, 30 s annealing at 54.4c, and 1 min extension at 72c, followed by a final extension step of 7 min at 72c. the pcr products were migrated on 1% agarose gel and were visualized by ethidium bromide. sequence alignment and phylogenetic analyses after sequencing, the sequences were edited using bioedit software ver. 7.0.9.0 (hall 1999) and then were aligned using the mesquite software (maddison & madtable 2. delphinium and aconitum species included in the molecular study (species used in its marker are shown with stars). species locality aconitum iranshahrii* mazandaran: polsefid, forest above village sangdeh, 1500-2500 m, assadi 73445. aconitum nasatum eeast azarbaijan: arasbaran protected area, doghrun mountain, 2500 m, assadi & sardabi 23945. delphinium aquilegifolium (boiss.) bornm. mazandaran: lar valley, 2450-2550m, wendelbo & assadi, 13264-tari. tehran: w of tehran, suleghun valley, 1500-2000m, assadi & mozaffarian 32699-tari. tehran:10 km from karaj, on chalus road, 1750m babakhanlu & amin 20004-tari. d. cyphoplectrum boiss.* fars: kazerun, komaraj,980m, forughi 7930-tari. khusestan: 74128-tari. khuzestan: 47 km to masjedsoleiman from haftgel, assadi & abohamzeh 38933-tari. d. crispulum rupr* ardabil: ca 9 km from khalkhal on the road to asalem, 2050m, assadi & shahsavari 66000-tari. west azerbaijan: khoy, hasan dehe-kan, 2500m, amini, 1716-tari. east azerbaijan: 35 km. ne of marand, kiamakidagh mt., assadi & olfat 68603, tari. east azerbaijan:23 km se of jolfa, near the geshlagh village, miaran, assadi & shahsavari 65786, tari. d. carduchorum chowdhuri & davis west azerbaijan: urumieh, mavana, kuhe dare rash, 2100-2700m, mozaffarian 74872-tari. d. dolichostachyum* d. dasycarpum stev. ex dc. kurdestan: baneh, 1650m, maroofi & fani 6959-tari. east azerbaijan: sahand mt., 2200m assadi & mozaffarian, 30641tari. d. dasycris= d. dasycarpum × d. crispulum east azerbaijan: 60 km n.e of maragheh, chagh-chagh pasture, 1850m, benvan 25028-tari. 158 masoomeh hasanbarani, fariba sharifnia, mostafa assadi dion 2010). some sequences were obtained from the genbank (table 3). the basis for the selection of taxon from the gene bank was the geographical distribution. phylogenetic relationships were assessed using bayesian inference (bi). the substitution model was obtained using the program mrrmodeltest ver. 2.3 (nylander 2004). gtr + g + i for nuclear dna and gtr + g for trnl-f region were identified as the best model for the dataset. the program mrbayes version 3.2 (ronquist & huelsenbeck 2003) was used for the bayesian reconstruction. after drawing several trees with different outgroups from ranunculaceae, the best results were obtained from these outgroups (nigella damascena for its marker and helleborus niger for trnl-f marker). species locality d. elbursense var. elbursense rech.* mazandaran: polesefid, forest above village sangdeh, 1500-2500m, assadi 73521& 73451-tari golestan: kurdkuy, 5-10 km from radkan to kurdkuy, 2200m, mozaffarian 78137-tari. mazandaran: polesefid, forest above village sangdeh, 1500-2500m, assadi 73521-tari. d. elbursense var. gymnobotrys rech. mazandaran: ramsar, s of javaherdeh, 2600-3200m, masassumi 56821-tari. mazandaran: siahbisheh, chalus valley, 2120m, sabeti 2056-tari. mazandaran: siahbisheh, chalus valley, 2100m, sabeti 1785-tari. mazandaran: siahbisheh.chalus valley, 2300m, sabeti 7964-tari. d. ilgazense p.h. davis* azerbaijan: tabriz, ahar road, 22 km to ahar, 1900-2000m, mozaffarian & mohammadi 37587-tari. d. khorasanicum sharifnia & hasanbarani khorassan: north west of neyshabur, bar fall, 2004 m, sharifnia and hasanbarani 16155 iaunt. d. laxiusculum (boiss.) rouy west azerbaijan: gooshchi pass, 1800m, siami & zehzad 7019-tari. ardabil: 45km from namin to germi, 220m, mozaffarian & nowrozi 34598-tari. ardabil:40 km from razi to germi, 1700m, mozaffarian & nowrozi 34762-tari. azerbaijan: kaleybar to jananloo, kiaragh, 1200m, hasanbarani 16785-iaunt. d. lanigerum boiss. & hohen. hamedan: alvand mt., 2700m, assadi & mozaffarian, 2700m 36809-tari. hamedan: near ganjnameh, 2100m, assadi & mozaffarian 36784-tari. tehran: shemiran, darband & passghale, 2000-2500m, mozaffarian & jamzad 43742-tari. d. micranthum boiss. & hohen. kurdestan: from baneh to saghez, kalawarash, 1900m, fattahi & hatami 2539-tari. kurdestan: saghez to baneh, nacarouz mt., 2500m, maroofi & mohammadi 6590-tari. 85470-tari. d. ochrolecum stev. ex dc. ardabil: 9km from diviation of kivi to ardebil road, above meresht village, 2000m, mozaffarian & nowrozi 34391-tari. west azerbaijan: urumieh, marmishu vally, 1737m, mozaffarian 87255-tari. d. pallidiflorum fyen* esfahan: fereydunshahr, near the village sibak, 2800m, assadi & khatamsaz 76521-tari. d. peregrinum l.* fars: nurabad, 22 km from fahilan to rashk, 900-1200m, mozaffarian 45975-tari. fars: 15 to 20 km from shiraz to esfahan, 1600-1900m, assadi & ranjbar 82991-tari. d. quercetorum boiss. & hausskn. east azerbaijan: ca. 20km w of marand, mountain above the village orlan, mishoudagh, 2000-2500, assadi & shahsavari 65472-tari. kurdistan: marivan, dizil,expose to iraq frontier, 2350m, maassumi & nickchehre, 80189-tari. kurdistan: 34km from chenareh to baneh, 1922m, assadi 85087-tari. d. schmalhausenii alboff kurdistan:kurdestan, ca. 17 km from baneh to marivan, 1740m, mozaffarian 87400-tari. d. speciosum m. b.* semnan: between shahrud and azadshahr, kuhe abr, 2600m, assadi & maassumi 21523-tari. golestan: n gorgan, ca 20 km charbagh toward gorgan, 1550m, assadi d. turkmenum lipsky semnan: touran protected area. 22 km from ghazaran to miandasht, 1240m, feritagh & jadidi 28987tari. khorassan: north west of neyshabur, bar fall, 2004 m, sharifnia and hasanbarani 17003iaunt. d. tuberosum auch. ex boiss. * west azerbaijan: ca. 15 km to maku on road from marand, 1200-1400m, assadi & mozaffarian 30110tari. hamedan 64503-tari. zanjan 29393-tari. east azerbaijan: kaleybar to jananloo, kiaragh, 1200m, hasanbarani 16798-iaunt. d. ursinum rech. gorgan: tanghegol forest, 700-1000m, wendelbo & forughi 12766-tari. mazandaran: 32592-tari. tehran: between ushan & tehran, 1730m, assadi & shahsavari 69764-tari. d. venulosum boiss.* lorestan: nowjian, (between khoramabad & keshvar) 1850m, runemark & lazari 26112-tari. ilam: 10 km n.w. of islam abad, ilam road, 1550m, seraj 24666-tari. 159molecular insights on some iranian species of delphinium l. and aconitum l. (ranunculaceae) results and discussion the bayesian analysis result for the trnl-f region with posterior probabilities (pp) is shown as consensus tree in fig. 1. the length of the trnl-f sequences included in the final matrix ranged from 950 to 1050 base pair. helleborus niger is taken as an outgroup. this cladogram has several groups: species of annual delphinium (clade d), perennial delphinium (clade e), consolida (clade c) and aconitum (clade b). this result is congruent with the achievement of the study of jabbour & renner (2011). clade (a) includes the aconitum, delphinium and consolida (delphinieae tribe); jabbour & renner (2012) have revealed the monophyly of delphinium and aconitum. delphinium species (both annual and perennial species) make a clade with a pp= 0.63 in which the annual and perennial species create two distinct groups as subgenus delphinium (d) and subgenus delphiniastrum (e). the bayesian analysis result for the its region is shown in fig. 2. nigella damascena was considered as an outgroup. the length of the its sequences included in the final matrix ranged from 600 to 700. there were several groups in consensus tree, similar to the results of trnl-f marker: annual delphinium (clade e), perennial delphinium (clade d), aconitum (clade b), and consolida (clade c). by examining the results of chloroplast and nuclear marker, d. dasycarpum (only in chloroplast tree), d. speciosum, d. crispulum, d. elbursense var. elbursense and d. elbursense var. gymnobotrys (only in chloroplast tree) were close to each other, in spite of the fact that they are distinct species. in the ussr flora, there are two subgenera: consolida and eudelphinium,. eudelphinium includes 3 sections: kolobopetala, elaptosis and diedropetala (komarov 1970). according to ussr flora, d. speciosum, d. crispulum and d. dasycarpum belong to the elaptosis section similar to our molecular study (chloroplast tree) which are all in the same group. these species have cylindrical root, dark blue flowers, black anther and lower petals which are black with yellow barbate. delphinium turkmenum, d. laxiusculum, d. quercetorum, d. schmalhausenii, d. szowitsianum, d. ochrolecum, table 3. genbank accession number taken from ncbi. species trnl-f genbank its genbank delphinium halteratum jf331737 delphinium leroyi jn73564 aconitum baicalense jf331723 aconitum ciliare jf331724 ab004952 aconitum delphinifollium jf331725 af258681 aconitum ferox jf331726 ab004961–2 aconitum pendulum jf331728 ay150235 aconitum pentheri jf331729 jf331905-18 aconitum racemolusom af258652 ay150233 2 aconitum septentrionale jf331730 af216552 aconitum tanguticum jn573573 ay15023 consolida ajacis jf331687 jf33188 consolida axilliflora jf331692 consolida flava jf331695 jf331887 consolida orientalis jf331707 jf331896 delphinium pyramidale jn573581 delphinium afgahnicum jn573529 delphinium albocoeruleum jn573530 delphinium bakeri af258652 af258697 delphinium balansae jf331732 delphinium bicolor af258711 delphinium brachycentrum jn573515 delphinium cardinale af258740 delphinium crassifolium jn573540 delphinium cuneatum jn573542 delphinium dasycaulon jn573544 species trnl-f genbank its genbank delphinium decorum af258744 delphinium delavayi af258705 delphinium dubium jn573568 delphinium elatum jn573549 delphinium favargeri jf331679 delphinium fissum jn573552 delphinium flexosum jn573553 delphinium gracile jf331736 af258763 delphinium gypsophilum af258721 delphinium hesperium af258772 delphinium hirschefeldianum jf331988-95 delphinium incisum jn573558 delphinium kohatense jn573561 delphinium maakianum jn573573 delphinium macropetalum jf331996-2000 delphinium muscosum jn573572 delphinium oreophilum jn573576 delphinium suave jn573596 delphinium verdunanse jn573596 delphinium virgatum jf332030-1 delphinium viscosum jn573597 delphinium wendelboie delphinium staphisagria jn573598 jf332022 helleborus niger aj413290 nigella damascene ay150260 160 masoomeh hasanbarani, fariba sharifnia, mostafa assadi figure 1. bayesian tree for chloroplast dna (trnl-f region). abbreviations: h. niger= heleborus niger; a. iran= a. iranshahrii; a. pubi= a. pubiceps aconitum pubiceps (rupr.) trautv. is a synonym of aconitum nasutum fisch. ex rchb. ; a. septen= a. septentrionale; a. tangu= a. tanguaticum; a. race= a. racemolusom; c. orien= c. orientalis; d. cris= d. crispulum; d. dasyc=d. dasycarpum;d. elb var. elb= d. elbursens var. elbursense; d. speci= d. speciosum; d. elb var. gy= d. elbursense var. gymnobotrys; d. lanige= d. lanigerum; d. ilgaz= d. ilgazense; d. dolico= d. dolichostachyum; d. card= d. carduchorum; d. micran: d. micranthum; d. schmal= d. schmalhausenii; d. bite= d. biternatum; d. semiba; semibarbatum; d. ochrol= d. ochrolecum; d. szowits= d. szowitsianum; d. turkm= d. turkmenum; d.cypho= d. cyphoplectrum; d. tuber= d. tuberosum; d. laxiusc=d. laxiusculum; d. pallidi= d. pallidifl orum; d. querc= d. quercetorum; d. aquil= d. aquilegifolium; d. khora= d. khorasanicum; d.pereg= d. peregrinum; d. venulo= d. venulosum, d. virga= d. virgatum, d. albocoe=d. albocoeruleum; d. viscos= d. viscosum; d. gris= d. griseum; d. sanicu= d. saniculifolium; d. dasycaul= d. daycaulon; d. macrost= d. macrostachyum; d. kurdi= d. kurdicum; d. shmal= d. schmalhausenii; d. kohaten= d. kohatense; d. dasycris= d. dasycarpum × d. crispulum. 161molecular insights on some iranian species of delphinium l. and aconitum l. (ranunculaceae) d. biternatum, and d. semibarbatum are placed in the diedropetala section (komarov 1970) and in fig. 1, and except for d. schmalhausenii the other species are placed in one group. delphinium schmalhausenii is very similar to d. kurdicum and d. fi ssum but diff ers in fl ower color (d. shmalhausenii is brown-violet), and there seems to be a new position for d. schmalhausenii as a variety of d. kurdicum instead of a being a species. also in diedropetala section, d. cyphoplectrum, d. pallidifl orum, d. laxiusculum, d. quercetorum and d. tuberosum (complex species) are closely related to each other (iranshahr 1992). in fl ora of iraq, d. tuberosum is synonymous with d. cyphoplectrum, d. quercetorum, d. pallidifl orum and d. laxiusculum (townsend & evan 1974). based on the molecular study (trnl-f marker), the separation of these species is confi rmed. d. elbursense is an endemic species in iran and rechinger has announced two varieties for this species that were distributed in azerbaijan and hyrcanian region (iranshahr 1992). in our research, the separation of these two varieties based on chloroplast marker was approved (fig 1). based on the molecular result, it is suggested that the taxonomic level of d. elbursense var. gymnobotrys be elevated to a higher level. moreover, the results of micromorphological tepal epidermal patterns study confi rmed that d. elbursense var. elbursense and d. elbursense var. gymnobotrys are different in the tepal epidermal patterns (hasanbarani et al. 2016). annual taxa in the genus delphinium are arranged in delphinium subgenus and from the morphology point of view they are diff erent from perennial species (lower petals in this subgenus are without lobe, whereas they are accompanied by lobe and barbate in perennial species), and based on its and trnl-f trees they are classifi ed as clade e and clade d, respectively. subgenus delphinium is divided into two section: sect. anthriscifolium w.t wang and sect. delphinium. th e figure 2. bayesian tree for nuclear dna (its marker). abbreviations: a. iran= a. iranshahrii; a. septen= a. septentrionale; d. kamao= d. kamaoense; d. cris= d. crispulum; d. elb= d. elbursene var. elbursense; d. speci= d. speciosum; d. tricho= d. trichoporum; d. ilgaz= d. ilgazense; d. cypho= d. cyphoplectrum; d. pallidi= d. pallidifl orum; d. dolicho= d. dolichostachyum; d. tuber= d. tuberosum; d.pereg= d. peregrinum; d. venulo= d. venulosum; d. balcan= d. balcanicum; d. hirschfel= d. hirschfeldianum; d. anthirisi= d. anthriscifolium; d. balan= d. balansae. 162 masoomeh hasanbarani, fariba sharifnia, mostafa assadi geogeraphic distribution of the two sections of subg. delphinium is disjunct; delphinium section is distributed in the irano-turanian region, whereas anthriscfolium section is distributed in the warm zone of central and southern china and northern vietnam (xiang et al. 2017); the same results are confirmed in fig. 2. in iran, only d. venulosum and d. peregrinum are in the subgenus delphinium and their morphological differences are in the form of lower petal; their separation is clearly evident in the molecular tree. our other research on delphinieae tribe has shown that the genus delphinium, aconitum and consolida are distinct base on morphological features (hasanbarani et al. 2020). pollen studies in iranian species of the genus delphinium prove that if the two species are morphologically similar, it does not mean that the two species are close pollen type (hasanbarani et al. 2019). for example, the d. venulosum and d. pregrinum, which form a clade in molecular studies, differ in shape of the pollen. d. cyphoplectrum and d. tuberosum which are separate in molecular studies were also different in pollen studies. in the study of the flower morphology in delphinium, annual taxa like morphological studies were placed in separate morphological phenogram (hasanbarani et al. 2018). some species that are similar in flower morphology studies were included in a separate phylogenetic study. new record for iran d. dolichostachyum chowdhuri & p. h. davis in notes r.b.g. edinb. 22: 408 (1958). locality: iran. kurdistan: baneh, kochar cemetery, 1650 m, maroofi & fani, 6959 tari. d. dolichostachyum was originally described from turkey (davis 1965). this species was collected from kurdistan (baneh) and is morphologically related to d. carduchorum, but differs from it mainly considering the following characters: bract length, spur length, flower color and plant length (table 4). according to the distribution area and morphological character, it may seem that this species is d. carduchorum at first sight. delphinium dolichostachyum image and the type specimen are presented in fig. 3 and 4. conclusion the present molecular data provide strong support for the monophyly of delphinium, aconitum and consolida, and therefore d. elbursense var. gymnobotrys could be at high taxonomic level as distinct species. d. dolichostachyum is newly recorded for the flora of iran. the separation of d. tuberosum and d. cyphoplectrum (controversial species) is confirmed by molecular results. acknowledgment the authors are grateful to tari herbarium for providing the samples. table 4. morphological characters useful in separating delphinium carduchorum and delphinium dolicostachyum. characters d. dolicostachyum d. carduchorum plant length 60 cm 100 cm bract length 5 mm 40 mm bract form linear trisect inflorescence panicle raceme spur form cylindrical attenuate spur length 9-10mm 15-16mm color of sepal pale blue dark blue color of petal white yellow figure 3. image of d. dolichostachyum (this species is available in tari). 163molecular insights on some iranian species of delphinium l. and aconitum l. 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testing of bovine lymphocytes exposed to epoxiconazole using alkaline and neutral comet assay. caryologia 73(4): 99-109. doi: 10.13128/caryologia-984 received: june 25, 2020 accepted: september 24, 2020 published: may 19, 2021 copyright: © 2020 s. koleničová, b. holečková, m. galdíková, v. schwarzbacherová, m. drážovská. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. genotoxicity testing of bovine lymphocytes exposed to epoxiconazole using alkaline and neutral comet assay simona koleničová1, beáta holečková1,*, martina galdíková1, viera schwarzbacherová1, monika drážovská2 1 university of veterinary medicine and pharmacy in košice, department of biology and genetics, slovak republic 2 university of veterinary medicine and pharmacy in košice, department of epizootiology and parasitology, slovak republic *corresponding author. e-mail: beata.holeckova@uvlf.sk abstract. epoxiconazole belongs in the class of azoles which have been developed to protect crops from fungal diseases. the mechanism of action of these fungicides is to inhibit the specific cytochrome p450 enzyme (cyp), cyp51 (lanosterol 14α-demethylase) which contributes to ergosterol biosynthesis. since ruminants and cattle are exposed to contaminants during grazing, they are a suitable experimental model for genotoxicity testing. in our experiment, epoxiconazole (epx) (active agent, 99% of purity) was tested in vitro for its potential genotoxic and cytotoxic effects on bovine lymphocytes, isolated from whole peripheral blood. we exposed the lymphocytes to epx at concentrations of 2.5, 5, 10, 25, 50 and 100 μg/ml by two different ways: immediately after isolation of lymphocytes during 2 h in rpmi 1640 medium (without phytohaemagglutinin, pha) as well as on the last 2 h of 48-h culture (with pha). in a second case, we chose 48 h culture because the lymphocytes usually start dna replication 24 h after the start of the cultures; therefore, we incubated the cells longer to obtain dividing (proliferating) cells. the levels of dna damage were measured using alkaline and neutral comet assays. the results of alkaline comet assay showed the significantly increased percentage of dna breaks in both lymphocytes in medium without pha (2 h of exposure; non-proliferating cells) and lymphocytes cultured during 48 h in medium with pha (exposure for the last 2 h of cultivation; proliferating cells). similarly, neutral comet assay showed dose-dependent elevation of the dna migration induced in both non-proliferating and proliferating lymphocytes treated with epx when compared with negative controls. our results suggest that epoxiconazole fungicide is capable of causing damage to the genetic material of the bovine cells. keywords: epoxiconazole, genotoxicity, cattle, comet assay. introduction pesticides are a significant source of environmental pollution due to their wide-ranging application in agriculture and forestry. exposure to these pollutants can have both acute and chronic effects on target and non-target 100 simona koleničová et al. organisms (berenzen et al. 2005). long-term exposure and chronic poisoning with pesticides can trigger genotoxic and epigenetic processes through various pathways, interactions and doses resulting from the intensive use of pesticides and can cumulatively lead to genetic change in humans, covertly and without clinical evidence (bull et al. 2006). as later indicated by kaur and kaur (2018), occupational exposure to pesticides in agricultural workers has been associated with an increased incidence of various diseases such as cancer, parkinson’s disease, alzheimer’s disease, reproductive disorders, and birth defects. conazoles are a class of azole-based fungicides which are widely used as pesticides in the cultivation of crops despite their suspected endocrine disrupting properties (roelofs et al. 2014) but also as human and veterinary pharmaceuticals for the treatment of oropharyngeal, vaginal as well as systemic candida and mycosis infections (kjaerstad et al. 2010). these fungicides act by inhibiting a specific cytochrome p450 (cyp) enzyme, cyp51 (lanosterol 14α-demethylase), which mediates a critical step in the biosynthesis of ergosterol, a steroid required for the synthesis of the fungal cell wall (zarn et al. 2003). for this reason they are called demethylation inhibitor (dmi) or ergosterol-biosynthesis-inhibiting (ebi) fungicides. besides their effects on fungal cyp51, triazole-based conazoles have the potential to interact with the mammalian cytochrome p450 (cyp) system, e.g. via inhibition of aromatase (cyp19) which can lead to numerous toxicological effects (chambers et al. 2014). as reported by roelofs et al. (2013) conazoles also cause catalytic inhibition of the cyp17 enzyme, responsible for the conversion of pregnenolone and progesterone to androgen precursors. exposure to these compounds from multiple environmental matrices can cause many negative effects including carcinogenicity hepatotoxicity, reproductive and developmental toxicities (goetz and dix 2009; hester et al. 2012; heise et al. 2015; mu et al. 2016; heise et al. 2018). in spite of the large production and extensive usage of many conazoles, accurate data on human exposure levels are scarce. besides occupational and pharmaceutical exposure, individuals can also be exposed to conazoles through environmental, food, resident or bystander exposure. this is confirmed by the increasing concentrations of conazole pesticides found in surface and waste waters (kahle et al. 2008). epoxiconazole (epx) belongs in the triazole class of pesticides and is used worldwide as a fungicide for plant protection. it is known to combat various target fungal diseases in cereals, rice, sugar beets, bananas, coffee, and soybeans (passeport et al. 2011). this dmi fungicide was effectively used for the control of fusarium head blight of wheat in china (chen et al. 2012). in europe and australia, the epoxiconazole is part of several commercially successful one-compound fungicide formulations (epic, opus) or two-compound formulations composed from combinations of epoxiconazole with different pesticide (splice, swing gold, tango super, venture etc.). glyphosate, ddts and the broad-spectrum fungicides boscalid, epoxiconazole and tebuconazole were the most frequently found in agricultural soil samples of 11 member states of the european union (eu) (silva et al. 2019). these findings confirmed the previous study of hvĕzdová et al. (2018), where conazoles showed the second most frequent occurrence among currently used pesticides (cups) in central european arable soils. in the czech republic, vašíčková et al. (2019) determined that epoxiconazole was one of the main contributors to the overall pesticide mixture toxicity: the measured levels and its frequent presence in soils represented a risk for the agroecosystems. this contribution might be a result of low biodegradability and photochemical stability of the epx molecule that makes it very persistent in soil and aquatic sediment (passeport et al. 2011) and allows entering multiple environmental media through spray drift or surface runoff (potter et al. 2014). bovine farm animals are exposed to chemical agents through grazing, so they are the first in which adverse effects of pesticides might occur (drážovská et al. 2016). for this reason, in this study, we would like to present new data from an experiment where dna damage was investigated after exposure of bovine peripheral lymphocytes to epoxiconazole. both alkaline and neutral comet assays were used as the methods of choice for detection of single-strand and double-strand dna breaks. materials and methods blood samples were collected by means of jugular venipuncture from two healthy bulls (slovak indigenous cattle, 6 month old). the animals were kept in healthy conditions, not treated with any drugs and fed with clean feed. the study was conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. lymphocytes isolated from whole blood were used for the comet assays. epoxiconazole (cas registry number 133855-98-8, 99% purity, sigma, st. louis, mo, usa) was dissolved in dimethyl sulfoxide (dmso, sigma, st. louis, mo, usa) and introduced into culture flasks at concentrations of 2.5, 5, 10, 25, 50 and 100 μg/ml. the fungicide doses were chosen according to study of šiviková et al. (2018), where the fungicide cytotoxicity level was identified at a 101genotoxicity testing of bovine lymphocytes exposed to epoxiconazole using alkaline and neutral comet assay concentration of more than 100 μg/ml. the final dmso concentration was 0.1% in both the treated and untreated (negative control) cells. hydrogen peroxide (h2o2, mikrochem, sr, 250 μm) was used as a positive control agent. cell cultivation and treatment for comet assay, lymphocytes were immediately isolated from bovine whole blood using the histopaque®-1077 (sigma-aldrich, st. louis, mo, usa) separation medium. isolated lymphocytes were treated with epoxiconazole for 2 h in two different ways: immediately after isolation (non-proliferating lymphocytes) and for the last 2 h of 48 h cultivation (i.e. pre-cultivation of lymphocytes before 2 h treatment to obtain proliferating lymphocytes). medium for non-proliferating lymphocytes consisted from 4 ml rpmi 1640 medium supplemented with l-glutamine and 15 μm hepes, 1 ml bovine foetal serum (bofes) and 40 µl antibiotic/antimycotic mixture (100 u/ml penicillin, 0.1 mg/ml streptomycin and 0.25 µg/ml amphotericin) (sigma-aldrich, st. louis, mo, usa). immediately after isolation lymphocytes were added to the medium and exposed to the test fungicide for two hours (2 h) (i.e. concurrently with their addition to the medium) according the procedure of calderón-segura et al. (2012). in the experiment with proliferating lymphocytes phytohaemagglutinin (pha-l, 20 µg/ml, pan biotech, germany) was added to the above-described culture medium. the isolated lymphocytes were subsequently incubated at 37°c for 48 h and exposed to epoxiconazole for the last 2 h of cultivation. the cells of positive controls were treated with h2o2 (250 mm) for 5 minutes (horváthová et al. 2006). cytotoxicity after exposure completed, the cells were washed twice with phosphate-buffered saline (dulbecco a, ph 7.4) and resuspended to a final volume 1ml with pbs. cytotoxic effects on the bovine peripheral lymphocytes were evaluated using the trypan blue dye exclusion staining (0.4% trypan blue), where the number of viable (shiny) and dead (blue) cells were scored (viability test). alkaline comet assay the alkaline comet assay procedure was the same for both non-proliferating and proliferating lymphocytes. each concentration tested was represented on special microscope comet slides (cometslidestm 2-well, trevigen, gaithersburg, maryland, us) treated to promote agarose adherence, in this case ready‒to‒use low melting point agarose (lmpa). the cells were mixed with 0.75% lmpa in pbs. the cell suspension was pipetted onto the agarose layer, fitted with a cover slip and left to set at 4°c. after removal of the cover slips, the microscope slides were immersed in cold lysing solution (2.5 m nacl, 0.1 m na2edta, 10 mm tris, plus 1% triton x-100) for 1 h at 4°c. the slides were then transferred to a horizontal gel electrophoresis tank with electrophoresis solution (0.3 m naoh, 1 mm na2edta, ph>13) for 40 min unwinding at 4 °c, and then electrophoresis was conducted at 25v and 300ma for 30 min. the slides were neutralized two times for 10 min with 0.4 m tris-hcl (ph=7.4), stained with ethidium bromide (5 μg/ml ) on both sides, and fitted with cover slips. all of these steps were carried out in the dark and cold (4ºc) to prevent the occurrence of additional dna damage (collins 2002). neutral comet assay the slides were lysed in cold lysing solution (2.5 m nacl, 0.1m disodium ethylene diaminetetraacetic acid (edta disodium salt), 10 mm tris-hcl, ph=9.5, 1% n-lauroylsarcosine sodium salt, 1% tritonx-100) for 1h at 4°c. then the slides were moved to an electrophoretic tank with tbe buffer in which the “unwinding” was performed for 1 hour, followed by electrophoresis (20v) for 40 min. after electrophoresis, the slides were neutralized in blossom with neutralizing solution (0.4 m tris, ph=7.4) for 2 x 10 min. after drying, the glasses were stained with ethidium bromide (5 μg/ml) (gyori et al. 2014). dna damage evaluation comets were analysed with a nikon eclipse ni-u f luorescence microscope, equipped with a texas red single band pass filter. a total of 100 nucleoids per slide (three slides for each concentration 300 nucleoids) were scored visually and five classes of damage were recorded, from 0 (undamaged) to 4 (maximally damaged) according to dna fluorescence intensity in proportion comparing the comet tail and head. the scores 0-4 were attributed according to visual analysis of nucleoids. the overall score for each slide was therefore between 0-400 (collins 2002). the percentage of damaged cells and the extent of % dna damage in the comet tail were calculated. 102 simona koleničová et al. statistical analysis statistical analysis was performed using simple analysis of variance (anova, student’s t test), which was used to evaluate % dna breaks comparing treated and untreated groups (controls). results the results of our analysis of dna damage using both alkaline and neutral comet assays in non-proliferating (2 h exposure to fungicide) and proliferating (48h cultivation and exposure to fungicide for the last 2h) lymphocytes from bovine peripheral blood after exposure to epoxiconazole at concentrations of 2.5; 5; 10; 25; 50 and 100 μg/ml, are summarized in fig. 1a, b and fig. 2a, b. the percentage viability of non-proliferating and proliferating lymphocytes from bovine peripheral blood following exposure to epoxiconazole is shown in fig. 3a, b (alkaline comet assay) and fig. 4a, b (neutral comet assay). regarding the results of alkaline comet assay after 2h exposure of non-proliferating lymphocytes to epoxiconazole, increases in dna damage with statistical significance were found starting from concentration 5 µg/ ml in donor 1 (5 µg/ml * p<0.05; 10, 25, 50 and 100 µg/ ml ** p <0.01; anova and student’s t test; fig. 1a) as well as donor 2 (5 µg/ml * p <0.05; 10, 25, 50 and 100 µg/ml ** p<0.01; anova and student’s t test; fig. 1a). after 48h cultivation and exposure to epoxiconazole for the last 2 h, dna damage was observed in proliferating lymphocytes with statistical significance in donor 1 (5 µg/ml * p <0.05; 10, 25 µg/ml ** p <0.01; 50 and 100 µg/ml *** p <0.001; anova and student’s t test; fig. 1b) as well as donor 2 (10 µg/ml * p<0.05; 25, 50 µg/ml ** p<0.01; 100 µg/ml *** p<0.001; anova and student’s t test; fig. 1b). the viability of non-proliferating lymphocytes was greater than 95% in both donors (fig. 3a), and for proliferating lymphocytes it was greater than 94.7% in both donors, too (fig. 3b). statistically significant increases in dna damage with double-stranded breaks in proliferating and nonproliferating lymphocytes were detected using neutral comet assay after exposure to epoxiconazole (fig. 2a, b) at the same concentrations as for alkaline comet assay. lymphocyte viability is shown in fig. 4 a, b. figure 2. percentages of dna in tail estimated by means of neutral comet assay in bovine peripheral blood lymphocytes (non-proliferating) treated with epoxiconazole for 2 h (a) and in bovine peripheral blood lymphocytes (proliferating 48 h) treated with epoxiconazole for the last 2 h. nc (negative control): dmso; pc (positive control): h2o2 (250μm); a: p<0.05; b: p<0.01; c: p<0.001; mean ± sd. figure 1. percentages of dna in tail estimated by means of alkaline comet assay in bovine peripheral blood lymphocytes (non-proliferating) treated with epoxiconazole for 2 h (a) and in bovine peripheral blood lymphocytes ( proliferating 48 h) treated with epoxiconazole for the last 2 h (b). nc (negative control): dmso; pc (positive control): h2o2 (250μm); a: p<0.05; b: p<0.01; c: p<0.001; mean ± sd. 103genotoxicity testing of bovine lymphocytes exposed to epoxiconazole using alkaline and neutral comet assay dna damage results detected using neutral comet analysis after exposure of non-proliferating lymphocytes to epoxiconazole indicate statistically significant dna damage in donor 1 from the lowest concentration (2.5 and 5 µg/ml *p<0.05; 10 µg/ml ** p<0.01; 25, 50 and 100 µg/ml *** p<0.001; anova and student’s t test; fig. 2a) and in donor 2 from concentration 5 µg/ml (5 µg/ ml * p <0.05, 10, 50 µg/ml ** p <0.01; 25 and 100 µg/ ml *** p<0.001; anova and student’s t test; fig. 2a). proliferating lymphocytes showed statistical significance in donor 1 from starting from concentration 10 µg/ml (25 µg/ml ** p<0.01; 10, 50 and 100 µg/ml *** p<0.001; anova and student’s t test; fig. 2b) and in donor 2 starting from concentration 10 µg/ml (10, 50 µg/ml ** p<0.01; 25 and 100 µg/ml *** p<0.001; anova and student’s t test; fig. 2b). lymphocyte viability found in both donors after epoxiconazole exposure was higher than 95.8% (fig. 4a) in non-proliferating lymphocytes and higher than 90% in proliferating ones (fig. 4b). discussion comet assay (single-cell gel electrophoresis) is one of the most popular methods employed for the evaluation of dna damage and repair in eukaryotic cells (singh 2016; lu et al. 2017; moller 2018) this method is used to study processes dealing with dna damage in various fields, such as environmental toxicology, biological process monitoring, radiation biology, nutritional studies and cancer studies (olive 2009; wasson et al. 2008). this test has a wide spread in genotoxicity testing mainly due to advantages such as simplicity of the test, low cost and high sensitivity (hartmann et al. 2003; tice et al. 2000). the comet test is a universal and sensitive method measuring single-stranded and / or double-stranded dna breaks as well as photodimers (collins et al. 2008). there are two basic variants for determining dna damage using comet analysis under alkaline or neutral conditions (östling 1984; singh 1988). visual classification of nucleoids and calculation of percentage dna at the tail is commonly presented up today (collins et al. 2002; garcía et al. 2004; bruschweiler et al. 2016; hamdi et al. 2018) as an alternative to image analysis. in the present study, the possible genotoxic and cy totoxic effects of epoxiconazole fungicide were assessed in bovine lymphocy tes using alkaline and neutral variants of the comet assay. treatment was performed on non-proliferating and proliferating lymphocytes to evaluate whether the status of cells has an impact on the dna damage level. therefore, we evalufigure 3. viability of bovine peripheral blood lymphocytes used in alkaline comet assay. cells were treated with fungicide epoxiconazole for 2h (a) and for the last 2h of 48h cultivation (b). nc (negative control): dmso. figure 4. viability of bovine peripheral blood lymphocytes used in neutral comet assay. cells were treated with fungicide epoxiconazole for 2h (a) and for the last 2h of 48h cultivation (b). nc (negative control): dmso. 104 simona koleničová et al. figure 5. dnadamage was investigated after exposure of bovine peripheral lymphocytes to epoxiconazole. the cells were treated with the fungicide for 2 h (non-proliferating lymphocytes) and for the last 2 h of the 48-hour culture (proliferating lymphocytes). positive control was h2o2 (5 min). the results of the alkaline comet assay are shown in the first column (picture a, b, c) and the neutral comet assay in the second column (d, e, f ). negative control: a, d. selected concentration 50 μg/ml: b, e. positive control: c, f. 105genotoxicity testing of bovine lymphocytes exposed to epoxiconazole using alkaline and neutral comet assay ated two different experiments. the first one was with non-dividing (non-proliferating) lymphocytes exposed to epx immediately after isolation for 2 hours, as indicated by calderón-segura et al. (2012). the second with lymphocytes stimulated to divide by phytohaemagglutinin (pha) during 48 hours taking account the results of bausinger and speit (2014) who revealed that dna synthesis starts in t lymphocytes (similarly like in peripheral blood mononuclear cells, pbmc) around 24 h after stimulation with pha. therefore we chose 48-hour (24 h plus 24 h) cultivation allowing lymphocyte proliferation in the medium at least during one cell cycle. we treated the cultured lymphocytes with epx the last 2 h for the examination of the epoxiconazole ability to induce dna damage in proliferating cells. using alkaline comet assay we showed that epoxiconazole induced statistically significant dna damage in both non-dividing (without pha) and dividing (with pha stimulation) lymphocytes of cattle. epx induced dna migration in pha-stimulated cultured lymphocytes in the same range of concentrations like in non-stimulated ones but to a different extent; less dna migration was observed in pha-stimulated cells. it is likely that these results correspond with different capability of proliferating cells to repair dna damage. on the contrast to alkaline comet assay, neutral comet assay showed that dna breaks were induced in different ranges of concentrations in proliferating (dividing) lymphocytes (from 10 µg/ml) when compared with non-proliferating cells (from 2.5 µg/ml) (fig. 2a, b). one of explanation might be that the lower concentration did not induce dna damage or that neutral comet assay detects mostly double-strand breaks (lu et al. 2017), which were probably more effectively repaired in proliferating lymphocytes than in non-proliferating ones. the results of the comet assay can be affected by the exposure time which is one of a crucial factor. long incubation periods may not be appropriate for the comet assay because dna lesions may be repaired during the time that mutagens are inactivated, leading to false negative results (sekihashi et al. 2003). according to tice et al. (2000), an appropriate exposure time for chemical in vitro genotoxicity assessment should be around 3 to 6 hours; other papers refer 1 h, 2 h, 4 h or 24 h exposure times (lebaily et al. 1997; calderón-segura et al. 2012; želježić et al. 2016). it is known that cattle can accumulate foreign substances not only in the liver but also in the muscle (garcía-repetto et al. 1997), milk (pokorná et al. 1996) and fat (ferré et al. 2018) thereby increasing the genetic risk to humans through the food chain. guitart et al. (2010) reported that as a result of the application of fungicides in agricultural production, livestock poisoning may occur, the clinical manifestations of which are only rarely addressed. exposure of livestock to genotoxic substances may also induce mutations, lead to metabolic disorders, immunosuppression and decreased fertility. cattle are exposed to chemicals during grazing, so adverse effects may occur primarily in them. besides, some of the chemical agents have a long-term cumulative effect and can contribute to cancer through chronic exposure. genotoxicity assessment is an essential component of the safety analysis of all types of substances, ranging from pharmaceuticals, industrial chemicals, pesticides, biocides, food additives, cosmetics ingredients, to veterinary drugs, relevant in the context of international legislation aiming at the protection of human and animal health (ecvam 2013). as reported by bolognesi and morasso (2000) pesticides have been considered potential chemical mutagens. the genotoxicity of pesticides is generally considered to be the most serious of the possible side effects of their usage. the formation of highly reactive substances during oxidation processes, coupled with the ability to interact with dna, leads to a series of measurable changes, for example point mutations, chromosomal rearrangements, dna adducts, dna strand fragments and increased number of micronuclei (medina et al. 2007). there are several studies testing the genotoxicity of pesticides and mycotoxins on bovine lymphocytes (lioi et al. 2004; holečková et al. 2013; schwarzbacherová et al. 2017; ferré et al. 2020). šiviková et al. (2018) tested epoxiconazole in vitro in cultured bovine peripheral lymphocytes using chromosome aberrations, sister chromatid exchanges and micronucleus test. she found that epoxiconazole was not related to genotoxic and / or clastogenic / aneugenic effects, but had the ability to significantly affect cell-cycle kinetics and induce apoptosis. our results show that epoxiconazole can induce significant levels of dna damage in bovine lymphocytes, as revealed in both alkaline and neutral variants of the comet assay. on the other hand, no statistically significant dna damage was detected by drážovská et al. (2016), who investigated dna damage using alkaline comet assay after 2 h exposure of bovine lymphocytes to tango® super fungicide (epoxiconazole/fenpropimorph). potential genotoxic/cytotoxic effects of the epoxiconazole/fenpropimorph-based fungicide were also investigated by cytogenetic assays: chromosomal aberrations, sister chromatid exchanges, micronuclei and f luorescence in situ hybridization. the final results indicated that the tested fungicide was capable of evoking cytotoxic effect / cell-cycle delay in peripheral cattle lymphocytes. on the contrary schwarzbacherová et al. (2017) 106 simona koleničová et al. reported stimulation od dna-double strand breaks after 4 h exposure to epoxiconazole / fenpropimorph-based fungicide (tango super) using neutral comet assay. when compared with pure epx, the results of both studies mentioned above were probably affected by the presence of fenpropimorph and inert ingredients in the tested pesticide formulation, as well as by different exposure times and variants of comet assay. similarly to our observations, significantly increased percentages of comets and tail lengths were obtained after epoxiconazole treatment in the human colon carcinoma cell line (hct116) (hamdi et al. 2018). epoxiconazole was able to induce a range of cell damage in hct116 cells by generating ros, which in turn induces mitochondrial dna dysfunction and fragmentation leading to cell death, as confirmed by the attenuated death of cells treated with the antioxidant n-acetylcysteine (nac) prior to treatment with epoxiconazole. in the later study with f98 glioma cells the same author (hamdi et al. 2019) showed that epx induced cytotoxic effects, cell cycle arrest, cytoskeleton disruption, dna damage and apoptosis via caspases dependent signalling. in addition akram et al. (2019) confirmed that epoxiconazole was the potent inhibitor of 11-bhydroxylase (cyp11b1) and aldosterone synthase (cyp11b2) in hamster and human adrenal h295r cells; these enzymes catalyse the formation of cortisol and aldosterone in the adrenal cortex therefore in this study epoxiconazole seems to be an endocrine disruptor. similarly, taxvig (2007) concluded that disruption of a crucial enzyme such as cyp17, which is involved in steroid synthesis hormone, is one of the main endocrine-disrupting mechanisms of azole fungicides like tebuconazole and epoxiconazole. in our experiment, bovine lymphocytes were tested under in vitro conditions to obtain significant results, preceded by the testing of several methods and procedures to create optimal experimental conditions. treatment of cells with epoxiconazole was followed by determination of cell viability for each test concentration using the trypan blue exclusion method, where the percentage of viability represents the number of viable cells compared to the total number of cells surviving after treatment. the cell viability was greater than 90% at all concentrations tested. tice et al. (2000) recommended that in vitro treatment with chemicals should not reduce cell viability by more than 30%, and extended this similarly to in vivo experiments. other researcher maintain that the allowed cell viability after exposure should be at least 70-75% (želježić et al. 2018), or above 85 % (evans et al. 2016), and some also report up to 95% (lebailly et al. 2015) upon performing comet analysis. in general, the question of determining the sensitivity of both alkaline and neutral comet analysis is frequently discussed, so the comparison of sensitivity of individual methods is interesting from the practical point of view (afanasieva and sivolob 2018; azqueta and collins 2013; peycheva et al. 2009; collins et al. 2008). the alkaline variant demonstrates increased sensitivity in the investigation of agents causing dna strand breaks or inducing alkaline labile lesions of dna. currently, the first choice is to detect low levels of dna damage, either in lymphocyte samples or in genotoxicity testing in vitro and in vivo. according to other authors, the neutral variant is more sensitive than the alkaline one. for instance, afanasieva et al. 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d.m.a. brito, r.a. torres (2020) title. caryologia 73(2): 15-25. doi: 10.13128/ caryologia-672 received: october 23, 2019 accepted: march 27, 2020 published: july 31, 2020 copyright: © 2020 f. ito, d.j. gamamaia, d.m.a. brito, r.a. torres. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. gene flow patterns reinforce the ecological plasticity of tropidurus hispidus (squamata: tropiduridae) fernanda ito, danielle j. gama-maia, diego m. a. brito, rodrigo a. torres* lagea – laboratório de genômica evolutiva & ambiental, departamento de zoologia, centro de biociências, universidade federal de pernambuco, recife, brazil *corresponding author: rodrigotorres@ufpe.br abstract. the analysis of gene flow patterns can provide important insights into population dynamics in the context of landscape ecology. in lizards, this approach has been used to evaluate patterns related to climate change, habitat fragmentation, and taxonomic uncertainties. tropidurus hispidus is an ecologically plastic species, which presents some evidence of population structuring. in the present study, we investigated the potential structuring of t. hispidus populations across a gradient of tropical biomes, including the amazon and atlantic rainforests, the caatinga dry forest, the caatinga-atlantic forest transition zone (agreste), coastal restinga, and urban environments. nuclear issr markers were obtained by pcr/electrophoresis, and a number of population parameters were estimated and analyzed. despite the extreme environmental discontinuities found across the vast study area, the results revealed a high degree of genetic connectivity among the different demes. this pattern indicates that the species can be considered to be a single evolutionary taxon with gene flow among all populations, despite the marked environmental discontinuities. tropidurus hispidus clearly has a marked capacity for dispersal, which may be favored by its intrinsic genetic diversity. keywords: tropidurus hispidus, issrs, gene flow, dispersal capacity, population connectivity. introduction gene flow is one of the most important components in population structure because it can determine how much populations have evolved independently (slatkin 2018). therefore gene flow patterns can also provide important insights for studies on population ecology and also on population genetics based on a landscape ecology approach. the genetic admixture resulting from gene flow may contribute to a short-term increase in population fitness (facon et al. 2005) and adaptive potential (verhoeven et al. 2011). however, the approach usually focuses on micro-evolutionary phenomena and processes that lead to intraspecific discontinuities (holderegger and wagner 2006). 16 fernanda ito et al. genetic studies, especially in the neotropical region, and in particular for reptile species, have been more frequent in the last years focusing on questions related to climate change, habitat fragmentation, and taxonomic uncertainties (e.g. ricketts 2001; stow et al. 2001; berry et al. 2005; driscoll and hardy 2005; sumner 2005; hoehn et al. 2007; o’neill et al. 2008; tolley et al. 2009; freedman et al. 2010; levy et al. 2010; werneck et al. 2015; menezes et al. 2016; fazolato et al. 2017; cacciali and köhler 2018; oliveira et al. 2018). however studies focused on landscape genetics and on the gene flow patterns are still scarce in neotropical region. tropidurus hispidus is one of the largest species of the genus, reaching a rostrum-caudal length (rcl) of 114 mm (kolodiuk et al. 2010). it is found on a variety of substrates such as sand, tree trunks, and rocky outcrops, but it is primarily saxicolous, given that rocks provide space for foraging, shelter, nesting, and thermoregulation (pelegrin et al. 2017). also, this species is commonly found in urban areas, foraging and thermoregulating on walls and fences (rodrigues 1987; abreu et al. 2002; pelegrin et al. 2017). the ecological tolerance of t. hispidus allows this species to occupy a number of distinct morphoclimatic domains, such as the brazilian atlantic forest, coastal shrubby vegetation (restinga), transition areas between the caatinga scrub (in portuguese agreste), the atlantic forest, cerrado savanna, and rocky outcrops in the amazon basin (vanzolini et al. 1980; vitt 1995; abreu et al. 2002; carvalho 2013). the species is a habitat generalist, able to colonize a wide range of microhabitats (rodrigues 1987; vitt 1995; vitt and carvalho 1995; vitt et al. 1997; pelegrin et al. 2017). this species is also an opportunistic sit-and-wait predator with a diverse trophic niche, feeding mainly on arthropods, in particular ants, but in some areas they may include plant material in their diet, especially flowers (van sluys et al. 2004; ribeiro and freire 2011; pelegrin et al. 2017). differences in the composition of the diet among biomes reinforce the ecological plasticity of the species (pelegrin et al. 2017), but may also reflect distinct selective pressures on different populations. in addition to these dietary differences, there is some evidence of genetic structuring among populations. three distinct karyotypes have been found in six populations from different ecosystems in eastern brazil (kasahara et al. 1987; kasahara et al. 1996). all karyotypes had 2n = 36 and xx/xy sex chromosomes, but three variants (prominent, mild or absent) were found in a secondary constriction of the second chromosome pair, which appeared to be typical of specific sites, suggesting genetic variation on an inter-population level. however, specimens from the six populations are morphologically indistinguishable (kasahara et al. 1987; kasahara et al. 1996). also there is a clear evidence for cryptic diversity in t. hispidus as revealed by karyotype and dna barcode sequences analyses (matos et al. 2016). tropidurus hispidus is abundant across an extremely diverse ecological landscape (carvalho 2013). from the coast of pernambuco (north-eastern brazil) to the amazon basin there is a major shift in the geographical and ecological landscape, in which environmental variation may be reflected into distinct selective regimes, as previously suggested by the chromosomal and molecular evidences. then, given previous ecological, distributional, karyotypical, and molecular evidence, we tested for the hypothesis of the existence of population-level divisions in tropidurus hispidus along a highly diverse adaptive landscape in brazil, using nuclear dna markers adopting a gene flow approach. materials and methods a total of 155 specimens of tropidurus hispidus were captured at sites representing the distinct phytophysiognomic domains found across the landscape between the pernambuco and paraiba coasts in eastern brazil, and the amazon basin, in the north of the country (table 1; figure 1). the specimens were identified using the taxonomic key of rodrigues (1987). liver and muscle samples for dna analyses were collected from each specimen. these samples were immersed in 96% ethanol and stored in a freezer at -20°c. tissue was also obtained from three specimens of tropidurus torquatus from maricá, rio de janeiro, south-eastern brazil, and one eurolophosaurus divaricatus from alagoado, bahia, north-eastern brazil, for inclusion in the study as outgroups. dna extraction and issr amplification the extraction of dna was conducted using the sambrook and russell (2001) procedure. the integrity of the dna was checked by electrophoresis in agarose gel and the concentration was estimated by visual comparison with the intensity of the dna of the lambda phage. the dna was then diluted to a standard concentration of 5 ng/ul for the pcr-issr reactions. inter simple sequence repeats (issrs) are pcr-amplified nuclear genomic regions using primers anchored at microsatellite regions (ssrs) (gupta et al. 1994; zietkiewicz et al. 1994). these markers have been considered of low cost and highly reproducible (sarwat 2012), and very effective 17gene flow patterns reinforce the ecological plasticity of tropidurus hispidus (squamata: tropiduridae) in terms of studying the genetic variation and population cohesiveness in several biological groups (gamamaia and torres 2016; al salameen et al. 2018; hassaniem & al rashada 2019). the pcrs were carried out in a final volume of 20 µl in which consisted of 0.2 units of taq dna polymerase (new england/biolabs), 1x buffer, 50 mm mgcl2, 50 mm of primer, 0.2 mm dntp and 20 ng of genomic dna. the pcr reactions were run in a biocycler thermocycler and comprised a cycle of 4 min at 94°c, 39 cycles of 40 s at 94°c, 40 s at the specific temperature of each primer (table 2), and 120 s at 72°c, with a final annealing cycle of 7 minutes. all reactions were run with a negative control. horizontal electrophoresis was conducted in 1.8% agarose gel containing 0.5x tbe buffer diluted from an original 10x solution (0.89 m tris, 0.89 m boric acid and edta, 0.01m, ph = 8.3) for 4 hours at 60 volts. in each well of the gel we placed a solution containing 10 µl of the pcr product in 1.5 ml of gel loading dye blue (6x) and 1.5 ml of gel green (0.5 ml 10,000x in h20). to support the analysis of bands, we inserted 2 µl of 1 kb dna ladder marker with 1.5 ml of gel loading dye blue (6x) in one well. after the run, all gels were photographed using a transilluminator under an ultraviolet light source. data analyses initially, 17 different random issr primers were tested for their reproducibility and their degree of polymorphism. they were tested in five specimens from four sites using different pcr reagents from fermentas (thermo fisher scientific) and new england biolabs inc. (table 2). the 10 most polymorphic primers were then selected for the amplification of the dna of all the specimens (table 2), with the objective of generating at least 60 polymorphic loci, as recommended by telles et al. (2001) and nelson and anderson (2013). after photographic documentation, the gels were transformed into a binary matrix of presence and absence (0 = absent and 1= presence) of the dna bands. in order to avoid the misinterpretation of valid markers, only clear and welldefined bands were assigned as markers. it is important to note that to increase sample size, the animals sampled in the localities of caraguetama and tamandaré were treated as a single sample in all analyzes, since both areas represent the same adaptive landscape (named restintable 1. number (n) of tropidurus hispidus, t. torquatus, and eurolophosaurus divaricatus specimens captured in each site along the study area. species municipality* or state** geographic coordinates acronyms biome n camaragibe* 8º02’31”s 35º06’17”w af atlantic forest 24 canguaretama* 6º22’58”s 35º07’29”w rest restinga 2 gravatá* 8º16’02”s 35º27’35” w tz transition zone 25 tropidurus hispidus manaus* 3º09’34”s 59º36’10”w am amazon forest 19 petrolândia* 9º05’37”s 38º15’05”w ca1 caatinga 26 recife* 8º10’43”s 34º42’46”w uz urban zone 25 serra talhada* 7°59’7”s 38°17’34”w ca2 caatinga 30 tamandaré* 8º45’28”s 35º06’18”w rest restinga 4 total 155 tropidurus torquatus (og) rio de janeiro** 22º22’28”s 42º57’01”w 3 eurolophorus divaricatus (og) bahia** 13º07’07”s 38º28’50”w 1 figure 1. south america/brazil map depicting capture sites of t. hispidus. in evidence are the different capture sites in pernambuco (pe) brazilian state. the biomes accessed are written here within parentheses as follows: 1. manaus (amazon forest-am), 2. canguaretama-state of paraíba (restinga-rest), 3. serra talhada (caatinga-ca2), 4. petrolândia (caatinga-ca1), 5. gravatá (transition zone between caatinga and atlantic forest-tz), 6. camaragibe (atlantic forest-af), 7. recife (urban zone-uz), 8. tamandaré (restinga-rest). 18 fernanda ito et al. ga) (table 1). the overall genetic variation was measured in percentage by the proportion of the polymorphic loci having the total number of observed loci as 100%. to evaluate the existence of potential genetic and/ or evolutionary groupings among biomes, multi-dimensional scaling (mds) with neighbour-joining (nj) genetic distances was applied on local and regional scales through the simple matching technique (primer software) (clarke and gorley 2006). an additional nj topology was also obtained by using paup* v.4.0b10 (swofford 2000) in order to observe alternative groupings among sampled specimens. a maximum parsimony (mp) method was also used in order to test for hidden evolutionary diversity in t. hispidus across those different adaptive landscapes (biomes) having eurolophosaurus divaricatus and tropidurus torquatus as outgroups given their phylogenetic proximity to the study species (frost et al. 2001; passoni et al. 2008). these analyses were run in paup* v.4.0b10 (swofford 2000), in its graphic interface paupup v.1.0.3.1 (calendini and martin 2005). a maximum number of 100,000 random trees with 5000 replications were computed. the robustness of the branches was tested by the bootstrap method with 1000 random replicates. population structuring was tested by the bayesian approach using the structure 2.3.3 software (pritchard et al. 2000; falush et al. 2003, 2007; hubisz et al. 2009). in order to determine the number of populations (k) within the complete data set, ten independent runs for k= 1-10 and 100,000 mcmc (markov chains monte carlo) interactions after burn-in period were computed. the analysis was performed by using both the admixture model of population structure and allele frequencies correlated among populations. the number of populations (k) was estimated using the protocol described by evanno et al. (2005). in addition, we conducted an analysis of molecular variance (amova) to check for patterns of genetic isolation within and among local populations (excoffier et al. 1992) with arlequin v.3.5.1.2 (excoffier and lischer 2010). this method also permits the calculation of the global fixation index (φst). parameters of genetic differentiation among populations (gst) and the number of migrants per generation (nm – gene flow) were calculated with popgene 1.3.2 (yeh et al. 1999). results based on the 10 issr primers selected, a total of 283 loci were observed. overall, 99.2% of the observed loci were polymorphic. mean genetic neighbor-joinning distances among local populations varied from 0.12720 to 0.48763. table 2. issr primers tested in this study with sequence and annealing temperature. selected primers are marked with (*). primer sequence (5’ – 3’) annealing temperature (ºc) issr 1* (ag)8t 50,4 issr 2* (ag)8c 52,8 issr 3* (ga)8t 50,4 issr 4 (ga)8c 52,8 issr 5* (ct)8g 52,0 issr 6* (ag)8yc 52,8 issr 7 (ag)8ya 54,0 issr 8* (ga)8yt 52,8 issr 9* (ga)8yc 52,8 issr 10 (ga)8yg 54,0 issr 11 (ct)8ra 50,0 issr 12 (ac)8yg 54,0 issr 13* (ggac)3a 51,0 issr 14 (ggac)3c 51,0 issr 15 (ggac)3t 51,0 issr 16* (aacc)4 51,0 issr 17* (ggac)4 51,0 figure 2. multi-dimensional scaling plots of the genetic similarities (simple matching index) among the tropidurus hispidus populations sampled in the present study. see table 1 and text for acronyms. the graphs a/b show the different domains and localities respectively. 19gene flow patterns reinforce the ecological plasticity of tropidurus hispidus (squamata: tropiduridae) the simple matching mds analysis revealed a single grouping comprising all sampled populations on both regional and local scales (figure 2a-b). the nj topology showed also no particular genetic groupings among t. hispidus sampled from different biomes (figure 3). the maximum parsimony (mp) analysis revealed 2 constant and 281 informative characters. the majority-rule consensus topology (supplemental material) had a length (l) of 6236, a consistency index (ci) of 0.045, and a retention index (r) of 0.310. this topology also failed to identify any evolutionary differentiation among the populations analysed. the analysis identified a total of 283 loci and more than 90% were variable in terms of the proportion of polymorphic loci. this amount of molecular information satisfies the recommendation of nelson and anderfigure 3. neighbor-joinning topology from tropidurus hispidus specimens for issr markers. see table 1 and text for acronyms. 20 fernanda ito et al. son (2013) for the application of amova and bayesian structuring analyses. the amova indicated that 90.99% of the total genetic variance was found within populations and only 9.01% among populations (table 3). the bayesian structuring analysis revealed the existence of two genetic populations (k= 2; figure 4), and these genetic profiles were clearly distributed in all specimens throughout the geographic areas sampled. the global gst value was 0.07, while the nm was 6.59. the pairwise analyses showed values ranging from 0.03 to 0.06 for gst and from 7.75 to 15.03 for nm (table 4). discussion the genetic evidence of this study indicates strong connectivity among local t. hispidus groups, despite the intense ecological distinctiveness of the landscapes seen in the study area. the mds (figure 2), the nj topology (figure 3), and the mp topology (supplemental material) evidenced a lack of any genetic or evolutionary differentiation among t. hispidus groups, pointing to a high dispersive behaviour in this species. tropidurus hispidus is widely distributed in the caatinga and can also be found along the brazilian coast and in the amazon forest (carvalho 2013). the extent of its distribution range would suggest a high probability of differentiation due to strong and diverse evolutionary pressures imposed to the populations (kisel and barraclough 2010). however, the clustering produced by the bayesian analyses showed also no genetic structuring (figure 4). although there are two genetic populations, the analysis indicated a clear admixture of these two t. hispidus gene pools among the demes studied. the lack of genetic structuring resulted from an intense gene flow among populations as indicated by the degree of migrants per generation (table 4). the observed global nm value (6.59), as well as the pairwise ones (7.75 – 16.03), support the hypothesis of strong evolutionary cohesion, since nm ≥ 1 indicates a minimum amount of genetic migration capable of homogenizing demes within species (mills and allendorf 1996), including in lacertids (levy et al. 2010). this feature of a highly cohesive species could be also explained by a recent irradiation phenomenon. however this hypothesis requires a robust phylogeographic study offering coalescence-dating analyses. considering all the results, it is possible to argument in favour to the hypothesis of panmixia in t. hispidus, despite the discontinuity and historical changes seen in the biomes studied. this is surprising since t. hispidus individuals are sit-and-wait predators, territorialists, and oviparous that would suggest a tendency for structurings (prieto et al. 1976; van sluys et al. 2010; ribeiro and freire 2011). besides not dispersing through long distances (pontes et al. 2008), sit-and-wait predators are usually opportunists and can feed on a variety of food items according to the local availability (rodrigues 1987, 1988; vitt 1991; bergallo and rocha 1993; vitt 1995; vitt et al. 1997; pontes et al. 2008) suggesting ecological plasticity. ecological plasticity predicts high genetic diversity. higher genetic diversity tends to favour a better adaptation at the population, community and ecosystem levels (hughes et al. 2008). the feeding plasticity observed in t. hispidus (pelegrin et al. 2017) is related to its high success in attempts to colonize new sites (teixeitable 3. results of the amova for the tropidurus hispidus populations in the study area. (p < 0.01). source of variation degrees of freedom sum of squares components of variance % of variance among populations 6 729.782 3818.75 va 9.01 within populations 148 5709.625 38,578.55 vb 90.09 total 154 6439.407 42,397.30 100 φst 0.09007 figure 4. bayesian structuring analysis. the y axis indicates the probability-based assignments for the genetic composition of each specimen analyzed (vertical bars). note (1) ca2, (2) ca1, (3) af, (4) tz, (5) uz, (6) am, (7) rest (including specimens from canguaretama and tamandaré) . for a description of the sites, see table 1. table 4. pairwise gst (above diagonal) and nm (below diagonal) values recorded between tropidurus hispidus populations. for acronyms, please refer to table 1. ca2 ca1 tz af uz am rest ca2 0.0420 0.0363 0.0363 0.0322 0.0360 0.0527 ca1 11.4029 0.0457 0.0379 0.0368 0.0435 0.0511 tz 13.2673 10.4325 0.0383 0.0388 0.0458 0.0605 af 13.2673 12.7004 12.5522 0.0302 0.0341 0.0455 uz 15.0321 13.0902 12.3747 16.0316 0.0341 0.0505 am 13.3976 10.9953 10.4261 14.1587 14.1595 0.0492 rest 8.9877 9.2855 7.7588 10.4786 9.4000 9.6556 21gene flow patterns reinforce the ecological plasticity of tropidurus hispidus (squamata: tropiduridae) ra and giovanelli 1999) and to its capacity of expanding towards new habitats (levy et al. 2010; breininger et al. 2012). this hypothesis is supported by the high degree of genetic diversity observed herein in t. hispidus and this feature might be favouring historically the species to a better adaptation to different biomes. these combined evidences suggest the stepping-stone model of range expansion as a probable explanation for wide distribution of t. hispidus. molecular studies with species of tropidurus have revealed different patterns of evolutionary cohesion among populations, depending on the species studied. for instance, in tropidurus semitaeniatus and t. hygomi, populations tend to be highly structured, but due to different processes. in t. semitaeniatus the process of population structuring was mediated by the course of the river são francisco (northeastern brazil; werneck et al. 2015). in t. hygomi, the population structuring was associated to different marine transgression/regression events, which isolated or connected regions along the brazilian coastal plains (fazolato et al. 2017). tropidurus hispidus was expected to show the same pattern of genetically structured populations due to geographic isolation by different ecological pressures of morphoclimatic domains of humid forests and the caatinga (gonzález et al. 2011; matos et al. 2016). the data supporting these conclusions were the karyotypic structure and coi gene sequences, respectively (matos et al. 2016). however, our data failed to reinforce this idea and the analyses of the hypervariable regions of issr nuclear markers strongly pointed to panmixia. this occurred despite the geographical distances and the different selective pressures among studied biomes. a likely explanation for these contrasting evidences could be an intense dispersive behavior showed by t. hispidus males. indeed, the use of bi-parental genetic markers has been recommended as a strategy to understand patterns of gene flow and demography (goudet et al. 2002). the issrs markers analysed in this study agree with this recommendation and allow inferences about the gene flow among t. hispidus demes. cases of male-mediated dispersion in lizards have been documented in the literature in the last years (e.g. johansson et al. 2008; mouret et al. 2011; ferchaud et al. 2015). when considering mitochondrial markers, of female inheritance, populations seem structured, (matos et al. 2016) but when the male genetic pools is also analysed such structuring disappears, as seen here using the issrs markers. this supports the hypothesis that the expansion of t. hispidus distribution range, and therefore, new colonisations, would depend on a higher ecological ability of males to disperse farther than females. mark-recapture studies of males and females could confirm the explanations given herein. the occurrence of t. hispidus in urban areas, and its use of anthropogenic structures, (carvalho 2013; pelegrin et al. 2017) could lead to facilitated dispersion and extend its distribution range. human-facilitated dispersion occurs in other lizard species, including exotic and invasive species (vanzolini 1978; mausfeld et al. 2002; anjos and rocha 2008). the t. hispidus population of manaus (amazon), which was recently invaded by individuals from roraima (northern brazil), is an example of this phenomenon (ávila-pires 1995; carvalho 2013). however, this is speculative since we lack genetic data from roraima. on the other hand, the individuals from manaus had the same genetic profiles as the populations from pernambuco, and did not show any type of genetic structuring, corroborating the hypothesis of panmixia along our study area. our results revealed also that tropidurus hispidus has a genetic variation above 90%. this points to an excellent conservation status along the studied area, considering that low genetic variation would decrease this species’ ability to adapt to current and stochastic selective pressures (frankham and ralls 1998; frankham et al. 2002; allendorf and lundquist 2003). indeed, t. hispidus seems to have a high tolerance to habitat modifications (rodrigues 1987; ávilla-pires 1995), and it is a generalist regarding its microenvironmental requirements (vitt 1995; mendonça and moura 2011; pelegrin et al. 2017). therefore, our data reinforces this biological attribute (evolutionary potential), due to the high genetic variation observed. to conclude, according to our results, the sharing of a high genetic variation among the several t. hispidus population demes from different morphoclimatic domains seems to explain its ecological plasticity/evolutionary potential. according to vitt et al. (1997) and ivkovich et al. 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for obtaining metaphase chromosomes in six mangrove crab species alessio iannucci1, stefano cannicci1,2,*, zhongyang lin3, karen wy yuen3, claudio ciofi1, roscoe stanyon1, sara fratini1 comparison of the evolution of orchids with that of bats antonio lima-de-faria identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection mehdi zahravi1,*, panthea vosough-mohebbi2, mehdi changizi3, shahab khaghani1, zahra-sadat shobbar4 populations genetic study of the medicinal species plantago afra l. (plantaginaceae) saeed mohsenzadeh*, masoud sheidai, fahimeh koohdar a comparative karyo-morphometric analysis of indian landraces of sesamum indicum using ema-giemsa and fluorochrome banding timir baran jha1,*, partha sarathi saha2, sumita jha2 chromosome count, male meiotic behaviour and pollen fertility analysis in agropyron thomsonii hook.f. and elymus nutans griseb. (triticeae: poaceae) from western himalaya, india harminder singh2, jaswant singh1,*, puneet kumar2, vijay kumar singhal1, bhupendra singh kholia2, lalit mohan tewari3 population genetic and phylogeographic analyses of ziziphora clinopodioides lam., (lamiaceae), “kakuti-e kuhi”: an attempt to delimit its subspecies raheleh tabaripour1,*, masoud sheidai1, seyed mehdi talebi2, zahra noormohammadi3 induced cytomictic crosstalk behaviour among micro-meiocytes of cyamopsis tetragonoloba (l.) taub. (cluster bean): reasons and repercussions girjesh kumar, shefali singh* karyotype diversity of stingless bees of the genus frieseomelitta (hymenoptera, apidae, meliponini) renan monteiro do nascimento1, antonio freire carvalho1, weyder cristiano santana2, adriane barth3, marco antonio costa1,* karyotype studies on the genus origanum l. (lamiaceae) species and some hybrids defining homoploidy esra martin1, tuncay dirmenci2,*, turan arabaci3, türker yazici2, taner özcan2 determination of phenolic compounds and evaluation of cytotoxicity in plectranthus barbatus using the allium cepa test kássia cauana trapp1, carmine aparecida lenz hister1, h. dail laughinghouse iv2,*, aline augusti boligon1, solange bosio tedesco1 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(4): 111-120, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-966 caryologia international journal of cytology, cytosystematics and cytogenetics citation: m. husemann, d. sadílek, l.-s. dey, o. hawlitschek, m. seidel (2020) new genome size estimates for bandwinged and slant-faced grasshoppers (orthoptera: acrididae: oedipodinae, gomphocerinae) reveal the so far largest measured insect genome. caryologia 73(4): 111-120. doi: 10.13128/caryologia-966 received: june 10, 2020 accepted: september 24, 2020 published: may 19, 2021 copyright: © 2020 m. husemann, d. sadílek, l.-s. dey, o. hawlitschek, m. seidel. this is an open access, peer-reviewed article published by firenze university press (http://www. fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. new genome size estimates for band-winged and slant-faced grasshoppers (orthoptera: acrididae: oedipodinae, gomphocerinae) reveal the so far largest measured insect genome martin husemann1,*+, david sadílek2+, lara-sophie dey1, oliver hawlitschek1, matthias seidel1,3 1 centrum für naturkunde, universität hamburg, martin-luther-king-platz 3, de-20146 hamburg, germany 2 department of zoology, faculty of science, charles university, viničná 7, cz-12843 praha, czech republic 3 department of entomology, national museum in prague, cirkusová 1740, cz-19300 praha, czech republic *corresponding author. e-mail: martin.husemann@uni-hamburg.de +mh and ds equally contributed abstract. grasshoppers, specifically those of the family acrididae are known to have the largest genomes of all insects. however, less than 100 species of orthoptera have their genome size estimated so far. in the present study, we measured the genome size of five acridid species belonging to the two subfamilies oedipodinae and gomphocerinae. all of the genomes measured are large and range between 1c = 11.31 pg in the female of chorthippus dorsatus and 1c = 18.48 pg in the female of stethophyma grossum. the latter represents the so far largest measured insect genome. we further provide a summary of genome size estimates available for orthoptera. keywords: c-value, flow cytometry, stethopyhma, oedipoda, sphingonotus, chorthippus. introduction the genome has become one of the most important targets of interest for biologists. in times of high throughput sequencing, projects like i5k generate data of entire genomes are at a daily base (robinson et al. 2011; li et al. 2019). however, we still have little data and a limited understanding of the variance in genome size across organisms. especially for insects, the most diverse group of organisms on earth, data of only about 1,300 of the expected diversity of several million species are available (sadílek et al. 2019a; gregory 2020). generating new data on genome sizes is important, e.g., for choosing the adequate ngs applications for genomic sequencing (rodríguez et al. 2017). yet, genome size can also be a taxonomic feature 112 martin husemann et al. and can be used for species determination (sadílek et al. 2019b). for many applications taxa with specifically large genomes still remain a difficult target, especially if no complete genome sequence is available. further, in order to understand why some species or species groups have specifically large genomes, whereas others are rather small requires comprehensive data across a large range of taxa. while the so far largest genome of any organism was estimated in a plant, the monocot paris japonica franchet with 1c = 152.23 pg (pellicer et al. 2010), the largest genome sizes in insects have been measured in orthoptera, specifically caelifera, with 1c values of 16.93 pg in podisma pedestris (linnaeus, 1758) (podisminae) and 16.34 pg in stauroderus scalaris (fischer von waldheim, 1846) (gomphocerinae) (gregory 2020 for a list). however, there is also a lot of variation within orthoptera with genome sizes as small as 1c = 1.55 pg found in the cricket hadenoecus subterraneus (scudder, 1861) (rasch and rasch 1981). nevertheless, a clear trend for larger genomes in the short-horned grasshoppers is observed, and specifically in the family acrididae. in the present study, we were able to locate only 85 published genome size estimates from all orthoptera (e.g. gregory 2020). to better understand the evolution of genome size in orthoptera, especially the huge genomes of grasshoppers of the acrididae family, it is obligatory to generate additional information. hence, we provide new genome size information for members of the acrididae family, i.e. three species of the subfamily oedipodinae and two species of the gomphocerinae. we present, to our knowledge, the so far largest genome size of any insect and summarize the knowledge on genome sizes in orthoptera. material and methods sampling eight specimens from five species (table 1), all of the family acrididae, were collected for our analyses in september 2019 in hamburg, georgswerder (germany, 53.5097°n 10.0301°e). specimens were collected by hand and kept alive until further processing. we included two species of the subfamily gomphocerinae: chorthippus dorsatus (zetterstedt, 1821) and a species of the chorthippus biguttulus (linnaeus, 1758) group (a group of three species c. biguttulus, c. brunneus (thunberg, 1815), c.  mollis (charpentier, 1825), which can only be identified with certainty by male song patterns; our specimen is a female, but according to morphological traits most likely represents c. biguttulus), as well as three species of the subfamily oedipodinae: oedipoda caerulescens (linnaeus, 1758), sphingonotus caerulans (linnaeus, 1767), and stethophyma grossum (linnaeus, 1758) (table 1, 2). reference specimens are deposited in the zoological museum hamburg (zmh), part of the center of natural history (cenak) under the accession zmh 2019/21. genome size analysis nuclear dna content (2c) was measured by the f low cytometry method (fcm) as in sadílek et al. (2019a, b) at the department of botany of charles university, prague. the muscle tissue of one hind femur was used for fcm analysis against the plant-internal standard pisum sativum l. “ctirad” (fabaceae) with 2c = 9.09 pg (doležel et al. 1998; doležel and greilhuber 2010). fresh tissue was homogenized and mixed with a leaf of table 1. diploid chromosome number, 2c genome size, sample/standard ratio of both dapiand pi-stained samples and gc content of grasshopper species studied. samples were measured against p. sativum standard with 2c = 9.09 pg. f = female, m = male, 2n = male diploid chromosome number, 2c = nuclear dna content for nuclei with diploid chromosome number, cv = average coefficient of variation for each stain used. species 2n sex 2c (pg) sample/ standard dapi ratio sample/ standard pi ratio gc content (%) sample cv dapi pi sphingonotus caerulans 22+xx f 26.63 2.424 2.930 42.14 2.70 2.95 sphingonotus caerulans 22+x0 m 25.12 2.321 2.764 41.87 2.71 2.81 oedipoda caerulescens 22+xx f 28.39 2.621 3.123 41.88 3.71 5.62 chorthippus dorsatus 16+xx f 24.14 2.359 2.656 40.82 2.58 2.64 chorthippus biguttulus 16+xx f 22.62 2.149 2.488 41.35 2.50 4.07 stethophyma grossum 22+xx f 36.95 3.326 4.065 42.35 3.41 4.23 stethophyma grossum 22+x0 m 34.72 3.172 3.820 42.08 2.19 2.84 113new genome size estimates for band-winged and slant-faced grasshoppers reveal the so far largest measured insect genome ta bl e 2. g en om e si ze s of o rt ho pt er a so fa r m ea su re d. th e te m pl at e of t he t ab le w as e xt ra ct ed fr om g re go ry ( 20 20 ); it w as c om pl em en te d w ith o ri gi na l r ef er en ce s an d ad di tio na l s tu die s. r ef er en ce s w ith a n * in di ca te t ha t th e or ig in al r ef er en ce c ou ld n ot b e ac ce ss ed a nd d at a ar e ex tr ac te d on ly f ro m g re go ry ( 20 20 ). 1 r el at iv e ge no m e si ze m ea su re d w ith t he d a pi fr om m or ga nr ic ha rd s (2 00 5) . m = m al e, f = fe m al e, 2 c = g en om e si ze o f t he d ip lo id c el l, 2n = d ip lo id c hr om os om e nu m be r (i f s ex is n ot d et er m in ed , k ar yo ty pe o f t he m al e is p re se nt ed ; i n al l s pe ci es t he s ex d et er m in in g sy st em is x x /x 0, o nl y m al es o f p od is m a pe de st ri s ca n be v ar ia bl e w ith x y /x 0) , n .a . = n ot a va ila bl e; f d = f eu lg en d en si to m et ry , f c m = fl ow cy to m et ry m et ho d; a n = a nt en na , b r = b ra in , h e = ha em oc yt es , m s = m us cl e, o v = o va ri es , s = s pe rm , t s = te st es ; a c = a lli um c ep a (1 c = 1 6. 50 p g) , b o = b os ta ur us ( 1c = 3 .7 0 pg ), bp = b el lis p er en ni s (1 c = 1 .7 6 pg ), d m = d ro so ph ila m el an og as te r (1 c = 0 .1 8 pg ), d v = d ro so ph ila v ir ili s (1 c = 0 .3 4 pg ), g d = g al lu s do m es tic us ( 1c = 1 .2 5 pg ), h s = h om o sa pi en s (1 c = 3 .5 0 pg ), lm = l oc us ta m ig ra to ri a (1 c = 5 .5 0 pg ), m d = m us ca d om es tic a (1 c = 0 .9 0 pg ), m m = m us m us cu lu s (1 c = 3 .3 0 pg ), o m = o nc or hy nc hu s m yk is s (1 c = 2 .6 0 pg ), pa = p er ip la ne ta a m er ic an a (1 c = 3 .4 1 pg ), ps = p is um s at iv um ( 1c = 4 .5 5 pg ), sg = s ch is to ce rc a gr eg ar ia ( 1c = 8 .7 0 pg ). fa m ily su bf am ily sp ec ie s se x 1c [ pg ] 2n m et ho d c el l t yp e st an da rd s p. r ef er en ce s su bo rd er : c ae lif er a a cr id id ae a cr id in ae a cr id a co ni ca n. a. 12 .5 5 23 fd h e g d , o m r as ch 1 98 5* a cr id id ae a cr id in ae a cr id a co ni ca m 10 .8 2 23 fd t s g d r ee s et a l. 19 78 a cr id id ae a cr id in ae c al ed ia c ap tiv a m 10 .9 23 fd t s g d r ee s et a l. 19 78 a cr id id ae a cr id in ae c ry pt ob ot hr us c hr ys op ho ru s m 9. 37 23 fd t s g d r ee s et a l. 19 78 a cr id id ae a cr id in ae sc hi zo bo th ru s fla vo vi tt at us m 7. 5 n. a. fd t s g d r ee s et a l. 19 78 a cr id id ae c at an to pi na e m ac ro to na a us tr al is m 8. 49 23 fd t s g d r ee s et a l. 19 78 a cr id id ae c at an to pi na e pe ak es ia h os pi ta m 10 .4 7 23 fd t s g d r ee s et a l. 19 78 a cr id id ae c at an to pi na e ph au la cr id iu m v itt at um m 10 .7 3 23 fd t s g d r ee s et a l. 19 78 a cr id id ae c yr ta ca nt ha cr id in ae sc hi st oc er ca c an ce lla ta m 9. 49 23 fd t s lm jo hn a nd h ew itt 1 96 6 a cr id id ae c yr ta ca nt ha cr id in ae sc hi st oc er ca g re ga ri a n. a. 8. 96 23 fd v m m fo x 19 70 * a cr id id ae c yr ta ca nt ha cr id in ae sc hi st oc er ca g re ga ri a m 8. 71 23 fd t s m m w ilm or e an d br ow n 19 75 a cr id id ae c yr ta ca nt ha cr id in ae sc hi st oc er ca g re ga ri a m 8. 55 23 fd t s lm jo hn a nd h ew itt 1 96 6 a cr id id ae c yr ta ca nt ha cr id in ae sc hi st oc er ca g re ga ri a m 8. 74 23 fd s n. a. c am ac ho e t a l. 20 15 a cr id id ae c yr ta ca nt ha cr id in ae sc hi st oc er ca p ar an en si s m 8. 63 23 fd t s lm jo hn a nd h ew itt 1 96 6 a cr id id ae c yr ta ca nt ha cr id in ae va la ng a ir re gu la ri s m 9. 44 23 fd t s g d r ee s et a l. 19 78 a cr id id ae ey pr ep oc ne m id in ae ey pr ep oc ne m is p lo ra ns m 9. 7 23 fd s lm r ui zr ua no e t a l. 20 11 a cr id id ae ey pr ep oc ne m id in ae h et er ac ri s ad sp er su s m 6. 34 23 fd t s a c g os al ve z et a l. 19 80 a cr id id ae g om ph oc er in ae g om ph oc er us s ib ir ic us m 8. 95 17 fd t s a c g os al ve z et a l. 19 80 a cr id id ae g om ph oc er in ae c ho rt hi pp us a pi ca lis n. a. 12 .6 1 17 fd t s g d b el da e t a l. 19 91 * a cr id id ae g om ph oc er in ae c ho rt hi pp us b ig ut tu lu s f 11 .3 1 18 fc m m s ps th is s tu dy a cr id id ae g om ph oc er in ae c ho rt hi pp us b in ot at us n. a. 10 .9 1 17 fd t s g d b el da e t a l. 19 91 a cr id id ae g om ph oc er in ae c ho rt hi pp us c f. bi no ta tu s n. a. 10 .3 5 17 fd t s g d b el da e t a l. 19 91 a cr id id ae g om ph oc er in ae c ho rt hi pp us b ru nn eu s m 10 .1 5 17 fd t s a c g os al ve z et a l. 19 80 a cr id id ae g om ph oc er in ae c ho rt hi pp us b ru nn eu s m 9. 46 17 fd t s m m w ilm or e an d br ow n 19 75 a cr id id ae g om ph oc er in ae c ho rt hi pp us b ru nn eu s m 8. 55 17 fd t s lm jo hn a nd h ew itt 1 96 6 a cr id id ae g om ph oc er in ae c ho rt hi pp us d or sa tu s n. a. 8. 34 17 fd t s g d b el da e t a l. 19 91 a cr id id ae g om ph oc er in ae c ho rt hi pp us d or sa tu s f 12 .0 7 18 fc m m s ps th is s tu dy a cr id id ae g om ph oc er in ae c ho rt hi pp us ja co bs i n. a. 10 .8 4 17 fd t s g d b el da e t a l. 19 91 a cr id id ae g om ph oc er in ae c ho rt hi pp us ju cu nd us n. a. 11 .8 8 17 fd t s g d b el da e t a l. 19 91 a cr id id ae g om ph oc er in ae c ho rt hi pp us lo ng ic or ni s m 8. 58 17 fd t s a c g os al ve z et a l. 19 80 a cr id id ae g om ph oc er in ae c ho rt hi pp us n ev ad en si s n. a. 11 .5 3 17 fd t s g d b el da e t a l. 19 91 114 martin husemann et al. fa m ily su bf am ily sp ec ie s se x 1c [ pg ] 2n m et ho d c el l t yp e st an da rd s p. r ef er en ce s a cr id id ae g om ph oc er in ae ps eu do ch or th ip pu s pa ra lle lu s n. a. 14 .7 2 17 fd t s g d b el da e t a l. 19 91 a cr id id ae g om ph oc er in ae ps eu do ch or th ip pu s pa ra lle lu s n. a. 13 .8 3 17 n. a. n. a. n. a. pe tit pi er re 1 99 6 a cr id id ae g om ph oc er in ae ps eu do ch or th ip pu s pa ra lle lu s m 13 .3 6 17 fd t s m m w ilm or e an d br ow n 19 75 a cr id id ae g om ph oc er in ae ps eu do ch or th ip pu s pa ra lle lu s m 12 .3 1 17 fd t s lm jo hn a nd h ew itt 1 96 6 a cr id id ae g om ph oc er in ae c ho rt hi pp us s ca la ri s n. a. 14 .7 2 17 fd t s g d b el da e t a l. 19 91 a cr id id ae g om ph oc er in ae c ho rt hi pp us v ag an s m 8. 68 17 fd t s a c g os al ve z et a l. 19 80 a cr id id ae g om ph oc er in ae c ho rt hi pp us v ag an s n. a. 8. 64 17 fd t s g d b el da e t a l. 19 91 a cr id id ae g om ph oc er in ae m yr m el eo te tt ix m ac ul at us n. a. 13 .3 8 17 n. a. n. a. n. a. pe tit pi er re 1 99 6 a cr id id ae g om ph oc er in ae m yr m el eo te tt ix m ac ul at us m 12 .6 6 17 fd t s m m w ilm or e an d br ow n 19 75 a cr id id ae g om ph oc er in ae m yr m el eo te tt ix m ac ul at us m 12 .1 4 17 fd t s lm jo hn a nd h ew itt 1 96 6 a cr id id ae g om ph oc er in ae o m oc es tu s vi ri du lu s m 13 .1 6 17 fd t s lm jo hn a nd h ew itt 1 96 6 a cr id id ae g om ph oc er in ae st au ro de ru s sc al ar is n. a. 16 .3 4 17 n. a. n. a. n. a. pe tit pi er re 1 99 6 a cr id id ae m el an op lin ae c am py la ca nt ha o liv ac ea f 6. 98 n. a. fc m br g d h an ra ha n an d jo hn st on 2 01 1 a cr id id ae m el an op lin ae c am py la ca nt ha o liv ac ea m 6. 15 n. a. fc m br g d h an ra ha n an d jo hn st on 2 01 1 a cr id id ae m el an op lin ae m el an op lu s di ffe re nt ia lis m 6. 79 23 fc m br pa h an ra ha n an d jo hn st on 2 01 1 a cr id id ae m el an op lin ae m el an op lu s di ffe re nt ia lis n. a. 6. 23 23 fd h e g d , o m r as ch u np ub l. * a cr id id ae m el an op lin ae m el an op lu s di ffe re nt ia lis n. a. 3. 84 23 fd o v, t s b o sw ift a nd k le in fe ld 1 95 3* a cr id id ae m el an op lin ae m el an op lu s di ffe re nt ia lis f 7. 26 24 fc m br pa h an ra ha n an d jo hn st on 2 01 1 a cr id id ae m el an op lin ae m el an op lu s sa ng ui ni pe s n. a. 5. 83 23 fd h e g d , o m r as ch u np ub l. * a cr id id ae m el an op lin ae po di sm a pe de st ri s m 16 .9 3 23 /2 4 fd s sg w es te rm an n et a l. 19 87 a cr id id ae o ed ip od in ae a ilo pu s th al as si nu s m 6. 68 23 fd t s g d r ee s et a l. 19 78 a cr id id ae o ed ip od in ae a us tr oi ce te s pu si lla m 6. 29 23 fd t s g d r ee s et a l. 19 78 a cr id id ae o ed ip od in ae g as tr im ar gu s m us ic us m 9. 01 n. a. fd t s g d r ee s et a l. 19 78 a cr id id ae o ed ip od in ae h um be te nu ic or ni s m 8. 21 23 fd t s lm jo hn a nd h ew itt 1 96 6 a cr id id ae o ed ip od in ae c ho rt oi ce te s te rm in ife ra m 7. 22 23 fd t s m m w ilm or e an d br ow n 19 75 a cr id id ae o ed ip od in ae c ho rt oi ce te s te rm in ife ra m 5. 99 23 fd t s g d r ee s et a l. 19 78 a cr id id ae o ed ip od in ae lo cu st a m ig ra to ri a f 6. 44 24 fc m n. a. m m w an g et a l. 20 14 a cr id id ae o ed ip od in ae lo cu st a m ig ra to ri a n. a. 6. 35 23 fd h e g d , o m r as ch 1 98 5 a cr id id ae o ed ip od in ae lo cu st a m ig ra to ri a n. a. 6. 27 23 fd v m m fo x 19 70 a cr id id ae o ed ip od in ae lo cu st a m ig ra to ri a m 6. 09 23 fd t s m m w ilm or e an d br ow n 19 75 a cr id id ae o ed ip od in ae lo cu st a m ig ra to ri a m 5. 47 23 fd t s g d r ee s et a l. 19 78 a cr id id ae o ed ip od in ae lo cu st a m ig ra to ri a n. a. 5. 28 23 fd s m d bi er a nd m ül le r 19 69 * a cr id id ae o ed ip od in ae o ed ip od a ca er ul es ce ns f 14 .2 24 fc m m s ps th is s tu dy a cr id id ae o ed ip od in ae sp hi ng on ot us c ae ru la ns m 12 .5 6 23 fc m m s ps th is s tu dy a cr id id ae o ed ip od in ae sp hi ng on ot us c ae ru la ns f 13 .3 2 24 fc m m s ps th is s tu dy a cr id id ae o ed ip od in ae st et ho ph ym a gr os su m m 17 .3 6 23 fc m m s ps th is s tu dy a cr id id ae o ed ip od in ae st et ho ph ym a gr os su m f 18 .4 8 24 fc m m s ps th is s tu dy m or ab id ae m or ab in ae w ar ra m ab a vi rg o n. a. 4 15 fd br g d w hi te a nd w eb b 19 68 115new genome size estimates for band-winged and slant-faced grasshoppers reveal the so far largest measured insect genome fa m ily su bf am ily sp ec ie s se x 1c [ pg ] 2n m et ho d c el l t yp e st an da rd s p. r ef er en ce s m or ab id ae m or ab in ae w ar ra m ab a vi rg o n. a. 3. 75 15 n. a. n. a. n. a. pe tit pi er re 1 99 6 su bo rd er : e ns ife ra a no st os to m at id ae d ei na cr id in ae h em id ei na c ra ss id en s 1 m 5. 4 15 fc m a n bp m or ga nr ic ha rd s 20 05 a no st os to m at id ae d ei na cr id in ae h em id ei na c ra ss id en s 1 f 6. 01 16 fc m a n bp m or ga nr ic ha rd s 20 05 a no st os to m at id ae d ei na cr id in ae h em id ei na th or ac ic a 1 m 5. 95 15 fc m a n bp m or ga nr ic ha rd s 20 05 a no st os to m at id ae d ei na cr id in ae h em id ei na th or ac ic a 1 f 6. 53 16 fc m a n bp m or ga nr ic ha rd s 20 05 g ry lli da e g ry lli na e a ch et a do m es tic us n. a. 2. 38 11 fi a h e d m k os hi ka w a et a l. 20 08 g ry lli da e g ry lli na e a ch et a do m es tic us n. a. 2 11 fd h e g d , o m r as ch 1 98 5 g ry lli da e g ry lli na e a ch et a do m es tic us n. a. 2 11 fd o v, t s m m , h s li m ade -f ar ia e t a l. 19 73 g ry lli da e g ry lli na e a ch et a do m es tic us n. a. 2 11 fc m br d m g re go ry u np ub l. g ry lli da e g ry lli na e a ch et a do m es tic us n. a. 2 11 fi a h e g d g re go ry u np ub l. g ry lli da e g ry lli na e g ry llu s pe nn sy lv an ic us n. a. 2. 68 11 n. a. n. a. n. a. pe tit pi er re 1 99 6 g ry lli da e g ry lli na e g ry llu s pe nn sy lv an ic us n. a. 2. 06 21 fd s m d bi er a nd m ül le r 19 69 g ry lli da e g ry lli na e g ry llu s pe nn sy lv an ic us n. a. 2 21 fd h e g d , o m r as ch 1 98 5 g ry lli da e o ec an th in ae o ec an th us n iv eu s n. a. 1. 71 n. a. fc m br d v h an ra ha n an d jo hn st on 2 01 1 g ry llo ta lp id ae g ry llo ta lp in ae n eo sc ap te ri sc us b or el lii n. a. 3. 41 n. a. fc m br g d h an ra ha n an d jo hn st on 2 01 1 r ha ph id op ho ri da e c eu th op hi lin ae c eu th op hi lu s st yg iu s n. a. 9. 55 n. a. fd h e g d , o m r as ch a nd r as ch 1 98 1 r ha ph id op ho ri da e c eu th op hi lin ae h ad en oe cu s su bt er ra ne us n. a. 1. 55 n. a. fd h e g d , o m r as ch a nd r as ch 1 98 1 te tt ig on iid ae c on oc ep ha lin ae c on oc ep ha lu s sp . m 2. 65 33 fc m br g d h an ra ha n an d jo hn st on 2 01 1 te tt ig on iid ae c on oc ep ha lin ae c on oc ep ha lu s sp . f 3. 03 34 fc m br g d h an ra ha n an d jo hn st on 2 01 1 te tt ig on iid ae c on oc ep ha lin ae n eo co no ce ph al us tr io ps m 7. 29 n. a. fc m br g d h an ra ha n an d jo hn st on 2 01 1 te tt ig on iid ae c on oc ep ha lin ae n eo co no ce ph al us tr io ps f 7. 93 n. a. fc m br g d h an ra ha n an d jo hn st on 2 01 1 tr id ac ty lid ae  n .a . un kn ow n sp . n. a. 2. 63 n. a. fc m br d v h an ra ha n an d jo hn st on 2 01 1 tr ig on iid ae tr ig on id iin ae la up al a ce ra si na n. a. 1. 93 n. a. fc m br g d pe tr ov e t a l. 20 00 116 martin husemann et al. the standard in 500 μl of 4°c cold otto buffer i. the suspension of released cells was then filtered through a 42 μm nylon mesh and divided in two parts. one part was stained with 1,000 μl dapi solution (stock: 25 ml otto buffer ii, 1 ml dapi (0.1 mg/ml), 25 μl 2-mercaptoethanol (2 μl/ml)); the second part was stained with 1,000 μl propidium iodide (pi) solution (stock: 25 ml otto buffer ii, 1 ml rnase (1 mg/ml), 1 ml pi (1 mg/ml), 25 μl 2-mercaptoethanol) (doležel et al. 2007). for dapi analysis, the partec cyflow instrument (partec gmbh, münster, germany) with uv led chip and for pi analysis the partec sl instrument with a green solid-state laser (cobolt samba, 532 nm, 100 mw) were used. each sample was stained for several minutes before measurement, and 3,500 to 5,000 particles were recorded in each fcm analysis. fcm data were analysed with the partec flomax v. 2.52 software (partec gmbh, münster, germany). combined dapi and pi measurement results of the same sample express the at/gc ratio of the genome of the species, the gc content (e.g. šmarda et al. 2008; sadílek et al. 2019a, b). the gc content of p. sativum is 38.50% (e.g. barrow and meister 2002; šmarda et al. 2008) and the gc content of the analysed samples was calculated with the microsoft excel macro from šmarda et al. (2008). results dapi-stained samples yielded a lower coefficient of variation (cv) than pi-stained samples, on average cv = 2.83% and 3.59% respectively. all the analysed species of oedipodinae reached higher genome size values than the analysed species of gomphocerinae. we were able to measure the genome size of both sexes only in two species (s. caerulans and s. grossum). there, the female/male genome size values clearly reflected the xx/ x0 sex determination system differences. due to this sex determination system it is generally preferred to report genome size in 2c values rather than the commonly used 1c value. however, to allow for better comparability, we here report both values. all analysed species of oedipodinae had distinct genome size (table 1). the male of s.  caerulans had 2c = 25.12 pg (1c = 12.56 pg); the female had 2c = 26.63 pg (1c = 13.32 pg). the female specimen of o. caerulescens exhibited a 2c value of 28.39 pg (1c = 14.20 pg). the largest genome size was recorded in s. grossum, where the male reached 2c = 34.72 pg (1c = 17.36 pg) and the female 2c = 36.95 pg (18.48 pg). both closely related gomphocerinae species showed very similar genome sizes (table 1): 2c = 22.62 pg (1c = 11.31 pg) in the c. cf. biguttulus female and 2c = 24.14 pg (1c = 12.07) in the female of c. dorsatus. the sample/standard ratio of samples stained with pi was always higher than in dapi-stained samples of the same specimen, ranging from 11% difference in the female of c. dorsatus to 18% difference in the female of s. grossum. this trend is observable also in the gc content, where c. dorsatus had only 40.82% and the female of s. grossum had 42.35% (table 1). however, the gc content differences among all species analysed were minimal. discussion we present new genome size estimates for five species of acrididae, one of which represents the largest genome of all insects measured so far, the genome of the female of stethophyma grossum with 2c = 36.95 pg (1c = 18.48 pg). we also measured a female of c. dorsatus with 2c = 24.14 pg (1c = 12.07 pg). this species was measured before using the feulgen densitometry method with 1c = 8.34 pg (belda et al. 1991). however, the more recent method of flow cytometry we used is considered more accurate for genome size estimations (e.g. doležel and greilhuber 2010). furthermore, we collected all previous estimates from gregory (2020) and added few additional resources to provide some basic visualization of the genome size variation in the different subfamilies of orthoptera (fig. 1). in total, we gathered 92 (our new data included) estimates of genome sizes belonging to 54 species (table 2, fig. 1). these data included 68 estimates for caelifera (43 species) and 17 for ensifera (11 species). they ranged from 1c = 3.75 pg for warramaba virgo (key, 1963) (morabidae) (petitpierre 1996) to 1c = 18.48 pg for stethophyma grossum (oedipodinae, present study) in caelifera and from 1c = 1.55 pg for hadenoecus subterraneus to 1c = 9.55 pg for ceuthophilus stygius (scudder, 1861) (both cave rhaphidophoridae) in ensifera (rasch and rasch 1981). average 1c values in ensifera and caelifera are 3.16 pg (± 2.18 pg) and 9.83 pg (± 3.32 pg) respectively. further analyses at the family and subfamily level are difficult, as most data comes from acrididae with 66 measurements (78%). the average genome size in acrididae is 10.01 pg (± 3.19 pg). within acrididae, most estimates came from 26 measurements of gomphocerinae and 17 of oedipodinae with average genome sizes of 1c = 11.52 pg (± 2.17 pg) and 9.13 pg (± 4.20 pg) respectively (table 2, fig. 1). generally, the short-horned grasshoppers (caelifera) appear to have larger genomes compared to the long117new genome size estimates for band-winged and slant-faced grasshoppers reveal the so far largest measured insect genome horned grasshoppers (ensifera). however, this is not correlated with the number of chromosomes. despite their relatively low male number of chromosomes of 2n = 17 (most of other acrididae have 2n = 23; e.g. sylvester et al. 2019), gomphocerinae have some of the largest genome sizes. their average genome size is 1c = 11.52 pg ranging from 1c = 8.34 pg in c.  dorsatus (belda et al. 1991) to 16.34 pg in stauroderus scalaris (petitpierre 1996; gregory 2020). moreover, they show large intraspecific variation in genome size evident from different studies (table 2), for example: 1c = 12.31 pg to 14.72 pg for pseudochorthippus parallelus (zetterstedt, 1821) (john and hewitt 1966; wilmore and brown 1975; belda et al. 1991; petitpierre 1996) or 1c = 8.55 to 10.15 pg for c. brunneus (john and hewitt 1966; wilmore and brown 1975; gosalvez et al. 1980). all studies of the two species mentioned above share the method of feulgen densitometry and used testes to measure genome size. hence it remains unclear whether this variation is natural or the result of methodological differences. however, it is more likely that the large intraspecific differences are a result of a combination of multiple factors: different populations analysed, lack of chromosome observations, various standards used and also different instrumentation could play some role. the variation in genome size is even higher in oedipodinae with a minimum of 1c = 5.28 pg for locusta migratoria (linnaeus, 1758) (bier and müller 1969) and a maximum of 1c = 18.48 pg in stethophyma grossum. hence, s. grossum represents the so far largest measured confirmed insect genome. a study by schielzeth et al. (2014) measured much larger genome sizes for the gomphocerinae species c. biguttulus with 1c up to 236.05 pg. due to the enormous variation of the estimates in the study and critical methodological issues, camacho (2016) suggested that these estimates cannot be considered reliable. hence, we consider our estimate of the s. grossum genome size as the current upper size of insect genomes. since only very few species have been measured so far, it is expected that this is not the upper bound for genome sizes in grasshoppers or for insects in general. the reasons for the large size of caelifera genomes remain largely unknown. however, a recent paper by shah et al. (2020) suggests that repetitive dna and especially the expansion of satellite dna may be a main reason for the large genomes in orthoptera. the most likely causes are genome duplications at the basis of the acrididae, which would also explain their specifically high rates in nuclear mitochondrial pseudogenes (numts, figure 1. relative fluorescence histograms for samples stained with pi. 2c peaks represent diploid cells and 4c peaks represent cells in the g2 phase of the cell cycle. with replicated dna. standard used: p. sativum 2c = 9.09 pg. (a) s. grossum  female with 2c = 36.95 pg. (b) c. biguttulus female with 2c = 22.62 pg. 118 martin husemann et al. bensasson et al. 2000; song et al. 2008) posing difficulties to species identification using dna barcoding and to phylogenetic reconstruction (hawlitschek et al. 2017, song et al. 2018). it may also explain why only a single incomplete genome is available to date (wang et al. 2014). grasshopper genome sizes remain a major obstacle to genomic research, and many further studies will be required to understand genome size variation and evolution in orthoptera. acknowledgement we thank torsten demuth for providing locality access and help with sampling. we also thank martin fikáček (charles university, prague, czech republic) for financial support for processing the samples in fcm laboratory of tomáš urfus (charles university, prague, czech republic) from the botany department. data availability statement all data generated and used in this article is included as tables and figures. geolocation information all sampling for this study was performed 2019 in hamburg, georgswerder (germany, 53.5097°n 10.0301°e). references barrow m, meister a. 2002. lack of correlation between at frequency and genome size in higher plants and the effect of non-randomness of base sequences on dye binding. cytometry 47:1–7. belda je, cabrero j, camacho jpm, rufas js. 1991. role of c-heterochromatin in variation of nuclear dna amount in the genus chorthippus (orthoptera, acrididae). cytobios 67:13–21. bensasson d, zhang d-x, hewitt gm. 2000. frequent assimilation of mitochondrial dann by grasshopper nuclear genomes. mol biol evol 17:406-415. bier k, müller w. 1969. dna-messungen bei insekten und eine hypothese über retardierte evolution und besonderen dna-reichtum in tierreich. biol zentralblatt 88:425–449. camacho jpm, ruiz-ruano fj, martin-blázquez r, cabrero j, lorite p, cabral-de-mello dc, bakkali m. 2015. a step to the gigantic genome of the desert figure 2. genome size variation in the different subfamilies of orthoptera visualized as a boxplot. provided is the number of measurements (n) and the number of species (sp) these measurements were derived of (some of the species were measured repeatedly by different authors). most of the data excerpted from database gregory (2020) completed with another original data comprehended in table 2. *unknown species genome size was analysed, determined only on family level. 119new genome size estimates for band-winged and slant-faced grasshoppers reveal the so far largest measured insect genome locust: chromosome sizes and repeated dnas. chromosoma 124:263–275. camacho jpm. 2016. comment on schielzeth et al. 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mm. 2018. evolution, diversification, and biogeography of grasshoppers (orthoptera: acrididae). insect syst div 2:3;1-25. swift h, kleinfeld r. 1953. dna in grasshopper spermatogenesis, oögenesis, and cleavage. phys zool 26:301– 311. sylvester t, blackmon h. 2019. idiosycratic patterns of chromosome evolution are the rule not the exception. https://evobir.shinyapps.io/polyneopteradb/ current version of the database is 0.1 last updated 12 august 2019. wang x, fang x, yang p, jiang x, jiang f, zhao d, li b, cui f, wei j, ma c, wang y, he j, luo y, wang z, guo x, guo w, wang x, zhang y, yang m, hao s, chen b, ma z, yu d, xiong z, zhu y, fan d, han l, wang b, chen y, wang j. 2014. the locust genome provides insight into swarm formation and long-distance flight. nat comm 5:2957. westerman m, barton nh, hewitt gm. 1987. differences in dna content between two chromosomal races of the grasshopper podisma pedestris. heredity 58:221– 228. white mjd, webb gc. 1968. origin and evolution of parthenogenetic reproduction in the grasshopper moraba virgo (eumastacidae: morabinae). aust j zool 16:647–671. wilmore pj, brown ak. 1975. molecular properties of orthopteran dna. chromosoma 51:337–345. caryologia. international journal of cytology, cytosystematics and cytogenetics 73(1): 3-10, 2020 firenze university press www.fupress.com/caryologiacaryologia international journal of cytology, cytosystematics and cytogenetics issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-863 citation: s. sadeghian, a. hatami, m. riasat (2020) karyotypic investigation concerning five bromus species from several populations in iran. caryologia 73(1): 3-10. doi: 10.13128/caryologia-863 received: april, 2019 accepted: february, 2020 published: may 8, 2020 copyright: © 2020 s. sadeghian, a. hatami, m. riasat. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. karyotypic investigation concerning five bromus species from several populations in iran sara sadeghian, ahmad hatami, mehrnaz riasat research division of natural resources department, fars agricultural and natural resources research and education center, areeo, shiraz, iran *corresponding author: s.sadeghian@areeo.ac.ir abstract. karyotypes of five taxa (fourteen populations) of the genus bromus from different geographic origins is presented: b. scoparius, b. japonicus, b. madritensis, b. rubens and b. tomentellus. the ploidy levels were different. b. scoparius and b. japonicus were found 2n=2x=14, b. madritensis and b. rubens were found 2n=4x=28 and b. tomentellus were found 2n=6x=42. detailed karyotype analysis allows us to group the different populations and to postulate relationships among them. keywords. bromus, chromosome, karyology, iran. introduction the genus bromus l. belongs tribe bromeae and poaceae family. the taxon includes about 160 annual and perennial species (acedo and liamas, 2001) distributed all over the world. bromus species are distributed in temperate regions and are always exist with rangeland species (verloove, 2012). it is an important rangeland plant species in iran, which are placed in 6 sections; bromus, genea, nevskiella, neobromus, ceratochla and pnigma (bor, 1970) (table 1). the genea section is the widest section of the bromus genus in terms of geographic distribution (sales, 1994). bromus species are known as the species with various intra-specific ploidy levels and form different ecotypes with various characteristics. hill (1965) recorded up to 112 chromosomes for b. erectus. devesa et al. (1990) indicates the importance of cytological studies for understanding the evolution of the genus bromus. naganowas ka (1993) used genetic distances estimated based on centromeric index and total chromosome length to investigate interrelationships of several species of bromus. yang and dunn (1997) recorded various levels of polyploidy in b. inermis leyss. martinello and schifino-wittmann (2003) studied 14 accessions of bromus auleticus. their accessions were all hexaploid and the high symmetry and homogeneity of the karyotypes made it difficult to detect possible intraspecific differences. massa et al. (2004) proposed a taxonomic treatment within bromus sect. ceratochloa of south america. their plant materials included 28 hexaploid 4 sara sadeghian, ahmad hatami, mehrnaz riasat (2n=6x=42) populations and 2 octaploid (2n=8x=56) populations. oja and laarmann (2002) also recorded different ploidy levels within species of bromus (2n=14, 28, 42 and 56). sheidai and fadaei (2005) studied ten populations of six bromus species and the species possess karyotypes varying from 2n = 2x = 14 (diploid) to 2n = 4x = 28 (tetraploid). mirzaie-nodoushan et al. (2006a) investigated karyotypic of some bromus species in iran and indicated that populations of the species were differed in their karyotypic characteristics and ploidy levels of the populations were varied from 2n=14 to 2n=84. mirzaie-nodoushan et al. (2006b) also reported evolutionary karyotypic variation in b. tomentellus populations in iran and confirmed the existence of high levels of ploidy as well as existence of dodecaploid karyotypes in the species. sadeghian and et al. (2010) studied nine populations of three bromus species (b. danthoniae, b. sterilis and b. tectorum) and reported that all species were diploid with 2n=2x=14. artico et al. (2017) also reported that the chromosomal number of b. linnaeus was 2n = 6x = 42. since the karyological information is the basic requirement of a breeding program, in this study, 14 populations of bromus were surveyed for the karyological data as a part of an ongoing work on the populations. cytogenetic studies play an important role in determining the relationship between species especially wild and native plants and as a first step in the analysis of the phylogeny and evolution of species is relative. considering that the species studied in different climates of southwest iran and fars province are abundant they are considered as the main vegetation cover of these areas. therefore, to investigate the relationship between species, these species have been used in this study. materials and methods fourteen populations of five bromus species: b. tomentellus (three populations) belong to pnigma section, b. madritensis (two populations) and b. rubens (three populations) belong to genea section and b. scoparius (three population) and b. japonicus (three population) belong to bromus section were studied (table 1). voucher specimens were deposited in the herbarium of fars research and education center for agriculture and natural resources and in gene bank rifr (research institute of forest and rangelands) of iran. root tip meristems from seedling obtained by the germination of ripe seeds collected from natural populations (14 populations, representing 5 species) on wet filter paper in petri dishes and left at 22°c temperature. when they reached 1-1.5 cm in length, rootlets were collected. the material was pretreated in %0.5 saturated α-bromo naphthalene at 4°c for 4 h, fixed in %10 formaldehyde and chromium trioxide (1:1 volume ratio) for 16 to 20 h at 4°c. then, the roots tips were rinsed for 3 h in distilled water. hydrolysis was carried out with naoh (1 normal) at 60ºc for 20-30 min (sadeghian et al. 2010)) and used hematoxylin-iron for chromosome staining for 1-2 h. squashed in a droplet of %45 acetic acid and lactic acid (10:1) (wittmann 1965). at least, five well-spread metaphase plates from different individuals were analyzed per population. the best metaphasical table 1. the origin of materials used in chromosome studies of bromus. species (population) section origin altitude herbarium code b. japonicus (16462) bromus golestan, maraveh tapeh, station 430 m 16462 b. japonicus (16525) bromus golestan, gomayshan, seidabad -15 m 16525 b. japonicus (16587) bromus golestan, tooskasetan 1216 m 16587 b. madritensis (3668) genea fars, shiraz, dasht-e arjan 2000 m 3668 b. madritensis (arjan) genea fars, shiraz rosd of dasht-e arjan to tange abolhayat, about kandee village 1300 m b. rubens (15169) genea fars, kazeroon, kotal dokhtar 1400 m 15169 b. rubens (15317) genea fars, fasa, mianjangal 1750 m 15317 b. rubens (2125) genea fars, kazeroon 530 m 2125 b. scoparius (5983) bromus gilan, talesh, subatan yelagh 1800 m 5983 b. scoparius (5984) bromus gilan, talesh, khotbesara, lapehkara 1800 m 5984 b. scoparius (5985) bromus gilan, masal 1900 m 5985 b. tomentellus (bavanat) pnigma fars, bavanat, simakan, lakposhti range 2300 m b. tomentellus (simakan) pnigma fars, simakan, lakposhti range 2350 m b. tomentellus (eghlid) pnigma fars, eghlid, dozkord, pasahlaki 2200 m 5karyotypic investigation concerning five bromus species from several populations in iran plates were selected and measured by micromeasure 3.3 software (reeves et al. 2000). in each mitotic metaphase (at least 5 plates) the arm’s length of each chromosome was measured. the following parameters were estimated in each metaphase plate to characterize the karyotypes numerically: long arm (la), short arm (sa), total length (tl), relative length percentage (rl %), arm ratio (ar), centromeric index (ci) (huziwara, 1962), value of relative chromatin (vrc). karyotype asymmetry was estimated by three different methods namely, total form percentage (tf %) (huziwara, 1962); difference of relative length (drl), intra-chromosomal asymmetry index (a1) and inter-chromosomal asymmetry index (a2). both indices (a1 and a2) (romero zarco, 1986) were independent to chromosome number and size. also karyotypic evolution has been determined using the symmetry classes of stebbins (sc) (stebbins, 1971). karyotype formula was determined by chromosome morphology based on centromere position according to classification of levan (levan et al. 1964). for each population, karyograms were drawn based on length of chromosome size (arranged large to small). in order to determine the variation between populations, one-way unbalanced anova was performed on normal data and parameter means were compared by duncan’s test. the principal components analysis (pca) was performed to evaluate the contribution of each karyotypic parameter to the ordination of species. clustering was performed using the unweighted pair group method with arithmetic (upgma) after calculation of cophenetic correlation coefficient (r) to examine karyotype similarity among populations. numerical analysis was performed using sas ver. 6.12 (1996), jmp ver. 3.1.2 (1995) and statistixl ver. 1.7 (2007) softwares. results there was no different among basis chromosome number of the species (x=7). the somatic chromosome numbers (2n), karyotype formula and parameters for the studied species are summarized in table 2. two species as b. scoparius and b. japonicus were diploid, two species as b. madritensis and b. rubens were tetraploid and one species as b. tomentellus was hexaploid. the studied species included metacentric (m) and sub-metacentric (sm) chromosomes regarding the chromosomal types (table 2). satellites were observed in one chromosomes pair in b. scoparius and b. japonicus and two chromosomes pairs in b. madritensis and b. rubens and for b. tomentellus species which has three chromosomes pairs having satellites (fig 1). according to the stebbin’s bilateral table, populations of b. rubens (15317) included the highest value regarding the intra-chromosomal asymmetry index (0.288) and was classified as group 1b and population of b. japonicus (16462) included the lowest value regarding the intra-chromosomal asymmetry index (0.180) and was classified as group 1a. the results of analysis of variance indicated that there was a significant difference (p≤1%) between the populations in terms of chromosomal traits (tl, la, table 2. karyotypic characters of different bromus taxa and population. taxon (population) 2n a1 a2 %tf drl vrc sc k.f. b. japonicus (16462( 2x=14 0.180 0.129 45.174 5.369 9.605 1a 12m+2sm b. japonicus (16525( 2x=14 0.197 0.145 44.700 6.131 8.704 1a 14m b. japonicus (16587) 2x=14 0.250 0.144 42.950 6.193 8.249 1a 14m b. madritensis (3668) 4x=28 0.206 0.213 43.022 4.353 5.358 1a 28m b. madritensis (arjan) 4x=28 0.238 0.199 41.988 4.757 5.329 1a 28m b. rubens (15169) 4x=28 0.252 0.241 41.486 5.583 6.114 1b 28m b. rubens (15317) 4x=28 0.288 0.256 38.797 5.332 6.013 1b 28m b. rubens (2125) 4x=28 0.257 0.230 39.904 5.389 6.390 1b 24m+4sm b. scoparius (5983) 2x=14 0.233 0.116 42.493 4.722 8.166 1a 14m b. scoparius (5984) 2x=14 0.227 0.124 42.113 5.722 7.929 1a 12m+2sm b. scoparius (5985) 2x=14 0.214 0.126 44.062 5.479 6.866 1a 14m b. tomentellus (bavanat) 6x=42 0.215 0.110 42.523 1.698 6.271 1a 38m+4sm b. tomentellus (simakan) 6x=42 0.204 0.122 44.354 2.278 6.894 1a 42m b. tomentellus (eghlid) 6x=42 0.225 0.140 41.278 2.146 6.878 1a 42m 2n: diploid chromosome numbers a1: intrachromosome asymmetry index, a2: interchromosome asymmetry index, tf%: total form percentage, drl: difference of relative length, vrc: value of relative chromatin, symmetry classes (sc) of stebbins and karyotype formula (k.f.). 6 sara sadeghian, ahmad hatami, mehrnaz riasat a d g j m b c e f h i n k l figure 1. representative mitotic plates of bromus – (a) b. scoparius (5983), 2n=2x=14, (b) b. scoparius (5984), 2n=2x=14, (c) b. scoparius (5985) 2n=2x=14, (d) b. japonicus (16462), 2n=2x=14, (e) b. japonicus (16525), 2n=2x=14, (f ) b. japonicus (16587), 2n=2x=14, (g) b. rubens (15169), 2n-4x=28, (h) b. rubens (15317), 2n=4x=28, (i) b. rubens (2125), 2n=4x=28, (j) b. tomentellus (bavanat), 2n=6x=42, (k) b. tomentellus (simakan), 2n=6x=42, (l) b. tomentellus (eghlid), 2n=6x=42, (m) b. madritensis (3668). 2n=4x=28, (n) b. madritensis (arjan), 2n=4x=28. 7karyotypic investigation concerning five bromus species from several populations in iran sa) which revealed large variations among the germplasms in regard to studied traits. symmetry type of stebbins (1971) and asymmetry indices of romero-zarco (1986) are given in table 2. difference in the relative length percentage (drl) of the highest and the smallest chromosomes varied from 6.19 in b. japonicus (16587) to 1.69 in b. tomentellus (bavanat). according to table 2, b. rubens (15317) was placed in 1b and had the highest values of intrachromosomal asymmetry index. similarly, high drl value leads to more changes in the construction of chromosomes. it had the lowest tf%. the tf% and a1 values had inverse ratio (table 2). the mean value of chromosome’s long arm was varied from 5.27 in b. japonicus (16462) to 2.92 in b. madritensis (3668). averages of chromosome’s short arm were different from 2.24 in b. madritensis (arjan) to 4.34 in b. japonicus (16462). the total length of the chromosome was varied from 9.61 in b. japonicus (16462) to 5.17 in b. madritensis (arjan) and the mean value of chromosome’s arm ratio was in range from 1.41 in b. rubens (15317) to 1.22 in b. japonicus (16462) (table 3). the results showed that the highest vrc amongst all populations was obtained for b. japonicus (16462) and the lowest was obtained for b. madritensis (arjan). based on intra-chromosomal asymmetry, some populations had the most asymmetrical and evolutionary karyotype. according to inter-chromosomal asymmetry, b. rubens (15317) had the most asymmetrical karyotype in all of the populations. the ratio of long arm/short arm chromosomes (ar) showed a high significant difference among some species belong to different sections, while other species are not clearly distinct (table 3). diploid species of b. japonicus (16462) for instance, had the lowest ar value (1.22), the highest tf% value (45.17) and the lowest a1 value (0.18), exhibiting the most symmetrically karyotypes, while b. rubens (15317) with the highest ar value (1.41), the lowest tf% value (38.80) and the highest a1 value (0.29) were introduced as the most asymmetrical karyotypes (table 3). the pattern of variation of a1 and a2 values has been compared with the pattern of stebbins’ system. the statistical comparison based on completely randomized design showed that there were significant differences among the populations for tl, la and sa traits (p ≤ %1) (table 4). the principal component analysis (pca) of the karyotypic parameter shows the first two principal components account for 0.81% of total variance. component one (0.59%) put emphasized on the a1, a2 and drl. while component two (0.23%) accentuates, chromosome total length, long arm length, short arm length and tf% values which had the highest coefficients of eigen vectors (table 5). the diagram of the population’s dispersion, based on two first components showed that the populations separated in four groups, which completely fits with the results obtained through the average grouping analysis method (fig. 2). the dendrogram obtained from the cytogenetic studies of 14 populations of bromus indicated the fortable 3. mean of chromosomes analysis of bromus population. populations tl la sa ar ci drl %tf a1 a2 b. japonicus (16462) 9.61 5.27 4.34 1.22 0.46 5.37 45.17 0.18 0.13 b. japonicus (16525) 8.71 4.82 3.90 1.24 0.45 6.13 44.70 0.20 0.15 b. japonicus (16587) 8.24 4.71 3.55 1.32 0.43 6.19 42.95 0.25 0.14 b. madritensis (3668) 5.23 2.92 2.31 1.27 0.44 4.35 43.03 0.21 0.21 b. madritensis (arjan) 5.17 2.94 2.24 1.32 0.43 4.76 41.99 0.24 0.20 b. rubens (15169) 5.91 3.38 2.54 1.33 0.42 5.58 41.49 0.25 0.24 b. rubens (15317) 5.62 3.29 2.34 1.41 0.41 5.33 38.80 0.29 0.26 b. rubens (2125) 5.97 3.42 2.55 1.35 0.42 5.39 39.90 0.26 0.23 b. scoparius (5983) 8.02 4.55 3.48 1.31 0.43 4.72 42.49 0.23 0.12 b. scoparius (5984) 7.62 4.29 3.34 1.29 0.43 5.72 42.11 0.23 0.12 b. scoparius (5985) 6.87 3.84 3.03 1.27 0.44 5.48 44.06 0.22 0.13 b. tomentellus (bavanat) 6.08 3.42 2.67 1.28 0.43 1.69 42.52 0.22 0.11 b. tomentellus (simakan) 6.90 3.84 3.06 1.26 0.44 2.28 44.35 0.20 0.12 b. tomentellus (eghlid) 6.52 3.68 2.84 1.30 0.43 2.15 41.28 0.22 0.14 tl: total length of chromosome, la: long arm, sa: short arm, ar: arm ratio, ci: centromeric index, drl: difference of relative length, tf%: total form percentage, a1: intra-chromosome asymmetry index, a2: inter-chromosome asymmetry index. 8 sara sadeghian, ahmad hatami, mehrnaz riasat mation of five clusters in the euclidean distance of 0.05. the first cluster consists of the b. tomentellus populations (simakan, eghlid and bavanat). the populations of bavanat and eghlid showed the most kinship, and the population of simakan is located in a relatively short distance to these two populations. the second cluster consists of b. rubens (15317) and b. rubens (2125) populations which showed a close kinship. the third cluster consists of the populations b. madritensis (3668, arjan) and b. rubens (15169), the two populations of 3668 and arjan are closely related to each other, with a relatively short distance from the population of 15169. the populations of b. scoparius (5983, 5984, 5985) and b. japonicus (16587) formed the fourth cluster. the populations of 5983 and 5984 are located close to each other with a relatively short distance from the population of 5985. the population of 5985 is located in a longer distance than the three mentioned populations. the two populations of b. japonicus (16462 and 16525) with a short distance from each other, formed the fifth cluster (fig. 3). discussion this study reveals a detailed picture of the chromosome features in five bromus species of iran. the knowledge of chromosome numbers, karyotype evolution, ploidy level and genome size can provide additional information that not only gives further insight into the functioning of the genome, but also have considerable predictive powers. in this genus, the basic chromosome number is x=7, as were found for fourteen populations of five species of bromus (2n=2x=14, 2n=4x=28 and 2n=6x=42). this study confirmed that the bromus species show great variations in the number of chromosomes. at the interspecific level, quantitative and qualitative data allowed us the differentiation of several of the taxa studied. among species, the most variable characters were the number of “m”, and “sm” chromosomes, as well as the number and position of satellites (table 2; fig. 1). as a result, the species also could be differentiated by the number, type and position of satellites. this study revealed that three populations of b. rubens and b. madritensis were tetraploid (2n=4x=28) species (table 2 and fig. 1). this is in agreement with the results of an investigation recorded by sheidai and fadaei (2005). three populations of b. tomentellus was the only hexaploid (2n=6x=42), but mirzaie-nodoushan et al. table 4. the results of variance analysis for karyotypic data based on crd design. s.o.v d.f tl mean of squares la sa ar ci drl tf a1 a2 populations 13 11.50** 2.677** 2.032** 0.014** 0.001** 10.383** 9.7ns 0.004ns 440.55** error 56 0.50 0.131 0.115 0.009 4.64e-04 3.987 6.869 0.002 86.161 %c.v. 2.57 0.61 0.48 0.01 5.51e-04 5.19 7.40 2.57e-03 152.93 **: significant at 1%. table 5. specific values of variance percentage and coefficients of specific vectors in analysing main components. name of traits first component second component sa 0.93 0.32 la 0.87 0.44 tl 0.90 0.38 ar 0.050.72 ci 0.89 -0.27 a1 0.820.52 a2 -0.77 0.22 drl 0.09 0.79 tf 0.89 -0.23 specific values 5.28 2.07 percentage of variance 0.59 0.23 cum percentage of variance 0.59 0.81 princ1 p ri n c2 5.02.50.0-2.5-5.0 4 3 2 1 0 -1 -2 b.t. eghlid b.t. simak anb.t. bav anat b.s. 5985 b.s. 5984 b.s. 5983 b.r. 2125 b.r. 15317 b.r. 15169 b.m. a rjan b.m. 3668 b.j. 16587 b.j. 16525 b.j. 16462 score plot figure 2. scatter plot of 14 populations for the first two principals. 9karyotypic investigation concerning five bromus species from several populations in iran (2006b) recorded different ploidy levels within this species (2n=42, 70 & 84). three populations of b. japonicus and b. scoparius species studied were diploid (2n=2x=14), supporting the earlier report of safari et al. (2017). the present study confirmed that the bromus species show great variations in the number of chromosomes both at inter and intra-specific levels. this kind of genetic and cytogenetic variability can confer an adaptive advantage against variable climate and other ecological elements in the region (mirzaie-nodoushan et al. 2006a). the duncan’s test applied to the chromosome morphometric traits (la, sa, tl, ar, drl, tf%, a1 and a2) showed a highly significant difference among all examined populations of different sections (table 3). the study revealed cytogenetic differences (p≥1%) in anova for karyological data as well as the ratio of long arms to short arms among populations. so these results indicate a significant quantitative change in amount of chromatin in bromus species diversification (tables 2 and 4). considering the changes of intrachromosome asymmetry index (a1) among diploid and tetraploid species, the lowest value exists in the diploid (b. japonicus, 16462) and the highest value exists in the tetraploid species (b. rubens, 15317) (table 2). the results of analysis of variance showed that except for a1 and tf%, there was a significant difference (p≤1%) between genotypes in terms of chromosomal traits (la, sa, tl, ar, drl and a2) which indicated large variations among the germplasms in regard to studied traits. cluster analysis based on chromosomal characteristics separated, the fourteen investigated populations of bromus species into two major groups consistent of statistical analysis of chromosome morphometric traits (fig. 3). the first group has eight populations of bromus species (b. madritensis, b.rubens and b. tomentellus) which are tetraploid and hexaploid (2n=4x=28 & 2n=6x=42) and belong to genea and pnigma sections. the second group has six populations of bromus species (b. scoparius and b. japonicus) that are diploid (2n=2x=14) and belong to bromus section. cluster analysis based on cytological data showed that the populations with the lowest metric distance may lead to use populations in crosses for inducing the highest genetic variations (fig.3). however, grouping of the bromus populations based on karyotypic data, agrees with either the taxonomic treatment of the genus bromus of the same species based on morphological characters. figure 3. dendogram of 14 populations of bromus by analyzing nine karyotipic parameters using ward ‘s cluster analysis method. 10 sara sadeghian, ahmad hatami, mehrnaz riasat these genomic differences could be used for breeding purposes. in general, cytological studies of the bromus species growing in iran indicate the importance of polyploidy, chromosome structural changes, presumably quantitative changes in the amount of dna and probably the role of growing sites in species diversification and suggest that such data may be used in the taxonomy and phylogenetic consideration of the genus (hesamzadeh and ziaei nasab 2010). acknowledgments: the authors are grateful to mrs ladan jowkar for drawing charts. references acedo c, liamas f. 2001. variation of morphological characters of lemma and palea in the genus bromus (poaceae). ann. bot. fennici., 38: 1-14 artico ll, mazzocato ac, ferreira jl, carvalho cr, clarindo wr. 2017. karyotype characterization and comparison of three hexaploid species of bromus linnaeus, 1753 (poaceae). comparative cytogenetics, 11(2):213-223. bor nl. 1970. bromus. in: flora iranica, rechinger, k.h. 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ruta corsica and ruta lamarmorae (rutaceae) marilena meloni1, caterina angela dettori2, andrea reid3, gianluigi bacchetta2,4,*, laetitia hugot5, elena conti1 cytogenetic effects of c6h4 (ch3)2 (xylene) on meristematic cells of root tips of vicia faba l. and mathematical analysis cihangir alaca1, ali özdemir1, bahattın bozdağ2, canan özdemir2,* clethodim induced pollen sterility and meiotic abnormalities in vegetable crop pisum sativum l. sazada siddiqui*, sulaiman al-rumman temporal analysis of al-induced programmed cell death in barley (hordeum vulgare l.) roots büşra huri gölge, filiz vardar* genetic diversity, population structure and chromosome numbers in medicinal plant species stellaria media l. vill. shahram mehri*, hassan shirafkanajirlou, iman kolbadi a new diploid cytotype of agrimonia pilosa (rosaceae) elizaveta mitrenina1, mikhail skaptsov2, maksim kutsev2, alexander kuznetsov1, hiroshi ikeda3, andrey erst1,4,* study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (ocimum basilicum l.) irina petrescu1, ioan sarac1, elena bonciu2, emilian madosa1, catalin aurelian rosculete2,*, monica butnariu1 chemical composition, antioxidant and cytogenotoxic effects of ligularia sibirica (l.) cass. roots and rhizomes extracts nicoleta anca şuţan1,*, andreea natalia matei1, eliza oprea2, victorița tecuceanu3, lavinia diana tătaru1, sorin georgian moga1, denisa ştefania manolescu1, carmen mihaela topală1 phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm bombyx mori induced by microsporidian infection hungund p. shambhavi1, pooja makwana2, basavaraju surendranath3, kangayam m ponnuvel1, rakesh k mishra1, appukuttan nair r pradeep1,* electrophoretic study of seed storage proteins in the genus hypericum l. in north of iran parisa mahditabar bahnamiri1, arman mahmoudi otaghvari1,*, najme ahmadian chashmi1, pirouz azizi2 melissa officinalis: a potent herb against ems induced mutagenicity in mice hilal ahmad ganaie1,2,*, md. niamat ali1, bashir a ganai2 population genetic studies in wild olive (olea cuspidata) by molecular barcodes and srap molecular markers rayan partovi1, alireza iaranbakhsh1,*, masoud sheidai2, mostafa ebadi3 in vitro polyploidy induction in persian poppy (papaver bracteatum lindl.) saeed tarkesh esfahani1, ghasem karimzadeh1,*, mohammad reza naghavi2 long-term effect different concentrations of zn (no3)2 on the development of male and female gametophytes of capsicum annuum l. var california wonder helal nemat farahzadi, sedigheh arbabian*, ahamd majd, golnaz tajadod a karyological study of some endemic trigonella species (fabaceae) in iran hamidreza sharghi1,2, majid azizi1,*, hamid moazzeni2 karyological studies in thirteen species of zingiberacaeae from tripura, north east india kishan saha*, rabindra kumar sinha, sangram sinha caryologia. international journal of cytology, cytosystematics and cytogenetics 74(1): 75-82, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-809 caryologia international journal of cytology, cytosystematics and cytogenetics citation: e. ansari, m. khosrowshahli, a. ashraf jafari, a. etminan (2021) induction of autotetraploidy and its effects on morphophysiological traits in some annual and perennial medics. caryologia 74(1): 75-82. doi: 10.36253/caryologia-809 received: january 03, 2020 accepted: april 26, 2021 published: july 20, 2021 copyright: © 2021 e. ansari, m. khosrowshahli, a. ashraf jafari, a. etminan. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. induction of autotetraploidy and its effects on morphophysiological traits in some annual and perennial medics elham ansari1, mahmood khosrowshahli2, ali ashraf jafari3, alireza etminan4 1 phd student, department of plant breeding, islamic azad university, science and research branch, tehran, iran 2 department of plant breeding, islamic azad university, science and research branch, tehran, iran 3 research institute of forests and rangelands, agricultural research education and extension organization (areeo), tehran, iran 4 department o plant breeding, islamic azad university kermanshah branch, kermanshah, iran *corresponding author. e-mail: mkhosrowchahli@yahoo.com abstract. in order to study of the effects of polyploidy on morph-physiological traits in some annual and perennial medics, five annual and three perennial diploid medics were subjected to different concentrations of colchicine solution (0.1%, 0.5%, 1% and 2%) in mitosis stage. the induced tetraploids were identified by counting stomata guard cells chloroplasts. the highest survival rate and tetraploidy induction with average values of 80.2% and 74.3% were obtained using 0.1% colchicine concentration. all of the 21 entries (5 annual diploids and their induced tetraploids as m. lupulina, m. radiata, m. rijidula, m. truncatula, m. turbinata, and 3 populations of perennial diploid of m. sativa ssp. caerulea (karaj1, karaj2 and tehran) and their induced tetraploids and 5 commercial tetraploid alfalfa cultivars (bami, hamadani, baghdadi, ghareyongeh and renger) grown in pots in a glasshouse experiment using completely randomized design with five replications in 2018 in ahvaz, iran. data were collected for leaf length, leaf width, shoot and root length, seedling weight, shoot weight, root weight, branch number, leaf area index (lai) and water use efficiency (wue). result of mean comparisons between three groups (2x, induced 4x and controls 4x), showed that both groups of induced and natural tetraploids had significantly higher mean values for all the traits except leaf length, shoot length and root length than that for diploids. for the latter traits there were no significant differences between 2x and induced 4x. on the overall, the induced 4x had 60%, 62%, 68%, 65%, 22%, 109% and 47% higher mean values than their parental 2x for seedling weight, shoot weight, root weight, lai, wue and branch number, respectively. it was concluded that increasing ploidy level provides plant breeders with a promising tool in the breeding improve new varieties suited for future climate scenarios. keywords: ploidy level, colchicine, morphological traits, annual medicago, perennial medicago. 76 elham ansari, mahmood khosrowshahli, ali ashraf jafari, alireza etminan introduction alfalfa (medicago sativa) is a strategic and important forage species for animal feeding. a limited number of medicago species are cultivated for animal uses or for breeding improved varieties. among these species, medicago sativa is widespread in the most parts of the world and its wild forms are rarely available. commercial varieties of m. sativa are perennial cross-pollinated autotetraploid (2n=4x=32), with tetrasomic inheritance. m. sativa has both diploid and tetraploid forms (lapiņa et al., 2011). the sub species of caerulea is a perennial and diploid form (2n=2x=16). theoretically, conventional alfalfa has been evolved by sexual polyploidy, which is equivalent to crossing non-reduced (2x) gametes present in diploid species (bauchan and hossain, 1997; rosellini et al., 2016; pfeiffer and bingham, 1983). caerulea’s germplasm has the unused potential of being selected as a perennial, sustainable, drought-resistant and soil improving and pasture rehabilitation (li et al., 2010). annual alfalfas are more resistant to plant pests than perennial alfalfa (bauchan and azhar, 1998) and can be used in breeding programs as a genetic source to fungal and pest diseases resistance (yaege and stuteville, 2000), cold resistance and resistance to adverse and acidic soil conditions (gillespie, 1989). annual alfalfas through fertility and improved physical quality,  nitrogen fixation, increased organic carbon of soils (dalal et al., 1995) have a positive effect on grain yield of further grain crops. in addition, with the development of annual alfalfa cultivation, significant amounts of forage can be obtained. annual alfalfa can grow in summer and fall and planted in rotation with wheat. they also prevent soil erosion (biederbeck et al., 1993; badaroddin and meyer, 1990; mirzaei nodoushan, 2001). for many years, common alfalfa has been improved based on the classic plant breeding program. alfalfa varieties are usually synthetic varieties; developed by crossing of selected heterozygous parents and their offspring have advanced over three or four generations of seed proliferation (rowe and hill, 1999). duplication of the plant genomes, or polyploidy induction, which leads to changes in some traits, especially in horticultural, pharmaceutical and agronomic plant species, is based on the application of different drugs such as colchicine, new dinitroanilines, phosphorothioamidates oryzaline, and triflurarin etc. (melnychuk et al, 2020; niazian et al, 2020; su-jin et al, 2020; touchell et al, 2020). colchicine still remains the most efficient and clearly the most preferred and the most used anti mitotic agents because of its widely successful mitosis inhibition ability (touchell et al, 2020), and for its advantages such as high percentage of viability (melnychuk et al, 2020) and because of its widely successful mitosis inhibition ability, high solubility in water and ethyl alcohol and heat stability, ability to be autoclaved and easily applied to plant tissues solutions. therefor reduce the use of additional solvents, it is heat-stable, and can be autoclaved and easily applied to plant tissues (touchell et al, 2020). in the context of successful sexual autopolyploidy induction (by crossing (in alfalfa species, a comprehensive research was conducted by rosellini et al. (2016). osborn et al., (2003) in their review concluded that a novel variation in polyploids could involve changes in gene expression through increased variation in dosageregulated gene expression, altered regulatory interactions, and rapid genetic and epigenetic changes. mirzaei nodushan, (2001) reported that induction of polyploidy in self-pollinated and annual species is likely to improve their morphological and physiological properties and probably can facilitate the adequate gene transfer. there are some reports on phylogeny of domestic and foreign populations of medicago genus in iran. ghanavati et al. (2005) in study of genetic diversity of 22 species of genus medicago collected from iranian natural habitat using rapd marker generated a phylogenetic tree with 5 main cluster. populations of m. aculeala, m.constricta, m.rigiduloidos and m. rigidula with hard pod walls and spongy texture were classified in one cluster. populations of m. sauvagei, m. laciniata and m. polymorpha which had soft and flexible pod walls were classified together in a separate cluster. in another experiment ghanavat (2010) in phylogenetic analysis of 23 species of medicago based on 90 morphological characteristics by maximum parsimony approach, observed the most relationships between m. rugosa and m. scutellata, m. sativa and m. lupulina, m. coronata and m. minima, m. rigidula and m. rigiduloides, m. polymorpha and m. arabica, m. tornata and m. turbinata. salimpour (2012) in phylogenetic analysis of 23 species of alfalfa, the most phylogenetical relationships was observed between m. rugosa and m. scutellata, m. sativa and m. lupulina, m. coronata and m. minima, m. rigidula and m. rigiduloides, m. polymorpha and m. arabica, m. tornata and m turbinata. this information can be used in determining the dgree of success of inter-specific hybridization between different species in medicago genus. there are many comparative cytogenetic analyses between diploid and tetraploid perennial medics (yu et al. 2017). but there are few published reports of comparative cytogenetic analysis between perennial and annual diploid medics and their induced tetraploids. the aim of this study was to generate the best populations of annual and 77induction of autotetraploidy and its effects on morphophysiological traits in some annual and perennial medics perennial diploid medics using polyploidy breeding after induction by colchicine treatment and to compare changes of morpho-physiological traits between diploids 2x and induced 4x species and commerical 4x alfalfa cultivars. materials and methods: in this study, seeds of five annual diploid alfalfa were provided from natural resource gene bank of the institute of forests and rangelands, tehran, iran (table 1). at the same time, seeds of three perennial diploid were provided from college of agriculture, tehran university and seeds of five commercial varieties were obtained from pakan seed company, isfahan, iran. prior to seed sowing, seeds were scarified mechanically with sand paper and then sterilized with a benomyl fungicide. seeds of five annual diploid medics (m. lupulina, m. radiate, m. rigidula, m. truncatula, m. turbinate) and three perennial diploid medicago sativa ssp. caerulea populations (karaj1, karaj2 and tehran) were sown in a mixture of peat moss and field soil at a ratio of 2: 1 in plastic pots with 17 cm diameter. pots were kept in 20-25°c glasshouses and were irrigated regularly. about three months after sowing, 20 rooted single node cuttings from each of the young plants of diploid seedlings in mitosis stage subjected to three concentrations of colchicine solution (0.1, 0.5, and 1%) using dropper method. a drop of colchicine in the lateral bud was put and was repeated for four days after drying or being absorbed and after 15 days, the viability of cuttings was evaluated. the survival rate of the plants was calculated based on the number of well-established plants on the total number of cuttings treated in all the species studied. the induced tetraploids were identified using the usual method of chromosomes counting in the metaphase cells of root tip meristems with having a minimum of 10 metaphase plate mitosis for each species/ populations. the highest induced tetraploidy (80.2%) was obtained using 0.1% colchicine concentration. then, all of the 21 entries: 5 annual diploids and 5 induced tetraploids, 3 perennial diploids and 3 induced tetraploids of medicago sativa ssp. caerulea and 5 commercial tetraploid alfalfa cultivars as control (baghdadi, bami, hmamadani, renger, ghareyounje) grown in pots in a glasshouse using completely randomized design with five replications in 2018 in ahvaz, iran. in diploid and induced tetraploids (using 0.1% colchicine) and commercial tetraploids, data collected for leaf length (mm), leaf width (mm), shoot length (cm), root length (cm), shoot weight per plant (g), root weight (g), seedling weight (g) and, branches number per seedling, leaf area index (lai) and water use efficiency (wue). after about two months after growth of seedlings and induction polyploidy plants, middle leaves of seedlings were selected for measurements. lai measurements were performed manually using leaf plot and shadow measurements using mm checkerboard paper. the wue was estimated as , where: y= water beneficially used (seedling dry weight) and w=total amount of water that was estimated for each plant during the growth period. the data were analyzed by completely randomized design experiment and means were compared using tukey’s test. the sas9.1 software was used for analysis of variance and mean comparisons. results in the present study the effects of polyploidy induction on morph-physiological traits in five annual and three perennial diploid medics were assessed and they were subjected to different concentrations of colchicine solution (0.1%, 0.5%, 1% and 2%) in mitosis stage. the highest survival rate (80.2%) and tetraploidy induction (74.3%) were obtained in 0.1% colchicine concentration. result of analysis of variance showed a significant difference between all of the 21 entries for all of traits (p<0.01). such differences were predictable due to the origin and genome differences and ploidy levels between populations (table 2). the maximum of coefficient of variation (cv%) was 21.77% for root length and minimum was 11.87% for seedling weight, indicating that good accuracy of the experiment in evaluating traits. table 1. name, specifications and origin of annual diploid alfalfa. scientific name code origin of seed province altitude m latitude longitude medicago turbinate 24646 kermanshah kermanshah 1200 34°08’00” 46°10’00” medicago rigidula 45078 dallahou kermanshah 1213 34°23’34” 46°03’31” medicago radiate 44137 izzeh khozestan 767 35°36’07” 39°03’78” medicago lupulina 20307 unknown medicago truncatula 20587 unknown 78 elham ansari, mahmood khosrowshahli, ali ashraf jafari, alireza etminan mean comparisons were made between average over three groups of 2x, induced 4x and controls cv. 4x (table 3). result showed that the higher values of all traits were observed in control (cv. 4x) followed by induced tetraploid in terms of leaf width, shoot length, shoot weight, seedling weight, wue and branches number. in the other word, there were no significant differences between control cv. 4x and induced tetraploids for the latter traits. however, for leaf length, root length, root weight and lai, the means of induced tetraploids were significantly lower than that for controls (cv. 4x). the overall means of diploids were ranked as third place, however, for leaf length and root length. there was no significant differences between 2x and induced 4x. for other traits as: leaf width, shoot weight, root weight, seedling weight, lai, wue and branch number, the means of induced tetraploids were significantly higher than diploids (table 3). a separate mean comparisons were made among only naturally tetraploids varieties (table 4). result showed baghdadi cv. had higher mean values for all of traits except root length and root weight. for these two traits, the higher values were observed in ghareyounja cv. and for other traits, the hamadani cv. ranked in the third place (table 4). mean comparison were made between populations separately for diploids and induced tetraploids (tables 5 and 6). for diploid populations, the higher values of wue, lai, root and shoot weight, seedling weight, root and shoot length, leaf size were obtained in m. truncatula 2x followed by m lupulina 2x as the second place. both species are annual (table 6). in comparisons between perennial diploids, there was no significant differences between tehran karaj1 and karaj2 2x for wue, seedling, shoot weight, root and shoot length and leaf width. for branch number the higher value was obtained in tehran 2x (table 5). similarly, in comparisons between perennial induced tetraploids tehran 4x, ranked in the first class than two other induced perennial populations in terms of the morphological traits (table 5 and 6). in comparisons between annual induced tetraploids, the highest values of all of traits except of lai and branches number were observed in m trancatula 4x. similarly, m. lupulina 4x in terms of leaf width, root weight, lai, branches number was ranked in the first class and for the shoot and root length, shoot weight and seedling weight it was ranked as the second class. in comparisons of total average of 2x and total average of induced 4x groups, result showed in m. radiate by increasing ploidy level,  leaf length and leaf width decreased by 12% and 15%, respectively. in contrast, for m. rigidula the leaf length and leaf width of the 4x, increased by 15 and 42% than 2x, respectively. in the other diploid species, the increase in ploidy level showed no significant effect on leaf size, but a decrease in root and shoot length was observed in all 2x species ranging from 7 to 30% for shoot length and between 15 and 51% for roots length. thus, root weight and wue value in 4x were higher than twice that of 2x. in contrast, low increase was observed for lai (table 5 and 6). in overall, there were no significant differences between 2x and 4x for leaf size and lai. the induced 4x had 25% and 38% lower values than 2x for shoot and table 2. analysis of variance studied traits in 23 medic species/populations. sov df leaf length leaf width shoot length root length seedling weight shoot weight root weight leaf area index water use efficiency branch number populations 20 30.17** 39.07** 1074.9** 966.5** 0.476** 0.712** 0.039** 3.26** 0.384** 583.7** error 79 4.75 3.73 8.63 13.30 0.006 0.012 0.002 0.11 0.010 13.08 cv% 15.49 17.49 14.01 21.77 11.87 12.51 21.07 14.87 16.52 15.99 ** =significant at the 1% probability level. table 3. means comparison of morpho-physiological traits based on total ploidy levels in all studied alfalfa species. ploidy levels leaf length mm leaf width mm shoot length cm root length cm seedling weight g/plant shoot weight g/plant root weight g/plant leaf area index water use efficiency branch number perennial 4x (control) 15.88 a 11.96 a 26.30 a 25.51 a 1.08 a 0.77 a 0.31 a 2.96 a 0.26 a 26.04 a diploids 2x 13.02 b 9.90 b 18.97 b 14.46 b 0.63 b 0.47 b 0.16 c 1.89 c 0.11 b 17.64 b induced tetraploids 14.23 b 11.70 a 20.33 ab 14.79 b 1.02 a 0.76 a 0.27 b 2.31 b 0.23 a 25.68 a means with similar letters in each column has no significant difference at 5% probability level by tukey test. 79induction of autotetraploidy and its effects on morphophysiological traits in some annual and perennial medics table 4. means comparison of morpho-physiological traits in perennial alfalfa cultivars. commercial varieties 4x leaf length mm leaf width mm shoot length cm root length cm seedling weight g/plant shoot weight g/plant root weight g/plant leaf area index water use efficiency branch number baghdadi 19.60 a 13.00 a 39.18 a 27.54 b 1.32 ab 1.07 a 0.25 b 3.43 a 0.42 a 37.80 a bami 14.10b 10.90 a 18.30 c 18.90 c 0.72 c 0.46 c 0.26 b 2.00 b 0.17 c 22.80 b ghareyounje 14.70b 11.50 a 29.18b 32.48 a 1.27 b 0.91 b 0.35 a 3.30 a 0.29 b 18.40 bc hamadani 15.60 ab 13.00 a 26.46b 27.68 b 1.45 a 1.07 a 0.38 a 3.25 a 0.31 b 35.00 a renger 15.40b 11.40 a 18.38c 20.96 c 0.63 c 0.35 c 0.28 b 2.82 ab 0.13 c 16.20 c means with similar letters in each column has no significant difference at 5% probability level by tukey test table 5. mean comparison of diploid medicago species and their induced tetraploids for leaf length, leaf width, shoot length, root length and seedling weight. populations leaf length mm leaf width mm shoot length cm root length cm seedling weight g/plant 2x 4x 2x 4x 2x 4x 2x 4x 2x 4x m. sativa (karaj1) 12.20 b 12.43 c 10.60 bc 10.57 b 14.64 b 11.30b 13.00 b 8.81b 0.59 c 0.75 cd m. sativa (karaj2) 17.00 a 16.86 a 15.00 a 14.86 a 15.54 b 10.83b 8.60 b 5.43b 0.53 c 0.88 bc m. sativa (tehran) 12.40 b 12.00 c 10.60 bc 10.83 b 16.78 b 12.47 b 9.50 b 5.92 b 0.60 bc 1.01 b m lupulina 12.60 b 12.86 c 13.60 ab 13.43 a 14.54 b 10.69b 11.92 b 5.77b 0.68 b 0.96 b m. radiata 13.60 ab 12.00 c 9.20 cd 7.80 c 16.78 b 11.86 b 11.28 b 7.80 b 0.61 bc 0.90 bc m. rigidula 11.60 bc 13.40 bc 6.60 de 9.40 bc 15.10 b 11.48 b 12.10 b 8.84 b 0.64 b 0.93 b m. truncatula 15.00 ab 15.75 ab 12.80 b 13.25 a 54.52 a 50.44 a 51.58 a 40.63 a 1.32 a 1.54 a m. turbinata 15.80 ab 16.25 a 8.80 d 9.50 bc 14.86 b 12.75 b 11.08 b 9.33 b 0.43 cd 0.61 d average 13.78 13.94 10.90 11.21 20.35a 16.48b 16.13a 11.57b 0.68b 0.95a means with similar letters in each column has no significant difference at 5% probability level by tukey test. table 6. mean comparison of diploid medicago species and their induced tetraploids for shoot weight, root weight, lai, wue and branch number. population name shoot weight g/plant root weight g/plant leaf area index water use efficiency branch number 2x 4x 2x 4x 2x 4x 2x 4x 2x 4x m. sativa (karaj1) 0.38 cd 0.48 c 0.20 b 0.27 ab 2.10 bc 2.18 d 0.08 de 0.17 c 15.00 c 17.14 c m. sativa (karaj2) 0.41 cd 0.63b 0.12 c 0.25 ab 2.21 bc 2.33 c 0.09 d 0.18 c 16.60 c 19.00 c m. sativa (tehran) 0.48 bc 0.74 b 0.12 c 0.27 ab 1.53 c 1.65 f 0.11 cd 0.21 bc 16.40 c 18.00 c m. lupulina 0.55 b 0.71b 0.13 c 0.25 ab 3.22 a 3.41 a 0.17 b 0.23 b 35.60 a 44.00 a m. radiata 0.47 bc 0.67b 0.14 bc 0.23 ab 1.35 c 1.45 g 0.10 d 0.18 c 13.20 c 18.60 c m. rigidula 0.46 bc 0.64b 0.18 bc 0.29 ab 1.32 c 1.42 g 0.13 cb 0.18 c 14.40 c 17.60 c m. truncatula 0.95 a 1.22 a 0.37 a 0.31 a 2.82 ab 2.96 b 0.24 a 0.37 a 28.80 b 37.08 b m. turbinata 0.29 d 0.40 c 0.14 bc 0.21 b 1.64 c 1.78 e 0.07 de 0.11 d 12.80 c 16.50 c average 0.50b 0.69a 0.18b 0.26a 2.02b 2.15a 0.12b 0.20a 19.10b 23.49a means with similar letters in each column has no significant difference at 5% probability level by tukey test. 80 elham ansari, mahmood khosrowshahli, ali ashraf jafari, alireza etminan root length, respectively. in contrast, the induced 4x had 40%, 38%, 44%, 65% and 23% higher values than 2x for seedling weight, shoot weight, root weight, wue and branch number, respectively (tables 5 and 6). discussion in this study, we found higher ploidy induction at the lower colchicine concentration (0.1%). colchicine still remains the most efficient and clearly the most preferred and the most used anti mitotic agents because of its widely successful mitosis inhibition ability (touchell et al, 2020). the others anti-mitotic agents such as dinitroanilines and etc.. may also increase the ploidy levels. in addition, increasing of the ploidy levels can probably be an appropriate solution for crossing of various alfalfa species and increasing their genetic diversity and transferring desirable traits. it seems polyploidy induction in self-pollinated annual medic species are likely to improve the morphological and physiological characteristics in these species. the result indicated that induced 4x had 25% and 38% lower values for the shoot and root length than 2x, respectively, averaged over all of species. the reduction in root length of m. truncatula was higher than that of other species. the results of the present study were in line with results of pickens (2004) showing shorter shoots of colchicine treated plants compared to nottreated controls. overall, the induced 4x had 60%, 62%, 68%, 65%, 22%, 109% and 47% higher mean values than their 2x for seedling weight, shoot weight, root weight, lai, wue and branch number, respectively. in m. truncatula, the seedling weight of induced 4x was 16% higher than 2x (1.32 vs. 1.54 g/p). this result was in agreement with the result of the polyploidy induction in catharanthus roseus using colchicine solution that significantly increased the seedling weight of tetraploid plants compared to diploid plants (hosseini et al. 2013). shoot weight was higher in all induced species than the diploids. the higher values of shoot weight among all of population were obtained in m. truncatula. in this species, the value of induced 4x was 28% higher than its parental 2x. this result was in line with the staji et al. (2017) that found polyploidy induction significantly increased shoot weight in salvia leriifolia. tavan (2014) in some endemic species of thyme genus in iran found that increase in ploidy level was associated with an increase in shoot weight. similarly, bagheri and mansouri (2015) in cannabis (cannabis sativa l.) found that the root and seedling weight of polyploid plants were significantly increased than diploid plants. in our study, the higher values of lai were obtained in m. lupulina. lai was higher in all induced species than the diploids. in the overall average over all species, the lai of induced 4x was 7% higher than 2x (2.02 vs. 2.15). as the ploidy levels increased, the average value of wue in all studied species increased up to 65%. contributing factors to wue are related to climate factors that are essential for water use (evapotranspiration) and water supply (atmospheric rain), management factors for cultivation and operations lead to evaporation reduction from the soil surface (kafi and damghani 2001). it seems that m. truncatula has probably been the best choice for polyploidy induction in annual species. ploidy changes in cell size are due to an increase in the number of copies of genes and thus an increase in the amount of protein produced (tsukaya 2013). nowadays, it has been found that control of morphological traits at the molecular level by ploidy level alteration via genetic (tsukaya 2013), transcriptomic (li et al. 2012) and epigenetic (zeng et al. 2012, aversano et al. 2012) modification occurs. for example, changes in the genomic dose of polyploids lead to changes in the expression of genes involved in cell cycle, photosynthesis, and cell metabolism (shi et al. 2015), as a result of autotetraploidy, alterations in the expression of genes related to stress response, hormonal signaling actions, and response to phytohormones are applied which may lead to a flexible and rapid response to external and internal stimuli (del pozo and ramirez 2014). slight but pervasive changes in gene expression are probably correlated with large phenotypic differences in autotetraploids (allario et al. 2013). in some reports, autotetraploidy has caused epigenetic alteration by dna methylation at the functional genes sites, encoding proteins and at the sequences involved in dna replication, electron transport chain, and transcriptional regulation (zeng et al. 2012). in some cases, methylation has been the result of cytosine methylation at the cg and chg sites (aversano et al. 2012). probably a combination of chromosome duplication, along with genetic, epigenetic, and transcriptomic effects, can produce morphophysiological differences due to polyploidy induction, as in our experiment (yang et al. 2011). conclusion in addition to the highest survival rate and most successful induction after colchicine treatment, the induced tetraploid of m. truncatula, among all of evaluated species, showed the highest levels of morpho81induction of autotetraploidy and its effects on morphophysiological traits in some annual and perennial medics logical indices. it seems that if the goal of breeding is to increase in economical properties such as seedling weight and wue etc. it seems that m. truncatula probably is the best choice for polyploidy induction in annual species. all perennial diploids did not show any un-uniformity by increasing ploidy level in this study, the increasing of the ploidy level of agronomic “perennial” tetrapleoids may be a more effective step to achieve superior new cultivars. in comparing diploid alfalfas, the annual species, showed higher mean values for morphological traits than perennial diploid alfalfas. in comparisons among the three perennial “diploid” populations, tehran had higher mean values for shoot length, shoot weight, seedling weight and wue. therefore, tehran population is advisable for breeding improve new variety of perennial tetraploid via induce polyploidy. as a result, increasing the 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the karyotype of t. ruficollis comprised 2 large metacentric, 2 large submetacentric, 2 large acrocentric, 8 small metacentric, 4 small submetacentric, zw sex chromosomes and 40 microchromosomes. the molecular cytogenetical features that were exhibited only on the male t. ruficollis chromosome included two microsatellites and telomeric sequences: two signals of d(ca)15 on two microchromosomes, one signal of d(gc)15 on one of the first pair, and signals of agggttn sequences on each telomeric region of all macroand microchromosomes. the karyotype formula was deduced as: 2n (70) = lm4 + ma2 + sm2 + ssm2 + 2 sex chromosomes (sm1/ssm1) + 58 microchromosomes for s. chinensis and 2n (60) = lm2 +lsm2 + la2 + sm8 + ssm4 + z (msm1) w (ssm1) + 40 microchromosomes for t. ruficollis. keywords: spilopelia chinensis, tachybaptus ruficollis, bird chromosome, bird karyotype. 102 isara patawang et al. introduction birds, also known as avian dinosaurs, are a group of endothermic vertebrates, characterized by many features. spilopelia chinensis (figure 1a), or spotted dove, is a small pigeon that is a common local breeding bird throughout its native range on the indian subcontinent and in southeast asia. the species belongs to the genus spilopelia, subfamily columbinae, family columbidae, order columbiformes, clade columbimorphae and class aves (gibbs et al. 2001). tachybaptus ruficollis (figure 1b) or little grebe, is native to europe, africa and asia. tachybaptus ruficollis is one of six grebe species in the genus tachybaptus, family podicipedidae, order podicipediformes, clade phoenicopterimorphae and class aves (birdlife international 2020). twenty-eight species of columbidae and three species of podicipedidae have been reported in thailand, which the genus spilopelia comprises three species (s. orientalis, s. chinensis and s. tranquebarica) and the genus tachybaptus has only one species (t. ruficollis) (pratumthong et al. 2011). columbiformes, one of three orders in the columbimorphae clade, and podicipediformes, one of two orders in the phoenicopterimorphae clade, are both classified to the same columbea group by genome analyses. paleobiology and molecular biology suggest that neoavians and placental mammals originated about 66 million years ago during the late cretaceous to early paleogene period. the evolutionary lines of columbimorphae, including mesites, sandgrouse and doves, and phoenicopterimorphae, comprising flamingos and grebes, divided about 70 million years ago during the late cretaceous period (pacheco et al. 2011; ksepka and boyd 2012; yuri et al. 2013; jarvis et al. 2014). few avian chromosomal data studies have been reported, because of their difficulty compared to other vertebrates, as avian chromosomes are highly conserved compared to other vertebrate groups. at present, about 10% of total 10,857 bird species that have been reported karyotypic study. approximately half the number of karyotyped birds (≈50.7%) have diploid number of 78 and 82 chromosomes, and about 21.7% have 2n=80. extraordinary diversity of bird chromosome ranges from 2n=40 in falco columbarius (falconiformes) to 2n=142 in corythaixoides concolor (musophagiformes). the number of chromosomes, karyotypic features and sex chromosomes have been preserved in the avian genome on the chromosomal level and shared across all avian species (degrandi et al. 2020). the diploid number of 2n=80 was proposed to the presumptive ancestral bird chromosome, which can be used to explain the chromosomal evolution of birds well (griffin et al. 2007). this classic chromosomal study of thailand populations of s. chinensis and t. ruficollis species is the new recorded; in addition, we are the first to report on the molecular cytogenetic features of the t. ruficollis species. materials and methods sample collection s. chinensis tissue samples were derived from whole embryo tissue from two eggs; t. ruficollis tissues samples were derived from the feather coat. the s. chinensis eggs were collected from ban hauyrai (15°51’23.1”n, 102°50’06.1”e), wang muang sub-district, paui noi district, khon kaen province, thailand. the t. ruficollis samples were collected from a nesting area at the wastewater treatment plant of khon kaen university, khon kaen province, thailand. chromosomes were prepared from the tissue samples using fibroblast cell culture. figure 1. general characteristics of spilopelia chinensis (a) and tachybaptus ruficollis (b); scale bars = 5 centimeter. 103some molecular cytogenetic markers and classical chromosomal features of spilopelia chinensis and tachybaptus ruficollis fibroblast cell culture and chromosome preparation the chromosomes were prepared in three steps. first, the half-period old of eggs life cycle of s. chinensis and feather coat tissue of t. ruficollis used in this research were collected from bird nests as noted in the section above. second, the embryos and feather coat tissue were isolated and washed three times with phosphate buffered saline (pbs). the tissue samples were then chopped into pieces of 1 mm3 and placed onto the surface of a tissue culture flask at 41°c in a humidified air atmosphere containing 5% of co2 for 3-4 h. dulbecco’s modified eagle’s medium (dmem) containing 10% fetal bovine serum was added into the inverted flask and cultured overnight. the medium was refreshed after 2-3 d. finally, colchicine was introduced and mixed for further incubation of 30 min. the cells were harvested at 80-90% confluence using 0.25% trypsin (m/v) solution; they were separated into culture flasks in ratios of 1:2 or 1:3. the cell mixtures were centrifuged at 3,000 rpm for 10 min. after discarding the supernatant, the cells were treated with 10 ml of hypotonic solution (0.075 m kcl) and incubated at room temperature for 30 min. the cells were centrifuged and the supernatant discarded. the cells were fixed by gradually adding fresh cool fixative (3 methanol: 1 acetic acid) up to 8 ml. after centrifuging, the cells were repeatedly fixed until the supernatant was clear. the cells were added to 1 ml fixative by dropping onto a clean cold slide and then air dried (bai et al. 2011; phimphan et al. 2015). chromosome staining the chromosomes were conventionally stained using a 20%-giemsa working solution for 30 minutes (patawang et al. 2017). d(ca)15 and d(gc)15 microsatellites and telomeric (ttaggg)n sequence were used as probes. these probes were generated by pcr (pcr dig-probe synthesis kit, roche) in the absence of a dna template. fluorescence in situ hybridization (fish) was performed under highly stringent conditions on mitotic chromosome spreads. metaphase chromosomes and non-metaphase cells on slides were incubated with rnase (40 lg/ml) for 1.5 h at 37 oc. after the chromosomal dna was denatured for 4 min in 70 % formamide/29 ssc at ph 7.0 and 70 oc, the hybridization mixture (2.5 ng/ll probes, 2 lg/ll salmon sperm dna, 50 % deionized formamide, and 10 % dextran sulphate) was dropped on the slides and the hybridization was performed for 14 h at 37 oc in a moist chamber containing 29 ssc. the first post-hybridization wash was performed with 29 ssc for 5 min at 65 oc, and a final washing was performed at room temperature in 19 ssc for 5 min. the microsatellite repeats and telomeric probe were detected using anti-digoxigenin-fitc. finally, the slides were counterstained with dapi and mounted in an antifade solution (getlekha et al. 2016). chromosome checking and classifying the lengths of short arm (ls) and long arm (ll) chromosomes were measured to calculate the length of the total arm chromosome (lt, lt = ls + ll). relative length (rl) and centromeric index (ci) were estimated. ci was also computed to classify the types of chromosomes according to chaiyasut (1989). all parameters were used in karyotyping and idiograming. results and discussion karyological characteristics of s. chinensis both embryo samples of s. chinensis showed a diploid number of 70. the two embryos exhibited the same type of sex chromosome – z and w, which lead to presumed female embryo. the autosome comprised of 10 macrochromosomes –v4 large metacentric, 2 medium acrocentric, 2 small metacentric, and 2 small submetacentric – and 58 microchromosomes (table 1 and figures 2a-b). the diploid number found here differed from previous reports in the genus spilopelia: 2n=80 in s. chinensis (you-sheng et al. 2008), 2n=66 in s. risoria (tange and nakahara 1938-1939), 2n=78; 10 macrochromosomes + two sex-chromosomes (zz/zw) + 66 microchromosomes and 2n=76; 16 macrochromosomes + 60 microchromosomes in s. decaocto (srivastava and misra 1971), and 2n=76; 16 macrochromosomes + 60 microchromosomes in s. orientalis orientalis (makino et al. 1956). chromosomal features of t. ruficollis t. ruficollis had a diploid number of 60 and fundamental number of 80 in both male and female (figures 3a-b). the karyotype comprised of 20 macrochromosomes –2 large metacentric, 2 large submetacentric, 2 large acrocentric, 8 small metacentric, 4 small submetacentric and two sex chromosomes – and 40 microchromosomes. the sex chromosomes of t. ruficollis were classified to the zz/zw system; z was a medium submetacentric chromosome and w was a small submetacentric chromosome (table 2 and figures 3a-b). also, 104 isara patawang et al. the karyotype showed the gradually series size of the 11th to 30th pairs of microchromosomes. our result differed from ebied et al. (2005), who found a diploid number of 58 in t. ruficollis from an egyptian population. however, many of the karyotypic features of these two populations of t. ruficollis were the same, including the number of macrochromosomes (18) and sex chromosomes (2), and the type and size of each. the molecular cytogenetical features in this report that exhibited only on the male t. ruficollis chromosome included two microsatellites and telomeric sequences. first, signals of d(ca)15 microsatellites showed two signals on two microchromosomes; these presented alike in interphase (figure 4a), prophase (figure 4b) and metaphase (figure 4c) cells. next, microsatellite d(gc)15 appeared on the sub-centromeric region of the long arm of one chromosome of the first pair macrochromosome (figure 4d), shown in the idiogram as pair 1a and 1b (figure 4e), which is same only one signal of both non-metaphase and metaphase cells. finally, agggttn sequence signals showed on each telomeric region of all macroand microchromosomes, which appeared as green signals on interphase, prophase and metaphase cells as shown in figures 4(f-h). ours is the first study of these markers in this species, and is one of only a few avian chromosomal reports. microsatellites, simple sequence repeats (ssr), short tandem repeats (str) and simple sequence length polymorphisms (sslp) are found in prokaryotes and eukaryotes. they are widely dispersed in the genome, especially in the euchromatin of eukaryotes, and coding and noncoding nuclear and organellar dna (vieira et al. 2016; kumar 2018). the signals of d(ca)15 microsatellites on two microchromosomes of male t. ruficollis showed the one functional that was needed to find the answer in the future study. the signal of the d(gc)15 microsatellite that exhibited on only one chromosome of the 1st pair is another issue that needs to be addressed. we used agggttn sequence probes to investigate the feature of the male t. ruficollis chromosome. agggttn are repeated sequences on the terminal end of the chromosome arm of general vertebrates, for example humans, mice and the xenopus frog (ichikawa et al. 2015). the agggttn signals that appeared on the interphase, prophase and metaphase cells of the male t. ruficollis showed the existence of this sequence in this species (figures 4f-h). overview of avian chromosome in birds, females are the heterogametic sex with z and w sex chromosomes; males are the homogametic sex, with zz sex chromosomes. studies of sex chromosome evolution in birds and other systems with female heterogamety are important, because they offer independent replication of observations from x–y species. we observed the heterogametic zw sex chromosomes in female t. ruficollis (figure 5a) and found heterogametic chromosomes in two embryonic s. chinensis samples (figure 5b) in this study; this agreed with other avian sex chromosome studies (ellegren 2000; shibusawa et al. 2004). most avian chromosome studies have shown conserved characteristics on three macrochromosome pairs, including the 1st (metacentric, m), 2nd (submetacentric, sm) and 3rd (acrocentric, a) pairs. in addition, the 4th pair (metacentric or submetacentric) have been shown to exhibit the semi-conserved characteristic typical of many avian species. these characteristics have been table 1. mean length of short arm chromosome (ls), long arm chromosome (ll), total arm chromosome (lt), relative length (rl), centromeric index (ci), and standard deviation (sd) of rl, ci from 20 metaphase cells of two female individuals spotted dove (spilopelia chinensis), 2n=70. ch.p ls ll lt rl±sd cl±sd ch.s ch.t 1 3.630 5.190 8.820 0.242±0.012 0.588±0.024 l m 2 2.690 3.920 6.610 0.181±0.010 0.593±0.030 l m 3 1.180 4.320 5.500 0.151±0.008 0.785±0.026 m a 4 1.770 2.260 4.030 0.110±0.008 0.561±0.032 s m 5 1.450 2.210 3.660 0.100±0.009 0.604±0.028 s sm 1st sex chro. 1.850 2.220 4.070 0.111±0.008 0.545±0.024 s m 2nd sex chro. 1.340 2.490 3.830 0.105±0.010 0.650±0.030 s sm 7-35 microchromosomes abbreviations: ch.p, chromosome pair; ch.s, chromosome size; ch.t, chromosome type; l, large size; m, medium size; s, small size; m, metacentric; sm, submetacentric; a, acrocentric. 105some molecular cytogenetic markers and classical chromosomal features of spilopelia chinensis and tachybaptus ruficollis observed in many species, for example agelaius phoeniceus [2n=76] (cox and james 1984), anas platyrhynchos [2n=80] (skinner et al. 2009), rupornis magnirostris [2n=68], buteogallus meridionallis [2n=68], and asturina nitida [2n=68] (de oliveira et al. 2013), lonchura punctulata [2n=72] (kaewmad et al. 2013), ara macao [2n=6264] (seabury et al. 2013), turdus rufiventris [2n=78], t. albicollis [2n=78] (kretschmer et al. 2014), gallus gallus [2n=78] (phimphan et al. 2015); with the 1st pair m, 2nd sm, 3rd a and 4th m/sm. we found the same conserved chromosome pair characteristics in the two species in our study as in these other avian reports. in addition, the microchromosome is one of many characteristics that has been conserved in the genome of all avian and many reptilian species. the archetypal avian chromosome comprises about 40 chromosome pairs figure 2. metaphase chromosome plates and standardized karyotypes of embryonic individual 1 (a) and embryonic individual 2 (b) spilopelia chinensis, 2n=70 by conventional staining. 106 isara patawang et al. and usually 30 small to tiny microchromosome pairs. this karyotypic feature perhaps evolved 100-250 million years ago (burt 2002). the s. chinensis and t. ruficollis in this study had a microchromosome number of 58 and 40, respectively, indicating the close evolutionary lines between these two species and other avian species. acknowledgements this research was financially supported by the chiang mai university, thailand. we would like to thank the cytogenetics and cytosystematics research laboratory of the department of biology, faculty of science, chiang mai university for their help. the institute of animals for scientific purpose development of the national research council of thailand (resolution u1-04491-2559) approved this project. references bai c, wang d, li c, jin d, li c, guan w, ma y. 2011. establishment and biological characteristics of a jingning chicken embryonic fibroblast bank. eur j histochem. 55(1):e4. birdlife international, species factsheet: tachybaptus ruficollis. 2020. cambridge: birdlife international; 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(caprifoliaceae), using scot molecular markers fengzhen chen1, dongmei li2,* , mohsen farshadfar3 the new chromosomal data and karyotypic variations in genus salvia l. (lamiaceae): dysploidy, polyploidy and symmetrical karyotypes halil erhan eroğlu1,*, esra martin2, ahmet kahraman3, elif gezer aslan4 cytogenetic survey of eight ant species from the amazon rainforest luísa antônia campos barros1, gisele amaro teixeira2, paulo castro ferreira1, rodrigo batista lod1, linda inês silveira3, frédéric petitclerc4, jérôme orivel4, hilton jeferson alves cardoso de aguiar1,5,* molecular phylogeny and morphometric analyses in the genus cousinia cass. (family asteraceae), sections cynaroideae bunge and platyacanthae rech. f. neda atazadeh1,*, masoud sheidai1, farideh attar2, fahimeh koohdar1 a meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (juglans regia l.) melika tabasi1, masoud sheidai1,*, fahimeh koohdar1, darab hassani2 genetic diversity and relationships among glaucium (papaveraceae) species by issr markers: a high value medicinal plant lu feng1,*, fariba noedoost2 morphometric analysis and genetic diversity in rindera (boraginaceae-cynoglosseae) using sequence related amplified polymorphism xixi yao1, haodong liu2,*, maede shahiri tabarestani3 biosystematics, fingerprinting and dna barcoding study of the genus lallemantia based on scot and remap markers fahimeh koohdar*, neda aram, masoud sheidai karyotype analysis in 21 plant families from the qinghai–tibetan plateau and its evolutionary implications ning zhou1,2, ai-gen fu3, guang-yan wang1,2,*, yong-ping yang1,2,* some molecular cytogenetic markers and classical chromosomal features of spilopelia chinensis (scopoli, 1786) and tachybaptus ruficollis (pallas, 1764) in thailand isara patawang1,*, sarawut kaewsri2, sitthisak jantarat3, praween supanuam4, sarun jumrusthanasan2, alongklod tanomtong5 centromeric enrichment of line-1 retrotransposon in two species of south american monkeys alouatta belzebul and ateles nancymaae (platyrrhini, primates) simona ceraulo, vanessa milioto, francesca dumas* repetitive dna mapping on oligosarcus acutirostris (teleostei, characidae) from the paraíba do sul river basin in southeastern brazil marina souza cunha1,2,*,#, silvana melo1,3,#, filipe schitini salgado1,2, cidimar estevam assis1, jorge abdala dergam1,* karyomorphology of some crocus l. taxa from uşak province in turkey aykut yilmaz*, yudum yeltekin variation of microsporogenesis in sexual, apomictic and recombinant plants of poa pratensis l. egizia falistocco1,*,+, gianpiero marconi1,+, lorenzo raggi1, daniele rosellini1, marilena ceccarelli2, emidio albertini1 caryologia. international journal of cytology, cytosystematics and cytogenetics 73(3): 111-120, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-880 caryologia international journal of cytology, cytosystematics and cytogenetics citation: v. ventura de souza, m. sossai spadeto, r. abreu guedes, w.r. clarindo, c.r. de carvalho, j.a. severi, t. da silva souza (2020) toxicity of aristolochia decoction: a relevant herbal in folk medicine. caryologia 73(3): 111-120. doi: 10.13128/caryologia-880 received: march 13, 2020 accepted: july 14, 2020 published: december 31, 2020 copyright: © 2020 v. ventura de souza, m. sossai spadeto, r. abreu guedes, w.r. clarindo, c.r. de carvalho, j.a. severi, t. da silva souza. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. toxicity of aristolochia decoction: a relevant herbal in folk medicine victor ventura de souza1, micheli sossai spadeto2, roselena abreu guedes4, wellington ronildo clarindo1,2, carlos roberto de carvalho3, juliana aparecida severi4, tatiana da silva souza1,2,* 1 departamento de biologia, centro de ciências exatas, naturais e da saúde, universidade federal do espírito santo, alegre – es, brasil 2 programa de pós-graduação em genética e melhoramento, centro de ciências agrárias e engenharias, universidade federal do espírito santo, alegre – es, brasil 3 departamento de biologia geral, universidade federal de viçosa, viçosa – mg, brasil 4 departamento de farmácia, centro de ciências exatas, naturais e da saúde, universidade federal do espírito santo, alegre – es, brasil *corresponding author. e-mail: tatianas.souza@hotmail.com abstract. ethnopharmacology studies report the use of aristolochia (aristolochiaceae) species as medicinal plants in various parts of the world. however, the acids aristolochic (aas), secondary metabolites present in all species of aristolochia, have cytogenotoxic activity and they are a potent carcinogen to rodents and humans. the aim of the current research was to perform to initial screening for the toxicity of aristolochia labiata and aristolochia triangularis decoctions through germination and growth rate, flow cytometry, mitotic index and cytogenetics analysis in allium cepa. the decoctions were prepared from 2, 4, 8, 16 and 32 g l-1. decoctions at concentrations 4 g to 16 g l-1 significantly reduced the germination rate of allium cepa. seeds exposed to 32 g l-1 decoctions did not germinate. all decoctions reduced the growth rate of onion seedlings. decoctions at 4 g l-1 to 16 g l-1 inhibited mitotic index. highest concentrations of decoctions (8 g l-1 and 16 g l-1 for aristolochia labiata; 16 g l-1 for aristolochia triangularis) showed statistically significant increase in frequency of allium cepa nuclei in the g0/g1 phase. both decoctions induced the formation of heteropycnotic nuclei. qualitative phytochemical prospecting of decocts were performed and alkaloids secondary compounds were the largest presence in both species, indicating that the aas may be related to the observed toxicity. caution is recommended in the consumption of decoctions from aristolochia labiata and aristolochia triangularis stems. keywords: aristolochic acid, cytogenotoxicity, flow cytometry, heteropycnotic nuclei, allium cepa test. introduction large part of the population of developing countries, especially traditional communities, depends on herbal medicine for primary health care 112 victor ventura de souza et al. (who, 1978). the use of medicinal plants has been stimulated by brazilian government aiming at the sustainable use of brazilian biodiversity and the improvement of the public health system (brasil, 2006). ethnopharmacology studies report the traditional knowledge on the use of aristolochia (aristolochiaceae) species as medicinal plants in various parts of the world (heinrich et al., 2009; michl et al., 2013). in brazil, albuquerque et al. (2007) verified that aristolochia labiata (flowers, leaves and whole plant) is used by traditional communities in the caatinga region to relieve or cure menstrual colic and uterine inf lammations. decoction, infusion and maceration of leaves and stems of aristolochia triangularis cham. have traditionally been used to treat gynecology and urinary, gastro-intestinal, respiratory and musculoskeletal and joint diseases (araujo & lemos, 2015; bolson et al., 2015). the ethnobotanical survey showed that the aristolochia triangularis is used as tea for the cure of diseases and/or culturally defined symptoms. also, the species is used for the cure of spiritual diseases in the form of baths (silva, 2008). araujo and lemos (2015) and bolson et al. (2015) document that aristolochia triangularis had the highest use values among the species cited by residents of traditional communities in the northeast and south of brazil, respectively. some medicinal plant compounds have their toxicological properties well documented in the literature. despite the therapeutic effects of aristolochia species, these plants present the aristolochic acids (aas) which are nitrophenanthrenes carboxylic acids. aristolochic acids i (aai) and ii (aaii) present known cytoand genotoxic activity and they are a potent carcinogen to rodents and humans (chang et al., 2007; slade et al., 2009; hwang et al., 2012; bunel et al., 2016; youl et al., 2020), causing a specific nephropathy associated with renal cancer (li et al., 2018; sborchia et al., 2019). as a consequence of their toxic effects, the consumption of aristolochia species and its derivatives is prohibited in many countries such as australia, canada and united kingdom (iarc, 2002; neinhuis et al., 2005). however, in brazil its consumption is not regulated, being common to find dry parts of the plant in popular markets (silva, 2008), pharmacies and stores of natural products, ready to be prepared. aas toxicity becomes clinically significant after long periods of ingestion (yamani et al., 2015). nevertheless, the knowledge concerning about toxic effect of decoctions of aristolochia species is very scarce (amat et al., 2002). allium cepa test is commonly used to evaluate the toxic potential of medicinal plants and their metabolites (akinboro & bakare, 2007; oloyede et al., 2009). the germination rate of the seeds and the final growth of the seedlings are used to evaluate the phytotoxicity of the several compounds (macedo et al., 2008). besides, allium cepa test has also been accomplished to detect chromosomal abnormalities generated during the cell cycle (vicentini et al., 2001). this analysis is possible because allium cepa has few chromosomes (2n = 16) with relatively large total length (grant 1982). still, allium cepa test shows a similar sensitivity to other systems, like human lymphocytes (fiskesjö 1985), and a correlation of 82% to carcinogenicity tests in rodents (rank & nielsen, 1994). flow cytometry (fcm) has broadly contributed to improve knowledge on the plants and animals’ cell cycle and has been employed in the biomedical field, pharmacology, oncology and ploidy level determination (jayat & ratinaud, 1993). currently, fcm applied in plants represent a very powerful tool to detect the cytotoxicity and dna damage caused by drugs and environmental contaminants (citterio et al., 2002; monteiro et al., 2010; andrade-vieira et al., 2012). considering the wide use of aristolochia labiata and aristolochia triangularis as medicinal plants, this study was conducted to analyze the toxicogenetic effects of their decoctions in root meristematic cells of allium cepa. from the preliminary results obtained in this study, renal and testicular histopathology on mice is ongoing. these data are expected to help elucidate the effects of aristolochia decoctions. materials and methods plant materials aristolochia labiata was collected in the forest garden of the universidade federal de são joão del-rei (ufsj), located in the city of são joão del rei, minas gerais state, brazil. aristolochia triangularis was collected in the health ministry located in the city of venda nova do imigrante, espírito santo state, brazil. a voucher specimen of the plants has been deposited at the herbarium of ufsj, under reference number ufsj132 (aristolochia labiata) and ufsj5571 (aristolochia triangularis). plant names were checked and updated with the royal botanic gardens, kew online website www. theplantlist.org. seeds of allium cepa (isla®; batch number: 774758; 90% of germination) were used as a test organism in current research because they are genetically and physiologically homogeneous (leme et al., 2008). 113toxicity of aristolochia decoction: a relevant herbal in folk medicine preparation of decoctions stems of aristolochia labiata and aristolochia triangularis were boiled for 5 min in 1 l of distilled water (dh2o). considering that 8 g l-1 is the dosage commonly used in popular medicine (simões et al., 1995), 2, 4, 8, 16 and 32 g l-1 concentrations were prepared. qualitative phy tochemical prospecting of both decoctions was carried out from the methodology proposed by matos (1997). for this, the presence of the following metabolites was evaluated: phenols and flavonoids (sulfuric acid test), tannins (iron chloride iii), saponins (foam index), coumarin (naoh solution), alkaloids (mayer and wagner reagent), anthraquinone (ethyl ether + ammonia) and terpenoids (liebermann–burchard test). seed germination and seedling growth rates to the test hypothesis that aristolochia decoctions are phy totoxic (affects seed germination and seedling growth) to allium cepa, onion seeds were exposed for 28 days to aqueous extracts. culture medium ms (murashige & skoog, 1962) were supplemented with: 30 g of sucrose of l-1, 0.4 g of l-glutamine, 0.04 g of l-cysteine, 7 g of agar-1, ph = 5.7 ± 0.1. culture media were autoclaved for 20 min at 121°c under 1 atm. immediately after autoclaving, the decoction extracts were filter-sterilized and added to the tissue culture media. colchicine at 0.025% was used as positive control (pc) and ms medium without addition of decocts as negative control (nc). in laminar flow, allium cepa seeds were disinfected in 70% ethanol solution for 30 s and then with 1.5% naocl2 solution for 20 min, and washed three times in autoclaved dh2o for 1 min (mursarurwa et al., 2010). fifteen seeds were inoculated in eight flasks containing 15 ml of the culture medium. the inoculated seeds were maintained in a growth room with photoperiod 16/8 hours in light under 24 ± 1ºc. seed germination (percentage of germinated seeds in each treatment after initial exposure) was recorded on the 28th day. in this time, the final length of the seedlings was measured for each treatment using a caliper rule. mitotic and chromosomal abnormalities onion seeds were germinated in petri dishes lined with moistened filter paper with the different concentrations of decoctions. in addition, dh2o was used as negative control (nc) and the 0.025% colchicine as positive control (pc). germination occurred in b.o.d. at 24°c for 72 h. allium cepa roots with 1–2 cm were excised, fixed in ethanol: glacial acetic acid (3:1, v/v) and stored at -20°c. after 24h, the roots were hydrolyzed in 1n hcl at 25°c for 20 min, and subsequently the root meristems were stained with 2% acetic orcein, covered with foil and crushing the material. the slides were examined under a light microscope with 100x objective. ten slides were prepared for each treatment being analyzed 500 cells/slide. cytotoxic potential of decoctions was evaluated by mitotic index (number of dividing cells/total observed cells). genotoxic effect was measured by the chromosomal abnormalities index (ca). mutagenicity analysis was performed by micronuclei (mn) frequency. flow cytometry the meristems of allium cepa root after germination for 72 hours in a petri dish were fixed in ethanol: glacial acetic acid (3: 1, v / v) were stored for 24 hours at -20 ° c, transferred to 70% ethanol and stored at -20°c. fcm analyzes were performed with 5 replicates for each decoction concentration and negative control (dh2o), being three root meristems for each replicate. root apical meristems were excised, washed 3 times for 10 min in dh2o. nuclei isolation and staining were performed according to the protocol proposed by silva et al. (2010). nuclei suspensions were analyzed by flow cytometry pas® partec flow cytometer (partec® gmbh, munster, germany) after calibration of the equipment. the frequency of nuclei in g0/g1, s and g2/m phases was measured to verify the cytotoxicity of the decoctions in each specific cell cycle period. statistical analyses statistical analyses were performed using the bioestat 5.3 software. the shapiro-wilk test was used to verify the normality of the samples. as normality criteria were not satisfied, the non-parametric kruskal-wallis test with subsequent dunn test (p<0.05) was performed. results qualitative phytochemical analysis of decoctions is shown in table 1. decoctions presented phenols (detected by sulfuric acid) and alkaloids (detected by mayer and wagner reagents) as secondary metabolites. since the alkaloid secondary compounds were the largest presence in both species, other metabolites were not detected. 114 victor ventura de souza et al. table 2 shows the effects of the decoctions of aristolochia labiata and aristolochia triangularis on seed germination and seedlings of allium cepa. seeds of the control group germinated satisfactorily. decoctions of both species at concentrations 4, 8 and 16 g l-1 significantly reduced the germination rate of allium cepa. seeds exposed to 32 g l-1 decoctions did not germinate. all decoctions concentrations reduced the onion seedlings growth rate (table 2, figure 1a, 1b). cell proliferation rate was 13.34% in the control group (table 3). decoctions at 2.0 g l-1 did not promoted significant reduction of mitotic index. decoctions at 4, 8 and 16 g l-1 inhibited cell proliferation in allium cepa (table 3). changes in allium cepa cell cycle table 1. qualitative phytochemical analysis of aristolochia labiata and aristolochia triangularis reagents metabolite class aristolochia labiata aristolochia triangularis sulfuric acid phenols + + sulfuric acid flavonoides iron chloride iii tannins foam index saponins naoh solution coumarin mayer reagent alkaloids +++ +++ wagner reagent alkaloids + + ethyl ether + ammonia anthraquinone lieberman burchard terpernóides (-) absence; (+) presense table 2. effects of decoctions of to aristolochia labiata and aristolochia triangularis on seed germination and seedling growth of allium cepa species samples germination (%) seedling growth (cm) aristolochia labiata ms 87.5 8.48 ±4.21 colchicine 0.025% 75.0 0.93± 0.25* 2 g l-1 87.5 1.46 ±0.92* 4 g l-1 62.5* 1.44± 0.37* 8 g l-1 62.5* 0.88±0.32* 16 g l-1 50.0* 0.67± 0.22* 32 g l-1 ng ng aristolochia triangularis ms 87.5 8.48 ±4.21 colchicine 0.025% 75.0 0.93± 0.25* 2 g l-1 75.0 0.53±0.19* 4 g l-1 50.0* 0.82 ±0.14* 8 g l-1 50.0* 0.45±0.50* 16 g l-1 50.0* 0.40±0.43* 32 g l-1 ng ng ms: negative control -culture medium (murashige and skoog, 1962). colchicine 0.025%: positive control. ng: did not germinate. * significant difference in relation to negative control (p <0.05) kruskal-wallis test. figure 1. seedlings of allium cepa after 28 days of exposure to the decoctions of (a) aristolochia labiata and (b) aristolochia triangularis. nc = negative control culture medium (murashige and skoog, 1962); pc = positive control (0.025% colchicine). 115toxicity of aristolochia decoction: a relevant herbal in folk medicine are also shown in table 4 and representative histograms are shown in figure 2. according to fcm analyses, decoctions of both species promoted a concentrationdependent increase in frequency of allium cepa nuclei in the g0/g1 phase. this increase was statistically significant for the highest concentrations of decoction (8 and 16 g l-1 for aristolochia labiata; 16 g l-1 for aristolochia triangularis). thus, the frequency of nuclei in the s and g2/m phase tended to decreased, parking cells in interphase. aristolochia labiata decoctions at 8 and 16 g l-1 caused a greater number of cell damage; the highest dose table 3. mitotic index (% percentage ± standard deviation) of allium cepa root meristem cells exposed to decoctions of aristolochia labiata and aristolochia triangularis species samples mitotic index % number of cells in each cell cycle phase interphase prophase metaphase anaphase telophase aristolochia labiata distilled water 13.34 ± 4.72 4333 335 126 137 69 colchicine 0.025% 10.52 ± 3.61 4286 343 149 150 72 2 g l-1 6.92 ± 3.56 4691 75 144 81 9 4 g l-1 1.02 ± 0.86* 4949* 9* 21* 15* 6* 8 g l-1 1.10 ± 2.30* 4941* 11* 29* 13* 6* 16 g l-1 0.76 ± 0.68* 4960* 12* 6* 15* 7* 32 16 g l-1 ng ng ng ng ng ng aristolochia triangularis distilled water 13.34 ± 4.72 4319 339 128 142 72 colchicine 0.025% 10.52 ± 3.61 4286 343 149 150 72 2 g l-1 8.38 ± 3.18 4951* 15 14 14 06 4 g l-1 2.20 ± 3.94* 4888* 51* 24* 24* 13* 8 g l-1 3.34 ± 3.69* 4803* 69* 73* 53* 02* 16 g l-1 3.84 ± 2.14* 4961* 16* 06* 11* 06* 32 16 g l-1 ng ng ng ng ng ng distilled water: negative control. colchicine 0.025%: positive control. ng: did not germinate.* significant difference in relation to negative control (p <0.05) kruskal-wallis test. figure 2. histogram representative of the meristem of allium cepa. a) histogram representing the negative control. note the presence of the g0/g1 peak (channel 100) g2/m (channel 200), and between the particles at different times is s phase of the cell cycle, showing that the negative control has particles at all stage of g0/m. b) histogram representing the treatment of 16 g l -1 of aristolochia labiata, observe particles in the peak (channel 100) demonstrating that the cells are in g0/g1, but there is no peak at (channel 200), which confirms the no progression of nuclei for the g2/m phase, indicating that the extracts prevent cell proliferation. table 4. frequency (%) of allium cepa nuclei in cell cycle phases after treatment with aristolochia labiata and aristolochia triangularis decocts species samples %g0/g1 %s %g2/m aristolochia labiata distilled water 70.08 ± 6.91 18.37 ± 4.03 11.00 ± 3.62 2 g l-1 88.96 ± 1.90 8.08 ± 1.36 3.00 ± 0.93 4 g l-1 91.34 ± 2.93 6.46 ± 2.63 2.00 ± 1.07 8 g l-1 97.16 ± 2.91* 2.57 ± 2.81* 0.27 ± 0.60* 16 g l-1 99.85 ± 0.32* 0.14 ± 0.32* 0.00 ± 0.00* 32 g l-1 ng ng ng aristolochia triangularis distilled water 74.08 ± 4.52 11.02 ± 4.17 14.89 ± 1.93 2 g l-1 84.72 ± 2.83 8.82 ± 1.86 6.46 ± 4.54 4 g l-1 86.23 ± 3.61 8.12 ± 1.47 5.63 ± 3.24 8 g l-1 87.18 ± 4.03 7.22 ± 2.84 5.59 ± 1.48 16 g l-1 92.28 ± 2.09* 4.34 ± 1.17* 3.38 ±1.24* 32 g l-1 ng ng ng distilled water: negative control. ng: did not germinate. * significant difference in relation to negative control (p <0.05) kruskalwallis test. 116 victor ventura de souza et al. reached a level of absence of core peak in g2/m phase. aristolochia triangularis decoction at 16 g l-1 significantly reduced the number of nuclei in s and g2/m phase. due to severe cytotoxicity, chromosomal abnormalities and micronuclei were not observed. heteropycnotic nuclei (figure 3) were analyzed separately. decoctions of both species at 4, 8 and 16 g l-1 significantly increased the frequency of this abnormality. aristolochia triangularis decoction at 2 g l-1 also induced heteropycnotic nuclei on the allium cepa root cells (table 5). discussion the genus aristolochia presents a wide range of physiologically active compounds classified in five main categories: terpenes, phenols, alkaloids, flavonoids and lignoids (pacheco et al., 2009). phytochemical analyses indicated mainly the presence of alkaloids in both decoctions studied and in a lower extent, phenols. phenols bring advantages to the plant, as they are related to attraction of pollinators and protection against herbivory among others (piesik et al., 2011). species of aristolochiaceae are rich in alkaloids (schmeiser et al., 2001), including aas that have attracted intense research interest because of cytoand genotoxic properties of aai and aaii (wu et al., 2005; chang et al., 2007; slade et al., 2009; bastek et al., 2019). the amounts of aai and aaii in decoctions were not determined. however, as the aas are slightly soluble in water (o’neil, 2001), we believe that possibly they are present in decoctions, which is the way by which people consume species of aristolochia. hwang et al. (2012) quantified aas in aqueous extracts of aristolochia manshuriensis kom. the genotoxicity of the extracts was detected by bacterial reverse mutation assay and micronucleus in mice bone marrow erythrocytes. according to the authors, the genotoxicity of aristolochia manshuriensis is directly related to the aas. allium cepa test has been used as first cytogenotoxic screening of medicinal plant extracts (dias & takahashi, 1994; fachinetto et al., 2007; meneguetti et al., 2014; mendes et al., 2012) because the results are reliable and similar to those performed with in mammals (rank & nielsen, 1994), contributing to the safe use of these herbs (mendes et al., 2012). studies concerning toxicological activity of aristolochia extracts are very scarce, in spite of their use in several countries (amat et al., 2002). in the current research, the toxic effects of decoctions of aristolochia labiata and aristolochia triangularis on allium cepa were evaluated. decoctions of both species at 32 g l-1 were phytotoxic because prevented the germination of onion seeds. the other concentrations tested promoted delayed germination and growth and mitodepressive figure 3. heteropicnotic nucleus (arrow) in allium cepa meristematic root cells after exposure to the decoctions of aristolochia labiata. 1000x magnification. table 5. percentage of chromosomal alterations (ca) and heteropycnotic nucleus observed in allium cepa cells exposed to decoction of aristolochia labiata and aristolochia triangularis species samples %ca %heteropycnotic nucleus aristolochia labiata distilled water 0.04 ± 0.12 0.00 ± 0.00 colchicine 0.025% 5.88 ± 2.81* 0.00 ± 0.00 2 g l-1 0.12 ± 0.16 0.00 ± 0.00 4 g l-1 0.00 ± 0.00 8.84 ± 1.53* 8 g l-1 0.00 ± 0.00 15.8 ± 13.03* 16 g l-1 0.00 ± 0.00 29.60 ± 29.02* 32 g l-1 ng ng aristolochia triangularis distilled water 0.04 ± 0.12 0.00 ± 0.00 colchicine 0.025% 5.88 ± 2.81* 0.00 ± 0.00 2 g l-1 0.00 ± 0.00 13.4 ± 7.95* 4 g l-1 0.00 ± 0.00 17.2 ± 19.57* 8 g l-1 0.00 ± 0.00 17.1 ± 13.02* 16 g l-1 0.00 ± 0.00 19.12 ± 22.2* 32 g l-1 ng ng distilled water: negative control. colchicine 0.025%: positive control. ng: did not germinate. * significant difference in relation to negative control (p <0.05) kruskal-wallis test. 117toxicity of aristolochia decoction: a relevant herbal in folk medicine effects on allium cepa root cells. akinboro and bakare (2007) also documented a relationship between macroscopic and microscopic parameters for allium cepa root cells exposed to toxic aqueous extracts herbs. the growth inhibition of the seedlings was always accompanied by the reduction of the number of cells in division. corroborating our results, gatti et al. (2004) showed that extracts of aristolochia esperanzae kuntze delayed seed germination and root growth of lactuca sativa l. and raphanus sativus l. according to baličević et al. (2015), extracts of aristolochia clematitis reduced the germination and growth of tripleurospermum inodorum l. (weeds), and the concentration of 100 g l-1 inhibited the germination of seeds of these plants. aqueous extract at 25 g l-1 of aristolochia triangularis presented antimitotic action to meristematic cells of allium cepa (amat et al., 2002). watanabe et al. (1988) documented that aas are potent inhibitors of seed germination. fcm analyses also showed a presence of compounds in the decoctions that delayed the progression of the cell cycle. the decoctions tested caused a concentrationdependent increase of allium cepa nuclei in g0/g1. consequently, aristolochia decoctions promoted a reduction of allium cepa nuclei in s and g2/m phases. these results could reflect the activation of g0/g1 checkpoints in response to dna damage. plant cells have a p53-independent control of proliferation (pelayo et al., 2003). the checkpoint pathways transduce antimitogenic signals that lead to the temporary interruption of the cycle. thus, the repair mechanisms can act before the irreversible transition to the subsequent cycle phase (pelayo et al., 2003; junqueira & carneiro, 2012). also, heteropycnotic nuclei were observed in response to decoctions exposure. these markers of cell death are characterized by condensation of the nucleus (andrade-vieira, et al., 2012), making it inoperative for failure of the enzyme synthesis (manjo & joris, 1995; levin et al., 1999). the activation of cell death mechanisms is the last resource to avoid proliferation of cells containing abnormal dna. in this way, we could infer that dna damage was not repaired in g1, and in response, cell death pathways were activated. some studies also documented that aristolochia extracts and aas promoted cell cycle arrest and cell death in mammalian cell lines. li et al. (2006) reported that aai may cause dna damage and cell cycle delay in porcine proximal tubular epithelial cell lines through a wild-type p53-independent pathway, prior to apoptosis or necrosis. chang et al. (2007) found that cell cycle distribution determined by flow cytometry showed an increase of human urinary tract epithelium cells in the g0/g1 phase after exposure to aas mixture (41% aa i and 56% aa ii). proteins levels that block the cell cycle (p53, p21 and p27) have increased. additionally, there was a decrease in cyclind1/cdk4 complex, which control proteins required for the progression of the cycle (chang et al., 2007). li and wang (2013) verified that methanol extract from aristolochia debilis siebold & zucc. stems inhibited proliferation of human colon cancer cells by inducing sub-g1 arrest. the authors also showed that aristolochia debilis induced apoptosis in ht-29 cells by upregulation of bax and corresponding downregulation of bcl-2 expression as well as ros production. allium cepa test was suitable for screening initial toxicity of aristolochia labiata and aristolochia triangularis decoctions. our studies report similar results for other test systems. in vivo research on renal and testicular histopathology of decoctions in mice is ongoing. conclusion allium cepa test was used to evaluate decoctions from aristolochia labiata and aristolochia triangularis stems. phytochemical analysis indicated mainly the presence of alkaloids in both decoctions studied. the decoctions promoted inhibition of onion seed germination and seedling growth. the mitodepressive effect of both decoctions was determined by mitotic index and fcm analyses. the induction of heteropycnotic nuclei suggests that decoctions promote cell death. we suggest that the aas may be related to the observed toxicity. caution is recommended in the consumption of decoctions from aristolochia labiata and aristolochia triangularis stems. acknowledgements the authors thank fundação de apoio à pesquisa e inovação do espírito santo for undergraduate scholarship. we also thank damielle leite figueiredo for technical support and ariane tonetto vieira for editing images. references akinboro a, bakare aa. 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c, el khattabi c, el mere s, declèves ae, pochet s, antoine mh. 2020. characterization of cytotoxic effects of aristolochic acids on the vascular endothelium. toxicology in vitro, 104811. caryologia. international journal of cytology, cytosystematics and cytogenetics 73(4): 17-26, 2020 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.13128/caryologia-949 caryologia international journal of cytology, cytosystematics and cytogenetics citation: f. tapia-pastrana, a. delgado-salinas (2020) first cytogenetic register of an allopolyploid lineage of the genus aeschynomene (leguminosae, papilionoideae) native to mexico. caryologia 73(4): 17-26. doi: 10.13128/caryologia-949 received: may 24, 2020 accepted: november 10, 2020 published: may 19, 2021 copyright: © 2020 f. tapia-pastrana, a. delgado-salinas. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid ftp: 0000-0003-0232-2110 first cytogenetic register of an allopolyploid lineage of the genus aeschynomene (leguminosae, papilionoideae) native to mexico fernando tapia-pastrana1,*, alfonso delgado-salinas2 1 facultad de estudios superiores zaragoza, universidad nacional autónoma de méxico, laboratorio de genecología, batalla 5 de mayo s/n esquina fuerte de loreto, col. ejército de oriente, iztapalapa, c.p. 09230, ciudad de méxico, mexico 2 instituto de biología, departamento de botánica, universidad nacional autónoma de méxico, apartado postal 70-233, 04510, cd. de méxico, mexico *corresponding author. e-mail: pasfer@unam.mx abstract. a conventional cytogenetics analysis revealed for first time an allopolyploid lineage of the genus aeschynomene in mexico. the hybrid condition is confirmed after all the prometaphase and metaphase nuclei of the hybrids exhibited only one pair of sat-chromosomes, confirming the existence of nucleolar dominance and amphiplasty. the karyotype formula for this lineage was 2n = 4x = 40 = 34 m + 6 sm with a total diploid chromosome length (tdcl) = 28µm and an average chromosome size (ac) = 1.40 µm. comparison of the karyotype and other chromosomal parameters with recent cytogenetics records for other species of the subgenus aeschynomene included in the nod-independent clade allows propose to aeschynomene evenia and a. scabra as possible progenitors. furthermore, other comparison of seedlings focused at the number of leaflets of the first four eophylls of the proposed parents and of the hybrid individuals allowed to observe coincidences that support the proposal made from the cytogenetic analysis. evidence of “gigas” effects on flowers and fruits of hybrids is also shown. keywords: cryptic taxa, cytotype, karyotype, nucleolar dominance, sat-chromosomes, secondary constrictions, seedlings. i. introduction aeschynomene linnaeus (leguminosae, tribe dalbergieae s. l.) is a diverse genus of subfamily papilionoideae (papilionoid legumes) distributed in the tropics and subtropics of the world (lavin et al. 2001, klitgaard and lavin 2005). it comprises herbaceous and woody species, annual, repetitive and perennial with different ecological requirements. several species contribute to supplement nitrogen to the soil through the production of nodular roots and stems in symbiosis with nitrogen fixing bacteria, so they are economically important as green manure (alazar and becker 1987; fernandes 1996; souza et al. 2012) and recently, aeschynomene evenia c. wright has been proposed as a model species in genetics to develop new agronomic 18 fernando tapia-pastrana, alfonso delgado-salinas strategies in the engineering of nitrogen fixing nodules that enhance rice production (arrighi et al. 2012, 2013). this taxon belongs to the group of 11 semi-aquatic species of aeschynomene that have the property of being nodulated by photosynthetic bradyrhizobium that lack the nodabc genes necessary for the synthesis of nod factors and are grouped into the so-called nod-independent clade (chaintreuil et al. 2013; brottier et al. 2018) and that correspond to the morphological series indicae and sensitivae (rudd 1955). the genus aeschynomene traditionally included in the aeschynomeneae tribe (polhill et al.1981) and currently circumscribed in the dalbergioid clade (lavin et al. 2001; wojciechowski et al. 2004) has evolved in different ecological niches and includes herbaceous forms, annual and perennial shrubs and trees up to 8 meters, with compound pinnate leaves and papilionoid flowers that are generally self-pollinated, although there is crosspollination by bees (rudd 1955; fernandes 1996; arrighi et al. 2014, carleial et al. 2015). other studies indicate that the genus aeschynomene is not monophyletic and taxa with basifixed stipules and a campanulate calyx (subgenus ochopodium vogel) are more related to the genera machaerium persoon and dalbergia linnaeus f. than to taxa with medfixed stipules and a bilabiate calyx (subgenus aeschynomene léonard) (ribeiro et al. 2007; cardoso et al. 2012). currently aeschynomene genus contains 170 (http:// w w w.theplantlist.org) to 180 species (klitgaard and lavin 2005) 231taxa and cytotypes at four ploidy levels: diploid (2x), tetraploid (4x), hexaploid (6x) and octoploid (8x) (index to plant chromosome numbers; kawakami 1930; bielig 1997; arrighi et al. 2012, 2014; chaintreuil et al. 2016, 2018; brottier et al. 2018). america, where most of the taxa are 2n = 20 diploids, has been proposed as the center of origin of the genus, with a secondary distribution in africa and asia where polyploid species and some cases of aneuploidy predominate (chaintreuil et al. 2018; tapia-pastrana et al. 2020). although it is clear that in the dalbergioid clade, diploid 2n = 20 genera predominate, with some polyploid and aneuploid species, in aeschynomene there is currently a renewed interest in knowing to what extent polyploidy has contributed to the diversification and radiation of the group. in this respect arrighi et al. (2014) revealed multiple hybridization/polyploidization events, highlighting the prominent role of allopolyploidy in the diversification of nod-independent clade. in addition chaintreuil et al. (2016) studied african aeschynomene species and their data support the idea that the whole african group is fundamentally tetraploid and revealed the allopolyploid origin of a. afraspera j. léonard (2n = 8x = 76) and a. schimperi hochst. ex a.rich. (2n = 8x = 56), where variations in the number of chromosomes also indicated possible dysploidy/aneuploidy events. in mexico, aeschynomene is represented by 31 species and infraspecific taxa including several endemisms. an investigation about the patterns of chromosomal evolution in mexican species, including six taxa of the nod-independent clade, showed the predominant of a basic 2n = 20 diploid structure and evolutionary patterns related to the corresponding morphological series (tapia-pastrana et al. 2020). in the present research, a conventional cytogenetic study was carried out to obtain the karyotype and analyze the level of ploidy in a mexican population initially described as aeschynomene scabra g. don, where the size of the flowers, fruits and seeds generated suspicions about a possible hybrid origin. in addition as the sampled individuals exhibited floral morphotypes similar to those of a. evenia c. wright and a. scabra, whose collection records in mexico would support their participation in the hybridization process, the growth pattern of the first four eophylls was also compared in putative hybrids and their parental assumptions. 2. material and methods 2.1 collection sites seeds of the putative hybrids were collected in the municipio de la huerta, estado de jalisco, mexico, 19°29´n; 105°01́ w (carleial s/n, mexu). the climate is semi-dry and warm. mean temperature in the area is 25.2 °c, and there is a well-defined rainy season (average annual precipitation: 1107  mm) occurring from june to october (garcía-oliva et al. 2002). the seeds of aeschynomene evenia and a. scabra were collected in the municipalities of coyuca de catalán (18° 19´ n; 100° 42´ w, jc soto 15333 (mexu)) and arcelia (18°18´54´́ n; 100°17´02´́ w, jc soto 15393 (mexu)) respectively, in the state of guerrero, mexico. both municipalities are part of the tierra caliente region. the predominant climate is warm subhumid with rains from june to september (average annual precipitation: 1100 to 1200 mm). the studied taxa are assigned to the infrageneric classification of neotropical aeschynomene sensu rudd (1955) series indicae of subgenus aeschynomene and are part of the nod-independent monophyletic clade (chaintreuil et al. 2013), whose taxa are nodulated on roots and stems by photosynthetic bradyrhizobium strains lacking the nod abc genes necessary for the synthesis of nod factors (giraud et al. 2007). 19first cytogenetic register of an allopolyploid lineage of the genus aeschynomene native to mexico 2.2 chromosome and karyotype procedures in putative hybrids seeds were collected in summer 2014 and from at least six plants. batches of 40 seeds from each plant were used. the seeds were scarified and germinated in petri dishes lined with a moist filter paper at room temperature and under natural light. chromosomes at metaphase and prophase were obtained following the splash method (tapia-pastrana and mercado-ruaro 2001). all meristems were collected from 2-4 mm long roots pretreated with 2 mm 8-hydroxyquinolin for 5 h at room temperature and fixed in the fixative (ethanol: acetic acid=3:1). they were then treated with a mixture of 20% pectinase (sigma) and 2% cellulase (sigma) in 75 mm kcl for 60 min at 37 °c. after centrifugation at 1500 rpm for 10 min, the cell pellet was transferred to 75 mm kcl solution for 13 min at 37 °c. after two successive rinses with the kcl solution, they were again fixed in the fixative and subsequently rinsed twice more. one or two drops of the suspension of pellet were placed on clean slides, air-dried and stained in 10% giemsa for 13 min. preparations were made permanent using a synthetic resin. at least ten metaphase plates of intact cells with well-spread chromosomes, no chromosome overlapping, and same contraction and ten prophase plates were photographed from each collection, using a microscope (axioscope, carl zeiss) and analyzed for chromosome number determinations. five photographs of metaphases with chromosomes having similar comparable degrees of contraction and centromeres clearly located were utilized to obtain the total diploid chromosome length (tdcl), total chromosome length (tcl), average chromosome length (ac), the difference in length between the longest chromosome and the shortest chromosome (range) and the longest/shortest chromosome ratio (l/s). the shapes of chromosomes were classified according to levan et al. (1964) and the tf was obtained following huziwara (1962). furthermore, prometaphase cells were analyzed to verify both the number of nucleoli, and the behavior of the sat chromosomes. the information thus obtained was compared with that recently recorded for aeschynomene evenia and a. scabra in another cytogenetic study where the same method was used for karyotype analysis in aeschynomene species and varieties (tapia-pastrana et al. 2020). 2.3 seedlings and eophylls in order to compare seedling morphology in individuals of the supposedly hybrid population with those of aeschynomene evenia and a. scabra, the development of 20 individuals grown in pots under greenhouse conditions was evaluated. interest was particularly focused on the number of leaflets and the presence of hairs on their edges until the complete development of the fourth leaf. eophylls at the first, second, third and fourth eophyllar nodes were referred to as e1, e2, e3 and e4, respectively following schütz et al. (2019). photographs of seedlings were taken with a canon sx700 hs camera. 3. results 3.1 karyotype analysis a total of 410 cells were analyzed in metaphase and 16 in prometaphase and all exhibited a 2n = 4x = 40 (fig. 1 a-c). tdcl was 28 µm and ac 1.40 µm. the chromosomal range was 0.56 µm, the ratio 1.48 and a tf = 42.46. the karyotype formula was 2n = 4x = 34m + 6sm (table 1). consistently, in all prometaphase and metaphase nuclei, only one pair of submetacentric chromosomes was observed having lax secondary constrictions and macrosatellites in short arms (sat-chromosomes) (fig. 1 a-c). the karyotype exhibits small chromosomes (1.72-1.16 µm) clearly discernible, with predominance of metacentric chromosomes (m) and lacking subtelocentric chromosomes (st). this arrangement is consistent with a tf that describes a slightly asymmetric karyotype (fig. 1d and table 1). occasionally the sat-chromosomes were observed immersed in a single nucleolus. 3.2 seedlings and eophylls the seedlings of the three taxa are illustrated in fig. 2 a-c. eophylls are stipulated, alternate, petiolate, pinnate, with alternate leaflets, have elliptic to oblong leaflets, a rounded apex, an entire margins, and one central primary vein in the three taxa under study. the leaf lets did not present trichomes; both adaxial and abaxial surfaces are glabrous. the number of leaflets in the first four eophylls in seedlings of individuals of aeschynomene evenia, a. scabra and putative hybrids are shown in tables 2-4 respectively. 4. discussion it is clear t hat t he entire da lbergioid clade (adesmia, dalbergia and pterocarpus subclades) is dominated by 2n = 2x = 20 species, with scattered polyploids and aneuploids (lavin et al. 2001). in addition an ances20 fernando tapia-pastrana, alfonso delgado-salinas tral state reconstruction performed in a phylogeny based on its + matk of the aeschynomene genus and related genera indicated that diploidy is the ancestral condition in the entire group reviewed (brottier et al. 2018). however, the role of allopolyploid speciation events in the origin of new taxa is now recognized (arrighi et al. 2014). as far as we know, the first assumption about of hybridization in aeschynomene is attributed to rudd (1955) who pointed out that the species with the widest distribution within the indicae series (nod-independent clade) tend to be more variable and intergrade with their neighbors. later, verdcourt (1971) suggested that specimens of aeschynomene rudis bentham (also into nodindependent clade) with large flowers could be of polyploid origin, without pointing out the possible duplication mechanism involved, auto or allopolyploidy. to date, several studies have shown that the clade of a. evenia is mainly diploid (2n = 2x = 20), however some species such as a. indica linnaeus (2n = 4x = 40, 2n = 6x = 60) seem to be of recent allopolyploid origin (arrighi et al. 2014; chaintreuil et al. 2018; tapia-pastrana et al. 2020). furthermore, it has been found that all species of the group a. afraspera are polyploid (2n = 4x = 28, 38, 40; 2n = 8x = 56, 76) and have a common ab genomic structure (chaintreuil et al. 2016). in facts phylogenetic relationships between diploids and polyploids elucidated from its sequences show that in the nod-independent clade, species such as a. evenia, a. scabra and a. rudis participate in the hybridization/polyploidization events and formation of polyploid complexes that have contributed to the radiation of this group (arrighi et al. 2014). figure 1. mitotic metaphase cells of hybrid aeschynomene 2n = 4x = 40. a-c, metaphase chromosome plates in optimal spread; d, karyotype 34m + 6sm. the chromosomes are aligned in decreasing order. arrows point to secondary constrictions and satellites on short arms of submetacentric chromosomes. table 1. average chromosome measurements obtained from five nuclei in metaphase of the hybrid population (2n = 4x = 40 = 34m + 6sm) under study. cp tcl (µm) lla ( µm) lsa (µm) r ct 01 1.72 0.96 0.77 1.24 m 02 1.63 0.89 0.73 1.21 m 03 1.59 0.89 0.69 1.28 m 04 1.55 0.81 0.72 1.12 m 05 1.53 0.81 0.70 1.15 m 06 1.50 0.82 0.66 1.24 m 07 1.48 0.83 0.63 1.31 m 08 1.46 0.79 0.66 1.19 m 09 1.44 0.79 0.63 1.25 m 10 1.42 0.79 0.61 1.29 m 11 1.38 0.74 0.64 1.17 m 12 1.36 0.89 0.46 1.93 sm* 13 1.34 0.78 0.55 1.41 m 14 1.31 0.70 0.59 1.18 m 15 1.28 0.72 0.55 1.30 m 16 1.24 0.83 0.40 2.07 sm 17 1.23 0.69 0.52 1.32 m 18 1.19 0.67 0.54 1.24 m 19 1.19 0.66 0.49 1.34 m 20 1.16 0.78 0.36 2.16 sm tdcl 28.00 ac 1.40 abbreviations: cpchromosome pair; tcltotal chromosome length; llalength long arm; lsalength short arm; rarm ratio; ct-chromosome type; tdcltotal diploid chromosome length; acaverage chromosome length; mmetacentric; smsubmetacentric; *satellite. abbreviations: cpchromosome pair; tcl total chromosome length; llalength long arm; lsalength short arm; rarm ratio; ct-chromosome type; tdcltotal diploid chromosome length; acaverage chromosome length; mmetacentric; smsubmetacentric; *satellite. 21first cytogenetic register of an allopolyploid lineage of the genus aeschynomene native to mexico in the present investigation, the chromosomal number obtained in all the nuclei analyzed from the individuals under study was 2n = 4x = 40, which undoubtedly shows that they are polyploid cells and that the individuals from which they come integrate a polyploid lineage not previously detected in mexico (rudd 1955; tapiapastrana et al. 2020). the origin of the polyploidy (auto or allopolyploidy) were established easily from the number of sat chromosomes unambiguously identified both in nuclei in prometaphase and metaphase and by their position in relation to the nucleolus. indeed, polyploidy, the process of genome doubling that gives rise to organism with multiple sets of chromosomes, is recognized as an important process in plant evolution, a major mechanism of adaptation and is often invoked as a driver of diversification (ramsey and figure 2. seedling morphology of aeschynomene under study until the complete development of the fourth eophyll a, aeschynomene evenia; b, a. scabra; c, hybrid of aeschynomene. table 2. number of leaflets up to the fourth eophyll in aeschynomene evenia. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 e1 10 10 10 10 10 10 10 10 10 10 10 9 10 9 8 10 9 10 10 8 e2 12 12 12 12 11 12 14 12 10 14 12 12 14 12 10 13 12 11 10 11 e3 15 16 15 14 14 16 17 15 14 16 16 15 17 12 12 16 16 12 14 12 e4 16 18 16 16 16 16 18 18 14 16 18 16 18 16 15 16 16 14 16 15 table 3. number of leaflets up to fourth eophyll in aeschynomene scabra.   1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 e1 10 8 10 10 10 10 10 8 10 9 10 10 10 10 10 8 8 8 8 10 e2 12 15 14 14 14 14 14 12 16 13 14 13 14 14 14 12 12 13 15 14 e3 20 20 22 18 18 18 19 19 20 17 17 18 18 16 18 19 19 19 20 16 e4 24 25 27 23 22 22 22 22 24 22 22 22 22 21 22 22 22 20 25 21 table 4. number of leaflets up to fourth eophyll in hybrids.   1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 e1 10 10 8 10 10 9 10 8 8 10 8 10 8 10 10 8 10 10 8 10 e2 14 14 12 14 13 14 14 14 14 14 14 14 14 16 14 14 14 14 14 10 e3 18 20 18 20 20 19 20 20 20 19 18 20 18 20 20 20 20 15 18 14 e4 21 25 22 23 23 26 23 24 23 24 23 26 22 27 22 22 23 22 23 14 22 fernando tapia-pastrana, alfonso delgado-salinas schemske 1998; soltis et al. 2009) and it is likely to be one of the most predominant mechanisms of sympatric speciation in plants (otto and whitton 2000). it can act alone, resulting in autopolyploidy, or in concert with hybridization, producing allopolyploids, and both modes lead to plant speciation. it should be mentioned that in the process of polyploidization by total gene duplication (autopolyploidy) the number of satellites present in a diploid species is also doubled, since this does not involve loss or suppression of the nucleolar function, the nor regions associated with secondary constrictions in sat-chromosomes are they show lax and therefore satellites are clearly appreciated. it is known that nors contain tandemly arranged highly reiterated ribosomal rrna genes coding for 18s-5.8s-26s rrna whose expression is under epigenetic control (pikaard 2000). for example, medicago sativa linnaeus, a recognized autotetraploid exhibits four macrosatélites in metaphase cells (falistocco 1987). in contrast, plants of allopolyploid origin as cotton (gossypium hirsutum linnaeus 2n = 4x = 52 aadd, endrizzi et al. 1985), wheat (triticum aestivum linnaeus 2n = 6x = 42, aabbdd, lacadena and cermeño 1985; friebre et al. 1995) and canola (brassica napus linnaeus 2n = 4x = 38 aacc; xiong and pires 2011) undergo inactivation of the regions of the nucleolar organizer (nor) of one of the parental genomes, silenced by the effect of nucleolar dominance (navashin 1934) and consequently a smaller number of satellites is recorded (doyle et al. 2008; ge et al. 2013). it is, rdna loci may be additive in number, but then exhibit differences in gene expression. interspecific hybrids often have rrna genes of one parent functionally dominant over the rrna of the other parent, and there are many examples of such regulation of rrna gene activity in allopolyploids (pikaard 2000; pires et al. 2004). comparative analyses of nucleolar organizer regions (nors) of somatic metaphase chromosomes made by phase contrast, c-banding and silver staining have demonstrated that the activity of the nors of certain chromosomes can be suppressed or partially inhibited by the presence of other sat-chromosomes. the nor competition is cytologically expressed as amphiplasty: a term proposed to denote morphological changes which occur in chromosomes following interspecific hybridization (rieger et al. 1976). the secondary constriction of the sat-chromosome of one of the parental species is missing in the hybrid and the satellite is retracted onto the chromosome arm as a consequence (lacadena and cermeño 1985). thus, in the hordeum murinum linnaeus complex (poaceae, triticeae), tetraploid and hexaploid cytotypes arising from hybridization exhibit only a pair of chromosomes with secondary and satellite constrictions (cuadrado et al. 2013). in fact, the inactivation or epigenetic silencing of ribosomal genes is one of the most common phenomena in hybrid and polyploid members of triticeae linnaeus (cermeño and lacadena 1985; carmona et al. 2016) and one of the first examples of differential gene expression discovered in plant hybrids nearly a century ago (navashin 1934; matyásӗk et al. 2007). in the present work, the repressive effects on nors from allopolyploid population are cytologically expressed (amphiplasty) as the suppression of a secondary constriction clearly observed in all their complements (fig. 1 a-d). the karyotype exhibited in hybrid individuals (34m + 6sm) (fig. 1d and table 1) coincides in several respects with that expected at a cross between a. evenia (2n = 2x = 7m + 3sm) and a. scabra (2n = 2x = 10 m) (fig. 2 in tapia-pastrana et al. 2020). for example, the number of sm chromosomes in a. evenia agrees with the 6sm in hybrid individuals. in addition to submetacentric chromosomes, these individuals exhibit metacentric chromosomes whose predominance is consistent with the karyotype formulas described in their putative relatives, whose complements lack subtelocentric chromosomes (tapia-pastrana et al. 2020). there is a coincidence between thc and ac and even the morphology of the sat-chromosomes (submetacentrics with macrosatellites in short arms) and their position in the karyotype is very similar to that recently described in a. scabra (tapiapastrana et al. 2020). therefore we propose to a. evenia and a. scabra as progenitors of the allopolyploid population (2n = 4x = 40 = 34m + 6sm) registered in this work. the reasoning is simple: if a diploid species is involved in the origin of a tetraploid cytotype, its chromosomes must be present in it. the same is true if tetraploid forms are involved in the origin of hexaploid forms (cuadrado et al. 2013). in mexico, recent collection data shows that populations of both species occupy overlapping ranges in some central areas of the country where a. evenia is considered an introduced species (arrighi et al. 2013; chaintreuil et al. 2018; tapia-pastrana et al. 2020). this new proposal is not surprising, since previously the indicae series species grouped within nod-independent clade, including a. evenia and a. scabra, have been identified as progenitors in allopolyploids and in the formation of polyploid complexes, although attempts at hybridization have failed to form fertile individuals (arrighi et al. 2014). regarding the identity of the allopolyploid taxon recorded here, it can be argued that a detailed review of its complete morphological characters (data not shown) suggests that it shares characteristics described for aeschynomene rudis particularly in the shape and size of flowers, fruits (hispidulous, verru23first cytogenetic register of an allopolyploid lineage of the genus aeschynomene native to mexico coses, or muricate at the center) and seeds (rudd 1955). however, it also recalls the robust version of a. scabra described by rudd (1955). the existence of cryptic taxa in aeschynomene as well as the need for broader sampling to detect new cytotypes has already been pointed out (brottier et al. 2018, chaintreuil et al. 2018) and the results of this study confirm this. regarding the results obtained from the seedling comparison, these seem to support a close relationship between the individuals of the three populations studied (fig. 2, tables 2-4). in principle, the observed intervals in the number of leaflets per eophyll (e1-e4) show some uniformity, particularly e1, whose interval (8-10 leaflets) was repeated in the three populations. in intermediate eophylls (e2-e4) a close concordance is observed between a. scabra and the hybrid population, while in a. evenia the number of leaflets was lower in correspondence with the taxonomic description of this species (rudd 1955). furthermore, the morphology of the eophylls was similar and in all populations the leaflets figure 3. floral morphotypes (above), dissected flowers and fruits (below) of the taxa under study. a, d and e, aeschynomene evenia; b, f and g, a. scabra; c, h and i, hybrid of aeschynomene. all three taxa exhibit typical pea or papilionoid flowers. these zygomorphic flowers comprise a standard (vexillum or banner) petal (adaxially placed), two lateral petals (wings) and two (usually partially fused and abaxially placed) keel petals, which conceal the androecium and gynoecium. the fruits have similar characteristics and are mainly differentiated by their size. above scale bar = 0.5 cm, below = 1.0 cm. 24 fernando tapia-pastrana, alfonso delgado-salinas exhibited entire margins, without trichomes and with a central primary vein. polyploids are known to often have novel phenotypes that are not present in their diploid progenitors or that exceed the range of parent species (“gigas” effects) (ramsey and schemske 2002; ramsey and ramsey 2014). in this sense, fig. 3 shows floral morphotypes, dissected flowers and fruits of the populations studied here, where similarities are observed, but the differences in size of such characters are highlighted. the results obtained in this study confirm that in the nod-independent lineage within the genus aeschynomene, hybridization and polyploidization play a relevant role in the formation of species and those taxa such as the polymorphics a. evenia and a. scabra actively participate in it. acknowledgements this study is part of the doctoral thesis of the first author, f t-p, carried out at the posgrado en ciencias biológicas of the universidad nacional autónoma de méxico (unam). the authors thank to dr. samuel carleial for seeds originally identified as a. scabra and to the division of postgraduate studies and research of the faculty of higher studies, zaragoza, unam for the support provided during 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abdul_shakoor954@yahoo.com abstract. the caryophyllaceae family is complex. several attempts have been carried out in the past to study caryophyllaceae members. this study mainly focused on allochrusa bunge to determine its genetic structure and used issr markers, its, and rps16 data to classify and differentiate allochrusa species. we collected 122 allochrusa specimens. our analysis included morphological and molecular method approaches. morphometry analysis indicated that floral characters could assist in the identification of allochrusa species. a. persica (boiss.) boiss. and a. versicolor fisch. & c.a.mey. showed affinity to each other. a. bungei boiss. formed a separate group. analysis of molecular variance showed significant genetic differentiation in allochrusa (p= 0.001). the majority of genetic variation was among the allochrusa population. we recorded minimum gene flow (nm=0.176) between allochrusa species. besides this, isolation by distance occurs in allochrusa members, as shown in the mantel test result (r = 0.01, p = 0.0002). structure analysis revealed three genetic groups. it is evident that a. persica, a. versicolor, and a. bungei differ genetically from each other. our current findings have implications in plant systematics and biodiversity management. keywords: allochrusa, issr–analysis, network, population structure, species delimitation. introduction caryophyllaceae contains 88 genera and 2,200 species. the caryophyllaceae family is subdivided into three subfamilies, ie. caryophylloideae, alsinoideae, and paronychioideae (greenberg and donoguhe 2011; pirani et al. 2014; hernandez-ledesma et al. 2015). the caryophyllaceae has a worldwide distribution, and this family is diverse. the mediterranean region is consid122 kun zhu et al. ered a hot spot or center of diversity for caryophyllaceae (harbaugh et al. 2010; greenberg and donoguhe 2011). allochrusa bunge has about eight species distributed in turkey, central asia, afghanistan, caucasus, transcaucasia, and iran (boissier 1867; schischkin 1936; cullen 1967; schiman-czeika 1988). according to flora orientalis by bunge (boissier 1867: 559), allochrusa includes three species in iran [a. versicolor boissier (1867: 559), a. bungei boissier (1867: 560), a. persica boissier (1867: 560)]. schischkin (1936) classified acanthophyllum c.a.mey. into two subgenera [euacanthophyllum (boissier, 1867: 561) schischkin (1936: 783) and allochrusa (bunge in boissier, 1867: 559). schischkin (1936: 799)] included two sections in the subgenus. four allochrusa species were reported in iran by schiman-czeika (1988). acanthophyllum meyer plant species are shrubs and perennial. the majority of acanthophyllum occurs in iran and central asian countries (ghaffari 2004; pirani et al. 2014; mahmoudi shamsabad et al. 2020). the caryophyllaceae family is a complex taxonomical family. therefore given the taxonomic complexity in caryophyllaceae, some studies were conducted to resolve taxonomical and classification issues. for instance, phylogenetic data on acanthophyllum supports the notion of inclusion of allochrusa within acanthophyllum (pirani et al. 2014). however, traditional taxonomical and morphological characters are dissimilar between acanthophyllum and allochrusa. henceforth, allochrusa is classified as a separate genus (pirani et al. 2014). according to madhani et al. (2018) the acanthophyllum clade includes allochrusa, gypsopgila herniarioides, and allochrusa species. they revealed that both markers (its) and the chloroplast gene rps16 does not allow allochrusa to differentiate from acanthophyllum. the species of the genus allochrusa were considered once as members of acanthophyllum subgenus. allochrusa (schischkin 1936) and molecular phylogenetic studies by madhani et al. (2018) corroborate the taxonomic treatment performed by pirani et al. (2014) and contradict the treatment by hernandez-ledesma et al. (2015), where it was recognized provisionally at the generic level. according to this concept, it is necessary to resurrect the generic name acanthophyllum for some taxa treated as allochrusa in recent taxonomic surveys (madhani et al., 2018). morphological characters such as leaves, f lower arrangement, or inflorescence are crucial characters to identify allochrusa species (boissier 1867; schischkin 1936; cullen 1967; schiman-czeika 1988). plant leaves are narrow and spiny. corymbose inflorescence, calyx tubular, petals 5, ovules 4‒5, and seed are reniform and curved in allochrusa (boissier 1867; schischkin 1936; cullen 1967; schiman-czeika 1988). based on morphological characters, new species, ie. allochrusa lutea falat. & mahmoodi was recorded in iran (mahmoodi and falatoury 2016). this species is limited to the northwestern part of iran. a. lutea differs from a. persica in stem length and flower symmetry and shape (mahmoodi and falatoury 2016). advent in molecular biology has paved our understanding to characterize genetic diversity and population structure in plant species (shakoor et al. 2021). molecular markers played a vital role in conservation biology and plant genetic resources (erbano et al. 2015; esfandani-bozchaloyi and sheidai 2018 ). molecular markers, including inter simple sequence repeats (issr) and its phylogenetic studies on the caryophyllaceae family, showed the significance of molecular methods to resolve the genetic and evolutionary relationship within the members of caryophyllaceae (greenberg and donoguhe 2011; korkmaz and yildirim 2015). allochrusa lutea is restricted to the zanjan province, while its closest relative species (a. persica) occurs in nw iran. the altitudinal range is 1300–1600 m a.s.l. a. lutea grows on low montane steppe life zone in open, disturbed, and dry areas with a high percentage of scree on the ground (mahmoodi and falatoury 2016). a. persica has been reported from iran, east azerbaijan province. a. bungei: turkey: kars, kaĝziman, tuzluça, 13 km west of tuzluça, 1060 m; iran: east azarbayejan, between marand and jolfa. a. versicolor: iran: east azarbayejan: 42–55 km w marand toward evowghli, 1000 m; marandkhoy; west azarbayejan: 60 km after makou to dasht-e zanganeh, 900 m; khoy road of marand; ca. 10 km from gharaziaeddin to marand, 8 km from babolabad, 982 m; maku, kulus bulaghi; between maku & khoy, evaghli, 1100 m.three species of allochrusa versicolor, a. bungei, and a. persica are found in iran. these species have almost similar morphological features. it is difficult to identify and separate these species on the basis of traditional taxonomy and morphology. therefore, due to complexity in identification, we only used issr markers to identify/ separate these species. the phylogenetic approach has been used on other accessions, and no unedited sequences were produced. our approach integrated morphological and molecular methods to analyze allochrusa species. materials and methods plants collection 122 plant samples were collected. overall, seven natural populations were sampled. five to eight specimens from each plant population were recorded. further details about the plant location are provided (table 1, 123morphological method and molecular marker determine genetic diversity and population structure in allochrusa figure 1). we carefully identified the plant species, i.e., allochrusa versicolor, a. bungei, and a. persica according to previous identification protocols (boissier 1867; schischkin 1936; cullen 1967; schiman-czeika 1988). dr. shahram mehri helped in plant collections. plant samples were deposited in the islamic azad university herbarium. we examined 38 morphological characters (10 qualitative, 28 quantitative). the details of morphological characters are provided (table 2). plant morphology analysis before morphometric analysis, we transformed data. mean and variance was coded as 0 and 1. to measure the similarity among plant individuals, we followed euclidean distance (podani 2000). multidimensional scaling (mds) and unweighted pair-group method with arithmetic mean (upgma) method to group the plant species (podani 2000). principal component analysis (pca) to find the variation in the morphological characters of allochrusa plant species. these analyses were done in the past software, version 2.17. (hammer et al. 2001). phylogenetic reconstruction two different nuclear and chloroplastidial dna markers (its an rps16 respectively) were prelimitable 1. location and herbarium accession numbers of of a. bungei, a. persica and a. versicolor sp pop locality latitude longitude altitude (m) voucher no. a. bungei 1 east azerbaijan,tabriz to sperkhan to sahand 36°43’20.25” 48°20’32.07” 1450-2000 pamh 3455 a. bungei 2 east azerbaijan, nematabad, near tabriz 36°44’22.38” 48°14’35.88” 1400 pamh 7896 a. bungei 3 east azerbaijan between marand and jolfa 36°65’86 48°38’65” 1800 pamh 6899 a. versicolor 4 east azerbaijan, marand-khoy 36°36’39 48°83’93” 1300 pamh 4187 a. versicolor 5 west azerbaijan, 10 km from gharaziaeddin to marand, 8 km from babolabad 36°87’77 48°90’10” 955 pamh 4629 a. persica 6 east azerbaijan,tabriz to sperkhan to sahand 36°19’22 48°34’88” 1500 pamh 4567 a. persica 7 east azerbaijan, tabriz, nematabad 36°30’97 48°90’10” 1200 pamh 6309 table 2. morphological characters of a. bungei, a. persica and a. versicolor populations. no characters no characters 1 plant height (mm) 20 fruit length (mm) 2 length of stem leaves petiole (mm) 21 bract length (mm) 3 length of stem leaves (mm) 22 bract width (mm) 4 width of stem leaves (mm) 23 bract length / bract width (mm) 5 length of stem leaves / width of stem leaves(mm) 24 pedicel length (mm) 6 width of stem leaves/ length of stem leaves (mm) 25 peduncle length (mm) 7 number of segment stem leaves (mm) 26 style length (mm) 8 length of basal leaves petiole (mm) 27 stamen filament length (mm) 9 length of basal leaves (mm) 28 number of flowers per inflorescence 10 width of basal leaves (mm) 29 phyllotaxy 11 length of basal leaves / width of basal leaves (mm) 30 vegetation-forms 12 width of basal leaves / length of basal leaves (mm) 31 leave shape 13 number of segment basal leaves 32 plant color 14 calyx length (mm) 33 shape of segments cauline leaves 15 calyx width (mm) 34 shape of calyx 16 calyx length/ calyx width (mm) 35 calyx apex 17 petal length (mm) 36 petal shape 18 petal width (mm) 37 leaf tips 19 petal length / petal width (mm) 38 shape of segments basal leaves 124 kun zhu et al. nary used to represent the phylogenetic relatedness of allochrusa versicolor, a. bungei and a. persica. for this purpose, no new sequences were produced, and we used accessions available in genbank (see the supplementary material s1). the phylogenetic inference was based on three different approaches; maximum parsimony (mp), maximum likelihood (ml), and the bayesian. maximum parsimony (mp) analysis was done in paup (swofford 2002). the heuristic search option was used for each of the two single region datasets, using tree bisection– reconnection (tbr) branch swapping, with 1,000 replicates of the random addition sequence. uninformative characters were excluded from the analysis. branch support values were calculated using a full heuristic search with 1,000 bootstrap replicates (felsenstein 2005), each with a simple addition sequence. the combinability of these two datasets was assessed by use of the partition homogeneity test (the incongruence length difference test (ild) of farris et al. (1995) as implemented in paup (swofford 2002). the test was conducted with invariant characters excluded (felsenstein 2005), using the heuristic search option involving 100 replicates of the random addition sequence and tbr branch swapping with 1,000 homogeneity replicates. the maximum number of trees was set to 500. the model of sequence evolution for each dataset was selected by use of the software mrmodeltest v. 2.3 (kumar et al. 2016) as implemented in mrmtgui based on the akaike information criterion (aic) (edgar 2004). all datasets were analyzed as a single partition with the kimura 2-parameters + g model by bayesian inference (bi) using the software mrbayes version 3.12(ronquist and huelsenbeck 2003) . posteriors on the model parameters were estimated from the data using the default priors. the analysis was performed with 4 million generations, using markov chain monte carlo search. mrbayes performed two simultaneous analyses starting from different random trees (nruns = 2) each with four markov chains trees sampled every 100 generations. no new sequences were produced. we downloaded the its and rps16 data on allochrusa species from national center for biotechnology information. accession numbers obtained from ncbi are provided in appendix. acanthophyllum mucronatum and acanthophyllum cerastioides (d.don) madhani & zarre were used as outgroup taxa. molecular marker assay (issr) we extracted dna from the fresh leaves of plants. plant dna was extracted according to a previous protofigure 1. location of allochrusa species in map. 125morphological method and molecular marker determine genetic diversity and population structure in allochrusa col (esfandani-bozchaloyi et al. 2019). the plant leaves samples were dried with the aid of silica gel. twenty-two issr primers from the university of british columbia were initially chosen for the issr assay. however, we selected 10 primers that could amplify the dna and yielded clear bands (table 3). the issr marker had a 16-18 bp nucleotide repeat sequence. dna amplification was done through pcr. a 25μl volume containing 10 mmtris-hcl buffer at ph 8; 50 mmkcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of single primer, 20 ng of genomic dna; and 3 u of taq dna polymerase were subjected to pcr reactions (bioron, germany). the pcr was carried out in techne thermocycler (germany). the initial denaturation stage of 5 minutes is 94°c. the initial denaturation step was followed by 36 cycles of 1 minute at 95°c, 1 minute at 50-52°c and 1 minute at 72°c. the final extension stage of 5-10 min at 72°c completed the reaction. the quality of the amplified product was checked on 1% agarose gel. ethidium bromide was used to dye the gel. we used a 100 bp molecular size ladder to compare the fragment size of the pcr product. we conducted genetic diversity, gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism analysis while following previous protocols (weising et al. 2005; freeland et al. 2011). neighbor-joining (nj) algorithm (saitou and nei 1987) was used to detect the evolutionary relationship between plant populations. we also performed network computation, i.e., tcs (clement et al. 2002), to construct the allochrusa plant population network. tcs analysis was done in the popart (population analysis with reticulate trees) (clement et al. 2002). the mantel test was performed in the past program ( hammer et al. 2001) to know the correlation between geographical and genetic distances between allochrusa plant population. we investigated the genetic differentiation between plant populations through the amova test (analysis of molecular variance) in genalex 6.4 (peakall and smouse 2006). the data was iterated1000 times to infer the statistical significance. to unveil allochrusa plant population genetic structure, we did genetic structure analysis through a bayesian-based model in structure software (pritchard et al. 2000). under the correlated allele frequency model, we used the admixture ancestry model. we ran twenty times markov chain monte carlo simulation to get the reliable results of k. besides this, the evanno test (evanno et al. 2005) was done to discern correct values of k. since our prime aim was to describe the genetic structure of the allochrusa plant population. therefore, gene flow analysis was carried out in popgene version 1.32 (yeh et al. 1999). results morphometry our clustering analysis showed the same results. upgma cluster results were generated based on morphological characters (figure 2). morphological characters failed to separate a. versicolor (2) and a. persica (3). the principal component results explained the morphological variation within species. overall first three table 3. details about the banding pattern revealed by issr primers. primers primers sequence (5’-3’) issr-1 dbdacacacacacacaca issr-2 ggatggatggatggat issr-3 gacagacagacagaca issr-4 agagagagagagagagyt issr-5 acacacacacacacacc issr-6 gagagagagagagagarc issr-7 ctctctctctctctctg issr-8 cacacacacacacacag issr-9 gtgtgtgtgtgtgtgtyg issr-10 cacacacacacacacarg figure 2. upgma dendrogram of allochrusa. abbreviations: 1-3. a. bungei (1); a. versicolor (2); a. persica (3). 126 kun zhu et al. components explained the majority of variation (74%) in allochrusa species. among three components, the first component described 55% of the total variation. floral characters such as calyx teeth, petals, and limb shape showed a positive correlation (> 0.70). the second pca component explained the variation in ovary shape, seed morphology. a. bungei, a. persica, and a. versicolor had morphological differences. phylogenetic tree the reconstructed phylogenetic tree is shown (figure 3). acanthophyllum mucronatum and acanthophyllum cerastioides constituted in a single clade, while other species were in two separate clades. its and rps16 data set supported separation of a. versicolor, a. persica and, a. bungei with high bootstrap value (> 0.98) (figure 3). the results show that allochrusa species are monophyletic. issr and genetic diversity we conducted detailed genetic diversity and other genetic parameters on the issr generated data (table 4). a. versicolor showed high polymorphism (57.53%), gene diversity (0.33), and shannon information index (0.30). a. persica plant population had low polymorphism and shannon information index (0.15). analysis of molecular variance showed population differentiation in allochrusa (p= 0.001). seventy-three percentage of genetic variation was among the allochrusa population. comparative less genetic variation, i.e., 27%, was reported within the population (table 5). fst pairwise analysis showed that allochrusa members are genetically dissimilar. minimum gene flow occurs (nm=0.176) between allochrusa species. a. versicolor and a. persica were genetically related (0.88). these species are more closely related to each other. a. versicolor and a. persica can exchange genetic material and hybrid with each other. the mantel test result indicated a positive correlation (r = 0.01, p = 0.0002) between genetic and geographical distances among allochrusa taxa. tcs network analysis and clustering results showed a similar clustering pattern (figure 4 a, b). issr molecular primer demonstrated its utility to divide allochrous species into different groups or clades, as evident in the ward tree (figure 4 a). it is evident that a. persica, a. versicolor, and a. bungei differ genetically from each other (different segment colors) (figure 5). structure analysis revealed three genetic groups. yellow and blue segments indicated individuals of a. bungei and a. versicolor. on the other hand, the green color segment highlighted a. persica specimens (figure 5). allochrous show genetic variability within taxa due to introgression (hybridization) between different spefigure 3. maximum likelihood phylogram based on the combined its – rps16 dataset, with acanthophyllum mucronatum and acanthophyllum cerastioides as outgroups. abrreviations: a1 = a. versicolor; b1= a. persica; c1-c3= a. bungei; d1= acanthophyllum mucronatum; e1-e2= acanthophyllum cerastioides; numbers above branches: maximum likelihood bootstrap support values, numbers below branches: bayesian posterior probabilities. table 4. genetic diversity parameters based on issr data allochrusa species. (n = number of samples, ne = number of effective alleles, i= shannon information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism). species n na ne i he uhe %p a. bungei 5.000 0.336 1.034 0.23 0.25 0.19 51.83% a. versicolor 4.000 0.344 1.042 0.30 0.33 0.20 57.53% a. persica 5.000 0.369 1.011 0.15 0.22 0.22 42.15% n = number of samples, ne = number of effective alleles, i= shannon information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism. table 5. analysis of molecular variance result. source df ss ms est. var. % φpt among pops 27 1501.364 95.789 18.154 73% 73% within pops 139 334.443 3.905 2.888 27% total 166 1955.807 20.060 100% φpt: proportion of the total genetic variance among individuals (p < 0.001). 127morphological method and molecular marker determine genetic diversity and population structure in allochrusa cies. henceforth, we performed horizontal gene transfer (hgt) analysis on issr and its data of the studied allochrous species (figure 6). we obtained two introgression events between a. persica and a. versicolor, and the same events happened between a. persica and a. bungei. allochrous species revealed 0.2-0.3 observed heterozygosity (ho) value. in addition to this, inbreeding depression showed high values (fis = 0.3-0.7). discussion we used traditional taxonomical and molecular methods to understand genetic and population structure in allochrusa. the current climate change scenario and biodiversity threats have emphasized the need to conduct genetic diversity studies. given the progress in molecular tools, several investigations have been done to analyze population structure in plants (pirani et al. 2014; erbano et al. 2015; esfandani-bozchaloyi et al. 2017; esfandani-bozchaloyi et al. 2018; shakoor et al. 2021). current morphological findings showed the importance of floral characters to explain the variation and difference among allochrusa species. pca analysis highlighted the significance of calyx teeth, petals, and limbs to identify the allochrusa species. in iran, a new allochrusa was reported based on f loral characters (mahmoodi and falatoury 2016). past and current ecological and taxonomical investigations have successfully implemented morphological characteristics to study plant species (neal et al. 1998; borba et al. 2002; mahmoodi and falatoury 2016; chen et al. 2020). however, the rationale for choosing molecular tools to study allochrusa was the overlapping of morphological characters in allochrusa. besides using issr markers, we also assessed the evolutionary relationship among allochrusa members. our results revealed genetic differentiation among studied species. a. persica and a. versicolor had a close genetic affinity between them. genetic association and relationship studies were conducted in caryophyllaceae ( fior et al. 2006; pirani et al. 2014; madhani et al. 2018). these studies recommended the use of its, cpdna, and matk to classify caryophyllaceae plant individuals. genetic diversity is a central theme in plant adaptability to cope with changing environments (tomasello et al. 2015). our analysis showed genetic diversity was low within the same individuals; however, comparative high genetic differentiation existed between different plant specimens of allochrusa. previous scientific data suggests that genetic diversity is linked with plant ability to endure against perturbation in the environment (booy et al. 2000). a. persica showed a low level of genetic diversity in our analysis. the reason for such finding could be the small number of populations. common logic suggests that population size correlates with genetic diversity (leimu-brown et al. 2006). present results (mantel test) figure 6. horizontal gene transfer (hgt) analysis based on issr and its data of allochrusa species. (dashed lines indicate introgression vents). figure 4. species delimitation in allochrusa species based on issr data. a = ward dendrogram, b = tcs network. figure 5. structure plot of allochrusa species. 128 kun zhu et al. about genetic and geographical distances indicated the distance isolation occurs in allochrusa species. we detected high inbreeding depression showed high values in the allochrusa population. high inbreeding depression reduces plant ability to survive against biotic and abiotic stress (ramsey and vaughton 1998). inbreeding depression occurs due to reduced population size (lonn and prentice 2002). inbreeding depression analysis is critical in the biodiversity management sector (neaves et al. 2015). molecular markers provide in-depth analysis and several genetic diversity parameters to describe inbreeding depression in plant species (glemin et al. 2006). current results showed limited gene f low in the allochrusa population. indeed, a low level of gene flow hinders the exchange of genetic material between species. it may pose survival threats to a small-sized plant population (booy et al. 2000). neighbor-joining and structure indicated three groups of allochrusa. genetic variation among the three groups had the same pattern. two hypotheses have been proposed in the past to explain the genetic variation pattern. genetic diversity is maintained through gene flow; another explanation is connectivity among plant populations (dostalek et al. 2010). a. bungei and a. versicolor had similar macro and micromorphological similarities. nonetheless, they are recognized as a separate taxon. the main differences noted in stem and calyx indumentum, pedicle size, the calyx teeth, petal apex, and limb shape were significant to separating the taxon. these findings are in accordance with mahmoodi and falatoury (2016). they also showed that a. lutea is close to a. persica morphologically. a. persica and a. lutea are similar in habit, leaves shape. a. bungei is a subshrub, covered with glandular hairs. a. persica is perennial herbs with thick woody caudex, without distinctive glandular hairs, petals white with purple striate on the claw (schischkin 1936; schimanczeika 1988). our findings suggest the use of plant morphology features and molecular data to identify allochrusa species. species identification and differentiation is an essential task for systematic and evolutionary studies. we showed that molecular markers have resolving power to solve the plant systematics complex questions. present results have applications in biodiversity and conservation management. acknowledgment this study was supported by the basic scientific research business research project of provincial colleges and universities in heilongjiang province (project number: 135409216). the funding agency had no role in designing the study. references boissier pe. 1867. flora orientalis, vol. 1. georg (genevae); p. 1017. booy g, hendriks rjj, smulders mjm., van groenendael jm, vosman b. 2000. genetic diversity and the survival of populations. plant biol. 2:379-395. borba el, shepherd gj, berge cvd, semir j. floral and vegetative morphometrics of five pleurothallis (orchidaceae) species: correlation with taxonomy, phylogeny, genetic variability and pollination systems. ann. bot. 90(2): 219-230. chen m, zuo x-a, zhao x-y. 2020. comparative floral characters, pollinator limitation, and pollination success in different habitats of caragana microphylla lam. front. ecol. evol. 8(170):1-11. clement m, snell q, walke p, posada d, crandall k. 2002. tcs: estimating gene genealogies. 6th, international parallel and distributed processing symposium; 2002; fort lauderdale, fl. ieee computer society. cullen j. 1967. allochrusa bunge. in: davis ph, editor. flora of turkey and the east aegean islands, vol. 2. edinburgh university press edinburgh; p. 174–175. dostalek t, munzbergova z, plackova i. 2010. genetic diversity and its effect on fitness in an endangered plant species, dracocephalum austriacum l. conserv genet. 11:773–783. edgar rc. 2004. muscle: multiple sequence alignment with high accuracy and high throughput. nucleic acids res. 32(5):1792-7. erbano m, schuhli gse, santos, epd. 2015. genetic variability and population structure of salvia lachnostachys: implications for breeding and conservation programs. int. j. mol. sci. 16(4):7839-7850. esfandani-bozchaloyi s, sheidai m, keshavarzi m, noormohammadi z 2017a. genetic diversity and morphological variability in geranium purpureum vill. 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evidence from a multi-locus species tree reconstruction. mol. phylogenetics evol. 82: 118-130. weising k, nybom h, wolff k, kahl g. 2005. dna fingerprinting in plants. principles, methods, and applications. 2nd ed. boca rayton: crc press; p. 472. yeh fc, yang r, boyle t. 1999. popgene. microsoft windows-based freeware for population genetic analysis. release 1.31. university of alberta, edmonton. appendix/supplementary data (s1) genbank accession numbers and nrdna its and cpdna rps16 sequence data of caryophyllaceae members. acanthophyllum mucronatum: kf924652.1 (madhani et al. 2018); mf401170.1 (madhani et al. 2018). acanthophyllum cerastioides: mf401122.1 (madhani et al. 2018); mf401168.1 (madhani et al. 2018). allochrusa versicolor: ay936270.1 (fior et al. 2006); kf924687.1 (fior et al. 2006). allochrusa bungei : kf924688.1 (pirani et al. 2014); kf924634.1 pirani et al. 2014). allochrusa persica: mn310763.1 (pirani et al. 2014); mn310916.1 (pirani et al. 2014). caryologia international journal of cytology, cytosystematics and cytogenetics volume 74, issue 1 2021 firenze university press caryologia. international journal of cytology, cytosystematics and cytogenetics 74(3): 77-89, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-886 caryologia international journal of cytology, cytosystematics and cytogenetics citation: songpo liu, yuxuan wang, yuwei song, majid khayatnezhad, amir abbas minaeifar (2021) genetic variations and interspecific relationships in salvia (lamiaceae) using scot molecular markers. caryologia 74(3): 77-89. doi: 10.36253/caryologia-886 received: march 23, 2020 accepted: september 24, 2021 published: december 21, 2021 copyright: © 2021 songpo liu, yuxuan wang, yuwei song, majid khayatnezhad, amir abbas minaeifar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid aam: 0000-0002-9371-1498 genetic variations and interspecific relationships in salvia (lamiaceae) using scot molecular markers songpo liu1, yuxuan wang1, yuwei song1,*, majid khayatnezhad2, amir abbas minaeifar3 1department of life science and biotechnology, nanyang normal university, nanyang, 473000, china 2department of environmental sciences and engineering, ardabil branch, islamic azad university, ardabil, iran 3department of biology. payame noor university. p.o. box19395-3697 tehran, iran *corresponding author. e-mail: nanyangyws@126.com; aaminaeifar@gmail.com abstract. the genus salvia includes an enormous assemblage of nearly 1000 species dispersed around the world. iran having 19 endemic species out of 61 is regarded as one of the important regions for salvia diversity in southwest asia. salvia species are herbaceous, rarely biennial or annual, often strongly aromatic. these species are of medicinal, commercial and horticultural value. due to the importance of these plant species, we performed a combination of morphological and molecular data for this species. for this study, we used 145 randomly collected plants from 30 species in 18 provinces. amplification of genomic dna using 10 primers produced 134 bands, of which 129 were polymorphic (97.78%). the obtained high average pic and mi values revealed high capacity of scot primers to detect polymorphic loci among salvia species. the genetic similarities of 30 collections were estimated from 0.61 to 0.93. according to the scot markers analysis, s. tebesana and s. verticillata had the lowest similarity and the species of s. eremophila and s. santolinifolia had the highest similarity. the aims of present study are: 1) can scot markers identify salvia species, 2) what is the genetic structure of these taxa in iran, and 3) to investigate the species inter-relationship? the present study revealed that scot markers can identify the species. keywords: iran, species identification, structure, salvia, scot (start codon targeted). introduction identifying the accurate boundaries of a species is critical to have a better perspective of any biological studies. therefore, species delimitation is a subject of extensive part of studies in the framework of biology (collard & mackill 2009, luo et al. 2011, wu et al. 2013). however, defining the criterion which could address the boundaries of species is different and the place of debates (jamzad 2012). among different populations, genetic diversity is 78 songpo liu et al. non randomly distributed and is affected by various factors such as geographic variations, breeding systems, dispersal mechanisms, life span, etc. change in environmental conditions often leads to variation in genetic diversity levels among different populations and populations with low variability are generally considered less adapted under adverse circumstances (falk & holsinger 1991, olivieri et al. 2016). most of the authors agree that genetic diversity is necessary to preserve the long-term evolutionary potential of a species (falk & holsinger 1991). in the last decade, experimental and field investigations have demonstrated that habitat fragmentation and population decline reduce the effective population size. in the same way, most geneticists consider population size as an important factor for maintaining genetic variation (turchetto et al. 2016). salvia l. is known as the largest genus in lamiaceae (mentheae-salviinae) with approximately 1000 species diversified in three regions of the world: central and south america (500 spp.), western asia (200 spp.) and eastern asia (100 species) (walker et al. 2004). iran having 19 endemic species out of 61 is regarded as one of the important regions for salvia diversity in southwest asia (jamzad 2012). salvia species are herbaceous, rarely biennial or annual, often strongly aromatic. these species are of medicinal, commercial and horticultural value (safaei et al. 2016). also, some salvia species have pharmacological properties, including antiplatelet, antiinflammatory and antithrombotic effects (hosseinzadeh et al. 2003, mayer et al. 2007; fan et al. 2010). some species of this genus are used in folk medicine, such as s. miltiorrhiza bunge , which is used for treatment of cardiovascular diseases (wang et al. 2007, 2009). salvia reuterana boiss. is an endemic species which grows in the highlands of central iran (jamzad 2012). its common name in persian is “mariam goli esfahani”, and the aerial parts of the plant are traditionally used as sedative and anxiolytic herbal medicine. in addition, the antibacterial, antioxidant, free radical scavenging and anti-anxiety properties of this herb have been proved in recent studies (erbano et al. 2015). the chemical composition of salvia strongly indicates that the herb has potential to become an important raw material for anti-inflammatory compounds and knowledge of the diversity of wild populations will therefore be important to inform the use and conservation of this genus (farag et al. 1986, li & quiros 2001). genetic surveys, in particular, are key measures to efficiently access the genetic resources of species of pharmacological interest. several markers have been previously applied to survey genetic variability within the genus salvia (song et al. 2010, wang et al. 2011). specifically, there are some important publications addressing s. miltiorrhiza, most of them utilizing dominant markers (wang et al. 2011). accordingly, some researchers have tried to assess this variability by issr and rapd techniques in different salvia species (song et al. 2010, wang et al. 2011, sepehry javan et al. 2012, zhang et al. 2013, peng et al. 2014, erbano et al. 2015). sepehry javan et al. (2012) mentioned that three major factors influencing genetic variations in salvia are: species, geographical distribution and selection. these factors along with cross-pollination make the taxonomy and genetic relationships of salvia species unclear (wang et al. 2011). morphological characteristics are easily affected by environment that makes identification of species more complex (chen et al. 2013). the conservation and suitable use of plant genetic resources require accurate monitoring of their accessions. so, genetic characterization is essential to manifest the extent of plant genetic diversity, and also to discover better genotypes; especially in the geographically differentiated genus such as salvia (song et al. 2010, peng et al. 2014, patel et al. 2014, kharazian et al. 2015). with the progress in plant molecular biolog y, numerous molecular marker techniques have been developed and used widely in evaluating genetic diversity, population structure and phylogenetic relationships. in recent years, advances in genomic tools provide a wide range of new marker techniques such as, functional and gene targeted markers as well as develop many novel dna based marker systems (esfandani-bozchaloyi et al. 2017 a, 2017b, 2017c, 2017d). start codon targeted (scot) polymorphism is one of the novel, simple and reliable gene-targeted marker systems. this molecular marker offers a simple dna-based marker alternative and reproducible technique which is based on the short conserved region in the plant genes surrounding the atg (collard & mackill 2009) translation start codon (collard & mackill 2009). this technique involves a polymerase chain reaction (pcr) based dna marker with many advantages such as low-cost, high polymorphism and extensive genetic information (collard & mackill 2009, luo et al. 2011, wu et al. 2013). the present investigation has been carried out to evaluate the genetic diversity and relationships among salvia species using new gene-targeted molecular markers, i.e. scot. this is the first study on the use of scot markers in salvia genus; therefore, we performed molecular study of 145 specimens of 30 salvia species. we try to answer the following questions: 1) is there infra and interspecific genetic diversity among studied species? 2) is genetic distance among these species correlated with their geographical distance? 3) what is the 79genetic variations and interspecific relationships in salvia (lamiaceae) using scot molecular markers genetic structure of populations and taxa? 4) is there any gene exchange between salvia species in iran? materials and methods plant materials a total of 145 individuals were sampled representing 30 geographical populations belong 30 salvia species (sp1= salvia aristata aucher ex benth; sp2= s. eremophila boiss; sp3= s. santolinifolia boiss; sp4= s. tebesana bunge; sp5= s. bracteata banks & sol; sp 6= s. suffruticosa montb. & aucher; sp7= s. dracocephaloides boiss.; sp8= s. hydrangea dc. ex benth.; sp9= s. multicaulis vahl.; sp10: s. syriaca l.; sp11: s. viridis l.; sp12= s. mirzayanii rech. f. & esfand.; sp13= s. macrosiphon boiss.; sp14= s. sharifii rech. f. & esfand.; sp15= s. reuterana boiss.; sp16= s. palaestina benth.; sp17= s. sclareopsis bornm. ex hedge; sp18= s. spinose l.; sp19= s. compressa vent.; sp20= s. sclarea l.; sp21= s. aethiopis l.; sp22= s. microstegia boiss. & bal.; sp23= s. xanthocheila boiss. ex benth.; sp24= s. limbata c. a. mey.; sp25= s. chloroleuca rech. f. & aell.; sp26= s. virgate jacq.; sp27= s. nemorosa l.; sp28= s. urmiensis bunge; sp29= s. oligphylla aucher ex benth.; sp30= s. verticillata l.) in east azerbaijan, lorestan, kermanshah, guilan, mazandaran, golestan, yazd, esfahan, tehran, arak, hamadan, kurdistan, ilam, bandar abbas, ghazvin, khorasan and ardabil provinces of iran during july-agust 2017-2019. out-group taxa are: marrubium anisodon k. koch and m. cuneatum banks & sol. for morphometric and scot analysis we used 145 plant accessions (five to twelve samples from each populations) belonging to 30 different populations with different ecogeographic characteristics were sampled and stored in -20 till further use. more information about geographical distribution of accessions are in fig. 1. morphological studies five to twelve samples from each species were used for morphometry (some endemic species were collected due to the rarity of 5 to 12 numbers). in total 22 morphological (9 qualitative, 13 quantitative) characters were studied. data obtained were standardized (mean= 0, variance = 1) and used to estimate euclidean distance for clustering and ordination analyses (podani 2000). morphological characters studied are: corolla shape, bract shape, seed color, seed shape, bract color, corolla latex, leaf surface, calyx shape, basal leaf shape, pedicel length, calyx length, bract length, filament length, anther length, corolla length, nut length, nut width, basal leaf length, basal leaf width, corolla color, stem leaf length and stem leaf width. dna extraction and scot assay fresh leaves were used randomly from three to twelve plants in each of the studied populations. these were dried by silica gel powder. ctab activated charcoal protocol was used to extract genomic dna (esfandani-bozchaloyi et al. 2019). the quality of extracted dna was examined by running on 0.8% agarose gel. a total of 25 scot primers developed by collard & mackill (2009), 10 primers with clear, enlarged, and rich polymorphism bands were chosen (table 1). pcr reactions were carried in a 25μl volume containing 10 mm tris-hcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). the amplifications, reactions were performed in techne thermocycler (germany) with the following program: 5 min initial denaturation step 94°c, followed by 40 cycles of 1 min at figura 1. map of iran shows the collection sites and provinces where salvia species were obtained for this study; sp1= salvia aristata; sp2= s. eremophila; sp3= s. santolinifolia; sp4= s. tebesana; sp5= s. bracteata ; sp 6= s. suffruticosa; sp7= s. dracocephaloides; sp8= s. hydrangea; sp9= s. multicaulis; sp10: s. syriaca; sp11: s. viridis; sp12= s. mirzayanii; sp13= s. macrosiphon; sp14= s. sharifii; sp15= s. reuterana; sp16= s. palaestina; sp17= s. sclareopsis; sp18= s. spinose; sp19= s. compressa; sp20= s. sclarea; sp21= s. aethiopis; sp22= s. microstegia; sp23= s. xanthocheila; sp24= s. limbata; sp25= s. chloroleuca; sp26= s. virgate; sp27= s. nemorosa; sp28= s. urmiensis; sp29= s. oligphylla; sp30= s. verticillata 80 songpo liu et al. 94°c; 1 min at 52-57°c and 2 min at 72°c. the reaction was completed by final extension step of 7-10 min at 72°c. the amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated by using a 100 bp molecular size ladder (fermentas, germany). data analyses morphological studies morphological characters were f irst standardized (mean = 0, variance = 1) and used to establish euclidean distance among pairs of taxa (podani 2000). for grouping of the plant specimens, the upgma (unweighted paired group using average) ordination methods were used (podani 2000). anova (analysis of variance) were performed to show morphological difference among the populations while, pca (principal components analysis) biplot was used to identify the most variable morphological characters among the studied populations (podani 2000). past version 2.17 (hammer et al. 2012) was used for multivariate statistical analyses of morphological data. molecular analyses scot bands obtained were coded as binary characters (presence = 1, absence = 0) and used for genetic diversity analysis. discriminatory ability of the used primers was evaluated by means of two parameters, polymorphism information content (pic) and marker index (mi) to characterize the capacity of each primer to detect polymorphic loci among the genotypes (powell et al. 1996). mi is calculated for each primer as mi = pic × emr, where emr is the product of the number of polymorphic loci per primer (n) and the fraction of polymorphic fragments (β) (heikrujam et al. 2015). the number of polymorphic bands (npb) and the effective multiplex ratio (emr) were calculated for each primer. parameter like nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism (p% = number of polymorphic loci/number of total loci) were determined (weising et al, 2005, freeland et al. 2011). shannon’s index was calculated by the formula: h’ = -σpiln pi. rp is defined per primer as: rp = ∑ ib, were “ib” is the band informativeness, that takes the values of 1-(2x [0.5-p]), being “p” the proportion of each genotype containing the band. the percentage of polymorphic loci, the mean loci by accession and by population, uhe, h’ and pca were calculated by genalex 6.4 software (peakall & smouse 2006). nei’s genetic distance among populations was used for neighbor joining (nj) clustering and neighbornet networking (huson & bryant 2006, freeland et al. 2011). mantel test checked the correlation between geographical and genetic distances of the studied populations (podani 2000). these analyses were done by past ver. 2.17 (hammer et al. 2012), darwin ver. 5 (2012) software. amova (analysis of molecular variance) test (with 1000 permutations) as implemented in genalex 6.4 (peakall & smouse 2006) were used to show genetic difference of the populations. gene flow was determined by (i) calculating nm an estimate of gene flow from gst by popgene ver. 1.32 (1997) as: nm = 0.5(1 gst)/gst. table 1. scot primers used for this study and the extent of polymorphism. primer name primer sequence (5’-3’) tnb npb ppb pic pi emr mi scot-1 caacaatggctaccacca 10 10 100.00% 0.36 4.86 9.55 3.45 scot-3 caacaatggctaccaccg 9 8 84.99% 0.43 4.91 7.43 4.85 scot-6 caacaatggctaccacgc 13 13 100.00% 0.44 4.34 11.55 3.44 scot-11 aagcaatggctaccacca 16 16 100.00% 0.37 3.88 8.56 1.65 scot-14 acgacatggcgaccacgc 20 20 100.00% 0.55 6.23 8.23 2.47 scot-15 acgacatggcgaccgcga 15 14 93.74% 0.47 5.66 7.56 3.67 scot-16 ccatggctaccaccggcc 13 12 92.31% 0.34 3.21 5.60 5.55 scot-17 catggctaccaccggccc 12 12 100.00% 0.47 4.32 9.55 3.45 scot-18 accatggctaccaccgcg 11 9 82.89% 0.43 5.56 6.34 2.11 scot-19 gcaacaatggctaccacc 15 15 100.00% 0.39 3.25 10.11 1.87 mean 13.4 12.9 97.78% 0.46 4.9 8.4 3.6 total 134 129 note: tnb the number of total bands, npb: the number of polymorphic bands, ppb (%): the percentage of polymorphic bands, pi: polymorphism index, emr, effective multiplex ratio; mi, marker index; pic, polymorphism information content for each of cbdp primers 81genetic variations and interspecific relationships in salvia (lamiaceae) using scot molecular markers this approach considers the equal amount of gene flow among all populations. results species identification and inter-relationship morphometry anova showed significant differences (p <0.01) in quantitative morphological characters among the species studied. in order to determine the most variable characters among the taxa studied, pca analysis has been performed. it revealed that the first three factors comprised over 63% of the total variation. in the first pca axis with 42% of total variation, such characters as seed shape, calyx shape, calyx length, bract length and basal leaf shape have shown the highest correlation (>0.7), seed color, leaf surface, corolla length, filament length, nut width, basal leaf length, were characters influencing pca axis 2 and 3 respectively. different clustering and ordination methods produced similar results therefore, pca plot of morphological characters are presented here (fig. 2). in general, plant samples of each species were grouped together and formed separate groups. this result show that both quantitative and qualitative morphological characters separated the studied species into distinct groups. in the studied specimens we did not encounter intermediate forms. species identification and genetic diversity ten issr primers were screened to study genetic relationships among salvia species; all the primers produced reproducible polymorphic bands in all 30 salvia species. an image of the issr amplification generated by scot11 primer is shown in figure 3. a total of 129 amplified polymorphic bands were generated across 30 salvia species. the size of the amplified fragments ranged from 100 to 2000 bp. the highest and lowest number of polymorphic bands were 20 for scot-14 and 8 for scot-3, on an average of 12.9 polymorphic bands per primer. the pic of the 10 scot primers ranged from 0.36 (scot-1) to 0.55 (scot-14) with an average of 0.46 per primer. mi of the primers ranged from 1.65 (scot-11) to 5.55 (scot16) with an average of 3.6 per primer. emr of the scot primers ranged from 6.34 (scot-18) to 11.55 (scot-6) with an average of 8.4 per primer (table 1). the primers figure 2. pca plots of morphological characters revealing species delimitation in the salvia sp1= salvia aristata; sp2= s. eremophila; sp3= s. santolinifolia; sp4= s. tebesana; sp5= s. bracteata ; sp 6= s. suffruticosa; sp7= s. dracocephaloides; sp8= s. hydrangea; sp9= s. multicaulis; sp10: s. syriaca; sp11: s. viridis; sp12= s. mirzayanii; sp13= s. macrosiphon; sp14= s. sharifii; sp15= s. reuterana; sp16= s. palaestina; sp17= s. sclareopsis; sp18= s. spinose; sp19= s. compressa; sp20= s. sclarea; sp21= s. aethiopis; sp22= s. microstegia; sp23= s. xanthocheila; sp24= s. limbata; sp25= s. chloroleuca; sp26= s. virgate; sp27= s. nemorosa; sp28= s. urmiensis; sp29= s. oligphylla; sp30= s. verticillata. 82 songpo liu et al. with the high emr values were considered to be more informative in distinguishing the genotypes. the genetic parameters were calculated for all the 30 salvia species amplified with scot primers (table 2). unbiased expected heterozygosity (h) ranged from 0.11 (s. syriaca) to 0.29 (s. virgata), with a mean of 0.19. a similar pattern was observed for shannon’s information index (i), with the highest value of 0.45 observed in s. virgata and the lowest value of 0.12 observed in s. syriaca with a mean of 0.26. the observed number of alleles (na) ranged from 0.214 in s. eremophila to 0.89 in s. aristata. the effective number of alleles (ne) ranged from 0.98 (s. multicaulis) to 1.440 (s. virgata). amova test showed significant genetic difference (p = 0.01) among studied species. it revealed that 66% of total variation was among species and 34% was within species (table 3) moreover, genetic differentiation of these species was demonstrated by significant nei’s gst (0.21, p = 0.01) and d_est values (0.177, p = 0.01). these results revealed a higher distribution of genetic diversity among salvia species compared to within species. marrubium anisodon and m. cuneatum (out-groups) were separated from the other species. two major clusters were formed in ward tree (fig. 4). the first major cluster (a) contained two sub-clusters: s. sharifii and s. macrosiphon are separated from the other studied species and join the others with a great distance and comprised the first sub-cluster. the second sub-cluster was formed by s. xanthocheila, s. limbata, s. aethiopis, s. sclarea and s. virgate. the second major cluster also contained two sub-clusters: eight species of s. multicaulis; s. syriaca; s. viridis, s. reuterana; s. palaestina; s. sclareopsis; s. spinose and s. oligphylla were placed close to each other, while close genetic affinity between other species. in general, relationships obtained from scot data agrees well with species relationship obtained from morphological. this is in agreement with amova and genetic diversity parameters presented before. the species are genetically well differentiated from each other. these results indicate that scot molecular markers can be used in salvia species taxonomy. the nm analysis by popgene software also produced mean nm= 0.167, that is considered very low value of gene flow among the studied species. mantel test with 5000 permutations showed a significant correlation (r = 0.13, p=0.0002) between genetic distance and geographical distance, so isolation by distance (ibd) occurred among the salvia species studied. nei’s genetic identity and the genetic distance determined among the studied species (table 4). the results showed that the highest degree of genetic similarity (0.93) occurred between s. eremophila and s. santolinifolia. the lowest degree of genetic similarity occurred between s. tebesana and s. verticillata (0.66). the low nm value (0.167) indicates limited gene flow or ancestrally shared alleles between the species studied and indicating high genetic differentiation among and within salvia species. discussion genetic diversity is a basic component of biodiversity and its conservation is essential for long term survival of any species in changing environments (mills & schwartz 2005, tomasello et al. 2015, miao et al. 2019; fig. 3. electrophoresis gel of studied ecotypes from dna fragments produced by scot-16. 1= salvia aristata; 2= s. eremophila; 3= s. santolinifolia; 4= s. tebesana; 5= s. bracteata ; 6= s. suffruticosa; 7= s. dracocephaloides; 8= s. hydrangea; 9= s. multicaulis; 10: s. syriaca; 11: s. viridis; 12= s. mirzayanii; 13= s. macrosiphon; 14= s. sharifii; 15= s. reuterana; 16= s. palaestina; 17= s. sclareopsis; 18= s. spinose; 19= s. compressa; 20= s. sclarea; 21= s. aethiopis; 22= s. microstegia; 23= s. xanthocheila; 24= s. limbata; 25= s. chloroleuca; 26= s. virgate; 27= s. nemorosa; 28= s. urmiensis; 29= s. oligphylla; 30= s. verticillata;l = ladder 100 bp, arrows are representative of polymorphic bands. 83genetic variations and interspecific relationships in salvia (lamiaceae) using scot molecular markers xu et al. 2021, zou et al. 2019, wang et al. 2020). this is very important in fragmented populations because are more vulnerable due to the loss of allelic richness and increased population differentiation by genetic drift (decreases heterozygosity and eventual fixation of alleles) and inbreeding depression (increases homozygosity within populations; frankham 2005). therefore, knowledge of the genetic variability and diversity within and among different populations is crucial for their conservation and management (e.g. esfandani-bozchaloyi et al. table 2. genetic diversity parameters in the studied salvia species. sp n na ne i he uhe %p s. aristata 6.000 0.892 1.138 0.221 0.141 0.165 38.63% s. eremophila 6.000 0.244 1.032 0.26 0.23 0.18 55.53% s. santolinifolia 4.000 0.314 1.044 0.16 0.18 0.23 43.38% s. tebesana 8.000 0.201 1.00 0.33 0.17 0.12 42.23% s. bracteata 5.000 0.341 1.058 0.24 0.27 0.20 53.75% s. suffruticosa 3.000 0.567 1.062 0.24 0.224 0.113 44.73% s. dracocephaloides 5.000 0.336 1.034 0.23 0.25 0.19 51.83% s. hydrangea 4.000 0.344 1.042 0.20 0.23 0.20 57.53% s. multicaulis 5.000 0.369 1.011 0.15 0.18 0.12 42.15% s. syriaca 8.000 0.566 1.014 0.45 0.10 0.11 32.58% s. viridis 9.000 0.432 1.049 0.18 0.22 0.25 55.05% s. mirzayanii 8.000 0.313 1.026 0.144 0.13 0.26 49.23% s. macrosiphon 12.000 1.244 1.322 0.28 0.284 0.192 50.91% s. sharifii 5.000 0.358 1.117 0.28 0.15 0.12 44.30% s. reuterana 6.000 0.458 1.039 0.28 0.18 0.23 49.38% s. palaestina 5.000 0.455 1.077 0.377 0.24 0.22 55.05% s. sclareopsis 8.000 0.499 1.067 0.14 0.101 0.14 49.26% s. spinose 9.000 0.261 1.014 0.142 0.33 0.23 43.15% s. compressa 6.000 0.555 1.021 0.39 0.25 0.28 43.53% s. sclarea 10.000 0.431 1.088 0.33 0.22 0.13 57.53% s. aethiopis 3.000 0.255 1.021 0.15 0.18 0.12 42.15% s. microstegia 3.000 0.288 1.024 0.23 0.15 0.17 64.30% s. xanthocheila 9.000 0.352 1.083 0.23 0.22 0.14 45.05% s. limbata 8.000 0.333 1.016 0.122 0.12 0.22 48.23% s. chloroleuca 12.000 1.247 1.199 0.271 0.184 0.192 55.91% s. virgata 5.000 0.358 1.440 0.114 0.30 0.29 66.50% s. nemorosa 6.000 0.299 1.029 0.231 0.18 0.23 44.38% s. urmiensis 5.000 0.462 1.095 0.288 0.25 0.22 62.05% s. oligphylla 8.000 0.399 1.167 0.259 0.234 0.133 32.88% s. verticillata 8.000 0.477 1.167 0.356 0.233 0.148 31.26% abbreviations: n = number of samples, na= number of different alleles; ne = number of effective alleles, i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations. table 3. analysis of molecular variance (amova) of the studied species. source df ss ms est. var. % φpt among pops 28 1801.364 75.789 12.154 66% within pops 129 334.443 3.905 2.888 34% 66% total 144 1955.807 14.060 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance; φpt: proportion of the total genetic variance among individuals within an accession, (p < 0.001). 84 songpo liu et al. 2018a, 2018b, 2018c, 2018d; salari et al. 2013; 2020; jahani et al. 2019). in the present study we used morphological and molecular (scot) data to evaluate species relationship in salvia. morphological analyses of the studied salvia species showed that they are well differentiated from each other both in quantitative measures (the anova test result) and qualitative characters (the pca plot result). in addition, pca analysis suggests that characters like bract length, stipule length, bract shape, calyx shape, petal shape, length and width of stem-leaf, length and width of petal could be used in species groups delimitation. this morphological difference was due to quantitative and qualitative characters. genetic structure and gene flow pic and mi characteristics of a primer help in determining its effectiveness in genetic diversity analysis. sivaprakash et al. (2004) suggested that the ability of a marker technique to resolve genetic diversity may be more directly related to the degree of polymorphism. generally, pic value between zero to 0.25 imply a very low genetic diversity among genotypes, between 0.25 to 0.50 shows a mid-level of genetic diversity and value ≥0.50 suggests a high level of genetic diversity (tams et al. 2005). in this research, the scot primers’ pic values ranged from 0.36 to 0.55, with a mean value of 0.46, which indicated a mid-ability of scot primers in determining genetic diversity among the salvia species. comparable but low pic values have been reported with other markers like rapd and aflp in african plantain (ude et al. 2003), issr and rapd in salvia species (yousefiazar-khanian et al. 2016), aflp in wheat (bohn et al. 1999) and scot markers (etminan et al. 2018, pour-aboughadareh et al. 2017, 2018). in heikrujam et al. (2015), cbdp markers were found to be more effective than scot markers with regard to the average pic which was higher. in our study, the scot markers were found to be effective in the estimation of different salvia species genetic diversity with regard to average percentage polymorphism (97.78%), average pic value of scot markers (0.46), average mi (3.6) and average emr of scot markers (8.4), which were higher than other reported markers on salvia (wang et al. 2009, song et al. 2010, yousefiazar-khanian et al. 2016, etminan et al. 2018, gholamin and khayatnezhad 2020 a, b, c, d). however, various marker techniques were found to have different resolution of the genome regions and the number of loci that cover the whole genome for estimating of genetic diversity (souframanien & gopalakrishna 2004). a diverse level of polymorphism in salvia species using issr, corap, srap, scot and rapd markers had been reported earlier by wang & zhang (2009), song et al. (2010), yousefiazar-khanian et al. (2016) and etminan et al. (2018). gene flow is inversely correlated with the gene differentiation but is very important for population evolution, and takes place by pollen and seeds between populations (song et al. 2010). in the current study, detected gene flow (nm) among salvia species was 0.167, showed low genetic differentiation among salvia species. as a general rule, insects are the pollinators of salvia in old world (claßen-bockhoff et al. 2004, khayatnezhad and gholamin, 2012a, b). at the lower elevations, bees and at the higher altitudes insects like flies are the dominate pollinators among bilabiate flowers such as salvia (pellissier et al. 2010). according to moein et al. (2019) genetic structure of srap marker showed that despite the presence of a limited gene flow, two distinct ecotypes were formed which may be the consequences of reproductive isolation figure 4. ward tree of scot data revealing species delimitation in the salvia. 85genetic variations and interspecific relationships in salvia (lamiaceae) using scot molecular markers ta bl e 4. th e m at ri x of n ei g en et ic s im ila ri ty ( g s) e st im at es u si ng s c ot m ol ec ul ar m ar ke rs a m on g 30 s al vi a sp ec ie s. sp 1= s al vi a ar is ta ta ; s p2 = s. e re m op hi la ; s p3 = s. s an to lin ifo lia ; sp 4= s . t eb es an a; s p5 = s. b ra ct ea ta ; sp 6 = s. s uff ru tic os a; s p7 = s. d ra co ce ph al oi de s; sp 8= s . h yd ra ng ea ; s p9 = s. m ul tic au lis ; s p1 0: s . s yr ia ca ; s p1 1: s . v ir id is ; s p1 2= s . m ir za ya ni i; sp 13 = s. m ac ro si ph on ; s p1 4= s . s ha ri fii ; s p1 5= s . r eu te ra na ; s p1 6= s . p al ae st in a; s p1 7= s . s cl ar eo ps is ; s p1 8= s . s pi no se ; s p1 9= s . c om pr es sa ; s p2 0= s . s cl ar ea ; s p2 1= s . a et hi op is ; s p2 2= s . m ic ro st egi a; s p2 3= s . x an th oc he ila ; s p2 4= s . l im ba ta ; s p2 5= s . c hl or ol eu ca ; s p2 6= s . v ir ga te ; s p2 7= s . n em or os a; s p2 8= s . u rm ie ns is ; s p2 9= s . o lig ph yl la ; s p3 0= s . v er tic ill at a sp 1 1. 00 0 sp 1 sp 2 0. 84 2 1. 00 0 sp 2 sp 3 0. 78 6 0. 93 3 1. 00 0 sp 3 sp 4 0. 76 7 0. 83 6 0. 84 2 1. 00 0 sp 4 sp 5 0. 82 3 0. 82 3 0. 78 6 0. 75 4 1. 00 0 sp 5 sp 6 0. 78 1 0. 76 6 0. 76 7 0. 75 7 0. 79 3 1. 00 0 sp 6 sp 7 0. 74 9 0. 68 3 0. 82 3 0. 75 9 0. 83 6 0. 86 2 1. 00 0 sp 7 sp 8 0. 68 1 0. 77 6 0. 78 1 0. 66 0 0. 82 3 0. 84 6 0. 92 8 1. 00 0 sp 8 sp 9 0. 81 7 0. 66 0 0. 74 9 0. 77 1 0. 76 6 0. 80 8 0. 87 5 0. 95 1 1. 00 0 sp 9 sp 10 0. 71 5 0. 88 4 0. 81 2 0. 82 0 0. 72 1 0. 61 8 0. 70 8 0. 70 4 0. 68 0 1. 00 0 sp 10 sp 11 0. 64 5 0. 75 4 0. 70 3 0. 72 5 0. 63 5 0. 81 6 0. 88 4 0. 81 2 0. 82 0 0. 72 1 1. 00 0 sp 11 sp 12 0. 74 5 0. 75 7 0. 71 7 0. 67 2 0. 63 2 0. 75 2 0. 75 4 0. 70 3 0. 72 5 0. 63 5 0. 83 9 1. 00 0 sp 12 sp 13 0. 83 9 0. 75 9 0. 70 9 0. 68 0 0. 66 7 0. 71 2 0. 77 9 0. 79 8 0. 83 4 0. 75 0 0. 79 9 0. 64 2 1. 00 0 sp 13 sp 14 0. 75 9 0. 85 9 0. 78 5 0. 77 5 0. 66 6 0. 73 7 0. 67 5 0. 80 8 0. 76 8 0. 67 5 0. 72 7 0. 72 8 0. 68 4 1. 00 0 sp 14 sp 15 0. 64 1 0. 87 2 0. 79 2 0. 77 3 0. 64 9 0. 80 7 0. 69 1 0. 66 5 0. 72 0 0. 68 1 0. 74 6 0. 79 6 0. 67 6 0. 72 2 1. 00 0 sp 15 sp 16 0. 76 7 0. 74 0 0. 67 1 0. 65 0 0. 61 7 0. 78 2 0. 73 4 0. 79 9 0. 82 9 0. 73 3 0. 80 0 0. 70 9 0. 77 0 0. 75 4 0. 77 0 1. 00 0 sp 16 sp 17 0. 78 4 0. 80 2 0. 75 7 0. 71 6 0. 77 8 0. 70 2 0. 74 4 0. 77 8 0. 81 6 0. 74 0 0. 78 5 0. 67 6 0. 69 9 0. 75 6 0. 73 5 0. 77 8 1. 00 0 sp 17 sp 18 0. 82 7 0. 81 7 0. 78 4 0. 77 0 0. 64 1 0. 81 4 0. 73 5 0. 70 6 0. 71 9 0. 95 3 0. 74 1 0. 75 8 0. 74 6 0. 75 3 0. 79 5 0. 79 9 0. 75 6 1. 00 0 sp 18 sp 19 0. 70 1 0. 80 0 0. 75 1 0. 77 4 0. 73 2 0. 79 0 0. 75 0 0. 79 7 0. 81 2 0. 77 4 0. 99 0 0. 72 2 0. 63 5 0. 81 6 0. 88 4 0. 81 2 0. 75 0 0. 79 9 1. 00 0 sp 19 sp 20 0. 76 4 0. 72 3 0. 68 3 0. 65 9 0. 67 9 0. 75 4 0. 77 9 0. 79 8 0. 83 4 0. 75 0 0. 79 9 0. 75 5 0. 63 2 0. 75 2 0. 75 4 0. 70 3 0. 67 5 0. 72 7 0. 75 5 1. 00 0 sp 20 sp 21 0. 75 4 0. 84 4 0. 80 4 0. 79 3 0. 69 5 0. 68 1 0. 68 9 0. 82 5 0. 77 8 0. 69 1 0. 74 4 0. 63 6 0. 66 7 0. 71 2 0. 77 9 0. 79 8 0. 68 1 0. 74 6 0. 68 4 0. 71 1 1. 00 0 sp 21 sp 22 0. 63 6 0. 82 6 0. 78 6 0. 77 2 0. 68 6 0. 75 6 0. 70 1 0. 67 6 0. 71 0 0. 68 8 0. 75 7 0. 70 3 0. 66 6 0. 73 7 0. 67 5 0. 80 8 0. 73 3 0. 80 0 0. 84 8 0. 77 4 0. 71 2 1. 00 0 sp 22 sp 23 0. 77 3 0. 69 1 0. 63 2 0. 61 5 0. 60 2 0. 75 1 0. 73 4 0. 79 9 0. 82 9 0. 73 3 0. 80 0 0. 68 1 0. 64 9 0. 80 7 0. 69 1 0. 66 5 0. 74 0 0. 78 5 0. 84 6 0. 75 7 0. 70 7 0. 98 0 1. 00 0 sp 23 sp 24 0. 78 4 0. 80 2 0. 75 5 0. 73 0 0. 61 4 0. 65 1 0. 74 4 0. 77 8 0. 81 6 0. 74 0 0. 78 5 0. 62 4 0. 61 7 0. 78 2 0. 73 4 0. 79 9 0. 95 3 0. 74 1 0. 69 0 0. 65 7 0. 64 5 0. 72 6 0. 73 5 1. 00 0 sp 24 sp 25 0. 84 4 0. 81 7 0. 78 4 0. 77 0 0. 64 1 0. 80 9 0. 80 2 0. 75 5 0. 73 0 0. 61 4 0. 84 3 0. 75 9 0. 59 9 0. 70 2 0. 74 4 0. 77 8 0. 77 4 0. 99 0 0. 77 8 0. 69 1 0. 74 4 0. 63 6 0. 66 7 0. 75 7 1. 00 0 sp 25 sp 26 0. 70 1 0. 81 2 0. 76 1 0. 76 2 0. 73 6 0. 79 0 0. 81 7 0. 78 4 0. 77 0 0. 64 1 0. 82 5 0. 72 2 0. 64 1 0. 81 4 0. 73 5 0. 70 6 0. 75 0 0. 79 9 0. 71 0 0. 68 8 0. 75 7 0. 70 3 0. 66 6 0. 69 0 0. 79 7 1. 00 0 sp 26 sp 27 0. 76 4 0. 71 2 0. 67 2 0. 67 0 0. 66 9 0. 75 5 0. 81 2 0. 76 1 0. 76 2 0. 73 6 0. 86 0 0. 75 9 0. 73 2 0. 79 0 0. 75 0 0. 79 7 0. 69 1 0. 74 4 0. 82 9 0. 73 3 0. 80 0 0. 68 1 0. 64 9 0. 67 3 0. 75 5 0. 76 8 1. 00 0 sp 27 sp 28 0. 75 4 0. 84 4 0. 80 4 0. 79 3 0. 69 5 0. 66 9 0. 71 2 0. 67 2 0. 67 0 0. 66 9 0. 72 6 0. 64 7 0. 67 9 0. 75 4 0. 77 9 0. 79 8 0. 68 8 0. 75 7 0. 81 6 0. 74 0 0. 78 5 0. 62 4 0. 61 7 0. 65 6 0. 76 7 0. 69 0 0. 70 4 1. 00 0 sp 28 sp 29 0. 70 9 0. 82 6 0. 78 6 0. 77 2 0. 68 6 0. 75 6 0. 84 4 0. 80 4 0. 79 3 0. 69 5 0. 85 8 0. 70 3 0. 69 5 0. 68 1 0. 68 9 0. 82 5 0. 73 3 0. 80 0 0. 73 0 0. 61 4 0. 84 3 0. 75 0 0. 79 9 0. 71 0 0. 68 8 0. 75 7 0. 70 3 0. 72 3 1. 00 0 sp 29 sp 30 0. 72 1 0. 79 4 0. 75 4 0. 71 7 0. 79 5 0. 75 1 0. 82 6 0. 78 6 0. 77 2 0. 68 6 0. 83 6 0. 68 1 0. 68 6 0. 75 6 0. 70 1 0. 67 6 0. 74 0 0. 78 5 0. 77 0 0. 64 1 0. 82 5 0. 72 2 0. 81 6 0. 74 0 0. 78 5 0. 62 4 0. 61 7 0. 65 6 0. 76 5 1. 00 0 sp 30 sp 1 sp 2 sp 3 sp 4 sp 5 sp 6 sp 7 sp 8 sp 9 sp 10 sp 11 sp 12 sp 13 sp 14 sp 15 sp 16 sp 17 sp 18 sp 19 sp 20 sp 21 sp 22 sp 23 sp 24 sp 25 sp 26 sp 27 sp 28 sp 29 sp 30 86 songpo liu et al. caused by altitude gradient and different niches through parapatric speciation. the heterozygosity (h) and shannon index (i) reflect diversity and differentiation among and within the germplasm collections, respectively (que et al. 2014), and the higher the indices, the greater the genetic diversity. the magnitude of variability among na, ne, h and i indices using studied scot markers demonstrated a high level of genetic diversity among and within salvia species. the similar results reported in salvia miltiorrhiza based on issrs (zhang et al., 2013) and other salvia species using aflp markers (sajadi et al., 2010) as 95% and 99% polymorphism, respectively. also, polymorphism index (pi) in rapd primers was higher; whereas, other indices like pic, emr and mi were somewhat high in issrs. on the other hand, rp index was approximately equal in both techniques. in general, small differences in terms of calculated indices showed that both techniques had similar efficiency to differentiate the closely related ecotypes of salvia. chen et al. (2013) reported pic values about 0.20 in ocimum species by issr and rapd markers and also showed the rp values as 1.39 and 5.13, respectively. pic analysis can be used to select the most appropriate markers for genetic mapping. also, the high mi reflects the marker efficiency to simultaneously analyze a large number of bands (powell et al., 1996; patel et al., 2014). the high average simpson’s coefficients (about 0.80) indicate high genetic variability among studied accessions of salvia, too. this finding was similar to the study by manica-cattani et al. (2009) on accessions of lippia alba by issr and rapd. in their study on salvia lachnostachys ecotypes by issr primers, erbano et al. (2015) showed a range of 0.66-0.86 for simpson’s index. comparison of nei’s similarity coefficients between issrs and rapds showed that both markers had high diagnostic capability. this is consistent with the results of issr markers in mint accessions by kang et al. (2013) and salvia miltiorrhiza germplasms studied by zhang et al. (2013); while the genetic similarity derived from sraps and issrs represented high proximity among salvia miltiorrhiza populations (song et al., 2010). cluster analysis could group all 21 ecotypes and the results showed reasonable congruency in rapd and issr in terms of species topology. zhang et al. (2013) showed five major clusters for s. miltiorrhiza germplasms based on nei’s similarity coefficient for issrs; which did not indicate any clear pattern according to their locations. patel et al. (2014) reported that in dendrograms of issr and rapd, the genotypes of each ocimum species were grouped, separately. similar studies in populations of s. japonica and some other salvia species (sudarmono and okada 2008) did not show correlation between morphological variations and allozyme and dna sequences. it was concluded that s. japonica is still at the early stage of speciation process sympatry or co-occurrence of closely related species can either result from a sympatric speciation process or from secondary contact due to range expansion after speciation. under the allopatric scenario, genetic variation tends to be uniform across the genome due to a large proportion of the genome changing through a combination of divergent selection, differential response to similar selective pressures and genetic drift (see for example strasburg et al. 2012). in contrast, in the extreme case of sympatric speciation, gene flow between the incipient species can homogenize most of the genome, except for loci that experience strong divergent selection pressures or regions that are tightly linked with these loci (see for example, strasburg et al. 2012, via 2012). in conclusion, the results of this study showed that to evaluate the genetic diversity of the salvia genus, the primers derived from scot were more effective than the other molecular markers. also, salvia ecotypes/species were clearly separated from each other in the dendrogram and mds, indicating the higher efficiency of scot technique in salvia species identification. acknowledgment this work was supported by the national natural science foundation of china (u1404303) and postgraduate education reform and quality improvement project of henan province(yjs2021jd17). references al-quran s. 2008. taxonomical and pharmacological sur vey of therapeutic plants in jordan. journal of natural products,l(1):10-26. doi: 10.1556/034.59.2017.3-4.3 bohn m., utz h. f. melchinger ae. 1999. genetic similarities among wheat cultivars determined on the basis of rflps, aflps and ssrs and their use for predicting progeny variance. crop sci. 39, 228-237. chen sy, dai tx, chang yt, wang ss, ou sl, chuang wl, cheng cy, lin yh, lin ly, ku hm 2013. genetic diversity among ocimum species based on issr, rapd and srap markers. australian journal of crop science 7(10):1463-1471. collard bcy, mackill dj. 2009. start codon targeted (scot) polymorphism: a simple novel dna marker technique for generating gene-targeted markers in plants. plant mol biol rep 27:86-93. 87genetic variations and interspecific relationships in salvia (lamiaceae) using scot molecular markers claßen-bockhoff r, speck t, tweraser e, wester p, thimm s. reith m. 2004. the staminal lever mechanism in salvia l. 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(asteraceae) species that around the van lake in turkey pelin yilmaz sancar1,*, semsettin civelek1, murat kursat2 molecular identification and genetic relationships among alcea (malvaceae) species by issr markers: a high value medicinal plant jinxin cheng1,*, dingyu hu1, yaran liu2, zetian zhang1, majid khayatnezhad3 genetic variations and interspesific relationships in salvia (lamiaceae) using scot molecular markers songpo liu1, yuxuan wang1, yuwei song1,*, majid khayatnezhad2, amir abbas minaeifar3 development of female gametophyte in gladiolus italicus miller (iridaceae) ciler kartal1, nuran ekici2,*, almina kargacıoğlu1, hazal nurcan ağırman1 colchicine induced manifestation of abnormal male meiosis and 2n pollen in trachyspermum ammi (l.) sprague (apiaceae) harshita dwivedi*, girjesh kumar statistical evaluation of chromosomes of some lathyrus l. taxa from turkey hasan genç1, bekir yildirim2,*, mikail açar3, tolga çetin4 use of chemical, fish micronuclei, and onion chromosome damage analysis, to assess the quality of urban wastewater treatment and water of the kamniška bistrica river (slovenia) peter firbas1, tomaž amon2 the interacting effects of genetic variation in geranium subg. geranium (geraniaceae) using scot molecular markers bo shi1,*, majid khayatnezhad2, abdul shakoor3,4 genetic (ssrs) versus morphological differentiation of date palm cultivars: fst versus pst estimates somayeh saboori1, masoud sheidai2, zahra noormohammadi1,*, seyed samih marashi3, fahimeh koohdar2 further insights into chromosomal evolution of the genus enyalius with karyotype description of enyalius boulengeri etheridge, 1969 (squamata, leiosauridae) cynthia aparecida valiati barreto1,*, marco antônio peixoto1,2, késsia leite de souza1, natália martins travenzoli1, renato neves feio3, jorge abdala dergam1 caryologia. international journal of cytology, cytosystematics and cytogenetics 74(2): 65-77, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-919 caryologia international journal of cytology, cytosystematics and cytogenetics citation: timir baran jha, biplab kumar bhowmick, partha roy (2021) analysis of cma-dapi bands and preparation of fluorescent karyotypes in thirty indian cultivars of lens culinaris. caryologia 74(2): 65-77. doi: 10.36253/caryologia-919 received: april 24, 2020 accepted: may 19, 2021 published: october 08, 2021 copyright: © 2021 timir baran jha, biplab kumar bhowmick, partha roy. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid tbj: 0000-0003-0900-8167 bkb: 0000-0001-6029-1098 analysis of cma-dapi bands and preparation of fluorescent karyotypes in thirty indian cultivars of lens culinaris timir baran jha1,*, biplab kumar bhowmick2, partha roy1 1 department of botany, maulana azad college, kolkata -700013, west bengal, india 2 department of botany, scottish church college, 1&3, urquhart square, kolkata 700006, west bengal, india *corresponding author. e-mail: tbjha2000@yahoo.co.in abstract. india holds a significant rank in production and consumption of the age old protein rich crop lentil with only one cultivated species and a large number of phenotypically similar cultivars. the need for a reliable and cost effective method of genetic characterization to unravel differences within the lentil cultivars was felt. the present paper adopted ema based chromosome preparation followed by staining with two contrasting fluorochrome dyes cma and dapi that bind directly to gc and at rich heterochromatic segments on chromosomes. analysis of fluorochrome banding pattern furnished a comparative account of genetic diversity within the cultivars that could not be achieved by traditional karyotyping. the marker pair of nucleolar chromosomes (4th and 3rd, majorly) occupied a pivotal position to intensify differences between cultivars in terms of banding patterns around secondary constrictions, suggestive of yet unknown variation in heterochromatin composition. our study has strengthened genetic background and relationships of lentil cultivars. we observed certain types of unusual fluorochrome bands that put forward the exclusivity of indian germplasm and have questioned the mainstream heterochromatin elements of plant chromosomes captured by cma-dapi stains. the comprehensive fluorescent karyotypes of 30 l. culinaris medik. cultivars prepared for the first time, serve as an archetype for the benefit of future breeding programmes. keywords: lentils, cma-dapi, chromosomal bands, fluorescent karyotype, heterochromatin. introduction lentil is one of the richest protein containing domesticated ancient crop with only one globally cultivated species lens culinaris medik. india is the second highest producer and biggest consumer of lentils. the genus belongs to the largest subfamily (papilionoideae) of fabaceae (azani et al. 2017), along with many economically important genera producing pulses and beans. being the single cultivated species, large number of cultivars is in cultivation in our country. the characterization of indian germplasm is 66 timir baran jha, biplab kumar bhowmick, partha roy needed to sustain conservation and programmable utilization of resources. chromosomal characterization is a cost effective method to provide foundational information on the genome and genetic conservation for any future breeding program of particular crop plants. cytogenetic studies of indian lentils through conventional method failed to provide uniformity on chromosome morphometric parameters (bhattacharjee 1953; sharma and mukhopadhyay 1963; sinha and acharia 1972; naithani and sarbhoy 1973; lavania and lavania 1983; nandanwar and narkhede 1991). on the other hand, we have published detailed karyotype analysis of more than thirty l. culinaris cultivars obtained from the indian institute of pulses (jha et al. 2015, 2017; jha and halder 2016) through ema based giemsa staining method. our results were found to have near similarities with the results obtained by ladizinsky (1979). however, lens chromosomes (2n=14) are nearly similar in morphology. considering the status of research, we question i) is there any karyotype variability across cultivars beyond chromosome number, morphology and ploidy? ii) is it possible to find visible chromosomal landmarks in accordance with the germplasm diversity? and iii) whether we can step forward towards molecular kayotype database for indian lentils. as ema based chromosome analysis (fukui 1996) is the basis of molecular cytogenetics, we decided to carry forward our work with two contrasting fluorescent stains dapi and cma on the same cultivars. having affinity towards specific base pairs of dna, these fluorescent dyes reliably identify heterochromatin rich sectors on chromosomes, differentiate morphologically alike chromosomes and improve karyotype characterization (schweizer 1976; guerra et al. 2000; yamamoto 2012; weiss‐schneeweiss and schneeweiss 2013). so, our objective is to address chromosomal behavior after application of base specific fluorochromes and compile cultivar specific fluorescent banding profiles. the present paper considers a fluorescent karyotype dataset of 30 indian l. culinaris cultivars for the first time, as an important kit for lentil breeders and genome researchers. materials and methods chromosome preparation and fluorochrome staining the fluorescent karyotype analysis was carried out on 30 cultivars of lens culinaris presented in table 1. except for two (barasat, micro type and barasat, macro type, table 1), all the cultivars of lentil were obtained from the indian institute of pulse research (iipr), kanpur. germination of seeds and chromosome processing through enzymatic maceration and air drying (ema) was carried out as per our earlier protocol (jha and yamamoto 2012; jha et al. 2015, 2017, 2020). for fluorescent staining with dapi and cma, we followed our protocol (jha 2019) with required modifications. for dapi staining, slides were kept for 30 min in mcilvaine buffer, stained with 0.1µg ml-1 solution of dapi for 10 min, counterstained with 0.25mg/ml of actinomycin d (amd) for 15min and then mounted in nonfluorescent glycerol and observed under carl zeiss axio lab a1 fluorescence microscope using carl zeiss dapi filter cassette. chromosome images were captured with ccd camera attached with microscope. the slides were destained and air-dried. the same slides were placed in mcilvaine buffer for 30 min followed by incubation in mcilvaine buffer with 5mm mgcl2 for 10 mins and then stained with 0.1mg ml-1 cma solution for 45-50 mins. the slides were again washed in mcilvaine buffer with 5mm mgcl2 and finally mounted with non-fluorescent glycerol and kept for maturation at 40c for 48-72 hrs. cma stained slides were observed under the abovementioned f luorescence microscope fitted with carl zeiss fitc filter cassette, images captured with attached ccd camera and signals were analyzed using the software prog res 2.3.3. statistical analysis of karyotype relations karyotype relations among the cultivars was evaluated with the help of cluster analysis for data matrix normalization by unweighted pair group method with arithmetic averages (upgma) based on euclidean distance using info stat 2017d (free version). here, only the fluorochrome banding pattern of the cultivars viz. types and numbers of cma and dapi bands were utilized to draw the phenogram. results fluorochrome banding pattern in cultivars of l. culinaris medik. somatic chromosome analysis of the 30 lentil cultivars based on fluorescence banding patterns has provided an interesting catalogue of chromosome diversity. the chromosomes took up dapi stain within 10 minutes of incubation while the incubation time for cma staining was about 45-50mins. the same cma and dapi staining protocol was followed for all the 30 cultivars of l. culinaris. interestingly, we have obtained different types of dapi and cma banding patterns within the studied 67analysis of cma-dapi bands and preparation of fluorescent karyotypes in thirty indian cultivars of lens culinaris figure 1. somatic metaphase chromosomes of lens culinaris cultivars stained with cma: (a) dpl-15, (b) dpl-62, (c) ipl-81, (d) ipl-406, (e) ipl-316, (f ) jl-1, (g) hul-57, (h) kls-210, (i) ec-70394, (j) ec-70403, (k) ec-70404, (l) ec-78452, (m) ec-78455, (n) ec-78461, (o) ec-78475, (p) ec-78498, (q) ec-78542-a, (r) ec–223188, (s) ec – 255491, (t) ec–267526, (u) ec-267569-a, (v) ec–267590, (w) ec–267877, (x) barasat micro type. white arrows indicate cma+ bands and arrowheads indicate cma0 bands. bars 5µm. 68 timir baran jha, biplab kumar bhowmick, partha roy cultivars. at least 10 plates stained with dapi and cma for each cultivar was considered for analysis of banding types. secondary constriction marked the nucleolar organizing region (nor) of most of the cultivars, showing cma+ bands with different intensities while some nors remained neutral and termed cma0 as per barros e silva and guerra (2010). dapi staining in most of the cultivars resulted a clear gap (dapi-) corresponding to cma+ band. however, few exceptional cultivars yielded dapi+ band in the nor regions. based on the cma and dapi fluorescent banding, we have categorized following types of somatic chromosomes. the chromosomes with cma+/dapiband in the nucleolar region is termed type ‘a’. type ‘b’ has cma+/dapinucleolar constriction followed by a dapi+/cma0 band below centromere. the ‘c’ type nucleolar chromosome has a distinct cma+/ dapi+ secondary constriction. the fourth type ‘d’ has neutral cma band in secondary constriction. chromosomes with centromeric cma+/dapi0 bands are termed type ‘e’ while those with centromeric dapi+/cma0 bands are termed type ‘f’. type ‘g’ chromosome contains intercalary dapi+/cma0 band. the chromosomes having no detectable bands were termed as type ‘h’. distribution of different types of fluorochrome bands among the cultivars is summarized in table 1. a detailed analysis of the fluorochrome stained metaphase plates (figures 1-3) was carried out to formulate the diagrammatic fluorescent karyotypes of the 30 cultivars under study (figures 4 and 5). cma-dapi banding patterns have revealed that in majority of l. culinaris cultivars (table 1), the marker secondary constrictions with cma+ signals are present in the 4th pair of chromosomes. however, the same in some cultivars are present in the 3rd and exceptionally in the 5th and 2nd pairs, as in two cultivars (ec-70394, ec-78542-a). the most abundant cma+ satellites (type a chromosomes) are found among 50% of the presently studied cultivars. in addition to cma+ satellites, existence of type b chromosomes is found in 8 different cultivars (hul-57, ec-70403, ec-78542-a, ec-267526, ec – 267877, barasat micro type, pl -1406, ec -78410, table 1) and type d chromosomes in 5 different cultivars (dpl15, jl-1, ec-78452, ec – 70306, ec – 78473, table 1). of special mention, are the two cultivars (ec-70404, ec267569-a, table 1) with cma+/dapi+ satellite (type c chromosome). three cultivars (ipl -316, ec-70394, ec – 267877) had centromeric cma+ bands (type e) (table 1). one of them (ipl -316) shows centromeric cma+ bands (type e) in every chromosome except the nucleolar pair (table 1). on the other hand, ec -78410 shows intense centromeric dapi+ bands (type f) in all non-nucleolar chromosome pairs (table 1). centromeric dapi+ bands are consistently found in the 2nd or the 3rd pair of chromosomes in 5 cultivars (hul-57, ec-70403, ec-267526, barasat, macro type, pl -1406, table 1). intercalary dapi+ band (type g) is seen only in ipl-406 (table 1). comparative statistical assessment of fluorochrome banding pattern statistical evaluation of karyotype relations among the 30 lentil cultivars was carried out using euclidean distance matrix on the basis of cma and dapi bands. the upgma phenogram presented relative karyotype affinities and distances with a cophenetic correlation of 0.986 as a good fit between the cophenetic value matrix and the average euclidean distance matrix (figure 6). there are three separate groups in the upgma phenogram of which group i consisted of cultivars that do not have close affinity with each other (figure 6). within this group, ec -78410 and ipl -316 have fluorescent banding pattern that are in contrast to each other. also, existence of intercalary dapi+ band makes ipl-406 distinct, placed at the extreme end of the phenogram. the next noticeable cultivars are ec-70404 and ec-267569-a with cma+/dapi+ secondary constriction (table 1) (figure 6). the group ii is large, composed of three subgroups mainly differentiated by nucleolar banding pattern in their marker chromosomes. the first subgroup comprised of 5 cultivars with neutral cmadapi bands in their satellites (type d) (table 1, figure 6). the second subgroup is largest, comprising of 13 cultivars with cma+/dapisatellite (type a). here, two cultivars (ec-70394 and barasat, macro type) show little distance from rest of the cultivars, because of different types of centromeric bands (table 1, figure 6). the third subgroup comprises of 7 cultivars with ‘b’ type nucleolar chromosomes. this subgroup shows heterogeneity because of variations in centromeric bands (table 1, figure 6). discussion cytogenetics of l.culinaris is traditionally acknowledged for species delimitations, crossing behavior, conservation and utilization of plant genetic resources (ladizinsky 1979; tadmor et al. 1987; ladizinsky et al. 1990; ladizinsky 1999; mishra et al. 2007). with the present approach, we have entered the modern karyotyping system to study chromosomal specialization in indian lentils. the diversity of fluorescent karyotypes can be indisputably attributed to the differences in underlying chromosomal heterochromatin of the samples since i) 69analysis of cma-dapi bands and preparation of fluorescent karyotypes in thirty indian cultivars of lens culinaris figure 2. somatic metaphase chromosomes of lens culinaris cultivars stained with dapi: (a) dpl-15, (b) dpl-62, (c) ipl-81, (d) ipl-406, (e) ipl-316, (f ) jl-1, (g) hul-57, (h) kls-210, (i) ec-70394, (j) ec-70403, (k) ec-70404, (l) ec-78452, (m) ec-78455, (n) ec-78461, (o) ec-78475, (p) ec-78498, (q) ec-78542-a, (r) ec–223188, (s) ec – 255491, (t) ec–267526, (u) ec-267569-a, (v) ec–267590, (w) ec–267877, (x) barasat micro type. white arrows indicate dapi+ bands and arrowheads indicate dapiand dapi0 bands. bars 5µm. 70 timir baran jha, biplab kumar bhowmick, partha roy figure 3. somatic metaphase chromosomes of lens culinaris cultivars. cma stained plates: (a) barasat macro type, (b) pl-1406, (c) ec-70306, (d) ec -78410, (e) ec-78451-a, (f ) ec-78473. dapi stained plates: (g) barasat macro, (h) pl-1406, (i) ec-70306, (j) ec -78410, (k) ec-78451-a, (l) ec-78473. white arrows indicate cma+ or dapi+ bands and arrowheads indicate cma0 or dapi0 and dapibands. bars 5µm. 71analysis of cma-dapi bands and preparation of fluorescent karyotypes in thirty indian cultivars of lens culinaris figure 4. fluorescent ideograms of lens culinaris cultivars based on cma/dapi banding pattern: (a) dpl-15, (b) dpl-62, (c) ipl-81, (d) ipl-406, (e) ipl-316, (f ) jl-1, (g) hul-57, (h) kls-210, (i) ec-70394, (j) ec-70403, (k) ec-70404, (l) ec-78452, (m) ec-78455, (n) ec-78461, (o) ec-78475, (p) ec-78498, (q) ec-78542-a, (r) ec–223188, (s) ec – 255491, (t) ec–267526, (u) ec-267569-a, (v) ec–267590, (w) ec–267877, (x) barasat micro type. cma+, dapi+, cma+/dapi+ and cma0 bands are highlighted with green, blue, red and grey colors on the chromosomes, respectively and the types are indicated above the chromosome diagrams. bars 5µm 72 timir baran jha, biplab kumar bhowmick, partha roy we have applied the same fluorochrome staining protocol for every cultivar, ii) the method is repeated a number of times before ascertaining banding pattern in a cultivar and iii) at least 5 best metaphase plates of each cultivar with scorable signals were considered for establishing the fluorescent karyotype. considering the nature of nucleolar chromosomes, molecular banding technique has shed light on chromosomal landmarks and possible differences in nors that were previously found to be similar in lens (mehra et al. 1986; jha et al. 2015, 2017; jha and halder 2016). the marker nucleolar chromosomes (4th, along with the 3rd, 2nd and 5th in few cases) have been confirmed with characteristic cma-dapi signals, corroborating to our previous report (jha et al. 2017). the cma+ signals are generally accepted as the gc heterochromatic elements of the nors in plant groups (guerra et al. 2000; barros e silva and guerra 2010; yamamoto 2012; olanj et al. 2015) and so in papilionoids such as vicia (fuchs et al. 1998), cicer (galasso et al. 1996) and crotalaria (mondin and aguiar-perecin 2011). previously, 18s-5.8s-25s rdna probes had been localised in a single pair of l. culinaris, near the centromere (balyan et al. 2002), corroborating to the observation of cma+ signals in our present study. however, we found that the intensity of the nucleolar cma signals (type a) varies in certain cultivars, suggesting differences in nors that influence affinity towards the stain. intraspecific rdna variation has been thoroughly worked out in phaseolus (moscone et al. 1999; pedrosa-harand et al. 2006) and vigna (bortoleti et al. 2012; she et al. 2015, 2020) of papilionoideae. a number of factors such as transposition, unequal crossing over, inversion or locus duplication, had been suggested to drive nor variation in plant groups, including papilionoideae (moscone et al. 1999; chung et al. 2008; raskina et al. 2008). we consider similar possibilities in the indian lentils, subject to future confirmation by agnor staining or rdna fish. the ty pe d chromosomes have satellites that respond indifferently to the cma stain. the cma0 satellites indicate gc neutral nature of heterochromatin (barros e silva and guerra 2010). the type d satellites are in sharp contrast to type a bands, marking cultivar distinction. the other unusual type was the cma+/ dapi+ satellites (type c). previously, the cma+/dapi+ satellites were suggested to be a ‘less common’ or ‘rare’ type of heterochromatin (barros e silva and guerra 2010), breaking the generality of gc rich composition of plant nors (schweizer 1976; guerra et al. 2000). we document the occurrence of cma+/dapi+ satellites for the first time in lens of papilionoideae. co-localized cma+/dapi+ satellites are so far reported in allium nigrum (maragheh et al. 2019) and cestrum (fernandes et al. 2009). it is difficult to ascertain the heterochromatin composition of this type. there is a possibility of having at and gc rich segments to be placed so close that the different chromatin bands cannot be distinguished in condensed mitotic chromosomes (maragheh et al. 2019). however, nucleolar heterochromatin composition of indian lens culinaris displays considerable variation, perhaps due to enormous cultivation practice and artificial hybridization, which is a yet unaddressed field of study. cultivar specific differences were also accentuated by the non-nucleolar dapi+ and cma+ bands. the type e centromeric cma bands are unique type figure 5. fluorescent ideograms of lens culinaris cultivars based on cma/dapi banding pattern: (a) barasat macro type, (b) pl-1406, (c) ec-70306, (d) ec -78410, (e) ec-78451-a, (f ) ec-78473. cma+, dapi+, cma+/dapi+ and cma0 bands are highlighted with green, blue, red and grey colors on the chromosomes, respectively and the types are indicated above the chromosome diagrams. bars 5µm. 73analysis of cma-dapi bands and preparation of fluorescent karyotypes in thirty indian cultivars of lens culinaris of heterochromatin rarely reported in plants. however, non-nucleolar gc-rich heterochromatin was previously characterized in centromeric as well as pericentromeric regions of papilionoid species belonging to dioclea (souza and benko-iseppon 2004), psophocarpus (chaowen et al. 2004), crotalaria (mondin and aguiar-perecin 2011), vigna (bortoleti et al. 2012; she et al. 2015, 2020), phaseolus (bonifácio et al. 2012), lablab (she and jiang 2015), and canavalia (she et al. 2017). she et al. (2020) suggested the centromeric or pericentromeric gcheterochromatin to be a relic of genomic evolution in the subfamily papilionoideae. other even rare heterochromatin blocks were the centromeric (type f), pericentromeric (type b) and intercalary (type g) dapi bands, constituting landmarks to differentiate karyotypes of certain lentil cultivars. terminal or intercalary dapi+ bands were documented in few plants (vanzela and guerra 2000; divashuk et al. 2014), including few species of cucurbitaceae (bhowmick and jha 2015, 2019). terminal dapi bands are found in crotalaria (mondin and aguiar-perecin 2011) of papilionoideae. centromeric dapi bands are yet rare to encounter. however, in case of l. culinaris and related species, at heterochromatic regions were mapped by repetitive sequence probe fish (galasso et al. 2001; galasso 2003). also in papilionoideae, at rich heterochromatin at centromere and pericentromeric regions are reported in vigna (bortoleti et al. 2012; she et al. 2020), lablab (she and jiang 2015) and figure 6. upgma dendrogram derived from average euclidean distance based on fluorochrome banding pattern of 30 indian lentil cultivars, cultivar names in the left of their serial numbers. 74 timir baran jha, biplab kumar bhowmick, partha roy ta bl e 1. a na ly si s of c m a a nd d a pi fl uo re sc en t b an ds in th ir ty i nd ia n cu lti va rs o f l en s cu lin ar is ( 2n = 1 4) . sl . n o. c ul tiv ar s o rd er o f nu cl eo la r pa ir c m a b an ds d a pi b an ds to ta l n o. of b an ds / 2n fl uo re sc en t k ar yo ty pe fo rm ul a (2 n) fi gu re no . n o. c hr om os om e pa ir /s ty pe */ in te ns ity fi gu re n o. n o. c hr om os om e pa ir /s ty pe */ in te ns ity fi gu re n o. 1 d pl 15 4t h 2 4t h d / ne ut ra l 1a 0 2a 2 2d +1 2h 4a 2 d pl -6 2 4t h 2 4t h a /lo w 1b 0 2b 2 2a +1 2h 4b 3 ip l -8 1 4t h 2 4t h a /lo w 1c 0 2c 2 2a +1 2h 4c 4 ip l40 6 3r d 2 3r d a /h ig h 1d 2 6t h g /h ig h 2d 4 2a +2 g +1 0h 4d 5 ip l -3 16 4t h 2 12 4t h 1s t -3 rd , 5 th -7 th a /h ig h e/ hi gh 1e 0 2e 14 2a +1 2e 4e 6 jl -1 4t h 2 4t h d / ne ut ra l 1f 0 2f 2 2d +1 2h 4f 7 h u l57 3r d 2 3r d b / lo w 1g 2 2 3r d 2n d b /lo w f/ hi gh 2g 4 2b +2 f+ 10 h 4g 8 k ls -2 10 4t h 2 4t h a /h ig h 1h 0 2h 2 2a +1 2h 4h 9 ec -7 03 94 5t h 2 2 5t h 4t h a /h ig h e/ hi gh 1i 0 2i 4 2a +2 e+ 10 h 4i 10 ec -7 04 03 4t h 2 4t h b /h ig h 1j 2 2 4t h 2n d b /h ig h f/ hi gh 2j 4 2b +2 f+ 10 h 4j 11 ec -7 04 04 4t h 2 4t h c /h ig h 1k 2 4t h c /lo w 2k 2 2c +1 2h 4k 12 ec -7 84 52 4t h 2 4t h d / ne ut ra l 1l 0 2l 2 2d +1 2h 4l 13 ec -7 84 55 4t h 2 4t h a /h ig h 1m 0 2m 2 2a +1 2h 4m 14 ec 78 46 1 4t h 2 4t h a /h ig h 1n 0 2n 2 2a +1 2h 4n 15 ec -7 84 75 3r d 2 3r d a /lo w 1o 0 2o 2 2a +1 2h 4o 16 ec – 78 49 8 4t h 2 4t h a /h ig h 1p 0 2p 2 2a +1 2h 4p 17 ec -7 85 42 -a 2n d 2 2n d b /h ig h 1q 2 2n d b /lo w 2q 2 2b +1 2h 4q 18 ec -2 23 18 8 4t h 2 4t h a /h ig h 1r 0 2r 2 2a +1 2h 4r 19 ec -2 55 49 1 4t h 2 4t h a /h ig h 1s 0 2s 2 2a +1 2h 4s 20 ec -2 67 52 6 4t h 2 4t h b /h ig h 1t 2 4 4t h 1s t , 3 rd b /lo w f/ lo w 2t 6 2b +4 f+ 8h 4t 21 ec -2 67 56 9a 3r d 2 3r d c /lo w 1u 2 3r d c /lo w 2u 2 2c +1 2h 4u 22 ec -2 67 59 0 4t h 2 4t h a /h ig h 1v 0 2v 2 2a +1 2h 4v 23 ec 2 67 87 7 4t h 2 2 4t h 5t h b /h ig h e/ lo w 1w 2 4t h b /h ig h 2w 4 2b +2 e+ 10 h 4w 24 b ar as at , m ic ro ty pe 3r d 2 3r d b /h ig h 1x 2 3r d b /lo w 2x 2 2b +1 2h 4x 25 b ar as at , m ac ro ty pe 4t h 2 4t h a /h ig h 3a 2 3r d f/ hi gh 3g 4 2a +2 f+ 10 h 5a 26 pl 14 06 4t h 2 4t h b /h ig h 3b 2 2 4t h 2n d b /lo w f/ lo w 3h 4 2b +2 f+ 10 h 5b 27 ec 7 03 06 4t h 2 4t h d / ne ut ra l 3c 0 3i 2 2d +1 2h 5c 28 ec 78 41 0 4t h 2 4t h b /h ig h 3d 2 12 4t h 1s t 3r d , 5t h 7t h b /h ig h f/ hi gh 3j 14 2b +1 2f 5d 29 ec – 7 84 51 -a 4t h 2 4t h a /h ig h 3e 0 3k 2 2a +1 2h 5e 30 ec 7 84 73 4t h 2 4t h d / ne ut ra l 3f 0 3l 2 2d +1 2h 5f *t yp es o f n uc le ol ar b an ds a : c m a + / d a pi sa te lli te , b : c m a + / d a pi s at el lit e an d d a pi + / c m a 0 ba nd in lo ng a rm , c : c m a + / d a pi + sa te lli te , d : c m a 0 sa te lli te ; c en tr om er ic b an ds e: c m a + / d a pi 0 , f: d a pi + / c m a 0 ; in te rc al ar y ba nd g : d a pi + / c m a 0 ; h : n o ba nd s. 75analysis of cma-dapi bands and preparation of fluorescent karyotypes in thirty indian cultivars of lens culinaris arachis (silvestri et al. 2020). nonetheless, occurrence of centromeric cma+ or dapi+ bands along with nucleolar cma+/dapi+ or cma0 bands certainly advocate atypical heterochromatin composition in lens. the non-uniform composition and rearrangements of heterochromatin had been observed repeatedly in papilionoideae species (moscone et al. 1999; souza and benko-iseppon 2004; pedrosa-harand et al. 2006; mondin and aguiarperecin 2011; she et al. 2020), which becomes apparent in our study once again. in view of the diversity in fluorochrome banding pattern, we attempted to resolve karyotype relationships by the upgma method. identification of distinct subgroups has opened further scopes to complement marker assisted analysis of genetic diversity across varied range of indian cultivars with valuable agronomic traits. application of fluorochrome banding method has therefore helped to i) break the perception of an overall similar karyotype of cultivated lentils as observed in giemsa plates (jha et al. 2015, 2017; jha and halder 2016) ii) serve as the chromosomal blueprint for cultivar discrimination, ii) statistically represent the status of chromosomal relationships, iii) highlight the uniqueness of certain indian cultivars by means of unconventional banding pattern, and v) construct a fluorescent karyotype dataset of indian lentil cultivars. conclusion being a crop ‘as old as agriculture’ (sandhu and singh 2007), an exclusive chromosomal database of lentils is essential to complement genomic research databases like legume information system (dash et al. 2016) and knowpulse (sanderson et al. 2019). as an extension of our study involving lentil cytogenetics, we have delved into the first molecular karyotypes of the country’s native cultivars. notably, the cultivars are hosted by world’s second largest ex situ lentil germplasm stock i.e. iipr of nbpgr, the first being icarda (muehlbauer and mcphee 2005; coyne and mcgee 2013). in future, molecular cytogenetic study of wild lens species of india can be expected to strengthen the base of chromosomal evolution in papilionoideae. in face of stern climatic changes that affect future cultivation, the indian cultivars with interesting karyotype features and relationships can be fluently tested for performance and productivity. thus, our findings complement traditional or marker assisted breeding and would undoubtedly bridge up the lacuna for a systematic chromosomal database of indian lentils. acknowledgements tbj acknowledges dr. s. dutta, principal, maulana azad college, kolkata for the facilities provided and indian institute of pulses 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mammalia; vespertilionidae) adriano silva dos santos1, karina de cassia faria2 analysis of cma-dapi bands and preparation of fluorescent karyotypes in thirty indian cultivars of lens culinaris timir baran jha1,*, biplab kumar bhowmick2, partha roy1 toxic and genotoxic effects of aqueous extracts of polygonum weyrichii fr. schmidt on the allium test taken as an example maria v. smirnova*, anna v. korovkina comparative cytogenetic analysis between species of auchenipterus and entomocorus (siluriformes, auchenipteridae) amanda de souza machado, samantha kowalski, leonardo marcel paiz, vladimir pavan margarido, daniel rodrigues blanco, paulo cesar venere, sandra mariotto, liano centofante, orlando moreira-filho, roberto laridondo lui* impact of bisphenol a on seed germination, radicle length and cytogenetic alterations in pisum sativum l. sazada siddqiui*, saad abdurahamn muhammad al amri, huda ahmed al ghamdy, wadha saad saeed alqahtani, sarah mohammed alquyr, habab merghani yassin first report on nucleolar organizer regions (nors) polymorphism and constitutive heterochromatin of moonlight gourami, trichopodus microlepis (perciformes, osphronemidae) patcharaporn chaiyasan1, sumalee phimphan2, teamjun sarasan1, sippakorn juntaree3, alongklod tanomtong1, sitthisak pinmongkhonkul4, weerayuth supiwong3,* morphological method and molecular marker determine genetic diversity and population structure in allochrusa kun zhu1,*, lijie liu1, shanshan li1, bo li1, majid khayatnezhad2, abdul shakoor3,4 genetic diversity and comparative study of genomic dna extraction protocols in tamarix l. species xiao cheng1,*, xiaoling hong1, majid khayatnezhad2, fazal ullah3,4 cytotoxic and genotoxic effects of methanol extracts of vegetative parts of some gypsophila l. species using allium cepa assay özgün tuna-gülören1, ferhan korkmaz2*, meltem erdir2, ebru ataşlar2 molecular techniques in the assessment of genetic relationships between populations of consolida (ranunculaceae) jing ma1, wenyan fan1,*, shujun jiang1, xiling yang1, wenshuai li1, di zhou1, amir abbas minaeifar2,* caryologia. international journal of cytology, cytosystematics and cytogenetics 74(1): 97-107, 2021 firenze university press www.fupress.com/caryologia issn 0008-7114 (print) | issn 2165-5391 (online) | doi: 10.36253/caryologia-968 caryologia international journal of cytology, cytosystematics and cytogenetics citation: s. ma, m. khayatnezhad, a. abbas minaeifar (2021) genetic diversity and relationships among hypericum l. species by issr markers: a high value medicinal plant from northern of iran. caryologia 74(1): 97-107. doi: 10.36253/ caryologia-968 received: june 14, 2020 accepted: september 24, 2021 published: july 20, 2021 copyright: © 2021 s. ma, m. khayatnezhad, a. abbas minaeifar. this is an open access, peer-reviewed article published by firenze university press (http://www.fupress.com/caryologia) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the author(s) declare(s) no conflict of interest. orcid sm: 0000-0002-9371-1498 genetic diversity and relationships among hypericum l. species by issr markers: a high value medicinal plant from northern of iran shuyan ma1,*, majid khayatnezhad2, amir abbas minaeifar3 1 tangshan vocational & technical college, department of agricalture and forestry engineering, hebei tangshan 063000, china 2 young researchers club, islamic azad university-ardabil branch, ardabil, iran 3 department of biology. payame noor university. p.o. box19395-3697 tehran. iran *corresponding author. e-mail: ngmashuyan@163.com; aaminaeifar@pnu.ac.ir abstract. hypericum l. species are generally known locally in iran with the names “hofariqun” which ebn sina (or bo ali sina) called it. plants of the genus hypericum have traditionally been used as medicinal plants in various parts of the world. hypericum perforatum l. is the source to one of the most manufactured and used herbal preparations in recent years, especially as a mild antidepressant. therefore, due to the importance of these plant species, we performed a molecular data for this species. for this study, we used 175 randomly collected plants from 17 species in 9 provinces. amplification of genomic dna using 10 primers produced 141 bands, of which 127 were polymorphic (95.78%). the obtained high average pic and mi values revealed high capacity of issr primers to detect polymorphic loci among hypericum species. the genetic similarities of 17 collections were estimated from 0.617 to 0.911. according to inter-simple sequence repeats (issr) markers analysis, h. androsaemum and h. hirtellum had the lowest similarity and the species of h. perforaturm and h. triquetrifolium had the highest similarity. the aims of present study are: 1) can issr markers identify hypericum species, 2) what is the genetic structure of these taxa in iran, and 3) to investigate the species inter-relationship? the present study revealed that issr markers can identify the species. keywords: iran, species identification, structure, hypericum, issr markers. introduction identifying the accurate boundaries of a species is critical to have a better perspective of any biological studies. therefore, species delimitation is a subject of extensive part of studies in the framework of biology (collard & mackill 2009, wu et al. 2013). however, defining the criterion which could address the boundaries of species is different and the place of debates (esfandani-bozchaloyi et al. 2018a, 2018b, 2018c, 2018d). wild relatives of crops contain genes with the great potential for use in breeding programs and constitute a part of their gene pool (pandey et al. 2008). in addition, the study of 98 shuyan ma, majid khayatnezhad, amir abbas minaeifar intra-specific levels of genetic variation and investigation of genetic structure of wild populations is crucial for development of effective conservation strategies. the genus hypericum (guttiferae, hypericoideae) is perennial, belonging to the hypericaceae family, having 484 species in forms of trees, shrubs, and herbs, distributed in 36 taxonomic sections (crockett and robson 2011). the species of the family are distributed worldwide in the temperate zones but are absent in extreme environmental conditions such as deserts and poles. iranian species of this genus grow mainly in north, northwest and center of iran and form floristic elements of hyrcanian mountainous areas, irano-turanian, mediterranean and zagros elements. they generally prefer steep slopes of rocky and calcareous cliffs and margin of mountainous forests (robson 1968; azadi 1999). robson (1968) introduced 21 species in the area covered by flora iranica. robson (1977) and assadi (1984) reported h. fursei n. robson and h. dogonbadanicum assadi as two endemics of north and south west of iran. in flora of iran, azadi (1999) identified 19 species, 4 subspecies arranged in 5 sections (comprising campylosporus (spach) r. keller, hypericum, hirtella stef., taeniocarpum jaub. & spach. and drosanthe (spach) endl.), and two doubtful species including h. heterophyllum vent. and h. olivieri (spach) boiss. hypericum species are generally known locally in iran with the names “hofariqun” which ebn sina (or bo ali sina) called it (rechinger, 1986). st. john’s wort (hypericum perforatum l.) is the most important medicinal species of the genus and its main uses in medicine includes treatment of mild and moderate depression, skin wounds and burns (barnes et al. 2001). the plant contains a vast array of secondary metabolites, among which naphthodianthrones (hypericin and pseudohypericin), acylphloroglucinols (hyperforin and adhyperforin) and essential oil can be mentioned (morshedloo et al. 2012; radusiene et al. 2005). molecular markers provide a powerful tool for studying the genetic diversity. among advanced genetic markers, random amplified polymorphic dna (rapd) and inter simple sequence repeats (issr) markers have been widely used for diversity analyses (pharmawati et al. 2004). rapd technique is quick, easy and requires no prior sequence information. the technique detects nucleotide sequence polymorphism using a single primer of arbitrary nucleotide sequence (moreno et al., 1998). issr marker involves pcr amplification of dna by a single 16-18 bp. long primer composed of a repeated sequence anchored at the 3’ or 5’ end of 2-4 arbitrary nucleotides. the technique is rapid, simple, inexpensive and more reproducible than rapd (esfandani-bozchaloyi et al. 2017a, 2017b, 2017c, 2017d), (collard & mackill 2009, wu et al. 2013). the present investigation has been carried out to evaluate the genetic diversity and relationships among different hypericum species using new gene-targeted molecular markers, i.e issr markers . this is the first study on the use of issr markers in hypericum genus; therefore, we performed molecular study of 175 collected specimens of 17 hypericum species. we try to answer the following questions: 1) is there infra and interspecific genetic diversity among studied species? 2) is genetic distance among these species correlated with their geographical distance? 3) what is the genetic structure of populations and taxa? 4) is there any gene exchange between hypericum species in iran? materials and methods plant materials a total of 175 individuals were sampled representing 17 geographical populations belong 17 hypericum species in east azerbaijan, lorestan, kermanshah, guilan, mazandaran, esfahan, tehran, hamadan and kohgiluyeh and boyer-ahmad provinces of iran during julyagust 2016-2019 (table 1). for issr analysis we used 175 plant accessions (five to twelve samples from each populations) belonging to 17 different populations with different eco-geographic characteristics were sampled and stored in -20 till further use. more information about geographical distribution of accessions are in table 1 and fig. 1. morphological studies five to twelve samples from each species were used for morphometry. in total 18 morphological (11 qualitative, 7 quantitative) characters were studied. data obtained were standardized (mean= 0, variance = 1) and used to estimate euclidean distance for clustering and ordination analyses (podani 2000). morphological characters studied are: corolla shape, bract shape, calyx shape, calyx length, calyx width, calyx apex, calyx margins, bract length, corolla length, corolla width, corolla apex, leaf length and leaf width, leaf apex, leaf margins, leaf shape, leaf gland and bract margins. dna extraction and issr assay fresh leaves were used randomly from one to twelve plants in each of the studied populations. these were dried by silica gel powder. ctab activated char99genetic diversity and relationships among hypericum l. species by issr markers coal protocol was used to extract genomic dna (esfandani-bozchaloyi et al. 2019). the quality of extracted dna was examined by running on 0.8% agarose gel. for the issr analysis, 22 primers from ubc (university of british columbia) series were tested for dna amplification. ten primers were chosen for issr analysis of genetic variability, based on band reproducibly (table 2). pcr reactions were carried in a 25μl volume containing 10 mm tris-hcl buffer at ph 8; 50 mm kcl; 1.5 mm mgcl2; 0.2 mm of each dntp (bioron, germany); 0.2 μm of a single primer; 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). the amplifications, reactions were performed in techne thermocycler (germany) with the following program: 5 min initial denaturation step 94°c, followed by 40 cycles of 1 min at 94°c; 1 min at 52-57°c and 2 min at 72°c. the reaction was completed by final extension step of 7-10 min at 72°c. the amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated by using a 100 bp molecular size ladder (fermentas, germany). table 1. voucher details of hypericum species in this study from iran. no section sp. locality latitude longitude altitude (m) sp1 campylosporus (spach) r. keller h. dogonbadanicum assadi kohgiluyeh and boyer-ahmad 38°52’37” 47°23’92” 1144 sp2 androsaemum (duhamel) godron h. androsaemum l. mazandaran, haraz road, emam zad-e-hashem 32°50’03” 51°24’28” 1990 sp3 hypericum h. tetrapterum fries. guilan, sangar, road sid 29°20’07” 51° 52’08” 1610 sp4 h. perforaturn l. esfahan:, ghameshlou, sanjab 38°52’37” 47°23’92” 1144 sp5 h. triquetrifolium turra lorestan, oshtorankuh, above tihun village 33°57’12” 47°57’32” 2500 sp6 hirtella stef. h. lysimachioides boiss. & noe in boiss. kermanshah, islamabad 34°52’37” 48°23’92” 2200 sp7 h. asperulum jaub. & spach. hamedan, nahavand 38°52’37” 47°23’92” 1144 sp8 h. scabrum l. azerbaijan, 78 km from mianeh to khalkhl. 35°50’03” 51°24’28” 1700 sp9 h. hirtellum (spach) boiss. lorestan, durood 36°14’14” 51°18’07” 1807 sp10 h. elongaturn ledeb. guilan, lahijan 32°36’93” 51°27’90” 2500 sp11 h. davisii n. robson east azerbaijan, arasbaran 37°07’02” 49°44’32” 48 sp12 h. apricum kar. & kir. azarbaiejan, 48 km from tabriz to marand 28°57’22” 51°28’31” 430 sp13 h. helianthemoides (spach) boiss. tehran, damavand 30°07’24” 53°59’06” 2178 sp14 h. vermiculare boiss. & hausskn hamedan, alvand 28°57’22” 51°28’31” 288 sp15 taeniocarpium h. hirsutum l. mazandaran, nowshahr 34°46’10” 48°30’00” 1870 sp16 h. linarioides bosse. azarbaiejan, west of tabriz 35°37’77” 46°20’25” 1888 sp17 h. armennm jaub. & spach, mazandaran, chalos 33°47’60” 46°07’58” 1250 figure 1. map of iran shows the collection sites and provinces where hypericum species were obtained for this study; sp1= h. dogonbadanicum; sp2= h. androsaemum; sp3= h. tetrapterum; sp4= h. perforaturm; sp5= h. triquetrifolium; sp 6= h. lysimachioides; sp7= h. asperulum; sp8= h. scabrum; sp9= h. hirtellum; sp10: h. elongaturn ; sp11: h. davisii ; sp12= h. apricum; sp13= h. helianthemoides ; sp14= h. vermiculare; sp15= h. hirsutum; sp16= h. linarioides; sp17= h. armennm. 100 shuyan ma, majid khayatnezhad, amir abbas minaeifar data analyses morphological studies morphological characters were f irst standardized (mean = 0, variance = 1) and used to establish euclidean distance among pairs of taxa (podani 2000). for grouping of the plant specimens, the upgma (unweighted paired group using average) ordination methods were used (podani 2000). anova (analysis of variance) were performed to show morphological difference among the populations while, pca (principal components analysis) biplot was used to identify the most variable morphological characters among the studied populations (podani 2000). past version 2.17 (hammer et al. 2012) was used for multivariate statistical analyses of morphological data. molecular analyses issr bands obtained were coded as binary characters (presence = 1, absence = 0) and used for genetic diversity analysis. discriminatory ability of the used primers was evaluated by means of two parameters, polymorphism information content (pic) and marker index (mi) to characterize the capacity of each primer to detect polymorphic loci among the genotypes (powell et al. 1996). mi is calculated for each primer as mi = pic × emr, where emr is the product of the number of polymorphic loci per primer (n) and the fraction of polymorphic fragments (β) (heikrujam et al. 2015). the number of polymorphic bands (npb) and the effective multiplex ratio (emr) were calculated for each primer. parameter like nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism (p% = number of polymorphic loci/number of total loci) were determined (weising et al, 2005, freeland et al. 2011). shannon’s index was calculated by the formula: h’ = -σpiln pi. rp is defined per primer as: rp = ∑ ib, were “ib” is the band informativeness, that takes the values of 1-(2x [0.5-p]), being “p” the proportion of each genotype containing the band. the percentage of polymorphic loci, the mean loci by accession and by population, uhe, h’ and pca were calculated by genalex 6.4 software (peakall & smouse 2006). nei’s genetic distance among populations was used for neighbor joining (nj) clustering and neighbor-net networking (freeland et al. 2011, huson & bryant 2006). mantel test checked the correlation between geographical and genetic distances of the studied populations (podani 2000). these analyses were done by past ver. 2.17 (hammer et al. 2012), darwin ver. 5 (2012) software. amova (analysis of molecular variance) test (with 1000 permutations) as implemented in genalex 6.4 (peakall & smouse 2006) were used to show genetic difference of the populations. gene flow was determined by (i) calculating nm an estimate of gene flow from gst by popgene ver. 1.32 (1997) as: nm = 0.5(1 gst)/gst. this approach considers the equal amount of gene flow among all populations. results species identification and inter-relationship morphometry anova showed significant differences (p <0.01) in quantitative morphological characters among the species studied. in order to determine the most variable characters among the taxa studied, pca analysis has been performed. it revealed that the first three factors comprised over 73% of the total variation. in the first pca axis with 57% of total variation, such characters as corolla shape, calyx shape, calyx length, bract length and leaf shape have shown the highest correlation (>0.7), leaf apex, corolla length, leaf length, leaf width were characters influencing pca axis 2 and 3 respectively. different clustering and ordination methods produced similar results therefore, pca plot of morphological characters are presented here (fig. 2). in general, plant samples of each species were grouped together and formed separate groups. this result show that both quantitative and qualitative morphological characters separated the studied species into distinct groups. in the studied specimens we did not encounter intermediate forms. species identification and genetic diversity ten issr primers were screened to study genetic relationships among hypericum species; all the primers produced reproducible polymorphic bands in all 17 hypericum species. an image of the issr amplification generated by issr-5 primer is shown in figure 3. a total of 127 amplified polymorphic bands were generated across 17 hypericum species. the size of the amplified fragments ranged from 200 to 3000 bp. the highest and lowest number of polymorphic bands was 18 for issr-2 and 7 for issr-6, on an average of 12.7 polymorphic bands per primer. the pic of the 10 issr primers ranged from 0.23 (issr-3) to 0.44 (issr-6) with an aver101genetic diversity and relationships among hypericum l. species by issr markers age of 0.36 per primer. mi of the primers ranged from 1.37 (issr-9) to 4.47 (issr-1) with an average of 3.8 per primer. emr of the issr primers ranged from 4.60 (issr-6) to 11.11 (issr-9) with an average of 8.9 per primer (table 2). the primers with the high emr values were considered to be more informative in distinguishing the genotypes. the genetic parameters were calculated for all the 17 hypericum species amplified with issr primers (table 3). unbiased expected heterozygosity (h) ranged from 0.10 (h. hirsutum) to 0.31 (h. elongaturn), with a mean of 0.21. a similar pattern was observed for shannon’s information index (i), with the highest value of 0.33 observed in h. elongaturn and the lowest value of 0.13 observed in h. hirsutum with a mean of 0.23. the observed number of alleles (na) ranged from 0.23 in h. linarioides to 0.56 in h. apricum. the effective number of alleles (ne) ranged from 1.01 (h. scabrum) to 1.38 (h. elongaturn). amova test showed significant genetic difference (p = 0.001) among studied species. it revealed that 63% of total variation was among species and 37% was within species (table 4) moreover, genetic differentiation of these species was demonstrated by significant nei’s gst (0.31, p = 0.001) and d_est values (0.167, p = 0.001). these results revealed a higher distribution of genetic diversity among hypericum species compared to within species. different clustering and ordination methods produced similar results therefore, upgma clustering are presented here (figure 4). in general, plant samples of each species belong to a distinct section, were grouped together and formed separate cluster. this result show figure 2. pca plots of morphological characters revealing species delimitation in th hypericum species; sp1= h. dogonbadanicum; sp2= h. androsaemum; sp3= h. tetrapterum; sp4= h. perforaturm; sp5= h. triquetrifolium; sp 6= h. lysimachioides; sp7= h. asperulum; sp8= h. scabrum; sp9= h. hirtellum; sp10: h. elongaturn ; sp11: h. davisii ; sp12= h. apricum; sp13= h. helianthemoides ; sp14= h. vermiculare; sp15= h. hirsutum; sp16= h. linarioides; sp17= h. armennm. figure 3. electrophoresis gel of studied ecotypes from dna fragments produced by issr-7. sp1= h. dogonbadanicum; sp2= h. androsaemum; sp3= h. tetrapterum; sp4= h. perforaturm; sp5= h. triquetrifolium; sp 6= h. lysimachioides; sp7= h. asperulum; sp8= h. scabrum; sp9= h. hirtellum; sp10: h. elongaturn ; sp11: h. davisii ; sp12= h. apricum; sp13= h. helianthemoides ; sp14= h. vermiculare; sp15= h. hirsutum; sp16= h. linarioides; sp17= h. armennm. 102 shuyan ma, majid khayatnezhad, amir abbas minaeifar that molecular characters studied can delimit hypericum species in two different major clusters or groups. in the studied specimens we did not encounter intermediate forms. in general, two major clusters were formed in upgma tree (figure. 4), populations of h. dogonbadanicum (sect. campylosporus) and h. vermiculare (sect. hirtella) were placed in the first major cluster and were placed with great distance from the other species. the second major cluster included two sub-clusters. plants of h. perforaturm and h. triquetrifolium (sect. hypericum) and h. androsaemum (sect. androsaemum) comprised the first sub-cluster, while plants of h. lysimachioides; h. asperulum; h. scabrum; h. hirtellum; h. elongaturn; h. davisii; h. apricum; h. helianthemoides (sect. hirtella) formed the second sub-cluster. in general, relationships obtained from issr data agrees well with species relationship obtained from morphological. this is in agreement with amova and table 3. genetic diversity parameters in the studied hypericum species. sp n na ne i he uhe %p h. dogonbadanicum 8.000 0.499 1.067 0.18 0.171 0.14 49.26% h. androsaemum 9.000 0.261 1.024 0.192 0.23 0.23 43.15% h. tetrapterum 6.000 0.555 1.021 0.29 0.25 0.18 43.53% h. perforaturn 10.000 0.431 1.088 0.23 0.22 0.23 57.53% h. triquetrifolium 3.000 0.255 1.021 0.15 0.18 0.12 42.15% h. lysimachioides 3.000 0.288 1.024 0.23 0.25 0.27 64.30% h. asperulum 9.000 0.352 1.083 0.23 0.22 0.14 45.05% h. scabrum 8.000 0.333 1.016 0.192 0.12 0.22 48.23% h. hirtellum 12.000 0.247 1.199 0.271 0.184 0.192 55.91% h. elongaturn 5.000 0.358 1.380 0.334 0.30 0.31 66.50% i. davisii 6.000 0.299 1.029 0.231 0.18 0.23 44.38% h. apricum 3.000 0.567 1.062 0.24 0.224 0.213 44.73% h. helianthemoides 8.000 0.499 1.067 0.14 0.181 0.14 49.26% h. vermiculare 9.000 0.261 1.034 0.142 0.13 0.13 33.15% h. hirsutum 6.000 0.545 1.021 0.13 0.10 0.10 23.53% h. linarioides 6.000 0.234 1.032 0.26 0.23 0.18 45.53% h. armennm 8.000 0.499 1.067 0.19 0.191 0.14 39.26% abbreviations: (n = number of samples, na= number of different alleles; ne = number of effective alleles, i= shannon’s information index, he = gene diversity, uhe = unbiased gene diversity, p%= percentage of polymorphism, populations). table 2. issr primers used for this study and the extent of polymorphism. primer name primer sequence (5’-3’) tnb npb ppb pic pi emr mi issr-1 dbdacacacacacacaca 12 12 100.00% 0.26 5.86 8.55 2.45 issr-2 ggatggatggatggat 10 9 84.99% 0.23 2.91 7.43 3.85 issr-3 gacagacagacagaca 15 15 100.00% 0.44 3.34 10.55 2.44 issr-4 agagagagagagagagyt 10 10 100.00% 0.37 3.88 6.56 1.85 issr-5 acacacacacacacacc 18 18 100.00% 0.35 5.23 6.23 4.47 issr-6 gagagagagagagagarc 15 14 93.74% 0.37 4.66 5.56 3.67 issr-7 ctctctctctctctctg 13 12 92.31% 0.34 4.21 4.60 3.55 issr-8 cacacacacacacacag 13 13 100.00% 0.27 3.32 9.55 3.45 issr-9 gtgtgtgtgtgtgtgtyg 11 7 82.89% 0.43 5.56 6.34 3.11 issr-10 cacacacacacacacarg 17 17 100.00% 0.29 3.25 11.11 1.37 mean 14.1 12.7 95.78% 0.36 4.8 8.9 3.8 total 141 127 note: tnb the number of total bands, npb: the number of polymorphic bands, ppb (%): the percentage of polymorphic bands, pi: polymorphism index, emr, effective multiplex ratio; mi, marker index; pic, polymorphism information content for each of cbdp primers 103genetic diversity and relationships among hypericum l. species by issr markers genetic diversity parameters presented before. the species are genetically well differentiated from each other. these results indicate that issr molecular markers can be used in hypericum species taxonomy. the nm analysis by popgene software also produced mean nm= 0.123, that is considered very low value of gene flow among the studied species. mantel test with 5000 permutations showed a significant correlation (r = 0.23, p=0.0002) between genetic distance and geographical distance, so isolation by distance (ibd) occurred among the hypericum species studied. nei’s genetic identity and the genetic distance determined among the studied species (table 5). the results showed that the highest degree of genetic similarity figure 4. upgma tree of issr data revealing species delimitation in the hypericum. table 4. analysis of molecular variance (amova) of the studied species. source df ss ms est. var. % φpt among pops 28 1901.364 73.789 12.154 63% 63% within pops 129 234.443 3.805 2.888 37% total 144 1955.807 13.060 100% df: degree of freedom; ss: sum of squared observations; ms: mean of squared observations; ev: estimated variance; φpt: proportion of the total genetic variance among individuals within an accession, (p < 0.001). table 5. the matrix of nei genetic similarity (gs) estimates using issr molecular markers among 17 hypericum species.sp1= h. dogonbadanicum; sp2= h. androsaemum; sp3= h. tetrapterum; sp4= h. perforaturm; sp5= h. triquetrifolium; sp 6= h. lysimachioides; sp7= h. asperulum; sp8= h. scabrum; sp9= h. hirtellum; sp10: h. elongaturn ; sp11: h. davisii ; sp12= h. apricum; sp13= h. helianthemoides ; sp14= h. vermiculare; sp15= h. hirsutum; sp16= h. linarioides; sp17= h. armennm. sp1 sp2 sp3 sp4 sp5 sp6 sp7 sp8 sp9 sp10 sp11 sp12 sp13 sp14 sp15 sp16 sp17 sp1 1.000 sp2 0.742 1.000 sp3 0.786 0.833 1.000 sp4 0.767 0.836 0.842 1.000 sp5 0.823 0.823 0.786 0.911 1.000 sp6 0.781 0.766 0.767 0.757 0.793 1.000 sp7 0.749 0.683 0.823 0.759 0.836 0.862 1.000 sp8 0.681 0.776 0.727 0.728 0.834 0.750 0.799 1.000 sp9 0.817 0.610 0.746 0.796 0.768 0.675 0.727 0.728 1.000 sp10 0.715 0.884 0.800 0.709 0.720 0.681 0.746 0.796 0.680 1.000 sp11 0.645 0.754 0.785 0.676 0.829 0.733 0.800 0.709 0.820 0.721 1.000 sp12 0.745 0.757 0.741 0.758 0.816 0.740 0.785 0.676 0.725 0.635 0.839 1.000 sp13 0.666 0.737 0.890 0.722 0.719 0.853 0.741 0.758 0.834 0.750 0.799 0.642 1.000 sp14 0.649 0.807 0.799 0.755 0.812 0.774 0.990 0.722 0.768 0.675 0.727 0.728 0.684 1.000 sp15 0.617 0.782 0.744 0.636 0.834 0.750 0.799 0.755 0.720 0.681 0.746 0.796 0.676 0.722 1.000 sp16 0.778 0.702 0.757 0.703 0.778 0.691 0.744 0.636 0.829 0.733 0.800 0.709 0.770 0.754 0.770 1.000 sp17 0.641 0.814 0.800 0.681 0.710 0.688 0.757 0.703 0.816 0.740 0.785 0.676 0.699 0.756 0.735 0.778 1.000 104 shuyan ma, majid khayatnezhad, amir abbas minaeifar (0.91) occurred between h. perforaturm and h. triquetrifolium. the lowest degree of genetic similarity occurred between h. androsaemum and h. hirtellum (0.61). the low nm value (0.123) indicates limited gene f low or ancestrally shared alleles between the species studied and indicating high genetic differentiation among and within hypericum species. discussion genetic diversity is an important role in biology of long-term evolution of a taxon or a population. the basis of existence, growth, and evolution of taxon. thus, the study of genetic diversity of taxon is fundamental to recognize the taxonomy, origin, and evolution of taxon. moreover, such research will provide a theoretical basis for the germplasm resource conservation, development, utilization, and breeding (lubbers et al., 1991). the present research, revealed interesting data about its genetic variability, genetic stratification and morphological divergence in north and west part of iran. degree of genetic variability within a species is highly correlated with its reproductive mode, the higher degree of open pollination/ cross breeding brings about higher level of genetic variability in the studied taxon (meusel et al., 1965). pic and mi characteristics of a primer help in determining its effectiveness in genetic diversity analysis. sivaprakash et al. (2004) suggested that the ability of a marker technique to resolve genetic variability may be more directly related to the degree of polymorphism. generally, pic value between zero to 0.25 suggest a very low genetic diversity among genotypes, between 0.25 to 0.50 shows a mid-level of genetic diversity and value ≥0.50 suggests a high level of genetic diversity (tams et al., 2005). in this research, the issr primers’ pic values ranged from 0.23 to 0.44, with a mean value of 0.36, which indicated a mid-level ability of issr primers in determining genetic diversity among the species of hypericum. all of 10 primer pairs showed a good polymorphism in taxon of hypericum. a total 141 alleles were recognized for the studied species. total number of bands per primers ranged from 7 to 18 polymorphic bands and the mean of the allele number in loci was 12.7. in most studies, population size is limited to several vegetative accession (meusel et al., 1965; uotila, 1996). this population could be showed genetic drift, whose effect are observed in the high level of fis and low level of genetic diversity. the isolation of the population and absence the gene flow led to fragmentation of the hypericum populations. between genetic diversity parameters and population size were showing positive correlations that confirmed various studies (leimu et al. 2006). there are two reasons for the positive correlation between genetic diversity and population size (leimu et al., 2006). 1a positive correlation could imply the presence of an extinction vortex, where the drop-in population size lowers genetic diversity, which leads to inbreeding depression. the second reason is the fact that plant fitness differentiates populations based on variations in habitat quality (vergeer et al., 2003). according to booy et al. (2000) the low levels of genetic diversity could reduce plant fitness and restrict a population’s ability to respond to changing environmental conditions through selection and adaptation. genetic diversity (37%) was obtained within populations, whereas 63% of genetic variation obtained between the evaluated populations. one of the key factors determining the distribution of genetic variation is the breeding system in plant species (duminil, 2007). couvet (booy et al., 2000) revealed that one migrant per generation cannot be existed to guarantee long-term survival of small populations and that the number of migrants is demonstrate through life history characters and population genetic (vergeer et al., 2003). genetic variances between the three groups were very similar, but statistically important. there are two hypotheses for the absence of differences between isolated populations. the first hypothesis explained that genetic diversity within and between populations demonstrate gene flow processes, which led to the fragmentation of larger populations (dostálek et al., 2010). the second hypothesis presented that geographically proximate populations are more efficiently connected through gene flow than populations separated by greater distance. a high level of variation among h. perforatum populations was also reported by percifield et al. (2007) which confirms results of the present study. similar results have been reported on this species using the rapd markers by hazler pilepic et al. (2008). the high genetic diversity of h. perforatum populations is as a result of its mating systems. in fact, propagation method(s) of plant species is considered as one of the most important factors determining their levels of genetic diversity (hamrick 1982; hamrick and godt 1989). self-incompatibility is a wide spread phenomenon in the genus hypericum (robson 1981), resulting in the high levels of genetic variability (borba et al. 2001). furthermore, this perennial plant produces a great number of seeds every year in favor of the high amounts of diversity in this species (zhao et al. 2007). since widespread species may possess the higher levels of genetic diversity than narrowly distributed plants (hamrick and godt 1996; singh et al. 1998), the wide 105genetic diversity and relationships among hypericum l. species by issr markers range of h. perforatum distribution is an important factor in this respect. considering the low level of gene flow rate among studied wild populations of h. perforatum, therefore, genetic drift might be inevitable. in h. perforatum, the low rate of gene flow may be due to factors such as prevailing apomixes and short distance of seed dispersal as stated by hazler pilepic et al. (2008). molecular markers have been used to investigate the genetic diversity, population structure, and reproductive biology of h. perforatum (arnholdt-schmitt, 2000; haluŝková and koŝuth, 2003; barcaccia et al., 2006; percifield et al., 2007). however, due to the lack of a specific marker system for these plants, most of the studies used marker systems such as rapd and issr. in the present work, we took advantage of the ubiquity and abundance of issr method in plant genomes and their role in genomic diversification to develop and apply retrotransposon markers based on the issr method for the first time to hypericum. high among-population variation was previously reported in hypericum species by percifield et al. (2007), pilepić et al. (2008), and farooq et al. (2014). high differentiation among populations is mostly coupled with limited gene flow among them. the low gene flow and the high differentiation among populations has been explained mainly by founder events such as time since colonization (jacquemyn et al., 2004). in conclusion, the results of this study showed that to evaluate the genetic diversity of the hypericum genus, the primers derived from issr were more effective than the other molecular markers. also, hypericum species were clearly separated from each other in the dendrogram and pca, indicating the higher efficiency of issr technique in hypericum species identification. acknowledgment the authors thank anonymous reviewers for valuable comments on an earlier draft. references arnholdt-schmitt b (2000). rapd analysis: a method to investigate aspects of the reproductive biology of hypericum perforatum l. theor appl genet 100: 906–911. azadi, r. 1999: guttiferae. in: assadi, m. 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